Tissue Engineering of Cartilage and Bone (Novartis Foundation Symposium 249)

Novartis Foundation Symposium 249 TISSUE ENGINEERING OF CARTILAGE AND BONE 2003 TISSUE ENGINEERING OF CARTILAGE AND ...

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Novartis Foundation Symposium 249

TISSUE ENGINEERING OF CARTILAGE AND BONE

2003


The Novartis Foundation is an international scienti¢c and educational charity (UK Registered Charity No. 313574). Known until September 1997 as the Ciba Foundation, it was established in 1947 by the CIBA company of Basle, which merged with Sandoz in 1996, to form Novartis. The Foundation operates independently in London under English trust law. It was formally opened on 22 June 1949. The Foundation promotes the study and general knowledge of science and in particular encourages international co-operation in scienti¢c research. To this end, it organizes internationally acclaimed meetings (typically eight symposia and allied open meetings and 15^20 discussion meetings each year) and publishes eight books per year featuring the presented papers and discussions from the symposia. Although primarily an operational rather than a grant-making foundation, it awards bursaries to young scientists to attend the symposia and afterwards work with one of the other participants. The Foundation’s headquarters at 41 Portland Place, London W1B 1BN, provide library facilities, open to graduates in science and allied disciplines. Media relations are fostered by regular press conferences and by articles prepared by the Foundation’s Science Writer in Residence. The Foundation o¡ers accommodation and meeting facilities to visiting scientists and their societies.

Information on all Foundation activities can be found at http://www.novartisfound.org.uk

Novartis Foundation Symposium 249


2003

Copyright & Novartis Foundation 2003 Published in 2003 by John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester,West Sussex PO19 8SQ, UK 01243 779777 National International (+44) 1243 779777 e-mail (for orders and customer service enquiries): [email protected] Visit our Home Page on http://www.wileyeurope.com or http://www.wiley.com All Rights Reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning or otherwise, except under the terms of the Copyright, Designs and Patents Act 1988 or under the terms of a licence issued by the Copyright Licensing Agency Ltd, 90 Tottenham Court Road, London W1T 4LP, UK, without the permission in writing of the Publisher. Requests to the Publisher should be addressed to the Permissions Department, John Wiley & Sons Ltd,The Atrium, Southern Gate, Chichester,West Sussex PO19 8SQ, England, or emailed to [email protected], or faxed to (+44) 1243 770620. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold on the understanding that the Publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional should be sought. Other Wiley Editorial O⁄ces John Wiley & Sons Inc., 111 River Street, Hoboken, NJ 07030, USA Jossey-Bass, 989 Market Street, San Francisco, CA 94103-1741, USA Wiley-VCH Verlag GmbH, Boschstr. 12, D-69469 Weinheim, Germany John Wiley & Sons Australia Ltd, 33 Park Road, Milton, Queensland 4064, Australia John Wiley & Sons (Asia) Pte Ltd, 2 Clementi Loop #02-01, Jin Xing Distripark, Singapore 129809 John Wiley & Sons Canada Ltd, 22 Worcester Road, Etobicoke, Ontario, Canada M9W 1L1 Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books. Novartis Foundation Symposium 249 ix+251 pages, 53 ¢gures, 6 tables Library of Congress Cataloging-in-Publication Data Tissue engineering of cartilage and bone / [editors, Gregory Bock, Jamie Goode]. p. cm. ^ (Novartis Foundation symposium ; 249) Includes bibliographical references and index. ISBN 0-470-84481-7 (alk. paper) 1. Bone regeneration. 2. Joints ^Diseases. 3. Cartilage ^Diseases. 4. Animal cell biotechnology. 5. Tissue culture. I. Bock, Gregory. II. Goode, Jamie. III. Series. RC930.T575 2003 617.4’72^dc21 2003041126 British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library ISBN 0 470 84481 7 Typeset in 1012 on 1212 pt Garamond by DobbieTypesetting Limited, Tavistock, Devon. Printed and bound in Great Britain by Biddles Ltd, Guildford and King’s Lynn.

This book is printed on acid-free paper responsibly manufactured from sustainable forestry, in which at least two trees are planted for each one used for paper production.

Contents Symposium onTissue engineering of cartilage and bone, held atthe Novartis Foundation, London, 9^11April 2002 This symposium is based on a proposal made by Anthony Hollander Editors: Gregory Bock (Organizer) and Jamie Goode Arnold I. Caplan

Chair’s introduction 1

L. Stefan Lohmander Tissue engineering of cartilage: do we need it, can we do it, is it good and can we prove it? 2 Discussion 10 Arnold I. Caplan Embryonic development and the principles of tissue engineering 17 Discussion 25 GordanaVunjak-Novakovic The fundamentals of tissue engineering: sca¡olds and bioreactors 34 Discussion 46 Jennifer H. Lee, John Kisiday and Alan J. Grodzinsky Tissue-engineered versus native cartilage: linkage between cellular mechano-transduction and biomechanical properties 52 Discussion 64 Ernst B. Hunziker Discussion 78

From the preclinical model to the patient 70

Frank P. Barry Mesenchymal stem cell therapy in joint disease Discussion 96

86

J. Huckle, G. Dootson, N. Medcalf, S. McTaggart, E.Wright, A. Carter, R. Schreiber, B. Kirby, N. Dunkelman, S. Stevenson, S. Riley,T. Davisson and A. Ratcli¡e Di¡erentiated chondrocytes for cartilage tissue engineering 103 Discussion 112

vi

CONTENTS

Hajime Ohgushi, Jun Miyake and TetsuyaTateishi Mesenchymal stem cells and bioceramics: strategies to regenerate the skeleton 118 Discussion 127 Ranieri Cancedda, Maddalena Mastrogiacomo, Giordano Bianchi, Anna Derubeis, Anita Muraglia and Rodolfo Quarto Bone marrow stromal cells and their use in regenerating bone 133 Discussion 143 R. J. Dekker, C. A. van Blitterswijk, I. Ho£and, P. J. Engelberts, J. Li andJ. D. de Bruijn Studying the e¡ect of di¡erent macrostructures on in vitro cell behaviour and in vivo bone formation using a tissue engineering approach 148 General discussion I 170 Anders Lindahl, Mats Brittberg and Lars Peterson chondrocytes: clinical and cellular aspects 175 Discussion 186

Cartilage repair with

Elizabeth O’Byrne,Theodore Pellas and Didier Laurent Qualitative and quantitative in vivo assessment of articular cartilage using magnetic resonance imaging 190 Discussion 198 Alessandra Pavesio, Giovanni Abatangelo, Anna Borrione, Domenico Brocchetta, Anthony P. Hollander, Elizaveta Kon, FrancescaTorasso, Stefano Zanasi and Maurilio Marcacci Hyaluronan-based sca¡olds (Hyalograft1 C) in the treatment of knee cartilage defects: preliminary clinical ¢ndings 203 Anthony P. Hollander, Sally C. Dickinson,Trevor J. Sims, Carlo Soranzo and Alessandra Pavesio Quantitative analysis of repair tissue biopsies following chondrocyte implantation 218 Discussion 229 General discussion II Tissue engineering using recombinant human BMP2 Final discussion and summing-up Index of contributors Subject index

244

242

239

234

Participants Frank Barry OsirisTherapeutics, 2001 Aliceanna Street, Baltimore, MD 21231-3043, USA Michael D. Buschmann Biometrical and Chemical Engineering, E¤cole Polytechnique de Montre¤ al, C.P. 6079, Station ßCentre-ville@, Montre¤ al, Que¤bec H3C 3A7, Canada Ranieri Cancedda Centro di Biotecnologie Avanzate, Istituto Nazionale per la Ricerca sul Cancro, Largo Rosanna Benzi 10, Genova, I-16132, Italy Arnold Caplan (Chair) Skeletal Research Center, Case Western Reserve University, 2080 Adelbert Road, Cleveland, OH 44106-7080, USA Steven Goldstein Department of Orthopaedic Surgery , University of Michigan, G0161 400 NIB, Ann Arbor, MI 48109-0486, USA Alan Grodzinsky Center for Biomedical Engineering, Massachusetts Institute of Technology, Room 38^377, Cambridge, MA 02139-4307, USA Tim Hardingham UK Centre forTissue Engineering, School of Biological Sciences, 2.205 Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK Jill Helms UCSF, Department of Orthopedic Surgery, 533 Parnassus Avenue, U-453, San Francisco, CA 94143-0514, USA Anthony Hollander University of Bristol, Academic Rheumatology, Avon Orthopaedic Centre, Southmead Hospital, Bristol BS10 5NB, UK James Huckle Smith & Nephew Group, Research Centre,York Science Park, Heslington,YorkYO10 5DF, UK Ernst B. Hunziker ITI Institute for Dental and Skeletal Biology, UniversitJt Bern, Murtenstrasse 35, Postfach 54, CH-3010 Bern, Switzerland

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PARTICIPANTS

Wa’el Ka¢enah (Novartis Foundation Bursar) University of Bristol, Academic Rheumatology, Avon Orthopaedic Centre, Southmead Hospital, Bristol BS10 5NB, UK Anders Lindahl Department of Clinical Chemistry, Sahlgrenska University Hospital, S-413 45 G˛teborg, Sweden Stefan Lohmander Department of Orthopaedics, University Hospital Lund, 22185 Lund, Sweden Ivan Martin Department of Surgery, ZLF, Room 405, University of Basel, Hebelstrasse 2, CH-4031 Basel, Switzerland Chris Murphy (Novartis Foundation Bursar) Kennedy Institute of Rheumatology, Imperial College, Charing Cross Campus, 1 Aspenlea Road, Hammersmith, London W6 8LH, UK Elizabeth O’Byrne Department of Arthritis Biology, Novartis Institute for Biomedical Research, 556 Morris Avenue, Summit, NJ 07901-1398, USA Hajime Ohgushi Tissue Engineering Research Center (TERC), National Institute of Advanced Industrial Science and Technology (AIST), 3-11-46 Nakouji, Amagasaki City, Hyogo 661-0794, Japan Alessandra Pavesio Fidia Advanced Biopolymers srl (FAB),Via Ponte della Fabbrica 3/B, 35031 AbanoTerme (PD), Italy Joseph Quintavalla Department of Arthritis Biology, Novartis Institute for Biomedical Research, 556 Morris Avenue, Summit, NJ 07901-1398, USA Anthony Ratcli¡e Advanced Tissue Sciences, 10933 NorthTorrey Pines Road, LaJolla, CA 92037-1005, USA Brian Richardson Novartis Pharma AG, Arthritis & Bone Metabolism, WSJ-386.10.09, CH-4002 Basel, Switzerland Herb Schwartz DePuyAcroMed Inc., 700 Orthopaedic Drive,Warwaw, IN 46581, USA StephenTrippel Department of Orthopaedic Surgery, Indiana School of Medicine, 541 Clinical Drive, Suite 600, Indianapolis, IN 46202-5111, USA

PARTICIPANTS

Clemens van Blitterswijk The Netherlands

ix

IsoTis BV, P.O. Box 98, 3720 AB Bilthoven,

GordanaVunjak-Novakovic Massachusetts Institute of Technology, Room E25-342, 77 Massachusetts Avenue, Cambridge, MA 02139, USA John Wozney Wyeth Research, 87 Cambridge Park Drive, Cambridge, MA 02140, USA

Chair’s introduction Arnold I. Caplan Skeletal Research Center, Department of Biology, Case Western Reserve University, 2080 Adelbert Road, Cleveland, OH 44106-7080, USA

I’d like to put this meeting in perspective: the Foundation receives some 200 proposals for meetings each year, but holds just eight; thus, this is quite a special Symposium. The goal of this meeting is to identify the areas that can be coalesced into articulatable principles and sound hypotheses. The ¢eld of tissue engineering of cartilage and bone has now matured from what started out to be an empirical activity in the scienti¢c community to one which now has some sound principles and interesting working hypotheses. There was a long incubation period where we conducted empirical experiments based on clinical need and new emerging technologies. Now there are sound principles and methods that provide the basis for propelling this ¢eld into a rather dramatic upturn. You, as participants, whether presenting formal papers or as discussants, have the opportunity to identify where we have been and where we should go. As chair, I will try to focus on the points made by the individual speakers and to attempt to identify the emerging ideas in this ¢eld from a broad perspective. There are experts on cells, matrices, growth factors and clinical applications. These areas are the focus of the tissue engineering strategies for the 21st century. Although animal models are the focus of many presentations, the ultimate goal of this area of scienti¢c inquiry is the translation of basic information into suitable clinical protocols. Within this context, we must understand the limitations of these individual animal models and use them only as tools for testing broader hypotheses. Clearly, the study on rats or rabbits does not have direct application to human healthcare management, but it does provide the testing stage for the development of new logics for tissue engineering. The long-term goal is the translation of new scienti¢c principles of tissue engineering into more e¡ective regeneration of tissues and improved human healthcare management. Tissue engineering of bone and cartilage provides both a challenging and realistic goal for developing a new generation of medically relevant technologies.

1

Tissue engineering of cartilage: do we need it, can we do it, is it good and can we prove it? L. Stefan Lohmander Department of Orthopedics, University Hospital Lund, SE-22185 Lund, Sweden

Abstract. Current treatments of osteoarthritis (OA) focus on pain and loss of joint function. When these interventions fail, the destroyed joint is replaced by implants of metal, plastic and ceramics. In the future, we need to detect cartilage loss before it is too severe, prevent further loss and stimulate regrowth of lost cartilage. Research in tissue engineering can help us understand the complex requirements for regeneration of joint cartilage. Results from animal experiments and small, uncontrolled, open series of human cartilage repair suggest that functional repair can be accomplished in some joints in some patients. However, outcome is inconsistent. Do we need to recreate the original hyaline joint cartilage or will something else work as well? It is far from clear what factors determine a successful repair or what method is best. The durability of repair tissue is uncertain. The cost^bene¢t equation is unresolved, and current surgical interventions are associated with signi¢cant cost and morbidity. What is the ‘number-needed-to-treat’ to prevent one knee/patient lost to early retirement or future OA? The outcome measures used to determine success or failure of the repair deal with cartilage, joint and patient. The relationship between these outcome dimensions is unclear. However, the outcome as judged by the patient using standardized measures is the gold standard. 2003 Tissue engineering of cartilage and bone. Wiley, Chichester (Novartis Foundation Symposium 249) p 2^16

Joint cartilage can withstand an astonishing amount of repetitive physical stress. After all, in spite of osteoarthritis (OA) being one of the most common a¥ictions of the elderly, most of our joints serve us well throughout a long lifetime of use, and sometimes abuse. Surprisingly then, adult articular cartilage is reluctant to spontaneously repair even minor injuries to its structure. This apparent inability provides the rationale for current e¡orts to develop techniques of tissue engineering of cartilage. Cartilage is composed of a rich intercellular matrix with thinly dispersed chondrocytes, accounting for only 1^2% of the tissue volume in the adult. The matrix, being an underhydrated gel of proteoglycans and matrix proteins 2

TISSUE ENGINEERING OF CARTILAGE

3

reinforced by a three-dimensional network of collagen ¢brils, is directly responsible for the unique functional properties of the cartilage and provides shape, resilience and resistance against compression and shear (Heinegrd et al 1998, Mow & Setton 1998). Adult joint cartilage is anisotropic, the di¡erent tissue layers with cells and matrix having a distinct morphology, composition and function (Wong & Hunziker 1998). The chondrocytes are responsible for a slow turnover of matrix components. In fact, the biological half-life of the bulk of the aggrecan in adult human joint cartilage is measured in months and years, while that for the collagen ¢bril sca¡olding is measured in years and decades. Normal chondrocytes rarely divide. Joint cartilage lacks a vascular supply, being nourished via the synovial £uid. In fact, uncalci¢ed healthy cartilage resists vascular invasion. Formation and development of cartilage in the embryo is directed by an array of morphogenic and growth factors acting in a concerted and exquisitely timed local sequence on speci¢cally positioned undi¡erentiated progenitor cells (Caplan et al 1997, Reddi 1998, Wozney & Rosen 1998). New members are being rapidly added to an already long list of players in the band while we are learning their role in developing and adult cartilage (Brunet et al 1998, Erlacher et al 1998). The progenitor cells are recruited from a population of stem cells. The availability of undi¡erentiated progenitor cells (or mesenchymal stem cells) in bone marrow or elsewhere (periosteum, perichondrium, synovium, etc.) is critical in the formation of new cartilage. Their number is suggested to sharply decrease from birth to the teens, and to further decrease with aging (Caplan 1994). The lack of a vascular supply, sparse and highly di¡erentiated cells, a relative lack of undi¡erentiated progenitor cells, extremely slow matrix turnover, unique biomechanical properties, tissue anisotropy and a demanding environment provide examples of factors which handicap joint cartilage in tissue repair and engineering.

Do we need cell-based cartilage repair? OA is responsible for a major proportion of the global burden of disease. It is also uncontroversial to state that joint injury (to ligaments, cartilage and bone) among the young and adult is responsible for a signi¢cant proportion of subsequent OA, at least for the knee (Felson & Zhang 1998). Unfortunately, surgical reconstruction of injured knee ligaments or menisci, as currently practised, has not been proven to decrease the risk of OA after joint injury (Lohmander & Roos 1994). In light of these comments, it is important to note that cartilage repair, as currently practised in the laboratory and clinic, is not a cure for OA, and no data yet support its use in this condition.

4

LOHMANDER

If cartilage repair cannot be used to cure OA, couldn’t it at least prevent the development of OA as a result of joint cartilage injury? Although a compelling argument, several related issues need to be critically reviewed. Firstly: speci¢cally which types of cartilage injury are we considering, how common are they and how do we identify them? Neither full-thickness or partial-thickness cartilage injuries appear to heal spontaneously in the adult. There is little evidence for the progression of partial-thickness lesions to OA, while the evidence for fullthickness lesions is somewhat con£icting (Newman 1998). The incidence of fullthickness joint cartilage injuries of the knee is poorly documented, and isolated lesions constitute perhaps 1 out of 100 arthroscopy cases (Messner & Maletius 1996, Curl et al 1997). A major proportion of cartilage lesions thus occur in connection with a trauma causing concomitant tears of the anterior cruciate ligament (ACL) and/or menisci, providing a complex set of confounding factors that in themselves lead to OA and which thus make it di⁄cult to ascertain the bene¢cial e¡ects of cartilage repair on future OA risk in this complex setting. The second stated rationale for current clinical practice is short-term relief from symptoms commonly associated with full-thickness articular cartilage lesions, such as pain, swelling, locking and catching. Again, the natural symptom history of these lesions is debated, and opinions vary, with some investigators providing evidence that these lesions are benign and only occasionally cause signi¢cant symptoms or function loss (Newman 1998, Messner & Maletius 1996, Curl et al 1997, Levy et al 1996). The lack of consensus and scienti¢c evidence on both short- and long-term consequences of joint cartilage lesions constitute a problem when we try to construct a solid basis for the clinical practice of cartilage repair. More good data are needed. Can we do it? Cell-based repair of articular cartilage requires the transfer (or migration) to the injured site of chondrocytes, progenitor cells (stem cells) or genetically modi¢ed cells capable of di¡erentiating into chondrocytes. Results of transplantation of perichondrium or periosteum are probably based on the division, migration and di¡erentiation of progenitor cells residing in these tissues. In order for the transplanted cells to remain at the site, the lesion may be covered with periosteum, perichondrium or other material, or the cells may be contained in a biological, synthetic or engineered matrix. This matrix may be degradable or not. Finally, we may need to direct, enhance and halt the cell di¡erentiation and matrix synthesis at the appropriate stage by the directed and timed addition of growth factors (morphogenic factors) or genes. These factors may need a speci¢c and local delivery system.


5

In the animal A considerable literature exists which shows that some degree of repair can be accomplished in animal models of isolated full-thickness lesions, and the reader is referred to recent reviews for more details and further references (Caplan et al 1997, Chen et al 1997, Messner & Wei 1998, Hunziker & Rosenberg 1997, Tyler & Hunziker 1998, Coutts et al 1997). These experiments have commonly used rabbits, guinea pigs, mini pigs, sheep or dogs with full-thickness drill hole defects of various sizes. Methods for repair that have been examined, and sometimes compared, include transplantation of cartilage, cartilage-bone, perichondrium, periosteum, chondrocytes in combination with periosteum or perichondrium, chondrocytes in synthetic or biological matrices, and stem cells, with or without the addition of growth factors. Tissues and cells have been autogenic or allogenic. Species, animal age, defect size, follow-up times, and methods for treatment and monitoring outcome have all varied, making it di⁄cult to draw conclusions with regard to the bene¢ts of one method over the other. It appears that most repair methods provide some bene¢t over untreated full-thickness defects in animals, although short follow-up times and deterioration of tissue quality with time are cause for concern. Other problem areas are the integration of repair ‘cartilage’ with existing joint cartilage, and a tendency for repair cartilage hypertrophy both with regards to volume and developmental stage.

In the patient Early work, published as uncontrolled case series, suggested that the transplantation of autogenic perichondrium or periosteum could, in some cases, provide temporary improvement from symptoms and signs of full-thickness joint cartilage lesions (Homminga et al 1990, Muckle & Minns 1990). However, the lack of appropriate controls and short follow-up compromised evaluation of these early reports, as did small, heterogeneous and incompletely characterized patient materials and non-validated outcome measures. The publication of another such patient series, in which the transplantation of autologous chondrocytes was combined with a periosteum transplant, provided great stimulus for further work in this ¢eld (Brittberg et al 1994). Several reviews summarize and discuss the results of cartilage repair in the human (Newman 1998, Messner & Gillquist 1996, Brittberg et al 1997, Buckwalter & Mankin 1998, Bentley & Minas 2000, Minas 2001). Patient series with isolated chondral defects have been presented, after no treatment or treatment with drilling, microfracture, abrasion, perichondrium transplant, carbon ¢bre implants, or a combination of autologous cultured chondrocytes and periosteum. Patient groups have frequently been incompletely

6

LOHMANDER

characterized, of a wide age range and with isolated lesions or with lesions combined with malalignment or ligament injuries, and with varying anatomical locations in the knee. Inclusion and exclusion criteria, dropouts, and outcome measures have often been insu⁄ciently described or validated. Appropriate controls and randomization or blinding have more often than not been lacking. Finally, the use of the unbiased evaluator is only too rare. Today, we require such standards from clinical trials of new drugs. Are there any compelling reasons why the standards for trials of new and demanding surgical interventions should be any di¡erent? Is it any good? Can we prove it? What conclusions can we draw regarding bene¢ts of cartilage repair at this time? Published reports on patient series have found generation of repair tissue (¢brocartilage- or hyaline cartilage-like) and clinical and symptomatic improvement. However, nothing ruins good results like follow-up. This may have more truth in it than we would like, especially in cartilage repair. Joint problems are notoriously intermittent, and progress in human OA development is measured in years and decades. Follow-up times in both animal and human trials thus need to be appropriately long. Loss to follow-up matters (Murray et al 1997). Patients lost to follow-up after hip arthroplasty have a worse outcome than those who continue to be assessed. There is no reason to believe that patients treated with cartilage repair behave di¡erently. Long term follow-up studies of cartilage repair with minimal loss are thus essential, and such reports are now beginning to appear in the literature (Peterson et al 2000, 2002). These reports suggest durability of good outcome in many patients, while other reports suggest a deterioration of results with longer follow-up (Bouwmeester et al 1997). In light of the methodological weaknesses of these studies, the results cannot yet be taken as unequivocal support of the bene¢ts of one method of cartilage repair over the other. The concept of randomized, controlled and blinded trials is today rigorously applied in drug treatment trials, and there is no reason why the same standards should not apply here. The alternatives may be easier and cheaper but they are not ethical and will not answer the question. The importance of blinding of the evaluator cannot be overemphasized. The potential bias and placebo e¡ect of a surgical intervention is quite considerable (Roos et al 1997, Ryd et al 1997). At the present state of knowledge, there should be no ethical problems with blinding the patient with regard to two alternative treatments in a controlled trial. The use of an independent masked evaluator would accomplish a doubleblinded trial, and in the case of the patient treated without surgery, a singleblinded trial.


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The choice of outcome assessments in these trials is critical. Methods of assessment need to be validated for the particular patient group. Both the patient, the joint and the cartilage need to be assessed. For patient assessment, patientadministered validated questionnaires form the core, and such instruments are available but may need to be further tested for this particular group of patients (Mohtadi 1998, Roos et al 1998, Bellamy 1998, Irrgang et al 2001). Plain radiographs are insensitive, and magnetic resonance imaging preferable, but longitudinal radiological studies are technically demanding. Cartilage assessment may need to be invasive, requiring repeat arthroscopy. Cartilage biopsy histology has been used as a read-out in previous human (and animal) trials, but histopathological grading of cartilage may be unreliable (Ostergaard et al 1997). Probes being developed for arthroscopic use to monitor mechanical and electromechanical properties of joint cartilage may have a use (Sachs & Grodzinsky 1995, Peterson et al 2002). Trials need to be adequately powered, and contain large enough homogenous and well characterized patient groups, so that they have a fair chance of reaching the primary objectives of statistical signi¢cance and clinical relevance. Recent recommendations for trial design in OA are in part applicable to trials on cartilage repair (Bellamy 1998, Altman et al 1996). What should the primary objective(s) of the clinical trial be? We may from time to time need to remind ourselves that we are treating the patient, not the cartilage. The relationship is weak between radiological ¢ndings, cartilage histopathology and biochemistry on the one hand, and patient symptoms and function on the other hand. The patient seeks help because of symptoms and impaired function. Long-term improvement in these dimensions should thus form a primary objective of the trial. As noted, that requires the use of valid outcome instruments, controls, randomization and blinding. Well designed trials in cartilage repair are very demanding, but anything less would be unethical when dealing with treatments that are resource consuming and still associated with a signi¢cant failure rate and morbidity. Therapy with these still experimental methods should be done within the context of randomized, controlled and masked clinical trials with several years of follow-up time. Such trials, and further work in the laboratory will provide valuable information that will help develop better treatment for this group of patients, and improve our understanding of life and death of articular cartilage. Is it worth it? The patient-base (market) for cartilage repair, if de¢ned as those young or middleaged patients with clearly de¢ned, traumatic symptomatic joint cartilage lesion, is limited. Even continued re¢nement of the current practice of a two-stage,

8

LOHMANDER

technically demanding procedure will allow only a limited increase over the current number of a few thousand cases per year. The evaluation of the true potential of this and similar techniques is further limited by their current use primarily as salvage procedures when all else has failed, creating a ‘catch 22’ situation. If, however, we allow ourselves to speculate and suggest that in the future e¡ective cartilage repair could be accomplished by the intra-articular injection of suspensions of mesenchymal stem cells or genetically modi¢ed autologous cells (Lee et al 2001), it is quite probable that the patient-base would increase by several orders of magnitude. This would most certainly be the case if the indications were expanded from repair of traumatic cartilage injuries, to repair of early-stage cartilage damage, ‘pre-osteoarthritis’, in the young and middle-aged. This would also expand its possible use from the knee to other joints. These perspectives of course assume that a relevant degree of patient bene¢t is proven, and the cost^bene¢t ratio is acceptable.

References Altman R, Brandt K, Hochberg M et al 1996 Design and conduct of clinical trials in patients with osteoarthritis: Recommendations from a task force of the Osteoarthritis Research Society. Results from a workshop. Osteoarthritis Cartilage 4:217^243 Bellamy N 1998 Design of clinical trials for evaluation of disease-modifying osteoarthritis drugs (DMOADS) and of new agents for symptomatic treatment of osteoarthritis. In: Brandt KD, Doherty M, Lohmander LS (eds) Osteoarthritis. Oxford University Press, Oxford p 531^542 Bentley G, Minas T 2000 Treating joint damage in young people. Br Med J 320:1585^1588 Bouwmeester SJ, Beckers JM, Kuijer R, van der Linden AJ, Bulstra SK 1997 Long-term results of rib perichondrial grafts for repair of cartilage defects in the human knee. Int Orthop 21:313^317 Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peterson L 1994 Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med 331:889^895 Brittberg M, Lindahl A, Homminga G, Nilsson A, Isaksson O, Peterson L 1997 A critical analysis of cartilage repair. Acta Orthop Scand 68:186^191 Brunet LJ, McMahon JA, McMahon AP, Harland RM 1998 Noggin, cartilage morphogenesis and joint formation in the mammalian skeleton. Science 280:1455^1457 Buckwalter JA, Mankin HJ 1998 Articular cartilage repair and transplantation. Arthritis Rheum 41:1331^1342 Caplan AI 1994 The mesengenic process. Clin Plast Surg 21:429^435 Caplan AI, Elyaderani M, Mochizuki Y, Wakitani S, Goldberg VM 1997 Principles of cartilage repair and regeneration. Clin Orthop 342:254^269 Chen FS, Frenkel SR, Di Cesare PE 1997 Chondrocyte transplantation and experimental treatment options for articular cartilage defects. Am J Orthop 26:396^406 Coutts RD, Sah RL, Amiel D 1997 E¡ect of growth factors on cartilage repair. Instr Course Lect 46:487^494 Curl WW, Krome J, Gordon ES, Rushing J, Smith BP, Poehling GG 1997 Cartilage injuries: a review of 31,516 knee arthroscopies. Arthroscopy 13:456^460


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Erlacher L, Ng CK, Ullrich R, Krieger S, Luyten FP 1998 Presence of cartilage-derived morphogenetic proteins in articular cartilage and enhancement of matrix replacement in vitro. Arthritis Rheum 41:263^273 Felson DT, Zhang Y 1998 An update on the epidemiology of knee and hip osteoarthritis with a view to prevention. Arthritis Rheum 41:1343^1355 Heinegrd D, Bayliss M, Lorenzo P 1998 Biochemistry and metabolism of normal and osteoarthritic cartilage. In: Brandt KD, Doherty M, Lohmander LS (eds) Osteoarthritis. Oxford University Press, Oxford p 74^84 Homminga GN, Bulstra SK, Bouwmeester PS, van der Linden AJ 1990 Perichondral grafting for cartilage lesions of the knee. J Bone Joint Surg Br 72:1003^1007 Hunziker EB, Rosenberg L 1997 Articular cartilage repair. In: Koopman WJ (ed) Arthritis and allied conditions. A textbook of rheumatology. Lippincott Williams & Wilkins, Baltimore p 2027^2038 Irrgang JJ, Anderson AF, Boland AL et al 2001 Development and validation of the international knee documentation committee subjective knee form. Am J Sports Med 29:600^613 Lee KH, Song SU, Hwang TS et al 2001 Regeneration of hyaline cartilage by cell-mediated gene therapy using transforming growth factor beta1-producing ¢broblasts. Hum Gene Ther 12:1805^1813 Levy AS, Lohnes J, Sculley S, LeCroy M, Garrett W 1996 Chondral delamination of the knee in soccer players. Am J Sports Med 24:634^639 Lohmander LS, Roos H 1994 Knee ligament injury, surgery and osteoarthrosis. Truth or consequences? Acta Orthop Scand 65:605^609 Messner K, Maletius W 1996 The long-term prognosis for severe damage to weight-bearing cartilage in the knee: a 14-year clinical and radiographic follow-up in 28 young athletes. Acta Orthop Scand 67:165^168 Messner K, Gillquist J 1996 Cartilage repair. A critical review. Acta Orthop Scand 67:523^529 Messner K, Wei X 1998 Healing chondral injuries. Sports Med 6:13^24 Minas T 2001 Autologous chondrocyte implantation for focal chondral defects of the knee. Clin Orthop 391:S349^S361 Mohtadi N 1998 Development and validation of the quality of life outcome measure (questionnaire) for chronic anterior cruciate ligament de¢ciency. Am J Sports Med 26:350^359 Mow VC, Setton LA 1998 Mechanical properties of normal and osteoarthritic articular cartilage. In: Brandt KD, Doherty M, Lohmander LS (eds) Osteoarthritis. Oxford University Press, Oxford p 108^122 Muckle DS, Minns RJ 1990 Biological response to woven carbon ¢bre pads in the knee. A clinical and experimental study. J Bone Joint Surg Br 72:60^62 Murray DW, Britton AR, Bulstrode CJK 1997 Loss to follow-up matters. J Bone Joint Surg Br 79:254^257 Newman AP 1998 Articular cartilage repair. Am J Sports Med 26:309^324 Ostergaard K, Petersen J, Andersen CB, Bendizen K, Salter DM 1997 Histologic/histochemical grading system for osteoarthritic articular cartilage: reproducibility and validity. Arthritis Rheum 40:1766^1771 Peterson L, Minas T, Brittberg M, Nilsson A, Sj˛gren-Jansson E, Lindahl A 2000 Two- to 9-year outcome after autologous chondrocyte transplantation of the knee. Clin Orthop 374:212^234 Peterson L, Brittberg M, Kiviranta I, Akerlund EL, Lindahl A 2002 Autologous chondrocyte transplantation. Biomechanics and long-term durability. Am J Sports Med 30:2^12 Reddi AH 1998 Role of morphogenetic proteins in skeletal tissue engineering and regeneration. Nat Biotechnol 16:247^252 Roos H, Roos E, Ryd L 1997 On the art of measuring. Acta Orthop Scand 68:3^5

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DISCUSSION

Roos EM, Roos HP, Lohmander LS, Ekdahl C, Beynnon BD 1998 Knee injury osteoarthritis and outcome score (KOOS) development of a self-administered outcome measure. J Orthop Sports Phys Ther 28:88^96 Ryd L, Krrholm J, Ahlvin P 1997 Knee scoring systems in gonarthrosis. Evaluation of interobserver variability and the envelope of bias. Score Assessment Group. Acta Orthop Scand 68:41^45 Sachs JR, Grodzinsky AJ 1995 Electromechanical spectroscopy of cartilage using a surface probe with applied mechanical displacement. J Biomech 28:963^976 Tyler JA, Hunziker EB 1998 Articular cartilage regeneration. In Brandt KD, Doherty M, Lohmander LS (eds) Osteoarthritis. Oxford University Press, Oxford p 94^108 Wong M, Hunziker EB 1998 Articular cartilage biology and mechanics. Sports Med Arthrosc Rev 6:4^12 Wozney JM, Rosen V 1998 Bone morphogenetic protein and bone morphogenetic protein gene family in bone formation and repair. Clin Orthop 346:26^37

DISCUSSION Caplan: I was struck by the fact that if a patient has an untreated anterior cruciate ligament (ACL) tear, there is a lower probability of OA than if the ACL is operated on. Is this something you believe to be endemic in current healthcare treatment plans, or is this speci¢c to ACL lesions? Lohmander: The data I showed relate to outcome after ACL injury and reconstruction. Published results show that there is at least as much OA in the reconstructed knees as in those which are not reconstructed. Yet there are at least 100 000 of these surgeries being done each year in the USA. For what reason? It is clearly not to prevent post-injury OA. The limitation of the data I showed is that it takes a decade for the radiographic changes in OA to develop, so they re£ect the clinical practice as performed 10 years ago. When I asked my orthopaedic surgeon colleagues about the quality of the surgery 10 years ago, they said it was much better than it was 20 years ago. If I ask them today, they say the same thing, and that we can ignore the results from 10 years ago. They are probably correct in that the surgical techniques keep improving, but the fact is we don’t have any data to show that ACL reconstructive surgery as practised today prevents OA. And as a comment in parallel: we don’t yet have any data to prove that cell-based cartilage repair as it is practised today prevents OA from developing. Lindahl: I’d like to comment on the same results. With the ACL trauma data, you need to bear in mind that in many cases there are other associated joint injuries, and they are not treated in those studies. The ACL surgery itself doesn’t repair the cartilage defect that is probably the cause of the OA. A lot of the explanation is the lack of cartilage treatment. Lohmander: That is a nice hypothesis. I would like to be able to treat the cartilage problem early on. But I haven’t shown that it helps, and neither have you. This is a nice hypothesis, but that is all.


11

Quintavalla: In the case where there is an ACL break that is surgically corrected, but where there is no OA yet, would you automatically start treating a patient with a drug on the assumption that 15 years from now that patient will get OA? Lohmander: This is a useful model for disease modi¢cation in OA, assuming two things. First, that you have a disease modi¢er, and second that you have a readout that enables you to conclude with a reasonable degree of certainty that you are intervening in a relevant pathway on a relevant target. Assuming that this would pan out in clinical development, I would be willing to treat these patients with a disease modi¢er or surgical cartilage repair. This is a di⁄cult patient group. They get OA in their 30s, while the rest of us who don’t ski or play soccer (and tear our ACL) get it in our 60s or 70s. It is quite a di¡erent problem having OA when young. Caplan: I want to challenge one aspect of your logic. You focused on the ACL. Forty years ago there were no reconstructive repairs of ACLs, and individuals such as me who ruptured our ACLs instead were instructed to strengthen our collaterals, to stabilize the joint by non-invasive mechanisms. This is still done in some cases. I would suggest that the real di⁄culty in making a judgement is that we don’t know the genotype of the individual with regard to the longevity of their cartilage under non-optimal load bearing. One of the challenges for modern medicine is to provide predictors of genetically susceptible cartilage with regard to OA. Two things can then happen. One is that you could introduce prophylactically drugs or treatments that could attempt to dampen the in£ammatory response or other complications of cartilage wear or tear. Or you could try to develop treatment plans for correcting the gene defects or susceptibility. Another possibility stems from the fact that cartilage is a tissue that lacks a ready supply of self-repair cells, and you could try to increase the number of these cells. This would require accurate population-based genetic predictors. Lohmander: I agree and alluded to this in my presentation, and we are doing such studies at present, in which we combine sports injury work with genetic studies to explore how genetic background interacts with environment in OA. In population-based genetic work, we and others are identifying susceptibility loci for OA. I am quite certain that within a few years we will have a number of rather speci¢c genetic variations that are much more common than the ones we know about today. We are combining this work with our sports medicine work, with exactly the thinking you are suggesting, combining the genetic background of the injured individuals with the outcome data. I think the genetic background will turn out to be quite important. Hunziker: You asked the theoretical question of whether it makes sense to induce repair of the articular cartilage tissue following traumas such as an ACL tear or meniscal lesions. You stated that ¢rst it would be necessary to realign the

12

DISCUSSION

joint in order to treat the cause for the articular cartilage lesions. However, after traumas that lead to osteoarthritic disease, it takes 10^15 years for OA to develop (and it will happen in 60^80% of the patients with these types of trauma). It thus takes a large number of years to develop an osteoarthritic lesion in the presence of a malalignment. It would therefore still make sense to induce repair of the articular cartilage lesion, even though the malalignment could not be treated, since the patient may have a period of 10^15 years without signi¢cant problems. Lohmander: I agree, it makes good sense. Hunziker: And another point. If you grow repair chondrocytes under very special biomechanical conditions (such as malalignment conditions), perhaps these chondrocytes and neoformed cartilage will adapt to these special biomechanical conditions and be able to persist for longer periods of time. Lohmander: I agree with you that if I can delay the need for joint replacement for someone from their mid-30s to the late-40s, that is a very signi¢cant gain. But if we are looking for treatment and a cure, then it’s not ideal. It is certainly a step forward from the current situation, though. Cancedda: You mentioned that the lack of meniscus is another factor in the development of OA. There are still several surgeons who, in the case of lesion of the meniscus, prefer to remove it completely. What do you suggest in this case? Lohmander: I have a parallel series of graphs looking at the consequences of various types of meniscal surgery. The interesting thing is that the consequences of meniscal injury with regard to OA, and surprisingly also patient-relevant outcome, are not that di¡erent from ACL injuries. It appears that the meniscus is much more important to the well being of both joint and cartilage (and patient) than we previously assumed. From the orthopaedic point of view, an arthroscopic partial meniscectomy is regarded as a trivial intervention. But if you ask the patients a year later, many of them do have signi¢cant remaining problems: lots of them get OA. Tissue engineering with regard to the meniscus is a target that is well worth considering. Ratcli¡e: From the tissue engineering perspective, so far we have been making things simply because we can make them. From the clinical point of view, you now have an opportunity to tell us what you would like us to be making, rather than what we can make. You talked about repair as part of the process, and you want replacement as part of the process. Can you give us some indication as to what you would like to see delivered to the patient from a tissue-engineering perspective? Lohmander: I think that what we are currently doing repairing cartilage defects is worthwhile from the patient’s point of view. The problem is that it is not a huge business from a global perspective, compared with drugs or orthopaedic implants. It’s a niche market. If you are looking to expand this, from both the patient and market perspective, it would be by treating joints and cartilage where things have gone a bit further from just a localized defect, which we could ¢ll


13

out with new cells and cover up. It would be nice to do the same tissue engineering in a joint with more cartilage changes. In particular, we would like to be able to do this in ‘young’ people in their 30s or early 40s to prevent OA from developing, if possible. When they come to us with joints with OA, the only thing we today can do is tell them that when their joint has fallen apart completely we will put in a new one for them. We don’t like to put arti¢cial joints into people in their 30s or 40s because they don’t last. The other thing on my wish list was tissue engineered reconstruction of a meniscus. This would be an interesting option, because this is an underrated problem. Ratcli¡e: How long would they have to last in terms of a repair or replacement, to make the surgery and rehabilitation worthwhile? Lohmander: As Ernst Hunziker pointed out, if we can show that we can delay the need for joint replacement by a decade, this will be a major accomplishment. Caplan: Is the criterion you use to decide whether to replace a joint the pain coe⁄cient of the patient, or the clinical analysis you do, for instance by X-ray? What is the driver? Lohmander: All of these factors are considered, but the major driver is the patientperceived pain and functional loss. What else should we use? Caplan: If someone comes in with a radiologically horrible joint, and you can prescribe a pill to decrease their perception of pain and save 10 years, is this in the end identical in ‘value’ to repairing the surface of the joint that will also give you 10 years before you have to replace the joint? I’m asking about patient perception versus physical reality. Lohmander: Assuming that your ability to replace that joint 10 years later is not severely compromised technically, then I would say that from the point of view of the patient a good pain-killer that will dampen the pain signi¢cantly and allow a good function, then many patients would be quite happy with that. The danger is that you will compromise your options down the line. Hollander: I have a comment relating to the question of blinded trials. It seems that the reason blinded trials are a problem with this surgery, compared with a normal drug trial, is that you are not talking in terms of just 6 months to a year you are talking about 10 years if the outcome measure is loss of function and joint replacement. Is this the right outcome measure, or can we simply use pain? If not, can we identify good predictors within the ¢rst 12 months of cartilage repair that will tell us what will happen to function 10 years later? If so, we could start doing blinded trials over a shorter period of time. Lohmander: The outcome of a long-term trial of cartilage repair methodology could be joint replacement or not. However, a worthy and more short-term goal is quality of life for the patient. We should be talking to the investigators actually doing these trials. There is an emerging understanding of some clinical predictors for the long-term outcome. More can be learnt by exploring the long-term

14

DISCUSSION

outcome data now becoming available from follow-up of several patient groups operated on 5^10 years ago. Cancedda: I agree that we need to have randomized trials of these therapies. We are doing some of these in collaboration with di¡erent centres, but it is not so simple in surgery. There are many problems. One is that we can perform some randomized trials, but it is hard to do them blind, comparing techniques that are completely di¡erent. It is not like giving a pill. The surgeon knows very well whether they are doing one procedure or the other. Lohmander: This is the reason why we should take the treating surgeon out of the evaluating loop after the surgery. You let a blinded assessor and the patient evaluate the results. Caplan: Are there any data to indicate that physicians as blinded assessors are any better than the patient in this regard? Lohmander: No, the patient is the best. Caplan: So are you saying that whether it is the surgeon who did the surgery or not, the physician assessor always o¡ers a more optimistic evaluation than the patient? Lohmander: I would say that several publications (H˛her et al 1997, Roos 2001) show that the surgeon involved in the treatment almost always has a more positive interpretation of the outcome than the patient being treated. This assumes that you let the patient respond to the questions you have outside of the clinical environment. Caplan: So is your ¢x for this problem to rely on the patients, or train physicians to be better evaluators? Lohmander: The latter is a lost cause! I say this with a smile, because it is not the treating physician’s job to do this in the context of a clinical trial. Instead, the treating surgeon should encourage the patient and tell them it is the greatest treatment in the world, whichever the treatment was. We need the support of the placebo e¡ect for all forms of treatment. The treating physician should thus be out of the evaluation loop. van Blitterswijk: If the surgeon is always overestimating the e¡ect of the treatment, then it is not important. If you want to compare di¡erent treatments, at least they are consistent in their over-assessment. Lohmander: I disagree. We ask the patient in our studies. We tend to think these are soft data, but if you compare assessing X-ray ¢lms with asking patients a battery of questions repeatedly over time, the repeatability, precision and consistency of responses are much better in the patient responses than in reading X-ray ¢lms. I didn’t believe this 10 years ago, but now I do. Pavesio: One of the objectives of this research e¡ort is to develop guidelines for clinical trial conduct. Such guidelines will need to consider long-term outcomes, with special emphasis on identifying early predictors of such long-term outcomes:


15

Also, we should take into account the inevitable limitation of conducting nonblinded trials in this surgical setting. Lohmander: It is possible to do blinded surgical trials. There was a famous one published on brain surgery in Parkinson’s disease (Freeman et al 2001). Surgeons did craniotomies on all patients, but only some of them received transplant cells. Pavesio: How many patients will consent? Lohmander: A very small number. But it can be done. We are involved in blinded surgical trials. Pavesio: Realistically, however, I suppose this will result in small patient numbers. Goldstein: It seems to me that while there is some overlap, Stefan Lohmander is discussing two populations. The ¢rst is a group of individuals in which trauma has caused signi¢cant alterations in their joints, such as ACL tears and meniscal ruptures. These conditions result in an acute form of arthritis. The other, larger (and more commercially viable) population is those who develop OA over a long period of time (wear and tear degeneration). It seems that if we don’t separate these groups we may be unclear about our objective function. For example, the objective for the 20^30 year olds with traumatic injuries would be to alter the post-traumatic joint function in an e¡ort to delay or prevent degeneration. So the goal might be to try to return the joint to functioning as normally as possible, to preserve the cartilage so that it is subjected to normal wear and tear. The treatment of ‘ageing’ degeneration joint conditions may vary signi¢cantly. Cartilage in older people may be di¡erent from cartilage in young people in terms of repair and/or replacement. We also need to think in terms of treating a whole joint, rather than just a focal injury. Lohmander: I agree that we need to use di¡erent approaches for these two endpoints. I’d be surprised if we used the same treatment for a young joint as opposed to one that has already begun to deteriorate. This also comes back to the milieu into which the transplanted cells are dropped, which will almost certainly be di¡erent in 25 year old cartilage from 60 year old cartilage. van Blitterswijk: I would argue that there are di¡erences between acute trauma and the OA joint of a 60 year old patient, but I’m not sure that this necessitates di¡erent therapeutic approaches. Many people would feel that we still want to treat the older patients as having focal defects. If you are doing this, it might be a very similar approach. Lohmander: If you can ¢nd them early enough, yes. Caplan: The surgical treatment of an eroding surface is to create a focal defect by cutting precise edges and then putting in an implant. You can transform an eroding defect to a focal defect, but if you don’t get at the intrinsic cause of that eroding defect, it will still be there. There will have to be a drug approach, or an osteotomy, or some other correcting event. There is a group of experiments from my lab in

16

DISCUSSION

collaboration with Victor Goldberg, which have never been published, in which we took rabbits and did partial menisectomy. We then created full-thickness defects and plugged them with mesenchymal stem cells to engineer cartilage repair. The menisectomy resulted in a destabilized joint, which is a model for OA, and the implanted plugs functioned as if they were in standard defects. All of the reparative events occurred much faster and more actively because the milieu of the destabilized joint involves growth factors and cytokines that are trying to correct this instability problem in an accelerated fashion. When osteophytes form, it is from an expression of cytokines and growth factors that provide the joint with the ‘opportunity’ to stabilize itself. Although the discussion of the arthritic joint is of a negative microenvironment, it may be exactly the opposite: it may be a highly reparative environment, and if you can correct the actual physical cause of the degeneration process, you may get a better repair in the OA joint than in the quiescent joint that has su¡ered trauma. We do not have good predictors of what to do when. Indeed, OA and this wearing away of cartilage at the joint surface probably has hundreds of di¡erent phenotypes: we never segregate the patients into these phenotypes, but just give them the same drugs and treatment. In addition to looking at genotypes in terms of treatment plans, perhaps we should also properly identify the phenotype of the particular OA that we encounter. To summarize, Stefan Lohmander has identi¢ed the lack of precise outcome measurements as a major problem. Although he focused on cartilage, we also need to apply this to aspects of bone repair. On the horizon we would encourage our scienti¢c colleagues to provide us with predictors of outcomes, and translate the sequencing of the genome into appropriate genetic predictors, which will account for the huge variability we see in outcomes of standardized treatment plans. The need for reparative strategies at a variety of di¡erent loci is there. Whether it is a small business or big business is probably not as important as ¢nding solutions for both small and large problems. The scienti¢c basis for making judgements of success or applicability is still being debated. References Freeman TB, Willing A, Zigova T, Sanberg PR, Hauser RA 2001 Neural transplantation in Parkinson’s disease. Adv Neurol 86:435^445 H˛her J, Bach T, Munster A, Bouillon B, Tiling T 1997 Does the mode of data collection change results in a subjective knee score? Self-administration versus interview. Am J Sports Med 25:642^647 Roos EM 2001 Outcome after anterior cruciate ligament reconstruction a comparison of patients’ and surgeons’ assessments. Scand J Med Sci Sports 11:287^291

Embryonic development and the principles of tissue engineering Arnold I. Caplan Skeletal Research Center, Department of Biology, Case Western Reserve University, 2080 Adelbert Road, Cleveland, OH 44106-7080, USA

Abstract. Tissue engineering has, historically, used empirical methods to devise reparative strategies for optimizing the repair of skeletal tissue defects. The acquired experience and observations indicate that several aspects of successful repair protocols involve the engineered recapitulation of certain embryonic events. A careful study of the details of embryonic limb formation and subsequent di¡erentiation events into its component skeletal tissues suggests that aspects of these tissue formation events can provide guiding principles for the tissue-engineered regeneration of skeletal tissues in adults. A thesis is developed in reviewing selected aspects of embryonic limb formation whereby one could articulate broad tissue engineering principles that should be followed in order to regenerate portions of excised or damaged skeletal tissues. Central to the regeneration of skeletal tissues is the conversion of progenitor cells and tissue into the desired specialized tissue. For mesenchymal tissues, this requires the conversion of groups of mesenchymal cells with their relatively modest extracellular matrix (ECM) into functional skeletal tissues characterized by a voluminous and specialized ECM. Because of the absence of the complex signalling cascade characteristic of early embryonic events, it is improbable that adult tissue reconstruction strategies can recapitulate distinctive morphologies while forming newly di¡erentiated skeletal tissues. Thus, tissue-engineered regeneration protocols must provide the sca¡olds and boundaries to establish the contours and edges of reparative tissues and then must functionally and molecularly integrate this neo-tissue with the surrounding host tissue. Consequently, such sca¡olds must provide the reparative cells or their progenitors or the speci¢c attachment or binding sites for endogenous reparative cells. The sca¡olds must also provide the signals to start the reparative process, the means and signals to expand the reparative cells, the space for the unique and oriented specialized ECM and, lastly, the capacity to functionally integrate this neo-tissue in a seamless manner with the host tissue. Several tissue-engineering principles based on the details of embryonic events provide guides for the development of scienti¢c logics for new reparative strategies for the regeneration of skeletal tissues. 2003 Tissue engineering of cartilage and bone. Wiley, Chichester (Novartis Foundation Symposium 249) p 17^33

Tissue engineering is a complicated, multi-component discipline that uses information and strategies from a number of scienti¢c ¢elds in order to 17

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CAPLAN

reconstruct adult tissues that are absent or malfunctioning. The goal is to provide the engineered regeneration of tissues, with the emphasis on regeneration rather than repair. Tissue repair is usually a rapid process that has been evolutionarily selected to allow the animal to rapidly escape from danger and consists of the use of scar tissue to join the edges of the fractured tissue or to ¢ll tissue voids. For skeletal tissues, such as cartilage and bone, scars are locations of mechanical weakness and, thus, of future tissue failure under normal or high loads. By comparison, regeneration is a slow process which recapitulates many of the steps observed when tissue initially forms in the embryo. Based on these observations, I assume that the key steps or principles that govern the embryonic development of bone and cartilage (for example, the long bones and joints of the limbs) should be important components of the tissue-engineered regeneration of these tissues in adults. Below, selected aspects of embryonic limb formation and subsequent bone and cartilage development are reviewed and, where appropriate, the principles that may be important to tissue engineers are articulated. Just as primitive humans ¢rst identi¢ed ¢re, water, earth, and air as an oversimpli¢ed list of the key elements of our physical world, the reader should take the text below in the same sense as an oversimpli¢ed listing of key parameters put forth in the primitive beginnings of the new discipline, tissue engineering. Sequence of embryonic events The beginning The focus of this treatise derives from the observations of embryonic limb (arms, legs) development primarily observed in the avian embryo (Hinchli¡e & Johnson 1980). Following the split of the mesoderm into lateral plate and somatic layers, areas (cell clusters referred to as ¢elds) in both can be identi¢ed that give rise to limb tissues. Importantly, the limb outgrowth or bud that forms, is derived from lateral plate mesenchyme whose mitotic activity is higher than surrounding £ank mesoderm, thus producing a bulge from the £ank of the embryo, the limb bud. By the time the ¢rst outgrowth of the limb bud can be observed, the dorsal^ ventral and anterior^posterior axes have been established and committed by a complex multicomponent molecular cascade (Johnson & Tabin 1997). Moreover, at this time, cells have exited the somites and will continue to migrate and reach the limb bud and limb ¢eld (Christ & Ordahl 1995). Thus, the forming limb bud is composed of at least three di¡erent mesenchymal progenitor cells: lateral plate mesenchyme that will give rise to cartilage and bone, somitic mesenchyme that will give rise to all of the muscles of the limb, and vascular endothelial progenitor cells that will contribute to blood vessel formation in the developing limb. A fourth group of cells, not discussed here, consists of neural

THE PRINCIPLES OF TISSUE ENGINEERING

19

elements that enter the limb later. Thus, from the outset, the uniform appearing mesenchyme of the developing limb bud is composed of cells from di¡erent origins with di¡erent precommitted developmental potentials that re£ect the di¡erent tissues that make up the fully formed limb. All of these precommitment events involve complex signalling^response reactions unique to embryonic events. Tissue engineering principle: the progenitor mesenchyme is composed of a heterogeneous grouping of cells that have a predisposition to form di¡erent end-stage phenotypes and, thus, very di¡erent tissues within the same developmental ¢eld. Changing signals (morphogenesis) Embryonic development is characterized by an ever-changing pattern of molecular signalling between di¡erent adjacent tissues (for example, epithelial^mesenchymal interactions) and ever-changing expressional patterns of the cells themselves which a¡ect that cell and/or its neighbours (Pearse & Tabin 1998). The importance of this signal^response continuum is that it imparts positional information on the cell. Such multi-component signalling accomplishes several purposes, including telling cells to divide (or not), to be developmentally £exible (or not), to continue living or to die, and to precisely establish edges of that tissue ¢eld. This later ¢eld e¡ect translates into unique morphologies (elbows versus knees, ¢ngers versus toes) and these become permanent (committed) long before the edges can be observed microscopically or macroscopically. Clearly, the cells on one side of an edge are di¡erent from those on the other side; this di¡erence stems from substantial di¡erences in the signal^response sequence experienced by individual cells or their thresholds for response (Hartmann & Tabin 2000). Not discussed here is the fact that these discrete signal^response events and subsequent morphologies occur at a scale of size that is very small compared to the tissue volumes required in adult tissue repair/regeneration sites. The sum of all of the sequential events and the subsequent response of the cells to generate edges is summarily referred to as ‘morphogenesis’ (Zwilling 1968). Tissue engineering principle: it is unlikely that the adult milieu will provide the appropriate signal^response cascade to allow regeneration of discrete morphologies and, thus, all engineered tissues must be provided with the appropriate edges and contours. Mitogens Cells in forming tissues have a number of di¡erent rates of cell division and a number of di¡erent molecules that stimulate these mitotic events. The relatively rapid rates of cell multiplication observed in embryonic tissues can be sustained for certain adult cells in culture. The emphasis is on cell culture, because tissue engineers often use this technology to generate adequate starting numbers of

20

CAPLAN

cells for cell-based therapies. The primary di¡erence between these two systems is that the embryonic milieu is ever-changing, while cell culture systems are relatively constant. Also, the embryonic tissue environment is at a relatively high cell density, while cell cultures are established at sparse plating densities to encourage mitotic expansion. As cells di¡erentiate in vivo, they decrease their rates of cell division and then rarely divide (Searls & Janners 1969). The major cell division activity in vivo is by undi¡erentiated, multipotent progenitors; in contrast, some adult di¡erentiated cells can be forced to divide in culture. The di¡erentiation potency of these dividing adult cells is assumed to be limited, but recent studies indicate that, although they can faithfully express their phenotype of origin, they also have the capacity to di¡erentiate into other phenotypes. This plasticity observed with dedi¡erentiated adult cells in culture is in sharp contrast to that observed in newt limb regeneration, where genetically marked chondrocytes enter the repair blastema, rapidly divide, but faithfully and exclusively re-di¡erentiate into only chondrocytes (Steen 1968). Importantly, for all of the above, the mitogens used in cell culture of adult cells may not be the same as those prominent in the embryo. Moreover, in the proximal to distal wave of mitotic events in the embryonic limb, it may be that those mitogens prominent at one end are not active or present at the other end (Pearse & Tabin 1998, Yang et al 1997). Tissue engineering principle: the mitogens used in cell cultures and those found at implantation sites in vivo may be quite di¡erent from those that function in embryonic events. Same signal, di¡erent response Embryonic limb bud mesenchymal cells have been shown to have a very di¡erent response to powerful cytokines/growth factors depending on a cell location in the limb bud and when in the sequence of developmental events a cell is exposed to that agent. A good example of this is that of the exposure of distal limb cells to BMP2. In the early phases of distal digit formation, BMP2 is generated by limb cells, and cells with the appropriate receptors di¡erentiate into chondrocytes that form the models for digits. Twenty-four hours later, exposure of inter-digit mesenchymal cells to BMP2 stimulates apoptosis of those cells and ‘programmed cell death’ of the tissues between the digits (obviously, this does not happen in animals, such as ducks, which have webbing between the digits). Thus, in one phase of limb development, BMP2 is a powerful di¡erentiation agent, while in another phase it causes the elimination of the responding cells (Zou & Niswander 1996). Tissue engineering principle: powerful embryonic molecular mediators of di¡erentiation may have diverse e¡ects on cells depending on the cells’ signalling history, the cells near the responders, and the cells’ location in the tissue.


21

Tissue di¡erentiation The initiation event The formation of a central cartilaginous rod in the core of an embryonic limb bud is the ¢rst macro-event observed. This is preceded by the condensation of mesenchymal progenitor cells with the disappearance of capillary networks within the con¢nes of the condensation (Yin & Paci¢ci 2001) and then the secretion of the cartilage-speci¢c extracellular matrix (ECM) containing type II collagen and aggrecan as major components. Importantly, the condensation of the core mesenchyme is preceded by the formation of a £attened cell layer outside of the zone of condensation (Hinchli¡e & Johnson 1980). These distinctive cells in the £attened cell layer will give rise to the periosteum, and the ¢rst bone-forming osteoblasts will di¡erentiate from the cells inside of this layer, which is often inappropriately referred to by some as the perichondrium (Pechak et al 1986). Thus, the initiation event of the di¡erentiation sequence is the distinctive formation of a collar of £attened cells which mark the outer edge of the central cartilaginous rod. These cartilage rods take on the relative positions and shapes of the bones that will form later and have been called the ‘anlagen’ or models of bone. The question, thus, arises as to whether the collar causes the core cells to condense and initiate the chondrogenic sequence and dissolution of the vasculature by squeezing down the cell mass or, alternatively, the core condenses to pull the collar together.

Cartilage does not beget bone Because the cartilage rods are miniature models of the bones that will be central to limbs, it was thought that the cartilage was replaced by bone. This is not correct, and in embryonic limb buds, in growth plate, and in ectopic endochondral bone formation, cartilage is ¢rst replaced by the eroding vasculature and then by marrow components (Caplan & Pechak 1987). Indeed, the initial cartilage anlagen of the limb long bones de¢nes, quite precisely, the initial marrow cavity of the subsequently developing bones (Caplan 1988). Just as is the case for growth plate cartilage, the cells of the central cartilage rods that form in the limb buds go through rapid hypertrophic changes which terminate in the death of the distinctive hypertrophic chondrocytes; this hypertrophic zone is then eroded by digestive cells and its volume occupied by vascular and marrow elements. This is in distinct contrast to the temporally later di¡erentiation of cartilage at the proximal and distal ends of these central rods; these cartilages eventually form the joint surfaces and the unique cushioning hyaline cartilage.

22

CAPLAN

Tissue engineering principle: the initial formation of embryonic cartilage rods is temporally and molecularly separate from the events which give rise to the cartilage which will function at the joint surface for load bearing.

Bone formation First bone forms from the internal side of the collar zone from a monolayer sheet of osteoblasts that secrete a dense and oriented layer of osteoid, which is composed predominantly of type I collagen and which eventually mineralizes with calcium phosphate (Pechak et al 1986, Caplan & Pechak 1987, Caplan 1988). Osteoblasts are oriented secretory cells with their backs to the vasculature that sits just beyond the collar zone. From their faces at the opposite end of the cells, osteoblasts secrete, assemble, and organize the osteoid matrix. The presence of vasculature is obligatory and, in these initial events and all subsequent events, appears to be the driver of bone formation. Indeed, if vasculature is absent, the same progenitor cells usually di¡erentiate into cartilage (Goshima et al 1991). Tissue engineering principle: the relationship of vasculature to groupings of progenitor cells will control the formation of bone versus cartilage. Without vasculature, there is no bone; exclusion of vasculature will induce cartilage formation.

The ECM Mesenchymal tissues like cartilage, bone, tendon, ligament and other connective tissues are fabricated by highly di¡erentiated cells which produce unique ECMs that eventually dominate and de¢ne the characteristics of a tissue. By contrast, liver, kidney and intestine are highly cellular with highly di¡erentiated sets of cells, each of which provides a unique metabolic function. The bulk of cartilage, bone, tendon, etc. is the ECM that carries, distributes and manages various loads by very di¡erent mechanisms. The embryonic progenitor cell ECM is quite di¡erent from the ECM of the di¡erentiated tissue that subsequently forms. While the adult tissue is characterized by its very high ECM to cell ratio, the embryonic mesenchyme is characterized by its high density of progenitor cells and sparse ECM. This embryonic ECM has mostly water, hyaluronan to structure the water, and a thin ¢brillar network of type I collagen and ¢bronectin (Toole 1997). The relatively sparsely populated adult tissues have ECMs that have relatively dense collagen and proteoglycan networks which determine the mechanical and functional properties of the tissues. Even hyaline cartilage, which has a relatively large amount of water, has an ECM that contains substantially higher quantities of molecular constituents compared to the embryonic mesenchymal ECM.


23

Tissue engineering principle: the progenitor cells in developing tissues are in relatively high abundance within a loose, highly porous ECM network. This progenitor tissue then di¡erentiates into a sparsely populated tissue whose functional tissue characteristics are dominated by the properties of its relatively massive and specialized ECM. All cartilages are not created equal Embryonic development is characterized by the ever-changing signalling pattern such that, although arms and legs both have the same array of general tissue types (i.e. cartilage, bone, tendon, ligament, muscle, nerve, vasculature, etc.), they are all of obviously di¡erent morphologies. Indeed, single gene knockouts in mice can exhibit missing ¢ngers, but intact toes or, conversely, knees with morphological problems, while elbows are quite normal (Takeuchi et al 1999). Likewise, the cartilages of the ear, nose, throat, sternum, distal femur, and meniscus are all quite di¡erent, not only in morphology, but in some of their molecular constituents (Naumann et al 2002). These cartilages all have type II collagen and aggrecan, but they synthesize these components with di¡erent chemistries and in di¡erent proportions to other so-called ‘minor’ constituents. The mechanisms controlling the fabrication of these di¡erent cartilages and their di¡erent array of molecular constituents are currently unknown. Tissue engineering principle: a tissue is de¢ned by its unique anatomic and microscopic location, its function, and its molecular constituents. Although each site may provide some molecular cueing as to the fabrication of that new tissue at that site, it can be expected that additional engineering adjustments will be required for each site to provide site-speci¢c tissue di¡erentiation. Embryonic mimetics In an attempt to imitate the embryonic microenvironment, we have used tissue engineering cell delivery sca¡olds made of hyaluronan (HA) (Solchaga et al 1999, 2000, Caplan 2000). The basis for this approach rests upon the observations by Toole (1997) and colleagues (Goshima et al 1991) that HA levels are high in embryonic chick limb buds during morphogenesis and decrease during speci¢c di¡erentiation events. The HA is degraded into small fragments called oligomers by the enzyme hyaluronidase. Importantly, we showed that high molecular weight HA can be chondro-inductive (Kujawa et al 1986), while others have shown it to be anti-angiogenic (Feinberg & Beebe 1983). Moreover, the oligomers of HA have been shown to be angiogenic (Deed et al 1997) and also capable of preventing aggrecan from forming aggregates on HA (Kimura et al 1980). Using bone marrow mesenchymal progenitor cells in porous HA sponge sca¡olds in a fullthickness femoral condyle defect, we have shown that initially these cells form

24

CAPLAN

chondrocytes throughout. The dissolution of the HA sca¡old triggers the conversion of the bottom cells into hypertrophic chondrocytes that undergo apoptosis and are replaced by the invading vasculature, which is brought into this sector by the HA oligomers; eventually, new bone is formed by osteoblasts derived from host progenitors that enter with vascular elements. The cartilage at the top of the joint space surface remains and functions in weight bearing for at least 24 weeks. The impressive observation is the integration of the neo-cartilage with the host cartilage, presumably facilitated by the oligomers of HA (Solchaga et al 1999, 2000, Caplan 2000). The result is an impressive regeneration of hyalinelike cartilage at the joint surface. Tissue engineering principle: sca¡olds can be constructed to mimic the inductive embryonic milieu to form cartilage. With the enzymic removal of high molecular weight HA to form oligomers, the top cartilage integrates with host cartilage, while the bottom cartilage is replaced by vasculature and marrow and eventually bone. Thus, tissue-engineering sca¡olds can be inductive to start and their dissolution can trigger new events. Importantly, the breakdown products themselves can add value to the regenerative process just as they may function during tissue di¡erentiation events in embryonic tissue formation.

Conclusion It is possible to use the study of embryonic events as a guide for regenerative tissue engineering strategies.

References Caplan AI 1988 Bone development. In: Cell and molecular biology of vertebrate hard tissues. Wiley, Chichester (Ciba Found Symp 136) p 3^21 Caplan AI 2000 Mesenchymal stem cells and gene therapy. Clin Orthop 379:S67^S70 Caplan AI, Pechak DG 1987 The cellular and molecular embryology of bone formation. In: Peck WA (ed) Bone and mineral research, vol 5. Elsevier, New York, p 117^184 Christ B, Ordahl CP 1995 Early stages of chick somite development. Anat Embryol (Berl) 191:381^396 Deed R Rooney P, Kumar P et al 1997 Early-response gene signaling is induced by angiogenic oligosaccharides of hyaluronan in endothelial cells. Int J Cancer 71:251^256 Feinberg RN, Beebe DC 1983 Hyaluronate in vasculogenesis. Science 220:1177^1179 Goshima J, Goldberg VM, Caplan AI 1991 The origin of bone formed in composite grafts of porous calcium phosphate ceramic loaded with marrow cells. Clin Orthop 269:274^283 Hartmann C, Tabin CJ 2000 Dual roles of Wnt signaling during chondrogenesis in the chicken limb. Development 127:3141^3159 Hinchli¡e JR, Johnson DR 1980 The development of the vertebrate limb. Oxford University Press, New York Johnson RL, Tabin CJ 1997 Molecular models for vertebrate limb development. Cell 90: 979^990


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Kimura JH, Hardingham TE, Hascall VC 1980 Assembly of newly synthesized proteoglycan and link protein into aggregates in cultures of chondrosarcoma chondrocytes. J Biol Chem 255:7134^7143 Kujawa MJ, Carrino DA, Caplan AI 1986 Substrate-bonded hyaluronic acid exhibits a sizedependent stimulation of chondrogenic di¡erentiation of stage 24 limb mesenchymal cells in culture. Dev Biol 114:519^528 Naumann A, Dennis JE, Awadallah A et al 2002 Immunochemical and mechanical characterization of cartilage subtypes in rabbit. J Histochem Cytochem 50:1049^1058 Pearse RV 2nd, Tabin CJ 1998 The molecular ZPA. J Exp Zool 282:677^690 Pechak DG, Kujawa MJ, Caplan AI 1986 Morphological and histological events during ¢rst bone formation in embryonic chick limbs. Bone 7:441^458 Searls RL, Janners MY 1969 The stabilization of cartilage properties in the cartilage-forming mesenchyme of the embryonic chick limb. J Exp Zool 170:365^375 Solchaga LA, Dennis JE, Goldberg VM, Caplan AI 1999 Hyaluronic acid-based polymers as cell carriers for tissue-engineered repair of bone and cartilage. J Orthop Res 17:205^213 Solchaga LA, Yoo JU, Lunberg M et al 2000 Hyaluronan-based polymers in the treatment of osteochondral defects. J Orthop Res 18:773^780 Steen TP 1968 Stability of chondrocyte di¡erentiation and contribution of muscle to cartilage during limb regeneration in the axolotl (Siredon mexanicum). J Exp Zool 167:49^78 Takeuchi JK, Koshiba-Takeuchi K, Matsumoto K et al 1999 Tbx5 and Tbx4 genes determine the wing/leg identity of limb buds. Nature 398:810^814 Toole BP 1997 Hyaluronan in morphogenesis. J Intern Med 242:35^40 Yang Y, Drossopolou G, Chuang PT et al 1997 Relationship between dose, distance and time in Sonic Hedgehog-mediated regulation of anteroposterior polarity in the chick limb. Development 124:4393^4404 Yin M, Paci¢ci M 2001 Vascular regression is required for mesenchymal condensation and chondrogenesis in the developing limb. Dev Dyn 222:522^533 Zou H, Niswander L 1996 Requirement for BMP signaling in interdigital apoptosis and scale formation. Science 272:738^741 Zwilling E 1968 Morphogenetic phases in development. Dev Biol 2:S184^S207

DISCUSSION Helms: I’m interested in the stack cell, which you described as being squeezed out of the vasculature. This implies a mechanical force. Caplan: We don’t know whether it is mechanical or chemical. This is work of Bodo Christ and his colleagues. They have shown that cells originating in the somite that have vascular potential are present in the core of the limb and then are not found there during the condensation event (Wilting et al 1997). These cells die, or are somehow lost, or dedi¡erentiate. Helms: There is a clearing of the vasculature in the place where a condensation forms, but which comes ¢rst? Caplan: The vascular tissue is excluded and the somite-positive cells disappear before you see the actual condensation of the mesenchyme at the core of the limb. Lindahl: Your point that we have to look at part of the embryology was very interesting. I have a question, though. Recent work in Cli¡ Tabin’s lab shows that the Wnt14 gene is expressed and causes apoptosis in a certain area during joint

26

DISCUSSION

formation (Hartmann & Tabin 2001). How do you envisage the cartilage forming there? Caplan: Frank Barry and his colleagues have data that there are molecular changes in the chondrocytes that set up the edges of the presumptive joint. What hasn’t been described is the signalling pattern which establishes the developmental programme within those edges. As a late event, the Wnt family is clearly involved. Ohgushi: You showed that cartilage is di¡erent from one anatomical location to another. For example, you said that the ¢rst name is ear or knee, and the second name is cartilage. How about bone? Is that di¡erent? Caplan: I don’t have any answer, except to say that it is clear that cortical and trabecular bone are di¡erent. Both are vascular driven. The data are not about long bones in the leg versus long bones in the arm, for example. Clearly, the weight-bearing aspects of these are very di¡erent. What is known is that developmentally the signalling cascades for di¡erent bones are quite di¡erent. The best example I know is that the Bmp5 knockout in mice corresponds to a mutation that has been worked on for 30^40 years in mice called ‘short ears’. It was kept at the Jackson lab as short ears, but now they know it is a Bmp5 knockout. It has two little spikes from two speci¢c vertebrae that are di¡erent, the sternum is di¡erent and it has short ears (Kingsley et al 1992). We know from the work of Cli¡ Tabin (Logan et al 1998) and others on limb development that the wing ¢eld and leg ¢eld in chicken are set up by means of di¡erent signalling patterns and transcription factors. I would say embryologically that there are di¡erent cascades which set up the bones in arm versus leg. I am not sure whether the bones are di¡erent. Ohgushi: How about the matrix? Caplan: To my knowledge, no one has looked at the detailed extracellular matrix of di¡erent bones with regard to minor constituents, in the same way that we have done with the di¡erent cartilages. Hardingham: You presented some challenging ideas, but overall I think there was a mixed message. You argued that the embryo was the model system that would guide us in some of our aims, but then you gave many reasons why it wasn’t valid for that application. We take the embryonic mesenchymal cell di¡erentiation as a model because it has been well studied. What hasn’t been well studied is mesenchymal cell di¡erentiation in the repair of adult tissue, which I think may well involve some of the same candidate signalling components, but they may be organized in a di¡erent way. In the embryo much of the positional information is arranged through gradients of signalling molecules. You commented on the microscopic scale of what goes on: it is a very di¡erent dimension from repair in the adult. It is easy to arrange gradients on a microscale from just a few cells and sources. It is much more di⁄cult to arrange comparable gradients on a larger scale. We still have lessons to learn about this scale factor and the types of signalling


27

mechanisms that will drive cell di¡erentiation in tissue repair in an adult. These mechanisms in the adult may show signi¢cant di¡erences from those in the embryo. Caplan: In cartilage, I suggest that chondrocytes have half-lives and drop dead. Therefore there must be a progenitor cell that gives rise to new chondrocytes. Is this progenitor cell coming up from the marrow or down from the top? That is, is the turnover of cartilage bottom^up or top^down? Data indicate that it is top^ down. If this is the case, what are the characteristics of that progenitor cell and how can we get more of them there? How can we a¡ect the kinetics of natural cartilage turnover? Development starts with the fertilization of an egg and continues throughout the entire lifespan. Laurie Glimcher has identi¢ed a knockout which is lacking a transcription factor important in B and T cell development, called NFATp (Ranger et al 2000). A graduate student lost the homozygous breeding female and found it when it was six months old. They noted that it was limping badly. They sacri¢ced this animal and sections of the hip and knee showed that cartilage was erupting into the joint space. She took these slides to her father, Mel Glimcher, and he sent them out to others for interpretation, including me. The eventual interpretation was that NFATp inhibits mesenchymal stem cell di¡erentiation into chondrocytes. But this is only late-onset, because the knockout developed absolutely normally. It was only after 6 months that this e¡ect was seen. I would propose that there are agents which control cartilage turnover in post-pubescent animals, and that these agents actually have an e¡ect on the molecular and cellular characteristics of cartilage. This is also another target for us to try to understand: what is this developmental programme, and what controls the di¡erentiation of progenitor cells into chondrocytes in individuals above the age of 50? This is a therapeutic target. Cancedda: You are very much a believer in the scheme of haematopoeitic stem cell di¡erentiation, with very precise branching. But in mesenchymal di¡erentiation one can also consider plasticity. With regard to adult progenitor stem cells, have they lost their intrinsic ability to di¡erentiate, or is it that we are not able to recreate the right microenvironment corresponding to that which they are exposed to during embryonic development? Is it the ability of the cells that is in question, or our inability to recreate the embryonic conditions? If you go back to your brilliant description of endochondral bone formation, there are other groups who think that perhaps hypertrophic chondrocytes may be making a small contribution to some of the bone formation. In vitro, hypertrophic chondrocytes have the capability to express genes that are markers of bone cells. It is much more di⁄cult to demonstrate whether this occurs in vivo or not. There is evidence that in at least some situations one can observe hypertrophic chondrocytes in some kind of transitional phase. They can express at the same time markers speci¢c for cartilage and markers such as type I collagen that are bone speci¢c.

28

DISCUSSION

Caplan: You have worked on this problem for many years. One thing for sure is that in mammals (not chicken) there is no question that hypertrophic chondrocytes make type I collagen, osteonectin and osteopontin: these are all considered to be osteoblast-speci¢c proteins. But they are made by a hypertrophic chondrocyte. I would interpret this di¡erently than some others. This cell fabricates a matrix that is going to be calci¢ed. This calci¢ed matrix is what we indeed see in endochondral bone formation, and we would call it bone-like in many ways. Rather than call this cell an osteoblast, because its hormone receptor pattern is that of a chondrocyte, I would still maintain that this cell is a hypertrophic chondrocyte. However, before it drops dead it is indeed making all those products that we would call bone. The more important point that you make regards something that we are going to see in the next three to ¢ve years: there is plasticity among di¡erentiated phenotypes in the mesenchymal lineage, quite di¡erently than what we see in haematopoiesis. For example, we can take adipocytes that have fat droplets in them and all the transcription factors suggesting that they are terminally di¡erentiated, and by changing its in vitro microenvironment we can make that cell into an osteoblast. This is plasticity: not de- and re-di¡erentiation. Helms: How do you de¢ne an ‘osteoblast’? Caplan: We are de¢ning it by the secretory products that it is making and the transcription factors that are activating those genes. Helms: But those are expressed in other cell types. I don’t understand why a hypertrophic chondrocyte, by that de¢nition, can be an osteoblast as well. Caplan: You could call it an osteoblast. But the point I want to make is that there is plasticity in these di¡erent phenotypes in the mesenchymal lineage, and therefore the mesengenic diagram I am showing will additionally require arrows that go horizontally. There are other people who say that it goes back to a stem cell state and then down another lineage. For me, it is simpler to suggest horizontal arrows. The plasticity is huge in mesenchymal tissues, at least experimentally. Whether this occurs in vivo is something we can argue about. Trippel: The amount of plasticity in these di¡erent cell populations is important for tissue engineering. If the plasticity is su⁄cient in an already di¡erentiated cell population to enable the cell to go back and start over again, then we can look to mature cells in our tissue engineering work. On the other hand, if it is insu⁄cient, then e¡orts to use mature cells will be destined to fail. We would then need to go back into embryonic cell populations to get enough plasticity. Do you have any sense about how far back we need to go to get enough plasticity? Caplan: I assume that plasticity is restricted. Certain di¡erentiated cell types can go into other phenotypes, but not all phenotypes. It is a big problem for all of us who are working on ‘stem cells’. The stem cell assay is now compromised, because the way we prove we have a stem cell is to put it in microenvironments that direct it towards other phenotypic pathways. If half the cells can be forced into another


29

pathway (the plasticity issue), even though they are not stem cells, then the assay for that pathway is not going to tell you whether or not you have a stem cell. This whole concept of stem cells is only as good as our assays. Some of our assays are showing how plastic the cells are, not whether they are stem cells or not. There are some pathways that are not open to cells showing plasticity, but which are open to undi¡erentiated progenitor cells. I would be provocative and say that because of plasticity, we don’t need embryonic stem cells to get most of the mesenchymal phenotypes. Trippel: With regard to your hypertrophic chondrocyte, is this a cell that is going to stay around and set up the process of bone manufacture, like other osteoblasts, or is it moribund? Caplan: From my perspective, the hypertrophic chondrocyte that is making bone proteins is a cell that is going to drop dead. It is never organized to do its function by vasculature. This is one of the ways I would de¢ne an osteoblast, as a vascular-oriented cell. There are no data showing that the hypertrophic chondrocytes are oriented by vasculature to make lamellar bone. They make these products and calci¢ed matrix, but it is not a vasculature-driven process. Barry: While we may or may not understand a lot about the detailed control of di¡erentiation of a stem cell into a chondrocyte, at least as important is the change from an immature chondrocyte into an adult chondrocyte. There are dramatic di¡erences between those two cells, which we understand very poorly today in terms of how they undergo senescence, how they proliferate in culture and so on. A fundamental question: when you isolate a mesenchymal stem cell from an adult tissue, to what extent is that similar to the mesenchymal cell that you can isolate from a limb bud? How do you assess the science today, comparing those two cells? Caplan: Those two cells don’t have the same receptors on their cell surface. In the end, this is the criterion one has to use to di¡erentiate an embryonic mesenchymal cell from an adult mesenchymal cell. The cells from the limb bud or limb ¢eld have receptor molecules and go through a transcription factor cascade that are intrinsically di¡erent. Embryonic haematopoietic stem cells and adult haematopoietic stem cells are also intrinsically di¡erent. An embryonic haematopoietic stem cell will make embryonic haemoglobin in its red blood cells, and will do so even when you put it under adult inductive situations. Again, the embryonic progenitors appear to have di¡erent characteristics from the adult progenitors. Wozney: I would even go a step further and ask precisely which adult MSCs are you speaking about? There are MSCs in di¡erent adult tissues, for example, bone marrow and muscle. However, it is not clear that they respond in the same way, or have the same abilities to di¡erentiate into the various mesenchymal lineages.

30

DISCUSSION

Caplan: I was very careful to say that this plasticity issue makes assaying stem cells very di⁄cult. But every blood vessel in your body has mesenchymal cells that are sitting outside of the endothelial cells, the pericytes. Some of them are absolutely mesenchymal progenitor cells. In this case, every vascularized tissue in your body is a source of potential progenitors that will certainly go to cartilage and bone. You can mince up muscle, fat or skin and you will get mesenchymal progenitor cells from them. Are they the same or di¡erent? It’s very di⁄cult to answer because we don’t have a complete analysis of each of these cells. The data from Jim Gamble and the people who worked on adipocyte-isolated mesenchymal progenitors say there are some subtle di¡erences between these and marrowderived MSCs (Zuk et al 2001). There are probably going to be di¡erences of these progenitor cells from source to source. There are also purity di¡erences, depending on the tissue. The most homogeneous population in our hands, from a variety of sources, is from marrow. We can get rid of the haematopoietic stem cells by di¡erential plating. There are other ways of getting mesenchymal progenitor cells and getting them relatively pure. Hardingham: On the issue of plasticity, don’t you feel that nuclear transfer experiments really open our minds as to what may be the potential in plasticity? When you say that X doesn’t di¡erentiate into Y, shouldn’t it be that it’s not yet been shown possible for that to occur? Isn’t it that we just don’t know the relevant signals? Caplan: Your point is well taken. Every nucleated cell in your body has a complete gene set. I would state that a more experimental or proper way of describing a cell is that there are going to be pathways for which there is transcriptional accessibility and non-accessibility. We have looked for liver proteins from mesenchymal progenitor cells, and we never see any of the transcription factors or liver signals. This means that we don’t know how to take that heterochromatin and make it into euchromatin. Every cell is a potential source of any other cell, but in reality in practical terms for tissue engineering it is going to be a long time until someone ¢gures out how to get a marrow-derived mesenchymal progenitor cell to be a hepatocyte. You have to also be able to do this quantitatively. You can’t have one in a million cells be a hepatocyte for that to be practical in a tissue engineering sense. Vunjak-Novakovic: A related issue that may be important is that of environmental conditions. We all know that biochemical and physical factors act in concert with genes and mediate their expression. There is a great deal of information in the literature about the environment of adult tissues, and of cartilage in particular. But very little is known about the environment of young, developing cartilage. What many of us are trying to do is to mimic in vitro some of the conditions present in the developing tissue and thereby stimulate the cells to undergo rapid chondrogenesis. How much is known


31

about the physical forces and biochemical factors (local or systemic) in the developing cartilage? Caplan: In terms of embryology, there is a detailed understanding about what the signalling cascades are to get certain di¡erentiation sequences. There are no pathways that are completely known. In embryos, as you well know, there is no load bearing. This brings up a completely di¡erent point. In muscle, which is well studied in embryology, neonatology and adults, there are myosin isoforms that change. The ¢rst heart muscle that forms has a skeletal muscle myosin, which in the formation of the heart is replaced by cardiac myosin. In the adult, the only place where cardiac myosin is expressed is in the heart. But in the embryo, the ¢rst heart forms with skeletal myosin and in skeletal muscle the ¢rst myosin produced is smooth muscle myosin. Here is the problem: molecules that you would call phenotype-speci¢c in adults are managed in the embryo in a very di¡erent call-up sequence and in a very di¡erent pathway. Skeletal muscle in the developing embryo has smooth muscle myosin in it. This just won’t do in later life. Cardiac myosin doesn’t come up till later. Isoformic transitions occur that change the character of these tissues. We are nowhere near understanding these details in a predictive way that we can use in tissue engineering. Goldstein: In fact there are mechanical forces occurring during fetal development. They are not load-bearing in the sense that the fetuses are ambulating, but these physical forces are playing a signi¢cant role. While some of the basic patterns seem to be there, with inappropriate mechanical forces the ¢netuning of the morphology and the structure seems to be substantially altered. In a chick, if the appropriate muscular contractions in the upper and lower extremeties are lacking during development, then the morphology of the skeleton and joint is dramatically a¡ected. Lindahl: Earlier on you asked whether cartilage is remodelled from the top or the bottom. This is an important issue in tissue engineering. It includes cell tra⁄cking. It is a scienti¢c challenge for all of us to try to understand this. If it is from the top, it gives some explanation for osteoarthritis: if there is a rift that might be a reason for the osteoarthritis, but this is still unproven. When we looked at our implanted chondrocytes, we looked at a couple of the early genes expressed in chondrogensis, WNT14 and SOX9. The chondrocytes expressed these when we cultured them. When we look at tissue repair, does the cell have any alternative to using the genes that it had during development? Do they use the same toolbox of genes they had in development also in repair, or are there some other genes? Caplan: It’s an open question. In muscle generation in rodent and chick there is a very precise isoform transition that takes many weeks and months, during normal developmental sequences. When a muscle is regenerated, the same transition takes place in just three weeks, rather than three months. Our impression is that in real regenerative events, there is a recapitulation of the entire sequence, but it is on a

32

DISCUSSION

hurry-up time frame. I think all of these tissues will be di¡erent. My bet is that cartilage doesn’t use this embryonic form for adult, but I don’t have any data. Hardingham: I should add for the record that Charlie Archer (Cardi¡ University, UK) presented an abstract at the 2002 Orthopaedic Research Society meeting that described chondrocyte progenitors in the super¢cial zone of growing articular cartilage (Archer et al 2002; see: http://www.ors.org/Transactions/48/0009.PDF). Caplan: I’d also add that there’s a study by Zvi Nevo and his colleagues in Israel suggesting that FGFR3 is a marker for chondroprogenitor cells that appear in a zone outside the cartilage and migrate on the top of it (Segev et al 2000). These may actually contribute to the growth plate, so that when a condyle is in a growing animal, it is a target for these cells because it is increasing in its broadest dimensions as well as the growth plate going in the north and south directions. There are at least two or three studies in the literature suggesting that FGFR3 is a receptor for these chondroprogenitor cells that are walking across the top of the condyle before diving into the cartilage. In tissue engineering experiments Ernst Hunziker and Larry Rosenberg inferred that there were cells walking across the top of the condyle and into the engineered matrices in a full-thickness defect (Hunziker & Rosenberg 1996). Hollander: Should we really be trying to understand how to take our MSCs (or our transdi¡erentiated cells) and work out how to push them into making a matrix that is as exact a replica as possible of the mature cartilage, or should we take the alternative view that we rely on the in vivo environment to push these cells into making the matrix we want? Is there signi¢cant clinical advantage to be gained from engineering a mature piece of tissue? Caplan: One of the mistakes my laboratory made 15 years ago when we started doing these experiments was that we thought if we put mesenchymal progenitor cells in a particular anatomical site that the locus would do all the signalling that was necessary. I think this is wrong. We have lots of data showing that it is how you put the cells in and whether you kick start them before putting them in that are critical. Hollander: I’d go along with that, but the question is, should we be looking at ways to manipulate that cell delivery type or looking at the mature cartilage formation? Caplan: I think we will do what we always do in science both! Huckle: Is the key not to see whether that graft will inhibit joint degeneration, rather than form a hyaline cartilage? Frank Barry will present results on meniscus in his paper. His tissue doesn’t form a meniscal tissue, but it forms a tissue that stops degeneration. What is good enough? This is the key question. Caplan: To summarize: there are important lessons to learn from the formative aspects in which embryos do business. Some of those lessons apply to adults; others separate what happens in embryos from what can happen in tissue-engineered


33

constructs in adults. Vasculature is a driver of bone formation, and the absence of vasculature is an important aspect of cartilage formation. The identity of the cartilage in particular, and its anatomical location, will be important aspects to tissue engineering. We may not be able to manipulate mesenchymal progenitors to give us all of the cartilage phenotypes. This is a note of caution to all those who work with mesenchymal progenitor cells. We may have to subject ourselves to work with biopsies from di¡erentiated tissues, no matter how unpleasant we may ¢nd that prospect. The important aspect for tissue engineering is to understand that mesenchymal progenitor cells may not be a solution for every bone and cartilage problem. The signalling cascade, whether it is done in vitro or in vivo, is going to have to be tightly regulated in order to get tissue outcomes that appropriately integrate into each of these surgical sites. Eventually, there will have to be a joining of phenotyping of the patient with the corrective tissue engineered event, in order for that to be individually successful. References Archer CW, Redman S, Bishop J, Boyer S, Dowthwaite G 2002 The identi¢cation and characterisation of articular cartilage progenitor cells. Proc Orthop Res Soc 48:9(abstr) Hartmann C, Tabin CJ 2001 Wnt-14 plays a pivotal role in inducing synovial joint formation in the developing appendicular skeleton. Cell 104:341^351 Hunziker EB, Rosenberg LC 1996 Repair of partial-thickness articular cartilage defects: cell recruitment from the synovial membrane. J Bone Joint Surg Am 78:721^733 Kingsley DM, Bland AE, Grubber JM et al 1992 The mouse short ear skeletal morphogenesis locus is associated with defects in a bone morphogenetic member of the TGFb superfamily. Cell 71:399^410 Logan M, Simon HG, Tabin C 1998 Di¡erential regulation of T-box and homeobox transcription factors suggests roles in controlling chick limb-type identity. Development 125:2825^2835 Ranger AM, Gerstenfeld LC, Wang J et al 2000 The nuclear factor of activated T cells (NFAT) transcription factor NFATp (NFATc2) is a repressor of chondrogenesis. J Exp Med 191:9^22 Segev O, Chumakov I, Nevo Z et al 2000 Restrained chondrocyte proliferation and maturation with abnormal growth plate vascularization and ossi¢cation in human FGFR-3(G380R) transgenic mice. Human Mol Genet 9:249^258 Wilting J, Eichmann A, Christ B 1997 Expression of the avian VEGF receptor homologues Quek1 and Quek2 in blood-vascular and lymphatic endothelial and non-endothelial cells during quail embryonic development. Cell Tissue Res 288:207^223 Zuk PA, Zhu M, Mizuno H et al 2001 Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 7:211^228

The fundamentals of tissue engineering: sca¡olds and bioreactors Gordana Vunjak-Novakovic Massachusetts Institute of Technology, Harvard-MIT Division of Health Sciences and Technology, Cambridge MA 02139, USA

Abstract. Tissue engineering has the potential to provide cartilaginous constructs capable of restoring the normal function of native articular cartilage following joint injury or degradation. One approach to functional tissue engineering of cartilage involves the in vitro cultivation of tissue constructs by using: (i) chondrogenic cells that can be selected, expanded, and transfected to overexpress the genes of interest, (ii) sca¡olds that provide a de¢ned three-dimensional structure for tissue development and biodegrade at a controlled rate, and (iii) bioreactors that provide the conditions necessary for the cells to regenerate functional cartilaginous tissues. Here we explore the paradigm of tissueengineered cartilage repair that is based on the generation of immature but functional constructs in vitro, and the remodelling and maturation of these constructs in vivo. 2003 Tissue engineering of cartilage and bone. Wiley, Chichester (Novartis Foundation Symposium 249) p 34^51

Functional restoration of articular cartilage remains a challenge, and none of the existing treatment regimens gives a consistently good outcome (Buckwalter & Mankin 1998). Orthopaedic tissue engineering has a potential to develop novel approaches to orderly and mechanically competent regeneration of compromised cartilage structures. Ideally, an engineered graft should re-establish normal function inherent to the tissue being replaced, over a long term. One approach to functional tissue engineering of cartilage involves the in vitro cultivation of cartilaginous constructs that would have a capacity to further develop following implantation, develop site- and scale-speci¢c structural and biomechanical properties, and integrate ¢rmly and completely to the adjacent bone and cartilage. It is thought that these goals can be met if the engineered graft has the ability to support and mediate matrix remodelling in a fashion similar to that present in immature tissue. We explore here the paradigm of tissue-engineered cartilage repair that is based on the generation of immature but functional constructs in vitro, and subsequent remodelling and maturation of these constructs following in vivo implantation. 34

FUNDAMENTALS OF TISSUE ENGINEERING

35

FIG. 1. Model system. Chondrogenic cells are seeded onto sca¡olds at a high and spatially uniform initial density and cultured in vitro under conditions that promote the formation of functional tissue constructs. Sca¡old provides a structural template for tissue development, and biodegrades at a known rate. Bioreactor facilitates mass transfer of nutrients and metabolites, and provides the necessary physical and biochemical regulatory signals.

Cell^sca¡old^bioreactor system The cell^sca¡old^bioreactor system (Fig. 1) was designed to utilize some of the factors present during normal tissue development, and has been extensively studied in vitro (e.g. Freed & Vunjak-Novakovic 2000a) and in vivo (e.g. Schaefer et al 2002). It involves an integrated use of chondrogenic cells (selected and/or expanded as required; transfected to express the genes of interest; seeded at high initial density), biodegradable sca¡olds (highly porous structural templates that biodegrade at a known rate) and bioreactors (specialized environments designed to promote chondrogenesis) towards the in vitro engineering of functional tissue constructs. This system can potentially be used as a physiologically relevant model for controlled studies of tissue development and function, and to grow engineered constructs with structural and functional properties similar to those of immature articular cartilage. Cells Rapid chondrogenesis (in vivo or in vitro) can be achieved only if the native environment of adult articular cartilage is manipulated so that the de¢ciencies

36

VUNJAK-NOVAKOVIC

inherent to slow healing are overcome (Newman 1998). This involves at least two factors: bringing in new biosynthetically active cells, and facilitating transport of nutrients, metabolites and regulatory molecules (Jackson & Simon 1999). The cells used thus far to engineer cartilage have varied with respect to donor age (embryonic, neonatal, immature or adult), di¡erentiation state (precursor or phenotypically mature), and the method of preparation (selection, expansion, gene transfer). High and spatially uniform cell density was associated with improved chondrogenesis in engineered constructs based on di¡erentiated chondrocytes and bone marrow-derived progenitor cells (Freed & VunjakNovakovic 2000a). Gene transfer of human insulin-like growth factor (IGF)1 into bovine articular chondrocytes markedly improved the synthesis rates and wet weight fractions of glycosaminoglycans and collagen, and the biomechanical properties of engineered constructs (Madry et al 2002). High concentration of biosynthetically active cells present in immature constructs was associated with their rapid integration with native cartilage explants (Obradovic et al 2001). These ¢ndings imply that the presence of biosynthetically active cells is likely to determine the capacity of an engineered graft for continued development and integration following implantation in vivo. Sca¡olds Most studies suggest that the sca¡old is essential for promoting orderly regeneration of cartilage, in vivo and in vitro. In general, a sca¡old is designed to provide a structural template for cell attachment and tissue development. Sca¡olds investigated to date vary with respect to material chemistry (e.g. collagen, agarose, synthetic polymers), geometry (e.g. gels, ¢brous meshes, porous sponges), structure (e.g. porosity, distribution, orientation and connectivity of the pores), mechanical properties (e.g. compressive sti¡ness, elasticity) and degradation (please see e.g. Freed & Vunjak-Novakovic 2000a for review). Sca¡old structure determines the transport of nutrients, metabolites and regulatory molecules to and from the cells, whereas the sca¡old chemistry may have an important informational role. Sca¡olds should be made of biocompatible and biodegradable materials, preferentially those already used in products approved by the US Food and Drug Administration. To achieve isomorphous tissue replacement, the sca¡old should biodegrade at a rate matching the rate of extracellular matrix deposition and without any toxic or inhibitory products. Mechanical properties of the sca¡old at various scales can determine the mechanotransduction within the developing tissue, and thereby the suitability of a particular sca¡old for a particular tissue engineering application. The patterns of chondrogenesis were quite di¡erent for cells embedded in gels (formation of cell clusters that accumulated matrix over time but remained


37

separated with matrix-free gel after 6 weeks of culture, Buschmann et al 1992) and cells seeded onto meshes or sponges (initiation of chondrogenesis in the high cell density region at the construct surface, with progressive apositional development of continuously cartilaginous matrix over 6 weeks of culture, Freed et al 1998). These di¡erences between the developmental patterns and matrix compositions in the two types of constructs may be important for their functional properties. Construct compositions and mechanical properties were generally better for ¢brous meshes (Vunjak-Novakovic 1999) than for agarose gels (Buschmann 1992), which may be due to the spatial continuity of the cartilaginous matrix formed in constructs based on ¢brous meshes. In contrast, mechanical stimulation improved construct compositions only for chondrocytes cultured in agarose gel (Mauck 2000) which may be due to the enhanced signal transduction and £uid £ow through the gel between the cell clusters. Bioreactors Ideally, a bioreactor for cartilage tissue engineering should provide an in vitro environment for rapid and orderly development of functional tissue structures by isolated cells on three-dimensional sca¡olds. In a general case, bioreactors are designed to perform one or more of the following functions: (a) Establish spatially uniform concentrations of cells within biomaterial sca¡olds. (b) Control conditions in culture medium (e.g. temperature, pH, osmolality, levels of oxygen, nutrients, metabolites, regulatory molecules). (c) Facilitate mass transfer between the cells and the culture environment. (d) Provide physiologically relevant physical signals (e.g. interstitial £uid £ow, shear, pressure, compression). Three representative culture vessels that are frequently used for cartilage tissue engineering are compared in Fig. 2. All culture vessels are operated in incubators (to maintain the temperature and pH) with continuous gas exchange and periodic medium replacement. Flasks contain constructs that are ¢xed in place by threading onto needles and cultured either statically or with magnetic stirring. Rotating vessels contain constructs that are freely suspended in culture medium between two concentric cylinders the inner of which serves as a gas exchange membrane. The rotation rate is adjusted to maintain each construct settling at a stationary point within the vessel relative to an observer on the ground. After 6 weeks of culture in static £asks, cartilaginous matrix accumulated mostly at the periphery, in contrast to mixed £asks where cartilaginous matrix accumulated in the inner tissue phase but was surrounded by a thick ¢brous

38

VUNJAK-NOVAKOVIC

FIG. 2. Representative bioreactors. Schematic presentation of construct cultivation in static £asks, mixed £asks and rotating vessels and the respective full cross-sections of tissue constructs cultured for 6 weeks. Stain, safranin O/fast green; scale bar, 1 mm.

capsule. Only in rotating vessels, constructs were uniformly cartilaginous throughout their entire cross sections (Fig. 2). These di¡erences in tissue morphology could be related to the respective di¡erences in £ow and mass transport conditions in these three culture vessels (Table 1). In static £asks, mass transport between culture medium and tissue constructs is slow as it occurs by molecular di¡usion only (therefore tissue formation on the construct periphery), and there is no hydrodynamic shear at construct surfaces. In mixed £asks, mechanical stirring generates £uid £ow that enhances mass transport in bulk medium, but also generates turbulent shear (therefore capsule formation at construct surfaces). In rotating vessels, construct settling generates dynamic £uctuations in laminar £uid £ow that enhance mass transport at construct surfaces without adverse hydrodynamic e¡ects (therefore spatially uniform chondrogenesis throughout the construct volume). Hydrodynamic factors present during in vitro culture can modulate chondrogenesis in at least two ways: via associated e¡ects on mass transport of biochemical factors between the developing tissue and culture medium (e.g. oxygen, nutrients, growth factors), and by direct physical stimulation of the cells (e.g. shear, pressure). In vivo, mass transfer within articular cartilage involves di¡usion in conjunction with £uid £ow that accompanies tissue loading and unloading. In vitro, mass transfer has been shown to determine the size and composition of engineered constructs and native cartilage explants (VunjakNovakovic et al 2002). The composition, morphology and mechanical properties of engineered cartilage grown in mechanically active environments were generally better than in static environments (Freed & Vunjak-Novakovic 2000b). In particular, the hydrodynamic stresses acting at the surfaces of constructs cultured


TABLE 1

39

Overview of the operating conditions for each bioreactor vessel type

Cultivation vessel

Static £ask

Mixed £ask

Rotating vessel

Vessel diameter (cm) Medium volume (cm3 ) Tissue constructs or explants (5 mm diameter2 mm thick discs) Medium exchange

6.5 120 Fixed in place; n ¼ 12 per vessel

6.5 120 Fixed in place; n ¼ 12 per vessel

14.6/5.1 110 Freely settling; n ¼ 12 per vessel

Batch-wise (3 cm3 per construct per day) Continuous, via surface aeration 0 Static £uid None

Batch-wise (3 cm3 per construct per day) Continuous, via surface aeration 0.83^1.25 Turbulent(1) Magnetic stirring

Batch-wise (3 cm3 per construct per day) Continuous, via an internal membrane 0.25^0.67 Laminar(2) Settling in rotational £ow Convection (due to tissue settling) Dynamic, laminar

Gas exchange Stirring/rotation rate (s1) Flow conditions Mixing mechanism

Mass transfer in bulk medium Molecular di¡usion Convection (due to medium stirring) Fluid shear at construct None Steady, turbulent surfaces

1 The smallest turbulent eddies had a diameter of 250 mm and velocity of 0.4 cm/s; estimated according to Cherry & Papoutsakis (1988) by Vunjak-Novakovic et al (1996). 2 Tissues were settling in a laminar tumble-slide regimen in a rotational ¢eld; estimated according to Clift et al (1978) by Freed & Vunjak-Novakovic (1995) and Neitzel et al (1998). For more details see Freed & Vunjak-Novakovic (2000b).

in dynamic laminar £ow of rotating bioreactors although di¡erent in nature and several orders of magnitude lower than the forces associated with joint loading, markedly enhanced in vitro chondrogenesis (Freed et al 1998, Vunjak-Novakovic et al 1999). In vitro chondrogenesis The progression of chondrogenesis in constructs based on bovine calf chondrocytes cultured on ¢brous polyglycolic acid sca¡olds in rotating bioreactors has been associated with temporospatial changes in local concentrations of the cells and matrix shown in Fig. 3A,B. Cells at the construct periphery proliferated more rapidly during the ¢rst 4 days of culture (Fig. 3d) and

40

VUNJAK-NOVAKOVIC

FIG. 3. In vitro chondrogenesis. (A) Full cross-sections of tissue constructs after (a) 3 days, (b) 10 days and (c) 6 weeks of culture. Stain, safranin O/fast green; scale bar, 1 mm. (B) Spatial pro¢les of cell distribution after (d) 3 days, (e) 10 days and (f) 6 weeks of culture (measured by image processing). (C) Spatial pro¢les of oxygen distribution after (g) 10 days and (h) 6 weeks of culture (model predictions). (D) Spatial pro¢les of glycosaminoglycan distribution after (i) 10 days and (j) 6 weeks of culture (data points measured by image processing; lines ¼ model predictions). Based on data reported in Obradovic et al (2000).


41

initiated the matrix deposition in this same region (Fig. 3a). Over time, chondrogenesis progressed apositionally, both inward towards the construct centre and outward from its surface. Cell density gradually decreased and became more uniform, as the cells separated themselves by newly synthesized matrix and the construct size increased (Fig. 3d,e,f). After 10 days of culture, cartilaginous tissue had formed at the construct periphery (Fig. 3b). By 6 weeks of culture, self-regulated cell proliferation and deposition of cartilaginous matrix yielded constructs that had physiological cellularities (Fig. 3f) and spatially uniform distributions of matrix components (Fig. 3c). To facilitate data interpretation, a mathematical model was developed which yielded the concentrations of oxygen and glycosaminoglycans as functions of time and position within the constructs (Obradovic et al 2000). Production of glycosaminoglycans was taken as a marker of overall chondrogenesis in light of prior association of glycosaminoglycan deposition with that of collagen type II, the other major component of cartilage tissue matrix (Freed et al 1998). The model accounted for time-dependent local rates of synthesis, consumption and di¡usion of oxygen and glycosaminoglycans. Oxygen consumption due to energy metabolism and matrix biosynthesis resulted in a gradual decrease of oxygen concentration from the construct surface towards its centre. Due to the higher total number of cells and larger di¡usional distances, the decrease in oxygen concentration was markedly faster in 6 week as compared to 10 day constructs (Fig. 3g,h). Model predictions for concentration pro¢les of glycosaminoglycans (Fig. 3i,j, lines) were qualitatively and quantitatively consistent with those measured via high-resolution (40 mm) image processing of tissue samples (Fig. 3i,j, data points). Models of this kind can be used to rationalize experimental data for in vitro chondrogenesis in engineered constructs, and to relate this data to earlier observations of the dependence of global (2^5 mm scale) tissue properties on cultivation conditions (the domain of focus in each case being limited by available analytical tools). Construct function has been correlated with overall construct composition, which itself depended on the conditions of bioreactor cultivation (Vunjak-Novakovic et al 1999). Empirical relationships like these are fundamentally instructive. However, Fig. 3a^h shows clearly that the spatial distributions of tissue components are highly non-uniform during most of the cultivation period. Therefore, the spatial averaging intrinsic to measurement of overall construct properties can ¢lter out potentially signi¢cant information regarding internal gradients and associated mass transfer limitations upon cell metabolism and/or tissue growth. In addition, for the model to serve as a predictive tool for functional tissue engineering of cartilage, it should incorporate other aspects of tissue development, and in particular biomechanical construct properties (Vunjak-Novakovic & Goldstein 2003).

42

VUNJAK-NOVAKOVIC

Duration of bioreactor cultivation With increasing cultivation time, the constructs more closely approximated articular cartilage both structurally and functionally (Freed et al 1997, VunjakNovakovic et al 1999). The changes in biomechanical construct properties correlated with the respective changes in biochemical construct compositions. The wet weight fraction of glycosaminoglycans increased progressively from very low at 3 days to signi¢cantly higher than physiological at 7 months (Fig. 4a), whereas the fraction of total collagen increased during the ¢rst 6 weeks but remained at this level for the duration of culture (Fig. 4b). Six-week constructs had 75% as much glycosaminoglycans and 40% as much collagen per unit wet weight, equilibrium moduli of approximately 0.175 MPa (Fig. 4d), and hydraulic permeabilities that were fourfold higher than those for native cartilage (Fig. 4c). In 7 month constructs, both the equilibrium modulus and the hydraulic permeability became comparable with those measured native cartilage (Fig. 4c,d). Importantly, the wet weight fraction of collagen, which was subnormal when compared to adult articular cartilage, corresponded to that measured for fetal cartilage, both at 6 weeks and 7 months of cultivation (Fig. 4b). Likewise, the equilibrium modulus and hydraulic permeability of 6 week constructs were in the range of values measured for fetal cartilage (Fig. 4c,d). The e¡ect of the duration of culture on the integration of engineered and native cartilage was studied under controlled in vitro conditions, using bioreactors (Obradovic et al 2001). Disc-shaped constructs or cartilage explants were sutured into cartilage rings made of intact or trypsin-treated cartilage, cultured for 1^8 weeks and evaluated structurally and functionally (compressive sti¡ness of the central disk, adhesive strength of the integration interface). Immature constructs integrated better than either more mature constructs or cartilage explants. Integration of immature constructs involved cell proliferation and the progressive formation of cartilaginous tissue (Fig. 5a,b), in contrast to the integration of more mature constructs or native cartilage that involved only the secretion of extracellular matrix components. Compressive sti¡nesses improved from immature to more mature construct and cartilage explants (Fig. 5c), whereas the adhesive sti¡ness at the disc^ring interface was markedly higher for immature constructs than for either more mature constructs or cartilage explants (Fig. 5d). Therefore, in order to optimize engineering of functional equivalents of native cartilage, construct cultivation time should be selected to achieve a desired combination of compressive sti¡ness and integrative potential. Large osteochondral defects in adult rabbits were repaired using composites based on in vitro engineered cartilage (Schaefer et al 2002). Articular chondrocytes were expanded, seeded on poly(glycolic acid) (PGA) sca¡olds in bioreactors, cultured for 4^6 weeks, sutured to an osteoconductive support, implanted and


43

FIG. 4. Compositions and mechanical properties of engineered cartilage. (Top) Biochemical compositions (percentage wet weight) at di¡erent times of bioreactor cultivation. (a) Glycosaminoglycan, and (b) total collagen (hydroxyproline). (Bottom) Mechanical behaviour in con¢ned compression: (c) Hydraulic permeability (1015 m4/Ns) determined from the static and dynamic compression data at 30% strain and 1 Hz frequency and (d) Equilibrium modulus determined in static con¢ned compression from the slopes of stress^strain curves at 10^40% strain (MPa) (based on data reported in Vunjak-Novakovic et al 1999; additional native cartilage data are from Chen et al 2001 and Williamson et al 2001).

evaluated histologically, biochemically and biomechanically (indentation). After 6 months, defects implanted with engineered constructs were repaired with cartilage that had normal thickness and characteristic architectural features including the tidemark and columnar arrangement of chondrocytes, and new subchondral

44

VUNJAK-NOVAKOVIC

FIG. 5. Integration of engineered cartilage. Discs of engineered cartilage (5 mm diameter 2 mm thick) were sutured into rings of native cartilage (10/5 mm diameter 2 mm thick) and cultured in bioreactors for 1^8 weeks. (A) Face sections of composites are shown after (a) 2 weeks of culture and (b) 4 weeks of culture; disc is in the centre (left) and on the right (right). Stain: safranin O/fast green. (B) Functional evaluation: (c) con¢ned-compression equilibrium modulus of the discs at the time of construct preparation, and (d) adhesive strength of the integration interface (data from Obradovic et al 2001).


45

bone. In contrast, defects implanted with cell-free sca¡olds or left untreated repaired with ¢brocartilage. Repair tissues based on engineered cartilage composites had Young’s moduli comparable to native articular cartilage. Integration with subchondral bone was excellent, whereas integration with cartilage was not consistently good. These results suggest that functional engineered cartilage based on articular chondrocytes can provide a mechanically stable tissue-like template that remodelled in vivo into osteochondral tissue with physiologically thick and sti¡ cartilage. Summary Functional cartilaginous tissue constructs can be grown in vitro by using chondrogenic cells, biomaterial sca¡olds and bioreactors. We have discussed the paradigm of tissue-engineered repair of articular cartilage based on the in vitro engineering of immature cartilaginous constructs, and the subsequent remodelling and maturation of these constructs following in vivo implantation. By 6 weeks of culture, engineered constructs achieved structural and biomechanical properties of immature cartilage, and approximated native articular cartilage both structurally and functionally with time in culture. However, their capacity for integration with native cartilage decreased with time of culture. The conditions and duration of in vitro cultivation are thus likely to be determined by the desired combination of compressive sti¡ness and integrative potential at the time of implantation for each speci¢c application of the tissue being engineered. References Buckwalter JA, Mankin HJ 1998 Articular cartilage repair and transplantation. Arthritis Rheum 41:1331^1342 Buschmann MD, Gluzband YA, Grodzinsky AJ, Kimura JH, Hunziker EB 1992 Chondrocytes in agarose culture synthesize a mechanically functional extracellular matrix. J Orthop Res 10:745^758 Cherry RS, Papoutsakis T 1988 Physical mechanisms of cell damage in microcarrier cell culture bioreactors. Biotech Bioeng 32:1001^1014 Chen AC, Bae WC, Schinagl RM, Sah RL 2001 Depth- and strain-dependent mechanical and electromechanical properties of full-thickness bovine articular cartilage in con¢ned compression. J Biomech 34:1^12 Clift R, Grace JR, Weber ME 1978 Bubbles, drops and particles. Academic Press, New York, p 16^29, 142^148 Freed LE, Vunjak-Novakovic 1995 Cultivation of cell-polymer constructs in simulated microgravity. Biotech Bioeng 46:306^313 Freed LE, Vunjak-Novakovic G 2000a Tissue engineering of cartilage. In Bronzino JD (ed) The biomedical engineering handbook, CRC Press, Boca Raton. Chapter 124:1^26 Freed LE, Vunjak-Novakovic G 2000b Tissue engineering bioreactors. In Lanza R, Langer R, Vacanti J (eds) Principles of tissue engineering, 2nd edn, Academic Press, San Diego, chapter 13:143^156

46

DISCUSSION

Freed LE, Langer R, Martin I, Pellis NR, Vunjak-Novakovic G 1997 Tissue engineering of cartilage in space. Proc Natl Acad Sci USA 94:13885^13890 Freed LE, Hollander AP, Martin I, Barry JR, Martin I, Vunjak-Novakovic G 1998 Chondrogenesis in a cell-polymer-bioreactor system. Exp Cell Res 240:58^65 Jackson DW, Simon TM 1999 Tissue engineering principles in orthopaedic surgery. Clin Orthop 367:S31^S45 Madry H, Padera R, Seidel J et al 2002 Gene transfer of a human insulin-like growth factor I cDNA enhances tissue engineering of cartilage. Hum Gene Ther 13:1621^1630 Martin I, Obradovic B, Treppo S et al 1999 Modulation of the mechanical properties of tissue engineered cartilage. Biorheology 37:141^147 Mauck RL, Soltz MA, Wang CC et al 2000 Functional tissue engineering of articular cartilage through dynamic loading of chondrocyte-seeded agarose gels. J Biomech Eng 122:252^260 Neitzel GP, Nerem EM, Sambanis A et al 1998 Cell function and tissue growth in bioreactors: £uid mechanical and chemical environments. J Japan Soc Microrg Appl 15:S602^S607 Newman AP 1998 Articular cartilage repair. Am J Sports Med 26:309^324 Obradovic B, Meldon JH, Freed LE, Vunjak-Novakovic G 2000 Glycosaminoglycan deposition in tissue engineered cartilage: experiments and mathematical model. AIChE J 46:1860^1871 Obradovic B, Martin I, Padera RF, Treppo S, Freed LE, Vunjak-Novakovic G 2001 Integration of engineered cartilage. J Orthop Res 19:1089^1097 Riesle J, Hollander AP, Langer R, Freed LE, Vunjak-Novakovic G 1998 Collagen in tissueengineered cartilage: types, structure, and crosslinks. J Cell Biochem 71:313^327 Schaefer D, Martin I, Jundt G et al 2002 Tissue engineered composites for the repair of large osteochondral defects. Arthritis Rheum 46:2524^2534 Vunjak-Novakovic G, Goldstein S 2003 Biomechanical principles of cartilage and bone tissue engineering. In: Mow VC, Huiskes R (eds) Basic orthopaedic biomechanics and mechanobiology, 3rd edn. Lippincott-Williams & Wilkins, in press Vunjak-Novakovic G, Freed LE, Biran RJ, Langer R 1996 E¡ect of mixing on tissue engineered cartilage. AIChE J 42:850^860 Vunjak-Novakovic G, Obradovic B, Bursac P, Martin I, Langer R, Freed LE 1998 Dynamic cell seeding of polymer sca¡olds for cartilage tissue engineering. Biotechnol Prog 14:193^202 Vunjak-Novakovic G, Martin I, Obradovic B et al 1999 Bioreactor cultivation conditions modulate the composition and mechanical properties of tissue-engineered cartilage. J Orthop Res 17:130^138 Vunjak-Novakovic G, Obradovic B, Martin I, Freed LE 2002 Bioreactor studies of native and tissue engineered cartilage. Biorheology 39:259^268 Williamson AK, Chen AC, Sah RL 2001 Compressive properties and function-composition relationships of developing bovine articular cartilage. J Orthop Res 19:1113^1121

DISCUSSION Wozney: Did you say that you got better integration with constructs that have been cultured in vitro for shorter periods of time? Vunjak-Novakovic: Yes. Wozney: So why don’t you just seed the cells directly onto the sca¡old and put them in? Vunjak-Novakovic: Because the mechanical properties would be poor. Freshly seeded constructs are extremely fragile, and can support a few kilopascals of


47

pressure; so low that we could hardly measure their sti¡ness. Besides, it is not just the duration of culture that a¡ects the integration, but also the concentration and metabolic activity of the cells, and the cell:matrix ratio. Biosynthetically active cells are needed for integration, whereas a formed tissue is needed for mechanical competence. The question is, how can we strike a balance between the two, and have engineered constructs that have some of both properties? Caplan: You didn’t answer John Wozney’s question completely. You assumed that mechanical properties are necessary for implanted cartilage, and that you couldn’t put active cells into a sca¡old, let them go for a short time and implant them, even though they don’t have the requisite mechanical properties. In most orthopaedic implantation in humans, the cartilage is not loaded for quite a while. It is a question of whether you could get the tissue to be mechanically responsible rapidly. Aside from your normal prejudices, could you envisage getting cells into a sca¡old where they are actively making the material, and implanting that long before they acquire the requisite composite mechanical properties that you are looking for? Vunjak-Novakovic: You could do this. There are many variations to what we do, depending on the goals of the treatment. If I have a bias, it is that an engineered graft should be mechanically functional to some extent at the time of implantation, in order to enable the patient at least some movement. It isn’t ideal to have the patient completely immobilized. Caplan: But the patients are usually immobilized. Vunjak-Novakovic: It depends. The initial properties of the tissue that is being implanted into the joint will determine how much activity and loading can be accepted. This is one aspect. The other is better cell localization in tissue constructs as compared to the use of cells alone or cell-seeded sca¡olds. Caplan: So you get the cells localized, and making matrix. Say this takes 2 weeks. You said that you absolutely need four weeks to make a tissue. Vunjak-Novakovic: It takes about four weeks to make a tissue that has appreciable mechanical properties. Caplan: Does it have appreciable mechanical properties su⁄cient to go into an unloaded joint after two weeks? Vunjak-Novakovic: Even at zero time constructs can go into an unloaded joint. But, again, the question is what are you trying to achieve? One school of thought is that isolated cells in conjunction with sca¡olds and growth factors are the basis of tissue engineering. My equation also has an in vitro culture component, for two reasons. One is that a pre-formed tissue can be handled more easily, better survive implantation, and accept mechanical loading sooner. Such tissue is already directed towards cartilaginous di¡erentiation. The other is that tissue engineering in vitro can provide control over various factors that may determine tissue development and function, in particular over those factors that cannot be

48

DISCUSSION

easily manipulated and measured in vivo. Whether it takes two, four or six weeks to engineer a cartilaginous construct depends on a range of parameters, related to the sca¡old, cells and cultivation conditions. For example, chondrogenesis is more rapid in the presence of growth factors. Four weeks is the duration of culture consistent with the conditions that we have tested so far, and with the data that I showed you today. Caplan: In your defence, you did say that you wanted to use these as model systems to look at a variety of variables. Lohmander: Arnold Caplan, why would two weeks necessarily be better than four weeks? Caplan: John Wozney picked up on the point that integration of the graft could be enhanced if the cells were in the earlier phase of their di¡erentiation. Lohmander: So it is really an issue of integration. Vunjak-Novakovic: The available experimental data that we and others have obtained suggest that a high concentration of undi¡erentiated cells is key for the integration of engineered constructs with native cartilage. Caplan: To me, the beauty of your model is that you take a plug of mature cartilage and put a plug of test material in it. This is a brilliant way of looking at integration. Martin: Just to follow up on this, do the surgeons here see a bene¢t in early postoperative function under physiological loading conditions? Trippel: Right now, many of these people are immobilized anyway. This is a problem, for two reasons. First, it is probably not good for their joints to be immobilized for a long period. More importantly, patients don’t like to be immobilized. This raises the problem of patient compliance. If you have a therapy that is dependent on the patient’s willingness to comply with an immobilization routine that they don’t want to do, that therapy will fail in a certain percentage of patients because these individuals won’t be compliant and won’t remain immobile. It would be much better to have a construct that you could put in and which would work while enabling early motion. Lindahl: We are implanting the cells into the cartilage defect in the joint as a suspension. The joint is not immobilized, but undergoes passive motion without loading. The time before full loading is eight to 12 weeks. van Blitterswijk: One of the things I liked about your approach is that you moved from comparing the mechanical properties of the graft with those of adult cartilage, and began comparing them with fetal cartilage instead. Perhaps you are making life di⁄cult for yourselves, though, because your goal is to replace ¢brocartilaginous tissue. We are always aiming for the properties of normal hyaline cartilage, but shouldn’t we be satis¢ed when we are doing a better job when compared with ¢brocartilage?


49

Vunjak-Novakovic: We have been trying to approach the properties of hyaline cartilage. The idea is to regenerate to some extent the structural and mechanical properties inherent to this tissue in constructs grown in vitro, and then let these constructs mature and integrate in vivo, rather than growing ¢brocartilage and expecting it to transform into articular cartilage. van Blitterswijk: I am not asking you to grow ¢brocartilage; I’m asking you to do what you do, but to then compare it with ¢brocartilage. As long as it is better, you can still consider using it clinically. Caplan: That raises a valid point. We aim for perfect regeneration. But is repair an acceptable solution in tissue engineering compared with regeneration? In this context, ¢brocartilage would be seen as a repair substance. How is that bad, in a practical sense, in terms of the longevity of the joint? Lohmander: The key question is, in ¢ve years will the patient be able to tell the di¡erence? Pavesio: And is there an improvement compared to currently available conservative surgical techniques that originate ¢brous cartilage? Vunjak-Novakovic: More information is needed to address this point. The situation is further complicated by the e¡ect of age and spatial position within the tissue on its mechanical properties. In adult cartilage, the con¢ned-compression equilibrium moduli at the tissue surface are 200^250 kPa, in the middle zone about 400^500 kPa, and the deep zone about 700^900 kPa. In the developing cartilage, these values are lower. What is our baseline? Do we need the engineered cartilage just to compare to the articular surface? In this case, we are already at 50% of normal after four weeks of culture. Or do we need it to be as good as the deep zone? It all depends on what our goals are, and what the subsequent remodelling will be. Is the construct going to integrate and remodel into a new tissue with site- and scale-speci¢c structural and mechanical properties of native articular cartilage? We don’t really know how much is enough, and what are the targeted mechanical properties for engineered cartilage. Lohmander: If we are to convince surgeons to use tissue-engineered cartilage, it has to be practically useful in the setting of the operating theatre. If it becomes too di⁄cult and technically demanding it is not sellable. People won’t buy it. Barry: I’d like to address the issue of chemistry versus structure in terms of properties of a sca¡old. We did some experiments several years ago where we seeded di¡erent sca¡olds with progenitor cells, including HYAFF111 and PGA felt. The observation we made was that in the HYAFF111 sponge the proportion of cells that remained viable was much higher than in the case of PGA. It also took much less time for the cells to di¡erentiate into chondrocytes in the case of the HYAFF111 compared with the PGA felt. Are there really ¢rm conclusions about the chemical nature of the sca¡old, and how this in£uences the engineered tissue?

50

DISCUSSION

Vunjak-Novakovic: No. I was careful to say that our results were within the conditions of the study that involved di¡erentiated chondrocytes. In fact, there is some previously published work showing that PLGA-PEG in the form of a sponge was a better substrate for the growth of mesenchymal progenitor cells than the PGA mesh. The chemical properties and the kinetics of sca¡old degradation are also important as they determine whether the sca¡old will collapse before the cells have the ability to synthesize su⁄cient amount of matrix. Pavesio: It seems to me that the e¡ect of the bioreactor was masking everything else. That is, an appropriate sca¡old geometry and bioreactor design can create good cartilage. Perhaps the key question is whether it is preferable to implant a tissue construct with abundant extracellular matrix, or a less mature construct delivering actively proliferating cells. Clearly, I am inclined towards a cell delivery approach, but I may be biased. Vunjak-Novakovic: My expectation is that the best candidate for tissue engineered cartilage repair will be an immature tissue, rather than just cells on a biomaterial. I’m not suggesting to use a completely formed tissue, but rather a construct that is immature enough to further develop, remodel and integrate in vivo, while being mature enough to have some mechanical properties at the time of implantation that can enhance survival and handling. There is no experimental evidence favouring one or the other approach at this time. Quintavalla: The problem I have in visualizing how well an implant works is that it is always going into a site where there are stem cells coming through. It is hard to see at the end of the operation which was the original implant and which tissue comes from the normal healing response. If the pieces of implant tissue were implanted subcutaneously or in the joint without loading, would they maintain themselves as cartilage? Or would it disintegrate or go to bone? Vunjak-Novakovic: Constructs implanted subcutaneously maintain themselves as cartilage. We have used subcutaneous implantation to demonstrate the maintenance of gene expression in constructs based on transfected cells, and the explants were cartilaginous. Engineered cartilage implanted into deep osteochondral defects remodelled into physiologically thick articular cartilage and subchondral bone. Quintavalla: Does the engineered cartilage become ¢rmer or harder? Vunjak-Novakovic: Over time, we see improvements in mechanical properties of the grafts. Hollander: One thing that is missing from this discussion is the size of the lesion and the size of the construct that you are making. Presumably, if you are making a larger construct then getting good mechanical properties in the centre is going to be harder. Therefore you may buy yourself very little compared with a cell delivery system. If you are going for small focal lesions, however, you may gain some advantage.


51

Vunjak-Novakovic: The constructs that we grow are typically 1 cm in diameter and 5 mm thick. The thickness, rather than area, is the critical parameter. Also, it is likely that the initial structural and mechanical properties will be more important for large defects than for focal defects. Ratcli¡e: Gordana Vunjak-Novakovic’s bioreactor work shows that the growth conditions can modulate tissue properties, whether they be biochemical or mechanical. This in itself is a tool. Her experiments indicate that we can go and do the experiments comparing the di¡erent tissues that can be made in this way to see which is best. This is where this type of work leads us. It comes back to a question of what we really want. Do we want a material that lasts just ¢ve years? This is a starting point. The surgeons want something they can manipulate and implant with relative ease. What you start to build here are some design criteria. It is critical that the clinicians give us good feedback in terms of what they really need. The point has been raised about resurfacing large areas. Thickness is a big issue here, so the issue there is not so much one of integration, but ¢xation. Gordana is introducing the concept that we can manipulate growth and generate lots of di¡erent types of properties and compositions, and at the other end we need to start thinking about what we really want. Then we can start doing the compare and contrast experiments.

Tissue-engineered versus native cartilage: linkage between cellular mechano-transduction and biomechanical properties Jennifer H. Lee*, John Kisiday* and Alan J. Grodzinsky*{{1 *Biological Engineering Division, Department of {Electrical Engineering and Computer Science, Department of {Mechanical Engineering, Massachusetts Institute of Technology, Cambridge MA 02139-4307, USA

Abstract. Recent studies demonstrate that chondrocytes in native articular cartilage and in tissue-engineered constructs respond to mechanical stimuli through multiple regulatory pathways. Responses of the cells are manifested by intra- and intercellular signalling, alterations in transcription level, protein translation, post-translational modi¢cations, and synthesis of intracellular and extracellular macromolecules. In addition, mechanical stimuli can alter the balance between anabolic and catabolic processes that are critically important to cell-mediated extracellular assembly and degradation of the tissue matrix and, therefore, to the survival of tissue engineered constructs. Chondrocyte mechanotransduction is therefore a critically important link to the biomechanical properties of native cartilage and to developing constructs. Since implanted cartilage repair tissue will be subjected to mechanical loads throughout its lifetime, it is essential that the resident cells respond appropriately to the range of static and dynamic compressive and shear deformations in vivo in a manner that enables adaptive remodelling and minimizes catabolic degradation. The in vivo environment should thereby signal tissue-speci¢c maturation and integration processes in order to achieve the most appropriate tissue morphology and biomechanical function. 2003 Tissue engineering of cartilage and bone. Wiley, Chichester (Novartis Foundation Symposium 249) p 52^69

Biomechanical environment of chondrocytes in normal articular cartilage Mechanical loading of human synovial joints results in a complex combination of compression and shear forces acting on articular cartilage (Fig. 1). As a result, peak 1This

paper was presented at the symposium by Alan J. Grodzinsky to whom correspondence should be addressed. 52

ENGINEERED VERSUS NATIVE CARTILAGE

53

FIG. 1. (A) Joint loading can cause (B) cartilage tissue compressive and (C) shear strains leading to cell and matrix deformation, intratissue £uid £ow, V, £uid-induced electric ¢elds, E, and associated streaming potential drops, pressure gradients, and associated forces and £ows. These physical signals can initiate cellular mechanotransduction mechanisms (D) involving signalling, and changes in gene transcription, protein translation, post-translational modi¢cations, and molecular secretion.

stress amplitudes acting on cartilage can reach 10^20 MPa (100^200 atm) during activities such as stair climbing (Hodge et al 1986). Cartilage compressions of 15^40% may occur in response to long-term or ‘static’ loads within the physiological range (Herberhold et al 1999). In contrast, compressions of only a few percent occur during normal ambulation (e.g. the ‘dynamic’ strains that occur at walking frequencies of 1 Hz). The ability of cartilage to withstand physiological compressive, tensile and shear forces depends on the composition and structural integrity of its extracellular matrix (ECM). In turn, the maintenance of a functionally intact ECM requires chondrocyte-mediated synthesis, assembly, and degradation of proteoglycans (PGs), collagens, non-collagenous proteins and glycoproteins, and other matrix molecules. The collagen ¢brillar network of the ECM is primarily responsible for the ability of cartilage to withstand tensile and shear deformation during joint loading. In contrast, aggrecan molecules contribute to equilibrium sti¡ness in compression and shear due, in part, to

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FIG. 2. Mechanical properties of tissue-engineered cartilage constructs can be measured using con¢gurations developed for native cartilage biomechanical characterization studies including (A) con¢ned and (B) uncon¢ned compression, (C) tension, and (D) shear.

electrostatic repulsion interactions between the glycosaminoglycan (GAG) chains covalently bound to the core protein of aggrecan. The negatively charged GAG chains also confer a high resistance to £uid £ow within the tissue (Maroudas 1979), which leads to pressurization of the intratissue £uid during dynamic loading and a concomitantly high dynamic compressive sti¡ness that increases with the rate of compression. Biomechanical properties of natural cartilage During the past decades, biomechanical researchers have developed experimental and theoretical modelling tools to quantitatively characterize the material properties of native and tissue engineered cartilage (Butler et al 2000). The properties of native cartilage can be viewed as the ‘standard’ to which the properties of tissue-engineered constructs can be compared. For example, the equilibrium compressive modulus of adult articular cartilage measured in uniaxial con¢ned and uncon¢ned compression is of order 1 MPa, the dynamic compressive sti¡ness at 1.0 Hz approximately 10 times higher, the equilibrium shear modulus 0.2^0.5 MPa, and the equilibrium tensile modulus 10^50 MPa (Fig. 2). These are order-of-magnitude average values that are known to vary anisotropically and inhomogeneously with tissue depth, location along the


55

joint surface and between species. For example, the equilibrium modulus of cartilage in con¢ned compression has been reported to vary with depth such that deep zone tissue may be 10 times sti¡er than surface zone tissue (Chen et al 2001). A novel sca¡old for cartilage tissue engineering Emerging medical technologies for repair of articular cartilage include delivery of cells or cell-seeded sca¡olds to facilitate tissue regeneration. Biocompatible sca¡olds assist in providing a template for cell distribution and ECM accumulation in a three-dimensional geometry. A variety of biologically derived and synthetic polymeric and hydrogel materials are actively under investigation as sca¡olds for cartilage tissue repair, as described in this book. We have developed a novel self-assembling peptide hydrogel sca¡old for cartilage repair and developed a method to encapsulate chondrocytes within the peptide hydrogel (Kisiday et al 2002). Peptides containing amino acid sequences of alternating hydrophobic and hydrophilic side groups are able to self-assemble into stable hydrogels at low (0.1^1%) peptide concentrations. These selfassembling peptides form stable b-sheet structures when dissolved in deionized water and exposure to electrolyte or tissue culture medium initiates b-sheet assembly into interweaving nano¢bres (Fig. 3). During 4 weeks of culture in vitro, chondrocytes seeded within the peptide hydrogel at 15106 cells/ml retained their morphology and developed a cartilage-like ECM rich in PGs and type II collagen, indicative of a stable chondrocyte phenotype (Kisiday et al 2002). Total GAG accumulation in peptide hydrogels using 10% FBS medium increased with time in culture, reaching 70 mg/plug by day 28 (Fig. 4). Time dependent accumulation of GAG-rich ECM was paralleled by increases in equilibrium compressive modulus, from 1 kPa at day 0 (i.e. the weak sti¡ness of an acellular peptide gel) to 30-fold higher by day 26 (Fig. 4). Interestingly, cells seeded into peptide gels at 30106 cells/ml and cultured in medium containing 1% ITS+0.2% FBS showed an even more profound increase in equilibrium modulus to 93 kPa by day 28, along with a corresponding dynamic sti¡ness at 1 Hz of 1.3 MPa (Fig. 4), both values being approximately one-¢fth to one-third those of native human and animal articular cartilages. Results to-date have demonstrated the potential of a self-assembling peptide hydrogel as a sca¡old for the synthesis and accumulation of a true cartilage-like tissue (Kisiday 2002). How similar must engineered cartilage be to natural cartilage? The time evolution of neo-ECM accumulation and the associated development of tissue biomechanical properties that is embodied in the data of Fig. 4 motivate

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FIG. 3. Self-assembling KLD-12 peptide hydrogel (0.4% peptide concentration) formed from b-sheets that further assemble into interweaving nano¢bres (Top). The entire assembly process can occur in the presence of cells around individual chondrocytes, forming a gel of any shape (Bottom) seeded here at 15^30 million cells per ml.

FIG. 4. GAG accumulation and equilibrium compressive modulus of disks of chondrocyteseeded self-assembling peptide hydrogel constructs versus time in culture for specimens seeded at 15 million cells per ml and cultured in DMEM with 10% fetal bovine serum (FBS) over 28 days, and specimens seeded at 30 million cells per ml cultured in 1% ITS with 0.2% FBS by day 35 (data adapted from Kisiday 2002).


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fundamental questions about the underlying assumptions of tissue engineering. Should a fully mature neo-tissue ¢rst be fabricated in vitro prior to implantation? Alternatively, is it su⁄cient to initiate appropriate biosynthetic and growth pro¢les in vitro and then implant at an earlier stage? The latter approach would enable the in vivo environment to signal tissue-speci¢c maturation and integration processes in order to achieve the most appropriate tissue morphology and function. If this latter approach is chosen, how long should the initial ECM biosynthesis and growth period be before implantation? To address these questions, end-point measures of construct biology and biomechanics are needed as a function of time. Since implanted cartilage repair tissue will be subjected to mechanical loads throughout its lifetime, it is essential that the resident cells respond appropriately to the range of static and dynamic compressive and shear deformations in vivo in a manner that enables adaptive remodelling and minimizes catabolic degradation. Mechanical regulation of chondrocyte metabolism Studies in vivo have shown that chondrocyte mechanotransduction is critically important in the cell-mediated feedback between joint loading, the molecular structure of newly synthesized ECM, and the resulting functional biomechanical properties of cartilage. Many physical forces and £ows that occur in cartilage during loading in vivo (Fig. 1) have been identi¢ed and quanti¢ed in vitro (see recent reviews by DiMicco et al 2002, Wang et al 2002). Dynamic compression of cartilage results in deformation of cells and extracellular matrix, hydrostatic pressurization of the tissue £uid, pressure gradients and the accompanying £ow of £uid within the tissue, and streaming potentials and currents induced by tissue £uid £ow. In addition, local changes in tissue volume caused by static compression lead to physicochemical changes including alterations in matrix water content, ¢xed charge density, mobile ion concentrations and osmotic pressure. Any of these mechanical, chemical, or electrical phenomena may a¡ect cellular metabolism. Recent studies suggest that there are multiple regulatory pathways by which chondrocytes can sense and respond to mechanical stimuli. These pathways include upstream signalling and changes at the level of gene transcription, protein translation, and post-translational modi¢cations of newly synthesized macromolecules. In vitro models for studying mechanical regulation of chondrocyte gene expression Cartilage explants. Since the mechanisms by which chondrocytes respond to mechanical stimuli are di⁄cult to quantify in vivo, models such as cartilage explant organ culture and three-dimensional chondrocyte-gel culture systems have been

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FIG. 5. (Top) Dynamic compression can stimulate proteoglycan synthesis in cartilage explants. Insulin-like growth factor 1 (IGF1) is a potent stimulator of synthesis and a concentration of 300 ng/ml results in maximal stimulation using this system. However, dynamic compression superimposed on 300 ng/ml IGF1 causes a further signi¢cant increase in biosynthesis and accelerates transport of the protein into the explants (data adapted from Bonassar 2001). (Bottom) Synthesis of total protein (proline incorporation) and proteoglycans (sulfate incorporation) was signi¢cantly increased by shear deformation above 1.5% dynamic strain amplitude at a frequency of 0.1 Hz (data from Jin 2001).

used. Cartilage explants preserve native tissue structure and cell^matrix interactions; therefore, they enable quantitative correlations between mechanical loading parameters and biological responses such as gene expression and biosynthesis. Studies using animal and human cartilage explants have shown that static compression causes a dose-dependent decrease in PG and protein biosynthesis, while low amplitude dynamic compression (Fig. 5) can stimulate biosynthesis and augment transport of soluble proteins such as growth factors (Bonassar et al 2001). Contrary to dynamic compression, dynamic tissue shear strain amplitudes of 1^3% over a wide range of frequencies (0.01^1 Hz) can stimulate collagen signi¢cantly more than PG synthesis in the presence of serum (Jin et al 2001). Application of a constant stress (0.025^0.5 MPa in load control) (Valhmu et al 1998) as well as constant 50% strain (in displacement control) (Ragan et al 1999) both lead to an initial increase by 1 hour in aggrecan expression during the initial transient phase of creep or stress relaxation, respectively. By 24 h of 50% static


59

compression, both aggrecan and collagen mRNA levels were decreased signi¢cantly compared to uncompressed controls (Ragan et al 1999). Inhibitor studies by Valhmu (Valhmu et al 1998, Valhmu & Raia 2002) showed that the transient changes in gene expression they observed were related to signal transduction mechanisms involving cAMP, phosphoinositol, inositol-1,4,5trisphosphate and Ca2+/calmodulin-dependent cascades. Interestingly, 25^50% static compression had been observed previously to inhibit synthesis of collagen and PG within 1 h (Sah et al 1989). Thus, although mechanical compression can rapidly alter expression of these genes, the decrease in synthesis appears not to be related solely to changes in expression. Lee et al (2002a) observed a fall in glycolytic rates in response to mechanical stress applied to explants, and also concluded that changes in matrix synthesis in cartilage can occur by other mechanisms than changes in gene expression. These studies reinforce the notion that a complex combination of pathways appears fundamental to cartilage mechanobiology. The coupling between mechanical deformation and cellular response may also occur via intermediate soluble factors. Vincent et al (2002) recently observed transient activation of ERK (increase in phosphorylated ERK) within 5 minutes after harvesting porcine cartilage explants, which subsided after 48 h in medium. The same result was obtained after cutting cartilage explants in situ in an opened joint. Further investigation led to the identi¢cation of ¢broblast growth factor 2 (FGF2) as the ERK activating factor, which was released from extracellular stores by the mechanical deformation caused by cutting of the cartilage. Chondrocytes in high-density monolayer. Investigators have cautioned that the use of isolated chondrocytes that are depleted of natural matrix must be approached with care regarding the relevance of such experiments to native tissue and the potential for chondrocyte dedi¡erentiation. Nevertheless, mechanical stimulation studies of high density monolayer cultures of chondrosarcoma-derived cell lines or chondrocytes isolated from native cartilage are relevant to aspects of tissue engineering. Cyclic stretching, applied hydrostatic pressure, and £uid shear forces have been shown to induce changes in cell biosynthesis and expression of ECM molecules, matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), and heat shock protein 70 (hsp70). While such studies aim to reproduce the responses that may be relevant to in vivo loading, the relation between £uid shear and tissue shear deformation, and between hydrostatic pressure and tissue compression, etc. must be interpreted carefully. For example, while cyclical stretching results in tensional forces at the macroscopic level, under the right conditions chondrocytes may be subjected to compressive forces at the cellular level if there is an appropriate pericellular matrix. DeWitt et al (1984) subjected high-density chick epiphyseal chondrocytes to 5.5% cyclical strain at 0.2 Hz. After 24 h, but not at 4 h, they found a 1.4-fold

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increase in GAG synthesis, a 2.4-fold increase in DNA synthesis, and a 2.2-fold increase in cAMP. More recently, Fujisawa et al (1999) used RT-PCR to quantify gene expression in rabbit chondrocytes and HCS-2/8 human chondrosarcoma cells in response to cyclical stretching. 5 or 15 kPa applied at 0.5 Hz inhibited DNA synthesis, proteoglycan and protein synthesis, but increased expression of interleukin (IL)1 and pro- and active-MMP9 within 3^6 h. Hydrostatic pressure has also been observed to alter chondrocyte synthesis and gene expression depending on the magnitude and frequency of the applied load. Dynamic pressure at 10 MPa amplitude, 1 Hz, increased GAG synthesis and expression of aggrecan and type II collagen measured by Northern blotting (Smith et al 1996). Takahashi et al (1997) found that constant hydrostatic loading of HCS-2/8 decreased sulfate incorporation at 10 and 50 MPa but increased incorporation at 1 and 5 MPa. RT-PCR showed an increase in transforming growth factor (TGF) b expression at 5 MPa and a decrease in expression at 50 MPa of applied pressure. Investigators have also focused on the e¡ects of hydrostatic pressure on transcription and translation of hsp70 in high-density monolayer cultures. Kaarniranta et al (1998, 2001) found that 30 MPa of constant hydrostatic pressure applied for 12 hours led to an increase in hsp70 mRNA and intracellular protein (by Northern and Western blotting), signifying a stress response of the chondrocytes to pressure. However, analysis of the transcription of new hsp70 mRNA by nuclear run-on assay showed that no new transcripts were produced by pressure, while the positive control of applied heat shock (43 8C for 1^ 5 h) did induce transcripts. Kaarniranta et al (1998, 2001) concluded that the increases in mRNA and protein level were due to stabilization of the hsp70 transcripts rather than induction of new synthesis of hsp70 molecules. This response of transcript stabilization in response to constant hydrostatic pressure was found to be unique to hsp 70 through use of a cDNA array. Stabilization was not seen to occur with other genes (Sironen et al 2002). Mechanobiology in 3D sca¡old cultures for tissue engineering Investigators have employed widely varying mechanical stimulation protocols to examine chondrocyte di¡erentiation, proliferation, gene expression and biosynthesis in 3D sca¡old cultures relevant to cartilage tissue engineering. Time courses have ranged from 24 h of continuous static or dynamic loading to more complex 8 week regimens consisting of periods of dynamic compression interspersed between resting periods, and results have varied widely depending on magnitude, duration, duty cycle and type of mechanical stimulation. Both inhibition as well as stimulation of cartilage-like properties in the 3D culture systems have been reported, consistent with the likelihood that there are multiple pathways involved in chondrocyte mechanotransduction.


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Buschmann et al (1995) and Mauck et al (2000) demonstrated that ECM biosynthesis by calf chondrocytes in agarose gel culture could be stimulated by dynamic compression over short and long term loading regimes, respectively. The ability of chondrocytes to respond to loading appeared to depend on the presence of a well developed pericellular matrix (Buschmann et al 1995). Davisson et al (2002) showed that 24 h static compression inhibited GAG and protein synthesis while 24 h dynamic compression could stimulate synthesis by newborn calf chondrocytes seeded into ¢brous, non-woven poly(glycolic acid) (PGA). Compression parameters in these studies were similar to those used in previous cartilage explant experiments (Sah et al 1989, Bonassar et al 2001). In contrast, when seeded into type I collagen gels, chondrocytes exposed to static compression showed inhibition of ECM synthesis and gene expression, though oscillatory compression had no e¡ect on gene expression (Hunter et al 2002). Chondrocyte proliferation in unloaded sca¡olds appears to be highly sensitive to the sca¡old material. For example, cell number in self-assembling peptide gel culture was about fourfold higher than that in agarose gel culture during the ¢rst 7^9 days, while the cells still maintained chondrocyte phenotypic expression. The addition of compression can further modulate proliferation, as exempli¢ed by the inhibition of proliferation of chondrocytes when transplanted onto devitalized cartilage and subjected to low magnitude static compression (Li et al 2000). The e¡ect of mechanical loading on di¡erentiation and chondrogenesis has important implications regarding the use of stem cells for cartilage tissue engineering. Basic studies on biomechanical regulation of limb bud cells as a model for chondrogenesis have shown the sensitivity of mesenchymal cells to compression. Takahashi et al (1998) used mouse embryonic limb bud cells seeded into 3D gels of type I collagen from calf skin and examined tissue morphology, gene expression and protein translation. After 5 days of a static load of 1 kPa, the compressed cultures showed more chondrogenic aggregates as well as more cartilaginous matrix. Quantitative RT-PCR showed a ¢vefold increase in type II collagen expression and a 3.4-fold increase in aggrecan over that seen in control uncompressed cultures. The induction of collagen II expression increased in a load/dose-dependent manner from 1 kPa to 2 kPa. Expression of the Sox9 gene in these mice in vivo increases to a peak at day 11 during development and then decreases through day 14. In free swelling conditions in vitro, Sox9 transcript levels increased until day 3 and then decreased. However, in compressed cultures, Sox9 was maintained at an elevated level through day 10 of culture while transcript levels of IL1b, a suppressor of type II collagen, were signi¢cantly lower by 25% compared to unloaded control cultures. In contrast, Elder et al (2000) state that intermittent loading generally enhances chondrocyte di¡erentiation, and suggest that the results of Takahashi et al (1998) are a notable exception in this regard. Therefore, Elder and colleagues seeded stage 23/24 chick limb bud mesenchymal

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cells into agarose gel culture to study the e¡ects of cyclic compression on the chondrogenesis. They found that 0.33 Hz loading at 9 kPa peak stress (in load control) resulted in twice as many cells entering the chondrogenic pathway as manifested by sulfate incorporation into GAG, compared to unloaded or statically loaded controls.

Non-mechanical stimuli Several non-mechanical stimuli have also been applied to 3D cultures of chondrocytes in an attempt to induce the formation of cartilage-like properties in the construct. However, the focus of this work has been mainly to determine optimal culture conditions for growing cartilage tissue. Stimuli tested include oxygen tension, medium perfusion, exogenous type II collagen supplementation and transfection with recombinant bone morphogenetic proteins (BMPs). Oxygen tensions between 5% and 20% (physiological range 1^10%) allow chondrocytes to remain viable and express the aggrecan gene. Anoxic conditions (pO250.1%) result in a lower rate of glycolysis and a decrease in 18S and 28S ribosomal RNA as well as an overall decrease in mRNA present in the cells (Grimshaw & Mason 2000). Medium perfusion inhibited incorporation of S-GAG and expression of aggrecan and collagen II. Though medium perfusion has been found to be bene¢cial for culture of other cell types, it can inhibit chondrogenesis by articular chondrocytes in 3D culture (Mizuno et al 2001). Using an alginate bead culture system with exogenous type II collagen added, Lee et al (2002b) observed through antibody studies that chondrocyte binding to collagen II occurs through the b1 integrin. This binding event modulates cell response to TGFb1 providing speci¢city to the cytokine-signalling cascade (Lee et al 2002b). Again using alginate beads, Kaps et al (2002) found that BMP7 induced a decrease in type I collagen expression while transcription of type II collagen remained stable. There was also an increase in matrix synthesis by cells transfected with BMP7 compared to other transfected cells (BMP2, BMP4, BMP5, BMP6) or control untransfected cells (Kaps et al 2002).

References Bonassar LJ, Grodzinsky AJ, Frank EH, Davila SG, Bhaktav NR, Trippel SB 2001 The e¡ect of dynamic compression on the response of articular cartilage to insulin-like growth factor-I. J Orthop Res 19:11^17 Buschmann MD, Gluzband YA, Grodzinsky AJ, Hunziker EB 1995 Mechanical compression modulates matrix biosynthesis in chondrocyte/agarose culture. J Cell Sci 108:1497^1508 Butler DL, Goldstein SA, Guilak F 2000 Functional tissue engineering: the role of biomechanics. J Biomech Eng 122:570^575


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Chen AC, Bae WC, Schinagl RM, Sah RL 2001 Depth- and strain-dependent mechanical and electromechanical properties of full-thickness bovine articular cartilage in con¢ned compression. J Biomech 34:1^12 Davisson T, Kunig S, Chen A, Sah R, Ratcli¡e A 2002 Static and dynamic compression modulate matrix metabolism in tissue engineered cartilage. J Orthop Res 20:842^848 De Witt MT, Handley CJ, Oakes BW, Lowther DA 1984 In vitro response of chondrocytes to mechanical loading. The e¡ect of short term mechanical tension. Connect Tissue Res 12:97^109 DiMicco MA, Waters SN, Akeson WH, Sah RL 2002 Integrative articular cartilage repair: dependence on developmental stage and collagen metabolism. Osteoarthritis Cartilage 10:218^225 Fujisawa T, Hattori T, Takahashi K, Kuboki T, Yamashita A, Takigawa M 1999 Cyclic mechanical stress induces extracellular matrix degradation in cultured chondrocytes via gene expression of matrix metalloproteinases and interleukin-1. J Biochem (Tokyo) 125:966^975 Grimshaw MJ, Mason RM 2000 Bovine articular chondrocyte function in vitro depends upon oxygen tension. Osteoarthritis Cartilage 8:386^392 Herberhold C, Faber S, Stammberger T et al 1999 In situ measurement of articular cartilage deformation in intact femoropatellar joints under static loading. J Biomech 32:1287^1295 Hodge WA, Fijan RS, Carlson KL, Burgess RG, Harris WH, Mann RW 1986 Contact pressures in the human hip joint measured in vivo. Proc Natl Acad Sci USA 83:2879^2883 Hunter CJ, Imler SM, Malaviya P, Nerem RM, Levenston ME 2002 Mechanical compression alters gene expression and extracellular matrix synthesis by chondrocytes cultured in collagen I gels. Biomaterials 23:1249^1259 Jin M, Frank EH, Quinn TM, Hunziker EB, Grodzinsky AJ 2001 Tissue shear deformation stimulates proteoglycan and protein biosynthesis in bovine cartilage explants. Arch Biochem Biophys 395:41^48 Kaarniranta K, Elo M, Sironen R et al 1998 Hsp70 accumulation in chondrocytic cells exposed to high continuous hydrostatic pressure coincides with mRNA stabilization rather than transcriptional activation. Proc Natl Acad Sci USA 95:2319^2324 Kaarniranta K, Holmberg CI, Lammi MJ, Eriksson JE, Sistonen L, Helminen HJ 2001 Primary chondrocytes resist hydrostatic pressure-induced stress while primary synovial cells and ¢broblasts show modi¢ed Hsp70 response. Osteoarthritis Cartilage 9:7^13 Kaps C, Bramlage C, Smolian H et al 2002 Bone morphogenetic proteins promote cartilage di¡erentiation and protect engineered arti¢cial cartilage from ¢broblast invasion and destruction. Arthritis Rheum 46:149^162 Kisiday J, Jin M, Kurz B et al 2002 Self-assembling peptide hydrogel fosters chondrocyte extracellular matrix production and cell division: implications for cartilage tissue repair. Proc Natl Acad Sci USA 99:9996^10 001 Lee RB, Wilkins RJ, Razaq S, Urban JP 2002a The e¡ect of mechanical stress on cartilage energy metabolism. Biorheology 39:133^143 Lee JW, Qi WN, Scully SP 2002b The involvement of beta1 integrin in the modulation by collagen of chondrocyte-response to transforming growth factor-beta1. J Orthop Res 20:66^75 Li KW, Falcovitz YH, Nagrampa JP et al 2000 Mechanical compression modulates proliferation of transplanted chondrocytes. J Orthop Res 18:374^382 Maroudas A 1979 Physicochemical properties of articular cartilage. In: Freeman MAR (ed) Adult articular cartilage, 2nd edn, Tunbridge Wells, England, p 215^290 Mauck RL, Soltz MA, Wang CC et al 2000 Functional tissue engineering of articular cartilage through dynamic loading of chondrocyte-seeded agarose gels. J Biomech Eng 122:252^260 Mizuno S, Allemann F, Glowacki J 2001 E¡ects of medium perfusion on matrix production by bovine chondrocytes in three-dimensional collagen sponges. J Biomed Mater Res 56:368^375

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Ragan PM, Badger AM, Cook M et al 1999 Down-regulation of chondrocyte aggrecan and typeII collagen gene expression correlates with increases in static compression magnitude and duration. J Orthop Res 17:836^842 Sah RL, Kim YJ, Doong JY, Grodzinsky AJ, Plaas AH, Sandy JD 1989 Biosynthetic response of cartilage explants to dynamic compression. J Orthop Res 7:619^636 Sironen RK, Karjalainen HM, Torronen K et al 2002 High pressure e¡ects on cellular expression pro¢le and mRNA stability. A cDNA array analysis. Biorheology 39:111^117 Smith RL, Rusk SF, Ellison BE et al 1996 In vitro stimulation of articular chondrocyte mRNA and extracellular matrix synthesis by hydrostatic pressure. J Orthop Res 14:53^60 Takahashi K, Kubo T, Kobayashi K et al 1997 Hydrostatic pressure in£uences mRNA expression of transforming growth factor-beta 1 and heat shock protein 70 in chondrocytelike cell line. J Orthop Res 15:150^158 Takahashi I, Nuckolls GH, Takahashi K et al 1998 Compressive force promotes sox9, type II collagen and aggrecan and inhibits IL-1beta expression resulting in chondrogenesis in mouse embryonic limb bud mesenchymal cells. J Cell Sci 111:2067^2076 Valhmu WB, Raia FJ 2002 myo-Inositol 1,4,5-trisphosphate and Ca2þ /calmodulin-dependent factors mediate transduction of compression-induced signals in bovine articular chondrocytes. Biochem J 361:689^696 Valhmu WB, Stazzone EJ, Bachrach NM et al 1998 Load-controlled compression of articular cartilage induces a transient stimulation of aggrecan gene expression. Arch Biochem Biophys 353:29^36 Vincent T, Hermansson M, Bolton M, Wait R, Saklatvala J 2002 Basic FGF mediates an immediate response of articular cartilage to mechanical injury. Proc Natl Acad Sci USA 99:8259^8564 Wang CC, Guo XE, Sun D, Mow VC, Ateshian GA, Hung CT 2002 The functional environment of chondrocytes within cartilage subjected to compressive loading: a theoretical and experimental approach. Biorheology 39:11^25

DISCUSSION Goldstein: I would like to know about the mechanical properties of engineered cartilage. I have always considered the properties of cartilage to be dominated by its ability to manage water. To some degree this is discounting the shear attribute of the collagen. The ability to manage this water is a function of the character of the proteoglycans: their size and also how they are organized relative to the collagen matrix. It seems to me that none of these studies really talk about the character of proteoglycans. Whether the proteoglycan synthesis is increased or decreased may be totally irrelevant, particularly if the synthesis is of smaller chain proteoglycans. In this case all you will get is increased swelling and have low modulus. Don’t you think our objective function should change, and some of the variables we look at should be di¡erent than just modulus? Structure^function is everything: maybe we should focus on the structure being synthesized as our objective function to know how we are doing in terms of generating these properties. Grodzinsky:I agree. The molecular structure is ultimately the key to success, in real cartilage as well as in tissue-engineered constructs. This issue of managing water as being an important aspect of material properties is certainly critical for the


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compressive sti¡ness. But for tensile and shear sti¡ness it is much less important. Normal cartilage has a tensile modulus largely due to the ¢brillar collagen structure and cross-linking. Very few attributes of water management are involved in tensile sti¡ness. Clearly, this is a mode of failure of cartilage as well as constructs. The same is true for shear. For the case of pure shear, there is essentially minimal intratissue water £ow. But for compressive loading, the ability of the GAG molecules to imbibe water and help resist compression is critical. Probably the most telling mechanical property that addresses the issue of water management is the di¡erence between dynamic sti¡ness and equilibrium sti¡ness. The reason why dynamic sti¡ness is so much higher in compression is due to matrix resistance to £uid £ow and the resulting £uid pressurization. If the molecular structure that is synthesized within a construct is loose enough, di¡erent constructs having di¡erent hydraulic permeabilities will have di¡erent dynamic sti¡nesses even though they may have the same total GAG content. This may play a role in tissue failure. Caplan: You didn’t address a key issue that Gordana Vunjak-Novakovic brought up in the discussion after her paper: this tissue called cartilage is heterogeneous in its zonality. Therefore the texture of the surface versus the texture of the deep cartilage are intrinsically di¡erent. Do you go for this in vitro, or do you go for this in vivo? If you go for this in vivo, is there an exercise regime or conditioning regime that will be obligatory for a half-done piece of cartilage versus a full-done one? Grodzinsky: The answer to all your questions is, or course, that I don’t know. But I can speculate. The order of magnitude values for moduli that I listed were simply average macroscopic values. One can look at spatial scales of hundreds of microns versus a millimetre, and even at the level of the cell or pericellular matrix. There is a whole hierarchy of molecular to cell to tissue mechanical properties that people are interested in studying. In a sense, we are back a step or two. We are trying to see whether we can get to ¢rst base, let alone second base. A lot of the material properties that we have been talking about are much lower than those of normal cartilage, let alone the ¢ne gradations associated with depth-dependent properties. I think we will have to address the ¢ne gradations, and this is one of the rationales for mechanical loading. Perhaps there is a signal that cells near the surface will receive that is di¡erent from cells in the deeper part of the construct. This is because deformation of the whole construct will cause a gradation of deformations within, as you are saying. Let’s say you start with a homogeneous distribution of cells. If locally the deformation at the top is higher than the middle or deep zone, the cells at the top will respond di¡erently as well. Perhaps this is related to the way that cells in native tissue respond. Ka¢enah: Does this extend to the alternate loading that you mentioned? Grodzinsky: All we have is observation. The question is, does the cell adapt, and if you maintain a continuous loading is that good? People in the bone area have

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DISCUSSION

experimented with discontinuous loading regimens. This is something I think we have to explore in order to achieve increased synthesis rates as well as good quality of molecules. Vunjak-Novakovic: There is a large body of literature showing that synthesis rates of proteoglycan and collagen are increased in response to speci¢c loading regimens. In contrast, there is hardly any evidence about improved tissue compositions and mechanical properties. You observed an improvement in GAG content, but it was only 25% as compared to the 200% increase in GAG synthesis rate. Does this suggest that the e¡ect on synthesis rate is only transient? Also, is the loading con¢guration important? The resulting £uid £ow within the tissue will certainly be di¡erent for uncon¢ned and con¢ned compression. Is there something we could do to translate the enhancement of synthesis rates into improved tissue compositions? Grodzinsky: I think the most important attribute of the question is the need for long-term studies. Initially, much of the work in mechanobiology by its very nature has involved short-term studies. The data I showed indicated an increase in synthesis of about 25% just during the loading period, of about two weeks. John Kisiday now has a 56 day loading experiment in progress (Kisiday et al 2002). Stefan pointed out that it might take 5, 10 or even 30 years for the development of progressive osteoarthritis. Perhaps a 20% increase over two weeks isn’t so bad, because if we are really thinking long-term, then perhaps the ability for the cells to re-synthesize an appropriate matrix will be critical, not just producing an abnormally high matrix content in a short time. Vunjak-Novakovic: This is because most of the newly synthesized matrix is lost into the medium. Grodzinsky: That can depend on a lot of factors. In this particular system only about 10% of the newly synthesized proteoglycans and proteins were lost during that two week loading period. These low loss rates are reproducible. But we need more long-term studies to be able to understand the behaviour of chondrocytes under loaded conditions. Hardingham: On the same issue, and related to what Steve Goldstein said about the molecular composition of constructs, in all the di¡erent constructs that people have developed in reacting systems, the singular feature that sticks out is that they are poor in collagen. I hate to say this, but aggrecan is worthless unless you have a ¢brillar sca¡old. The normal articular cartilage has at least four times the content of collagen as it does of aggrecan, and this is a very dense network of ¢brillar components and is a major contributor to the hydraulic permeability and the sort of redistribution of molecules that is possible under dynamic loading conditions. It has never been apparent in any of the data why there has been a paucity in collagen within many di¡erent systems that people have explored. This is an issue that as a group we probably need to put more e¡ort into understanding.


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Buschmann: I agree. One thing that is neglected in biochemical characterization of cartilage is the quality of the collagen component. The distance between two GAG chains in the concentrations that are present in cartilage is about 10 times the diameter of a water molecule. This is why it is so hard to push water through this kind of structure. But if this structure isn’t immobilized in space, it moves with the water and pressurization of the interstitial £uid is reduced. What immobilizes GAG-bearing proteoglycans in the cartilage is a cross-linked and integrated collagen network. When one talks about integration, it is not just histological integration at the borders, but it is integration of the collagen architecture including vertical ¢brils piercing the calci¢ed layer and then becoming anisotropic in middle regions, and ¢nally that very special £attened laminar structure at the top articular surface. Grodzinsky: This also gets back to the issue of failure in tension. At the molecular level collagen is critical. Goldstein: I was hoping that someone would raise this issue. The proteoglycans are nothing without the matrix. Looking at the studies in vitro that have attempted to create a bioreactor that applies a physical environment along with its biological environment, most have been pretty simplistic in terms of the mechanical environment. No studies have created constructs that have been subjected to the shear stresses that would create the surface con¢guration of the extracellular matrix the bending, tensile and compressive stresses necessary to create a collagen network that has normal architecture. Until we do this we will never create a construct of articular cartilage that resembles the 3D construct of native articular cartilage. If you could design a bioreactor that has that level of complexity, it might be likely that it won’t integrate well with native cartilage. Does this mean we have two divergent approaches? One would like an immature tissue and implant it into a patient and trying to replicate an exercise protocol that would further develop the appropriate 3D structure of the construct. The other is to try to create a construct in vitro that is almost fully formed, and maybe replacing a very large area (not just a bounded defect) so that integration isn’t an issue (only requires integration with the bone). Grodzinsky: I agree. I think it is critically important to duplicate the more complex loading pro¢le that cartilage would be subject to in the joint. There are some shear studies underway in our lab using that system I showed. Perhaps at the very least these di¡erent model systems that are available can allow us to address the question as to whether or not ultimately we can forget about loading in vitro and simply implant at an earlier stage, to duplicate the more complicated joint loading regimen that occurs. At the least we may learn part of the answer to that question from doing these in vitro studies. Caplan: There’s a technical point I wanted to raise. Bovine chondrocytes synthesize an enormous amount of matrix. Human chondrocytes are not quite so

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vigorous, even in a bioreactor. The di⁄culty in assessing the contribution of signalling to the cell is that it is dependent on whether it is a bovine or a human cell. Aggrecan isn’t worth anything without the collagen network. Hardingham: Can I just add that without the aggrecan the collagen network is useless! Caplan: Cartilage consists of a lot of components. The question is whether it is possible to set up model systems with animal cartilages to try to predict clinical responses. In our experience, you can work out whatever you want with rabbit or bovine cells, but when you start dealing with human cells it is like dealing with a whole new issue. Is there a correlation between the natural mechanical environment, seen by cows versus humans, related to the response pro¢le that one can get from the same mechanical loads? Grodzinsky: There are some initial data. We have done a set of extensive studies with human donor tissue aged about 14^85, which we get from the Regional Organ Bank of Illinois by way of a collaboration with Klaus Kuettner and his group at Rush Presbyterian, Chicago. Some of the attributes of cows are in people. That is, chondrocytes in normal human explants will respond to static and dynamic compression, similar to the same pro¢le found in both adult and newborn bovine. The ability of adult human chondrocytes to respond to dynamic compression with an increase in biosynthetic rates decreases with age. Above age 60^65 we don’t see the same stimulation. Below this age we have seen it. There are certain attributes that turn out to be similar. van Blitterswijk: We have been discussing mechanical data, and usually we use average values. How much spread is there between individuals? It might be that the average value is much too low for some individuals. How much variation is there culturing human chondrocytes, for example in proliferation rates? Grodzinsky: It’s even worse than just having to deal with person-to-person variation. If you simply take one explant and look at the mechanical sti¡ness as a function of depth, there is a factor of 10 variation from the super¢cial to the deep zones. There will also be variation in plugs from along the surface of the same joint. Mechanical properties, just like tissue biochemical composition can vary. The question is, at what point do you begin to tailor the construct to the individual? van Blitterswijk: If it was possible, you could take the exact same site, then how much variation would you typically encounter between individuals? Caplan: I think it is impossible to answer because there is no such thing as the same site. Pavesio: The variability in individuals is essentially limited to cell yield. Of some 700 biopsies that we have processed, only 10 failed to yield cells. Such biopsies had characteristic features: they were either harvested in close proximity to the injured area, or had a very swollen appearance. van Blitterswijk: Is there a big spread in the proliferation rate?


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Pavesio: No, it is quite consistent. Lindahl: We have cultured tissue from 1200 patients since 1987. Out of these we have had 8 failures, and these were all among the ¢rst 200. The cells grow well, and there is little di¡erence between the patients. Pavesio: Our experience is that biopsy location and dimension are important factors. Caplan: I will try to summarize. It is clear that there is tissue culture and bioreactor technology available for constructing tissue implants that have particular properties that are comparable to native cartilage, but not perfect. Whether these need to be optimized (meaning getting ‘real cartilage’ in a construct) is still debatable. The point that Steve Goldstein made will come back to haunt us when regulatory agencies ask for a variety of criteria by which release success is judged for implant material, as well as ‘¢nished’ product before it goes into the patient. This will clearly involve some detailed biochemical, cell survival and mechanical assessment, as well as judging implantability. I’m sure the regulatory agencies will ¢nd another three or four criteria that will also have to be met. We are at a very early stage in judging the relationship of the average mechanical properties of an implant with regard to the zonal and speci¢c properties required for successful implantation, integration and long-term suitability. Since this is one of the most mechanically stressed tissues in our body, particularly at the knee and hip, this further analysis of micromechanical properties of macro cartilage will be useful. Reference Kisiday J, Jin M, Kurz B, Hung H, Semino C, Zhang S, Grodzinsky AJ 2002 Self-assembling peptide hydrogel fosters chondrocyte extracellular matrix production and cell division: implications for cartilage tissue repair. Proc Natl Acad Sci USA 99:9996^10 001

From the preclinical model to the patient Ernst B. Hunziker ITI Institute for Dental and Skeletal Biology, University of Bern, Murtenstrasse 35, P.O. Box 54, CH-3010 Bern, Switzerland

Abstract. Tissue engineering studies are complex in nature and involve many stages of testing before an experimental construct is ripe for human clinical trials. Biocompatibility questions, toxicological problems, pharmacological aspects, safety issues and proof of the principle itself represent but a few of the concerns that must be addressed. This contribution deals with the di¡erent types of animal experiment that are usually undertaken in tissue engineering studies. During the initial phase, small animal models are used to screen all potential components of the proposed construct, such as matrix, cells and signalling substances. In the second, the principle itself is put to the test using an appropriate defect model in a suitable animal species. Careful consideration of these latter two aspects is of paramount importance, many factors having to be considered and special requirements satis¢ed for meaningful experimentation. Finally, clinically relevant models are set up in animals to furnish basic information respecting the construct’s suitability and ripeness for testing in human clinical trials. 2003 Tissue engineering of cartilage and bone. Wiley, Chichester (Novartis Foundation Symposium 249) p 70^85

The choice of an appropriate preclinical model to study the repair of articular cartilage defects is an all-important determinant of our ultimate success in treating patients who have such lesions. The term ‘model’ relates not only to the animal species selected for investigations in vivo but also to the geometry of the experimental defect created. Ideally, the animal model should be one in which the articular cartilage layer closely resembles that in adult humans, in terms both of structure and dimensions. These conditions being satis¢ed, the required mimicry of human defect geometry and microenvironment would follow as a matter of course. But this ¢rst requirement is a tall order; indeed, it is one that cannot be met amongst the range of mammalian species commonly used for experimental purposes. This being the case, the designing of an appropriate defect model becomes a very weighty matter. But even before the necessary choices are made and the various considerations taken into account, a great deal of preliminary work needs to be undertaken in vitro, 70

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with a view to engineering a construct of matrix, cells and signalling substances that is suitable for testing in vivo. One of two alternative approaches may be adopted (Jackson & Simon 1999, Glowacki 2000). According to the ¢rst, chondroprogenitor cells are di¡erentiated in vitro under the appropriate stimulation conditions and the fully matured construct then implanted within the experimental defect. According to the second, more popular route, a suitable matrix carrying chondroprogenitor cells and signalling substances entrapped within a slow-delivery system is deposited directly into the experimental defect and the di¡erentiation process e¡ected in situ. And even before embarking on these in vitro experiments, the investigator must decide whether he or she wishes to elaborate a construct for the treatment of partialor of full-thickness articular cartilage defects, since the choice of constituents (particularly signalling substances) and the mechanical conditions to be simulated will be governed by the tissue makeup of the defect surrounds. And ¢rst and foremost, the biological principles underlying spontaneous repair in each type of defect are fundamentally di¡erent. But these aspects will be dealt with later. Screening of components When the investigator has put together the basic building blocks of his construct, each of these components must be screened in small animal models, such as rats or mice, to assess its likelihood of eliciting an adverse response in human patients. Biocompatibility and biodegradability issues, as well as pharmacological and toxicological aspects, have to be evaluated (Festing 1993, Vohr 1995, Fu et al 1995). The ideal choice of site for testing is of course the orthotopic one (Halloran et al 1979, Werntz et al 1996), in that the provocation or absence of a response can be taken as being tissue speci¢c. Indeed, in the case of constructs destined for defects within articular cartilage, there is no option but to test at the orthotopic site, since, being an immunologically privileged tissue, its biological microenvironment is unique and any reaction elicited is thus peculiar to this bodily compartment. Pharmacology With respect to the matrix sca¡old, the excretory pathway and half-life of each of its degradation products must be determined and the long-term pharmacological and possible toxicological or carcinogenic e¡ects evaluated. The fate not only of allogeneic but also that of autogenic precursor cells used in a construct must be scrutinized (Koc et al 1999). Their bodily distribution, homing preference and localization, as well as their potential for unfavourable reactivity and uncontrolled proliferation must be assessed. The signalling molecules employed

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must likewise be tested, since even if they elicit a chondrogenic response in vitro, it does not necessarily follow that they will do so in vivo. Under culturing conditions, various types of growth factor have been demonstrated to support chondrogenic activities (Chimal-Monroy et al 1996), but in vivo, only members of the transforming growth factor (TGF)b superfamily are truly e¡ective in this capacity (Hunziker et al 2001). When these aspects have been addressed and the potential problems ironed out, then the functionality of the construct needs to be tested in an appropriate animal model. The small mammalian species that are useful for the screening of a construct’s individual components are not suitable for this purpose, in that their articular cartilage layers are far too thin to permit optimal positioning and mechanical ¢xation of the implant during surgery. Hence, the choice must be made from amongst the available range of moderate and large animal models, such as guinea pigs, rabbits, dogs, sheep, goats or miniature pigs (Hunziker 1999a, Hunziker 1999b, Pritzker 1994). Duration and controls After its surgical implantation, the construct’s fate must be followed for a length of time that permits the drawing of valid conclusions. The periods chosen will be in£uenced not only by the animal model selected for in vivo testing but also by the dimensions of the defect. The assets and disadvantages of the tissue engineering approach over conventional therapeutic measures need to be assessed systematically, and this involves setting up the appropriate controls. The functionality of each of the construct’s components must be assessed separately, and each replaced by an alternative either with or without biological activity to ascertain whether it contributes speci¢cally to the engineered system. For instance, the chondroprogenitor cell pool employed should be substituted by e.g. skin ¢broblasts, which are unable to di¡erentiate into chondrocytes. Microenvironment The biological and mechanical microenvironments must likewise be controlled; they are indeed known to have a signi¢cant bearing on the healing outcome (Williams & Brandt 1984, Shahgaldi et al 1991, Roeddecker et al 1994, Yetkinler et al 1999). Nevertheless, the in£uence of such environmental factors is frequently overlooked in the experimental design. One common oversight will serve to illustrate this point. After a construct has been deposited within a large, fullthickness articular cartilage defect, it is often covered with a periosteal £ap to prevent its subsequent loss into the joint cavity (Grande et al 1989, Brittberg et al 1994). But such an appendage cannot be blithely a⁄xed as if it is no more than an


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inert lid; the biological consequences of inserting this living tissue must be considered. Being a living material, it may well contribute to the repair response by furnishing a supply of connective tissue cells (Hunziker 1999b). Hence, its presence complicates the experimental design and renders di⁄cult a reliable assessment of the construct’s biological contribution to the healing process. Defect dimensions It was mentioned at the beginning of this article that an investigator needs to be particularly mindful of an articular cartilage defect’s dimensions relative to its tissue surrounds, and one of the ¢rst decisions he needs to make before embarking on a tissue engineering study is whether his construct is destined for the treatment of partial- or of full-thickness lesions. Even when employing moderately-sized or largish animals, the surgical creation of partial-thickness articular cartilage defects is fraught with technical di⁄culties which are not easily surmounted on a reproducible basis. And it is probably for this reason that most scientists in this ¢eld tend to work with full-thickness defect models, which embrace the entire depth of the articular cartilage layer and additionally penetrate the subchondral bone plate. In these latter models, typically more than 90% of the lesion void lies within the bony compartment (Hunziker 1999a). Although they span heights (2^3 mm) that are more on a par with those of partial-thickness ones in human patients (Fig. 1) than would be those of suitably scaled-down super¢cial lesions, the biological microenvironment is profoundly di¡erent, as indeed is the mechanical one. Bone, unlike cartilage, is a richly vascularized tissue. Any defect that penetrates the subchondral bone plate will become in¢ltrated with blood stemming from the bone-marrow and osseous tissue spaces. This blood carries a whole posse of di¡erent precursor cell types and signalling substances, which form the basis of a spontaneous repair response (Shapiro et al 1993). Defects con¢ned to the articular cartilage layer have no access to these blood-borne cells and signalling molecules and they consequently manifest virtually no signs of a spontaneous repair reaction (Hunziker & Rosenberg 1996, Hunziker 2001). Defect simulation In order to obtain useful information respecting the repair potential of an engineered construct in the context of purely cartilaginous human defects, these bony sources of cells and signalling substances must be cut o¡. This could be achieved by producing a ‘virtual’ partial-thickness defect (Fig. 2), which would involve treating the £oor and walls of a full-thickness one in such a manner as to render them impervious to blood-borne cells and signalling substances. A ¢brin glue (Silver et al 1995) containing this polymer at a high concentration (120 mg/ml

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FIG. 1. Light micrographs of adult human (A,D), goat (B,E) and rabbit (C,F) articular cartilage in the medial femoral condyle, each set (A,B,C) and (D,E,F) represented at the same magni¢cation. (A,B,C) Low-magni¢cation overviews, illustrating the tremendous di¡erences in tissue proportions between the three species. Arrowheads, tidemark; S, subchondral bone plate. (D) High-magni¢cation detail of the human upper radial zone and the entire articular cartilage layers in the goat (E) and rabbit (F), illustrating the di¡erences in numerical cell density between the three species. C, calci¢ed cartilage; S, subchondral bone plate. Thick section, surface-stained with toluidine blue and McNeil’s tetrachrome. Magni¢cations: A,B,C, 13; D,E,F, 65. See Hunziker (1999a).

compared with 2 mg/ml in serum) could be used e¡ectively in this capacity. Such an imitation of the human physiological scenery is essential for a meaningful testing of the functionality, survival rate and durability of a construct destined for clinical use. Not only the depth but also the lateral dimensions of a human defect must be simulated in the chosen animal model. In the human knee joint, a focal partialthickness defect such as is generated by trauma or during the initial stages of osteoarthritis would typically span 1^2 cm in this direction. Given that its height would be maximally 2^3 mm (i.e. the thickness of the human articular cartilage layer), it follows that the engineered construct needs to ¢ll a void of about 1 ml, which is a tremendously large volume. This scale of production cannot indeed be


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FIG. 2. Light micrograph of a full-thickness defect in mature bovine articular cartilage. A virtual partial-thickness defect has been created by lining its £oor and walls with ¢brin glue (arrows), which blocks cell migration and vascular invasion from osseous tissue and the bonemarrow spaces into the defect void for a considerable time (up to one week). C, articular cartilage; B, bone tissue; M, bone-marrow spaces. Thick section, surface-stained with McNeil’s tetrachrome, toluidine blue 0 and basic fuchsin.

achieved under conventional culturing conditions but requires the use of special technologies, such as bioreactor systems (Freed et al 1998, Freed et al 1999, VunjakNovakovic et al 1999, Wu et al 1999). Albeit that the manufacturing process is thereby rendered possible, the long-term maintenance, activity and functionality of such enormous constructs in vivo poses a set of problems and challenges on quite a di¡erent plane of reckoning to that encountered when dealing with small ones. Even if an engineered construct is successfully implanted within an experimental animal, such as a goat or sheep, the repair cartilage formed will at best be goat-like or sheep-like, but not human-like, unless measures are taken from the onset of the tissue engineering process to simulate the human characteristics. Human articular cartilage is characterized by an extraordinarily low cellularity (Fig. 1) and a highly anisotropic structure (Hunziker 1992), and if repair tissue is to be functionally competent, this architecture must be re-established. This, of course, is more easily said than done. But investigators should at least be aware of the qualitative and quantitative di¡erences existing between the articular cartilage layers of their chosen animal models (Hunziker 1999a) and those in humans, and give due consideration to ways and means of better imitating the structure to which they

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aspire. One possible avenue to pursue would be the manufacture of a matrix sca¡old which is not isotropically, but anisotropically, structured to mimic the human dimensional scale. Seeded cells might thus be encouraged to follow the topographic di¡erentiation pattern manifested in native articular cartilage tissue, which they appear to be unable to do when randomly dispersed.

Biomechanics In vitro simulation of the biomechanical conditions operative in native human articular cartilage tissue may also have a positive bearing on a construct’s functionality after implantation. And likewise during the postoperative healing phase, appropriate stimulation of the natural tissue’s biomechanical functions may in£uence a construct’s fate and the repair outcome. For this reason, the instigation of a well-de¢ned loading protocol is recommended (Mow & Rosenwasser 1988). Conclusions No single animal model can adequately simulate human articular cartilage pathology. Such being the case, investigators need to be a little canny in the wiles adopted to circumvent the di⁄culties encountered en route and above all be scrupulously methodical in the following of a systematic approach to experimentation. As long as they constantly bear in mind that they have to imitate the human situation as closely as is possible with the means at their disposal, in all respects and at each stage of their investigation, they will be in a position to better judge the likely bene¢ts to be derived by treating patients with their engineered construct. References Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peterson L 1994 Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med 331:889^895 Chimal-Monroy J, Bravo-Ruiz MT, Diaz de Leon L 1996 Regulation of chondrocyte di¡erentiation by transforming growth factors beta 1, beta 2, beta 3, and beta 5. Ann NY Acad Sci 785:241^244 Festing MF 1993 Genetic variation in outbred rats and mice and its implications for toxicological screening. J Exp Anim Sci 35:210^220 Freed LE, Hollander AP, Martin I, Barry JR, Langer R, Vunjak-Novakovic G 1998 Chondrogenesis in a cell-polymer-bioreactor system. Exp Cell Res 240:58^65 Freed LE, Martin I, Vunjak-Novakovic G 1999 Frontiers in tissue engineering In vitro modulation of chondrogenesis. Clin Orthop 367:S46^S58


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Fu ZD, Chen WR, Gu LJ, Gu ZW 1995 The in£uence of the extent of target organs on sensitivities of methods for screening rodent carcinogens. Mutat Res 331:99^117 Glowacki J 2000 In vitro engineering of cartilage. J Rehabil Res Dev 37:171^177 Grande DA, Pitman MI, Peterson L, Menche D, Klein M 1989 The repair of experimentally produced defects in rabbit articular cartilage by autologous chondrocyte transplantation. J Orthop Res 7:208^218 Halloran PF, Ziv I, Lee EH, Langer F, Pritzker KP, Gross AE 1979 Orthotopic bone transplantation in mice. I. Technique and assessment of healing. Transplantation 27:414^419 Hunziker EB 1992 Articular cartilage structure in humans and experimental animals. In: Kuettner KE, Schleyerbach R, Peyron JG, Hascall VC (eds) Articular cartilage and osteoarthritis. New York, Raven Press, p 183^199 Hunziker EB 1999a Biologic repair of articular cartilage. Defect models in experimental animals and matrix requirements. Clin Orthop S135^S146 Hunziker EB 1999b Articular cartilage repair: are the intrinsic biological constraints undermining this process insuperable? Osteoarthritis Cartilage 7:15^28 Hunziker EB 2001 Growth-factor-induced healing of partial-thickness defects in adult articular cartilage. Osteoarthritis Cartilage 9:22^32 Hunziker EB, Rosenberg LC 1996 Repair of partial-thickness defects in articular cartilage: cell recruitment from the synovial membrane. J Bone Joint Surg Am 78: 721^733 Hunziker EB, Driesang IM, Morris EA 2001 Chondrogenesis in cartilage repair is induced by members of the transforming growth factor-beta superfamily. Clin Orthop 391:S171^S181 Jackson DW, Simon TM 1999 Tissue engineering principles in orthopaedic surgery. Clin Orthop S31^S45 Koc ON, Peters C, Aubourg P et al 1999 Bone marrow-derived mesenchymal stem cells remain host-derived despite successful hematopoietic engraftment after allogeneic transplantation in patients with lysosomal and peroxisomal storage diseases. Exp Hematol 27:1675^1681 Mow V, Rosenwasser M 1988 Articular cartilage: biomechanics. In: Woo SLY, Buckwalter JA (eds) Injury and repair of the musculoskeletal soft tissues. Park Ridge, IL: American Academy of Orthopaedic Surgeons, p 427^463 Pritzker KP 1994 Animal models for osteoarthritis: processes, problems and prospects. Ann Rheum Dis 53:406^420 Roeddecker K, Muennich U, Nagelschmidt M 1994 Meniscal healing: a biomechanical study. J Surg Res 56:20^27 Shahgaldi BF, Amis AA, Heatley FW, McDowell J, Bentley G 1991 Repair of cartilage lesions using biological implants. A comparative histological and biomechanical study in goats. J Bone Joint Surg Br 73:57^64 Shapiro F, Koide S, Glimcher MJ 1993 Cell origin and di¡erentiation in the repair of fullthickness defects of articular cartilage. J Bone Joint Surg Am 75:532^553 Silver FH, Wang MC, Pins GD 1995 Preparation and use of ¢brin glue in surgery. Biomaterials 16:891^903 Vohr HW 1995 Experiences with an advanced screening procedure for the identi¢cation of chemicals with an immunotoxic potential in routine toxicology. Toxicology 104:149^158 Vunjak-Novakovic G, Martin I et al 1999 Bioreactor cultivation conditions modulate the composition and mechanical properties of tissue-engineered cartilage. J Orthopaed Res 17:130^138 Werntz JR, Lane JM, Burstein AH, Justin R, Klein R, Tomin E 1996 Qualitative and quantitative analysis of orthotopic bone regeneration by marrow. J Orthop Res 14:85^93 Williams JM, Brandt KD 1984 Temporary immobilisation facilitates repair of chemically induced articular cartilage injury. J Anat 138:435^446 Wu F, Dunkelman N, Peterson A, Davisson T, De La Torre, Jain D 1999 Bioreactor development for tissue-engineered cartilage. Ann NY Acad Sci 875:405^411

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Yetkinler DN, Ladd AL, Poser RD, Constantz BR, Carter D 1999 Biomechanical evaluation of ¢xation of intra-articular fractures of the distal part of the radius in cadavera: Kirschner wires compared with calcium-phosphate bone cement. J Bone Joint Surg Am 81:391^399

DISCUSSION Caplan: You made a strong assertion that for human patients, the Genzyme implanted material wasn’t adequately tested by this multicomponent criterion. Yet the regulatory agency approved its use. Do you have any comment of the con£ict of this approach? Hunziker: I don’t see this as a con£ict. The initial experiments were performed in animals and were done in good faith by the scientists. They were convinced that they were done correctly, and the regulatory agencies are unable to assess each experimental factor. Today, a few years later, it is practically 100% sure that in these early animal experiments all the £aps were lost into the joint cavity, and no one knew this at that time. We all had assumed previously that because these £aps were sutured to the cartilage that they would stay in place. When we started to use them in our own experiments, dealing with super¢cial (i.e. partial-thickness) defects, to our great surprise we found that they do not stay in place. It is really very hard to monitor the fate of such £aps in vivo, but we learned from our own experience that this is absolutely necessary in future experiments. With respect to human patients, Dr Hollis Potter (Hospital for Special Surgery, New York City) recently reported in a congress meeting that approximately 65% (if I remember correctly) of the £aps are partially or completely lost, and in spite of this ¢nding, still some positive cartilage repair results are observed, indicating that probably the repair response from the subchondral bone plate is of primary importance in this procedure. Lindahl: I seem to be the one who’s doing everything wrong here! Perhaps I should explain a bit about the history of our experiments. In the rabbit we used the patella cartilage because we can’t treat the defect on the femoral condyle it’s too thin. The patella cartilage however is 1^2 mm thick and the periosteal £ap comprises a part of the thickness only. We have been looking at other animal models. When we presented the human data 10 years ago, people commonly said to us that we needed a larger animal model before we continue in humans, but I don’t think there exists a good animal model for humans. The Genzyme data were based on our ¢rst 150 patients. They had very good clinical outcomes. This is why they were able to take the risk of accepting the treatment. I agree with the criticism that we should have tried to address additional issues in animal models if such a model existed. If you take therapy for burn patients, where we transplant cultured skin cells, which has been a successful treatment for over 1000 human patients, there is no animal model that works. Although the route through animal models


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is needed to show proof of principle, how many animal models do you have to deal with before the human? Hunziker: I don’t think that there is any speci¢c animal model available that you could use to obtain a reliability of a repair result that would prove that the method tested in the animal could also work with 100% reliability in the human patient. There will always be some remaining risk of failure or of unexpected adverse e¡ects. However, what you are obliged to do is to choose an animal model (or animal models) that mimic the defect or the method as close as possible to the situation in the human patient. For example, Dr Frank Luyten’s group in Belgium (Department of Rheumatology, University Hospital Leuven) recently reported an animal experiment in which they evaluated the contribution of transplanted autologous chondrocytes to the repair of articular cartilage defects in the knee joint of adult goats. In these experiments, this group covered the defect by an autologous periosteal £ap, as described in the literature, and immobilized the knee joints (to prevent the loss of the £aps). The group found that only a very small percentage of the transplanted (and labelled) autologous chondrocytes contribute to the formation of the cartilage repair tissue (in the order of 10^12%). This illustrates that appropriate testing can be done in animal experiments provided the adequate animal experimental design is established. I am not attacking you in any way! We should, however, not forget that the autologous chondrocyte methodology (ACI) is associated with two other potential sources for repair tissue formation. First, the periosteal £ap that is sutured to the defect top, with its cambium layer directed towards the bottom of the defect, could act as a source for repair tissue production, provided the £ap stays in place. Another potential source for repair is the subchondral bone tissue: in practically all surgeries that I have seen for the application of ACI, the surgeons penetrate the subchondral bone plate or bone tissue compartment during the intervention, or at least scratch the subchondral bone plate. It is obvious that following release of the tourniquet at the end of the surgery there will be profuse bleeding from the bony compartment into the defect space, ¢lling it up with a haematoma, up to the sutured £ap material. This may be the main source for repair tissue formation in the ACI methodology and contribute the most to the repair tissue. Animal experiments can be done in order to clarify these still-open questions of the three potential sources for repair tissue, and which of these is primarily responsible. However, so far these experiments still have not been done. Caplan: I don’t want this discussion to turn into a debate about the applicability of this commercial procedure. Rather, I’d like us to focus on Ernst Hunziker’s thesis that very systematic and rigorous testing of animal models can sort between selected variant treatments, and one can use this for translating this tissue engineering technology into human use. Although we can approximate

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single human conditions, there is no animal model that will give us the kind of complexity that occurs in human cartilage. We need to regenerate a huge amount of cartilage in humans, and there are no animal models that give this. The subject under consideration is the range of variables that Ernst brought up, and the suitability of animal models in general. Buschmann: I was intrigued and concerned by the e¡ects in goats that you showed side by side, with an identical preparation of an osteochondral defect. One resulted in the formation of a subchondral bone cyst while in the other goat there was an intermediate-stage of repair with complete cartilage ¢lling of the defect. To what extent is this kind of irreproducibility expected? Is it a function of age? Do you have any insight as to why and when subchondral cysts form? Hunziker: In our laboratories we always used adult goats of 3^5 years of age for our repair studies. They are retired milking goats. In these animals we observe a great variability in the spontaneous repair response. Doug Jackson’s group (Long Beach, California) recently published a paper on spontaneous healing e¡ect of large full-thickness defects in a mature goat model (Jackson et al 2001). This group also ¢nds a very great variability in the spontaneous repair outcome between individual goats. Caplan: We have carefully followed the progress of somewhere between 1000 and 1500 rabbits (mostly 4^6 months of age) with cartilage defects. What I can say with great con¢dence is that the MSC preparations from the rabbits are hugely variable in the number of cells that we get at ¢rst or second passage, and also the quality of their osteochondral potential. Each rabbit also has an innate repair potential, a characteristic MSC yield and a characteristic quality of the MSC preparation. None of these three variables are correlated, which is surprising. Huge numbers of experimental animals are required for looking at rabbit osteochondral defects. The only statistically relevant information that one can derive with reasonable con¢dence is left^right comparability within the same rabbit. These are rabbits from the same supplier. This observation is probably not restricted to rabbits, but is applicable to almost any animal model. In orthopaedics 10 years ago, if experiments were not done in dogs they were not accepted. They used mixed-bred dogs, and they would classify them by weight. Here again, there is huge variability from dog to dog. You could say, tongue in cheek, that this variability is a good model for human variability! Schwartz: We have tried to characterize empty defects out to six months in adult castrated male goats, to see how they heal. We created 7 mm defects in both the trochlear groove and medial femoral condyle to a depth of 1 mm into the subchondral bone, using specially designed instrumentation that controls the depth regardless of the cartilage thickness. So, we had access to the marrow components. In the femoral condyle we saw cyst formation at about 8 weeks; however, they began to resolve at three months. By six months the cysts were


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pretty well fully resolved. There was a nice ¢ll of tissue there, but histologically it was ¢brocartilage. Trippel: The variability from animal to animal has two implications. One is that we don’t understand the healing process. If we understood the process, we would be able to explain the variability. Looked at from that standpoint, perhaps we shouldn’t be working on humans yet. Perhaps we need to go back and begin to understand the cell biology and physiology of the joint better, so we can get better control over what we are doing. A second implication is that if there is a great deal of variability from rabbit to goat, or goat to sheep, or sheep to dog, then it should be no surprise that as we go to humans, the humans will be di¡erent than all these other animals. If this is the case, then there is only one relevant model for humans, and that is human. If we take this as our basis for where to go next, then after showing proof-of-concept and resolving issues of pharmacology, safety, toxicity and so on, e⁄cacy will have to be determined in humans. But if we are going to determine e⁄cacy in humans, we have to do it right. This means doing it di¡erently from the way that orthopaedic surgeons often tend to do things. We need to go in with a correctly designed, rigorously scienti¢c trial with appropriate controls and randomization, and good outcome measures that are properly evaluated. Only then will we know whether what we have got is a result of the treatment or some other variable. It is all too common for orthopaedic surgeons to get out multiple tools and to try to apply them all at the same time, because they don’t really know which tool is producing the desired e¡ect, in the hope that one of them will work. Caplan: There are two models now available to permit implantation of materials into humans. One is the American model, in which the FDA requires very large clinical trials to gain approval. This requires, for single indications, in the order of US$50^100 million investments. For drug development, the ¢gure is US$300^500 million for some of the current drugs. The other model is very interesting, which is the European one: if you prove something is safe, in certain markets you can use that material on humans and the healthcare systems will pay. What is required is rigorous and stringent follow-up, which then goes back to the regulatory agencies. In this case, smaller companies can conduct clinical trials. As a consumer, I see the advantages of both of these approaches. Is it su⁄cient to prove safety to humans without documenting e⁄cacy? Trippel: This approach leaves the physician and patient in a quandary. If the equivalent approach is to use multiple techniques simultaneously we will never know which of those techniques actually works. It is like giving a mixture of drugs to someone to try to achieve a desired e¡ect, and not actually discovering what the active ingredient is. Caplan: From my standpoint as someone who receives healthcare, if it works, I don’t care. One of the interesting things for me personally is that 10 years ago I

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DISCUSSION

would have been more vociferous than Dr Hunziker in requiring pre-animal testing. Now, having had huge experience with some animal tests, I’m a little less convinced of the applicability of that with regard to human. Your point is a good one. If we have no excellent animal model for human, and we can document safety, should we allow that as a principle? By these two systems, the European system and the US system, we have a con£ict of logic. I was very impressed with Genzyme’s presentation to the FDA and their approval. In essence, they changed my mental set about procedures that have e⁄cacy in humans. If you have a procedure that produces a ¢brocartilage ¢x, and this lasts 10 or 20 years, is this a good thing? The dogma has always been for me that ¢brocartilage is bad. But there’s clearly now some clinical evidence with follow-up that it isn’t so bad. Perhaps it is OK. We need to think about the relationship between the ideal situation and the real situation. Lohmander: I’m not so sure that the situation in Europe and the USA is as di¡erent as you are implying. But let’s assume that there is a di¡erence, and it could be used to the advantage of the approach you are suggesting. Then ethically, the only way that you could go forward would be by rigorous trial design, which includes a priori power calculations, making very sure that the outcome of the trial will actually provide the information that you hope it will. I don’t think this will allow small ‘garage’ or ‘back-yard’ trials with 25 patients here and 30 patients there. We will need large multicentre trials to prove e⁄cacy and to select the procedure that is best for the patient. Caplan: I would agree with that, but I would also say that the garage trials have huge utility in ¢ne-tuning the procedures that start out under an ideal hypothesis, for general applicability. van Blitterswijk: To some extent you are reinventing the wheel here. The garage trials are no longer there. Before you can get to a clinical trial in Europe, you at least have to show the biological safety of the drugs. Obviously, you also do a risk assessment. Then, you start with your safety trial. This tests on small numbers to see if the product is safe. Only after this can you start with big numbers and test e⁄cacy. If you are lucky, you will already have seen a bit of e⁄cacy in the garage trial. This is the only way you can do it. Another point is that we are talking about medical devices in the regulatory sense. One of the most successful medical devices in this ¢eld is the arti¢cial hip implant. This should be the ideal implant to do this rigorous trial, comparing a new implant with an already existing one. If you are an orthopaedic surgeon and you are going to take a new hip implant and compare it with a standard hip, you will already have experience with the standard hip. So how are you going to make a good trial? You will be comparing a technique you are familiar with, with a completely new operative approach. It sounds nice, and in an ideal world it is what you would like to do, but how will it work in practice?


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Caplan: Let’s add an extra complexity. Take an osteoarthritic knee missing about 80% of the cartilage, and I have a technique for resurfacing that joint. I also have individuals of high compliance, and I try to do this resurfacing and I fail. That patient is then going to get a knee replacement that they would have obtained in the ¢rst place. Although you can complain about the ethics, the patients will say that they want a biological ¢x before they put this piece of metal in their body. I am now being driven by the patients’ desire for a biological ¢x. In reality, the downside in these non-life-and-death issues is that the patient will get a metal knee just as they would have originally. Lohmander: Of course, John Charnley never did a formal clinical trial of his arti¢cial hip implant designs and was immensely successful. But we also know of all the subsequent failures in the business of arti¢cial joints. Who has paid the price of these failures? The patients. Surgery is associated with risks that are not trivial. There is also such a thing as the ethics review board. They go by the 2000 Helsinki declaration. They will not allow the patients to make up their own mind, in general, regarding this. They will be the guardians of the patients, and will not allow me to recruit the patients into a trial, even if the patients would accept what I suggest to them. Caplan: I have seen some ethical review boards approve exactly what I just said, and others that would not. It depends on which institution, and who is presenting. Ratcli¡e: Just to bring this discussion back to a certain level of reality, whether it be Europe or the USA, these kinds of experiments are being done so that you can move into human trials regulated by what ever body it happens to be. Certainly in the USA, while these types of products have previously been regulated as devices, they are now being moved into biologics. If they are not being moved into biologics they are being regarded as something in between. You may well have to meet both sets of criteria. They are quite di¡erent. Consider this as one issue. You still have to demonstrate safety and give some indication of e¡ectiveness or e⁄cacy. This is to satisfy the regulatory authorities and also to satisfy the clinicians who will do your studies. It is no longer a device. It is either a combination product or a biologic, depending on how the regulatory authorities feel. In some parts of Europe it is even coming more under the drug-type regulations. This is another level of complexity. What the animal studies really do is drive you through this process. They have to be designed so that they satisfy all of those needs. Ernst Hunziker covered a massive spectrum. If you did all of that, hopefully you would satisfy everyone. Clearly what you need to do is negotiate or discuss with whoever is going to regulate you. Caplan: I would state, however, that physician-initiated procedures that don’t start out associated with any commercial entity, have available to them huge opportunity, depending on their ethics review board and how they present it.

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The physician^scientist can bring forth new technologies and try them without going through this rigorous animal trial approach. Trippel: I very much like your scenario, because it has an aura of realism about it. This is how a lot of orthopaedic procedures are developed. A surgeon comes up with an idea, and he does a garage experiment in a bunch of cases. The di⁄culty is not that he came up with a new idea, but rather how he evaluates that idea. It didn’t make the situation any more complicated, the way you described it. In fact, it would be an elegant simpli¢cation of the situation if he had simply decided to do it right. He comes up with a new idea that he thinks might work better than what he is currently doing. So he randomizes his patients into two di¡erent groups: the new idea and the way he has been doing it up until now. He does it in a properly controlled fashion. The garage experiment in which a person has no randomization, no control, no blinding and no valid instrument for evaluating outcomes is to science as doing the operation in the garage is to orthopaedic surgery. You just shouldn’t do it! Caplan: Since there are drug company representatives here, I’d like to pose the following question to the tissue engineers. It is well known within the drug industry that if you have a massive clinical trial which is hugely successful, and you bring this drug to market, for insurance purposes you prepare yourself for a 1% failure rate, for example, of this drug. For tissue engineering, under the best conditions, what would be an anticipated failure rate of these technologies? van Blitterswijk: With implants there is around a 10% removal rate within a 10 year period, depending on the age of the patient. If we could do this with tissue engineering it would be nice. Pavesio: I think we are all aware that there is not a single best animal model or a perfectly designed clinical study. There are elements of animal models and prospective, controlled clinical studies that are very relevant. However, we should not forget the huge leap forward in our knowledge that has been provided by the Swedish clinical experience, despite its limitations. Goldstein: This is an important point. We are starting to lose sight of the fact that our goal here is to think of ways of making progress. Ersnt Hunziker raised a number of issues that we need to consider if we are trying to choose an animal model to evaluate aspects of the tissue-engineering construct. We all recognize that it is unlikely that there is a perfect animal model. A number of people have raised the point that there are probably attributes of di¡erent animal models that have the ability to test di¡erent attributes of tissue-engineered constructs. Nothing we do is ever going to be risk free. All we can do is carefully consider the critical design principles as we try to identify the animal model that tells us whether it is worthwhile proceeding from an ethical, safety and business perspective. Morphology, geometry and surgical applicability issues are very important. The only aspect we haven’t discussed are the outcome measures. In the animals models


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we have access to pretty good outcome measures. We can look at the implant histologically and we can make tissue cell and molecular assays. In humans we have limited ability to do this. One of the things to consider in developing animal models is to think about what surrogate measures will be needed in human trials, and whether or not the animal study can be used to validate them. Ratcli¡e: I agree. When we do all these studies we are great at assessing our outcome, but to date I haven’t yet seen a good de¢nition of success in a cartilage repair model. What do we really regard as success? I’ve yet to see this well de¢ned. Lohmander: I would like the people working with animal models to try to come up with some semblance of a responder criteria set that we could use in our human trials. Ratcli¡e: I agree. This is a critical issue for us to address. I am presently designing an animal study for this purpose. Caplan: Let me summarize. There are animal models that are available and which can test speci¢c principles of tissue engineering, as would be required for safety and development studies of a commercial nature. The outcome analysis of animal models involves a variety of mechanical measurements on the repair tissue compared with normal, and there are biochemical and morphological analyses. In a scienti¢c sense, we can establish a criterion for success as long as it is based on the intrinsic variability that one could expect from these animal models. The availability of bioreactors, cell culture techniques and di¡erent sca¡olds will allow the fabrication of implantable material that has characteristics that are approaching those of in vivo cartilage, but clearly the question can be raised as to whether perfect cartilage needs to be implanted, or implanted material that matures to perfect cartilage is the best approach. As I said at the beginning of the day in my introductory remarks, I see the area of tissue engineering leaving its long inductive and slow start and accelerating in its sophistication. We have a long way to go, but we are on our way. Reference Jackson DW, Lalor PA, Aberman HM, Simon TM 2001 Spontaneous repair of full-thickness defects of articular cartilage in a goat model: a preliminary study. J Bone Joint Surg Am 83:53

Mesenchymal stem cell therapy in joint disease Frank P. Barry Osiris Therapeutics Inc., 2001 Aliceanna Street, Baltimore, MD 21231, USA

Abstract. Mesenchymal stem cells have the capacity to di¡erentiate into a variety of connective tissue cells including bone, cartilage, tendon, muscle and adipose tissue. These multipotent cells have been isolated from bone marrow and from other adult tissues including skeletal muscle, fat and synovium. Because of their multipotentiality and capacity for self renewal adult stem cells may represent units of active regeneration of tissues damaged as a result of trauma or disease. In certain degenerative diseases such as osteoarthritis (OA) stem cells are depleted, and have reduced proliferative capacity and reduced ability to di¡erentiate. The delivery of stem cells to these individuals may therefore enhance repair or inhibit the progressive destruction of the joint. We have developed methods for the delivery of mesenchymal stem cell preparations taken from bone marrow to the injured knee joint. This treatment has the potential to stimulate regeneration of cartilage and retard the progressive destruction of the joint that typically occurs following injury. 2003 Tissue engineering of cartilage and bone. Wiley, Chichester (Novartis Foundation Symposium 249) p 86^102

Within the stromal compartment of adult bone marrow there exists a population of cells referred to as mesenchymal stem cells (MSCs, Friedenstein 1976, Owen & Friedenstein 1988, Haynesworth et al 1992a, Pittenger et al 1999) with the capacity to di¡erentiate along osteogenic (Jaiswal et al 1997), chondrogenic (Barry et al 2001a, Johnstone et al 1998, Mackay et al 1998) or adipogenic (Pittenger et al 1999) lineages both in vitro and when implanted subcutaneously in nude mice (Dennis et al 1992, Haynesworth et al 1992a). These cells may be isolated using standardized techniques and expanded in culture through many generations, while retaining their capacity to di¡erentiate when exposed to appropriate signals. This multipotent property of MSCs generates opportunities for the development of new therapeutic strategies for the repair of tissues damaged as a result of trauma or disease and protocols have been devised for the treatment of defects in articular cartilage (Wakitani et al 1994, 2002), bone (Bruder et al 1998a) and tendon (Young et al 1998). In addition stem cells have been used for the repair of myocardium 86

MSC THERAPY

87

following infarction (Shake et al 2002), for bone marrow stromal recovery (Koc et al 2000) and osteogenesis imperfecta (Horwitz et al 1999, 2001). MSCs are commonly isolated from an aspirate of bone marrow taken from the superior iliac crest. The cells are plated following initial fractionation by density gradient. During the period of primary culture the non-adherent haematopoietic cell fraction is removed. The adherent MSC population, which represents approximately 0.001% of the nucleated cells in marrow, forms colonies that can be expanded through many generations. Cell surface markers Several cell surface markers have been used for selection of MSCs by £ow cytometry. A number of monoclonal antibodies has been raised against human MSCs and used for their characterization. These include SB10 (Bruder et al 1997), SH2, SH3 and SH4 (Haynesworth et al 1992b), and Stro-1 (Tri⁄tt et al 2001). In several cases the antigens that are recognized by these antibodies have been puri¢ed by immunoprecipitation and identi¢ed using a combination of time-of-£ight mass spectrometry and protein sequencing (Barry et al 1999, 2001b, Bruder et al 1998b). The SB10 antigen was shown to be activated by leukocyte cell adhesion molecule (ALCAM, CD166), and the SH2 antigen was identi¢ed as endoglin (CD105). CD166 binds to CD6 and is involved in activation of leucocytes and endoglin is a soluble transforming growth factor (TGF)b receptor, commonly associated with endothelial cells. Using a similar approach we have shown that the SH3 and SH4 antibodies recognize distinct epitopes on the membrane-bound protein 5’nucleotidase (CD73, Barry et al 2001b), a molecule present on many cell types. While these antibodies still have application in testing MSCs in vitro and in distinguishing MSCs from haematopoietic cells, they are not useful in vivo markers. Thus the study of the distribution and migration of MSCs in vivo is limited by the lack of availability of speci¢c markers. Di¡erentiation Conditions for induction of di¡erentiation of these cells in vitro have been well described. In the case of chondrogenesis MSCs are formed into high-density three-dimensional cultures, either in a pellet format (Barry et al 2001a, Johnstone et al 1998, Mackay et al 1998) or in alginate (Kavalkovich et al 2000, Weber et al 2002). In the presence of TGFb there is a rapid progression from the undi¡erentiated cells with a ¢broblastic morphology to a mature chondrocyte with production of an abundant cartilaginous extracellular matrix. This is the case with MSCs from a number of mammalian species (Fig. 1) and the chondrogenic pathway involves the deposition of matrix components including

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BARRY

FIG. 1. Chondrogenic di¡erentiation of mesenchymal stem cells from human (A), goat (B) and rabbit (C) bone marrow. Cells were cultured in pellet format in the presence of TGFb3 for 21 days and proteoglycan was detected by toluidine blue staining. Expression of type II collagen (D), COMP (E) and keratan sulfate (F) by human cells. Original magni¢cation in A^D ¼ 100 and 400 for E and F.

type II collagen, cartilage oligomeric matrix protein (COMP) and keratan sulfate. The sequence of events surrounding synthesis and deposition of the matrix has been studied in detail in pellet cultures (Barry et al 2001a). Fibromodulin expression is detected before there is evidence of overt chondrogenesis. This proteoglycan is present in normal articular cartilage that binds to type II collagen. This is followed by expression of COMP and aggrecan and synthesis of type II collagen begins later (after approximately 7 days). There is some value in comparing the appearance of matrix components during chondrogenesis of MSCs in vitro with matrix synthesis during limb development in vivo (Fig. 2). During early development of the mouse glenohumeral joint (Murphy et al 1999a), prior to joint cavitation, the cells that reside in the future articular layer express large amounts of ¢bromodulin, distinguishing them from chondrocytes that progress through endochondral ossi¢cation and which do not synthesize ¢bromodulin at this stage. Conversely the cells of the bone layer express large amounts of cartilage matrix protein (CMP), a member of the thrombospondin family, while the cells in the cartilage layer do not. The expression of ¢bromodulin by MSCs during early chondrogenesis in vitro and the absence of CMP indicate the potential of these cells to be similarly programmed (Barry et al 2001a). When MSCs are maintained in monolayer in the presence of osteogenic supplements, including ascorbic acid-2-phosphate, dexamethasone and bglycerophosphate, there is a rapid increase in alkaline phosphatase and deposition of a mineralized matrix (Jaiswal et al 1997, Bruder et al 1997, Murphy

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FIG. 2. Immunolocalization of ¢bromodulin (Left) and CMP (Right ) during development of articular cartilage in the 14 d mouse embryo. S, scapula; H, humerus. Sagittal sections through the glenohumeral joint of a mouse at 14 days post-coitus, showing di¡erential localization of ¢bromodulin and CMP in the chondrogenous (CL) and intermediate (IL) layers of the interzone (IZ) in the joint. Bar, 100 mm. (From Murphy et al 1999a, reproduced with permission.)

et al 2002). Similarly, cells cultured in monolayer in the presence of dexamethasone, methyl-isobutylxanthine and insulin become adipocytes with the production of large lipid-¢lled vacuoles (Pittenger et al 1999, Murphy et al 2002). Adipogenic di¡erentiation of MSCs is accompanied by the up-regulation of the fat-speci¢c transcription factor PPARg as well as fatty acid synthetase (Pittenger et al 1999). Clonal selection experiments (Pittenger et al 1999) have shown that MSCs represent a homogenous population of multipotent cells rather than a mixture of progenitor cells with di¡erent potential. Stem cells in disease It has been suggested that adult stem cells, because of their multipotent nature and their ability to self renew, represent the natural units of repair of tissue damaged as a result of trauma or disease (Weissman 2000). Cells in the bone marrow or other tissues become mobilized in response to wound signals and colonize the site of injury. Under the in£uence of local biochemical (or mechanical) signals the cells di¡erentiate along the appropriate pathway, leading to regeneration of the tissue. A corollary of this hypothesis is that stem cells may be depleted in some degenerative diseases, leading to an inability to repair damaged tissue. This has been shown to be the case in osteoarthritis (OA). In a recent study (Murphy et al 2002) MSCs were isolated from bone marrow aspirates from patients undergoing total joint arthroplasty for OA. Samples were taken from both the iliac crest and from the surgical site (the femoral head in the case of hip OA and either the distal femur or proximal tibia in the case of knee OA). When compared to cell preparations taken from healthy donors there were signi¢cant di¡erences in

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terms of proliferative capacity and the ability to di¡erentiate along the chondrogenic and adipogenic pathways (Fig. 3). Cells from OA donors showed reduced proliferation in vitro and a lower rate of di¡erentiation along both pathways. The signi¢cance of these results is still unclear however, and this is due in part to the inability to study the cells in vivo, for the reasons outlined above. It may be that depletion of stem cells is an active element in disease, or it may equally be the case that the cells are altered by the environment of the disease, speci¢cally by elevated levels of in£ammatory cytokines. MSCs cultured in pellets in the presence of either interleukin (IL)1a or tumour necrosis factor (TNF)a fail to di¡erentiate into chondrocytes (J. Mosca, personal communication 2001) or chondrogenesis is inhibited by IL1 (Majumdar et al 2001). Similarly cells cultured in medium conditioned with synovial £uid taken from an OA joint show reduced ability to di¡erentiate (M. Murphy, personal communication 2001). Although there is evidence that MSCs are depleted and functionally altered in OA the role of endogenous pools of stem cells will require much further study. While the decline in the di¡erentiation capacity of chondroprogenitor cells in OA patients may play a role in the degradation of articular cartilage there are certainly other factors involved. The synthesis of aggrecan by chondrocytes decreases with age as does the rate at which it is incorporated into aggregates (Bayliss et al 2000). Changes such as these in chondrocyte function may be a primary factor in OA and the changes in progenitor cell function described here may be of secondary importance. However, in the context of cell therapy applications in OA these ¢ndings suggest that the preparation of an e¡ective dose of autologous cells would be more di⁄cult because of the reduced proliferative capacity. This conclusion may impact on clinical applications that involve the use of autologous cells for tissue engineering or for gene delivery. Cartilage repair Articular cartilage has a limited repair capacity and super¢cial injuries commonly lead to more extensive degeneration of the articular surface. When the injury FIG. 3. (Top) The proliferative capacity of human OA MSCs, derived from iliac crest (IC, n ¼ 12) marrow or from either the tibial or femoral (T/F, n ¼ 13) compartments, in passage 1 culture was signi¢cantly lower than that of MSCs cultured from iliac crest marrow obtained from normal, older donors (n ¼ 7). Data are presented as the median, 25th and 75th percentile (vertical boxes) and 10th and 90th percentile as error bars. The dashed line represents the mean. *P50.05. (Middle) Chondrogenic activity was signi¢cantly reduced in cells from iliac crest marrow (P ¼ 0.046) and from tibia/femur marrow (P ¼ 0.032). (Bottom) Adipogenic activity of MSCs from the bone marrow of aged normal donors (n ¼ 7, 443 years old) and from marrow samples taken from the iliac crest (n ¼ 6, IC) and tibia/femur (n ¼ 5, T/F) marrow of individuals with OA. *P50.05. (From Murphy et al 2002, reproduced with permission.)

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penetrates the subchondral bone there is often a repair response that involves formation of ¢brocartilage (Minas & Nehrer 1997). The nature of the response is dependent on age and the location and size of the defect. The reparative tissue di¡ers from normal articular cartilage in biochemical and biomechanical properties and ultimately becomes ¢brillated, with formation of ¢ssures and extensive degeneration. A number of surgical protocols are currently in use for the treatment of articular cartilage defects. In abrasion arthroplasty (Johnson 2001), for instance, the subchondral bone is punctured by drilling to promote bleeding into the defect, with the result that there is formation of bone and ¢brous repair tissue. In the micro-fracture technique (Blevins et al 1998) the exposed subchondral bone is ‘picked’ to promote localized bleeding. Another approach involves the use of allografts (Convery et al 1996) where cartilage lesions are ¢lled with grafts of donor-derived osteochondral fragments. In mosaicplasty (Coutts et al 2001, Hangody et al 2001) cylindrical osteochondral plugs are harvested from nonload-bearing sites in the a¡ected joint and pressed into place within the osteochondral defect, creating an autograft ‘mosaic’ to ¢ll the lesion. Autologous chondrocyte therapy (Brittberg et al 1994) represents another approach, where a chondral biopsy is taken from a donor site at the time of clinical presentation. Chondrocytes, enzymatically released from the retrieved tissue, are expanded in monolayer culture, and subsequently implanted in a second procedure beneath a periosteal cover. The periosteal cover is sutured to the cartilage adjacent to the defect and sealed with ¢brin glue. All of these approaches o¡er exciting opportunities for the repair of cartilage defects. However, the long-term outcome may be uncertain and there may be other disadvantages associated with the harvest site, even when it is some distance from the lesion. Implantation of cells with both chondrogenic and osteogenic potential derived from the bone marrow for the treatment of osteochondral lesions represents another approach that might result in persistent, functional repair of the articular cartilage. There are a number of approaches for the delivery of cells to osteochondral lesions. For instance, the cells may be combined with an appropriate biomaterial that provides support and space-¢lling capacity. A number of di¡erent materials may be suitable for his purpose and selections may be made on the basis of cell adhesion, biocompatibility, degradation time, handling properties and regulatory approval, amongst others. Another approach involves the retention of cells beneath a £ap of material, as in the case of autologous chondrocyte therapy described above. It may also be possible to deliver cells to a¡ected joints in the absence of a support or ¢xation material, simply relying on the adhesive properties of the cells to target the lesion site. Among the important issues in the use of cells for cartilage repair are the chondrogenic nature of the cells, the mode of delivery, the e¡ect of the local

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FIG. 4. Appearance of posterior medial neomeniscus at 26 week post meniscectomy/20 week post injection of a preparation of MSCs delivered by intra-articular injection as a suspension in dilute hyaluronan. The top panels show sections of neomeniscal tissue from the operated joint of G150 stained with safranin O (left) and type II collagen (right), (original magni¢cation 20). The bottom panels show sections of neomeniscal tissue from the operated joint of G168 stained with safranin O (left) and type II collagen (right), (original magni¢cation 20).

environment and the use of biomaterial supports. The chondrogenic activity of stem cells has already been discussed and their ability to adhere to sca¡olds such as HYAFF111 (Murphy & Barry 2000, Solchaga et al 2000) and Gelfoam (Ponticiello et al 2000) while retaining the ability to di¡erentiate has been described. Stem cell therapy in osteoarthritis The development of e¡ective therapies for OA has been slow and today most recommended medications alleviate symptoms without altering the course of the disease. In many cases, joint replacement surgery is the best option to restore joint function. The observations made above on the characteristics of stem cells

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from OA patients indicated that stem cell therapy might be an e¡ective approach in impeding the degenerative changes in the OA joint. A number of studies have been carried out involving the delivery of stem cells to the goat knee joint following medial meniscectomy, resection of the anterior cruciate ligament (ACL) or a combination of the two. This procedure in combination with a de¢ned exercise regime leads to the development of lesions in the joint that are characteristic of OA (Murphy et al 1999b). The injected cells were transduced with green £uorescent protein (GFP) and the general approach was to deliver the cells as a suspension by intra-articular injection, thus avoiding the need for arthrotomy and the placement of a sca¡old. Injected cells were retained within the joint and also recovered from synovial £uid in a viable form. The cells colonized soft tissue surfaces, primarily those of synovial origin rather than the articular cartilage. Injection of MSCs into destabilized, osteoarthritic joints resulted in marked remodelling of the medial meniscus, which had been totally removed during surgery. The neotissue formed has a hyaline-like appearance and focal areas of type II collagen similar to developing rabbit meniscus (Fig. 4, Bland & Ashhurst 1996). Related to this tissue regeneration is a marked chondroprotective e¡ect of the MSC injection. These observations highlight the potential therapeutic bene¢t of injected MSCs in an osteoarthritic joint by regenerating neomeniscal tissue to stabilize the joint and protect the articular surfaces against progressive degeneration. Acknowledgements The author thanks Karl Kavalkovich for excellent technical assistance and Dr Mary Murphy for critical review of the manuscript.

References Barry FP, Boynton RE, Haynesworth S, Murphy JM, Zaia J 1999 The monoclonal antibody SH-2, raised against human mesenchymal stem cells, recognizes an epitope on endoglin (CD105). Biochem Biophys Res Commun 265:134^139 Barry F, Boynton RE, Liu B, Murphy JM 2001a Chondrogenic di¡erentiation of mesenchymal stem cells from bone marrow: di¡erentiation-dependent gene expression of matrix components. Exp Cell Res 268:189^200 Barry F, Boynton R, Murphy M, Haynesworth S, Zaia J 2001b The SH-3 and SH-4 antibodies recognize distinct epitopes on CD73 from human mesenchymal stem cells. Biochem Biophys Res Commun 289:519^524 Bayliss MT, Howat S, Davidson C, Dudhia J 2000 The organization of aggrecan in human articular cartilage. Evidence for age-related changes in the rate of aggregation of newly synthesized molecules. J Biol Chem 275:6321^6327 Bland YS, Ashhurst DE 1996 Changes in the content of the ¢brillar collagens and the expression of their mRNAs in the menisci of the rabbit knee joint during development and ageing. Histochem J 28:265^274 Blevins FT, Steadman JR, Rodrigo JJ, Silliman J 1998 Treatment of articular cartilage defects in athletes: an analysis of functional outcome and lesion appearance. Orthopedics 21:761^768

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Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peterson L 1994 Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med 331:889^895 Bruder SP, Horowitz MC, Mosca JD, Haynesworth SE 1997 Monoclonal antibodies reactive with human osteogenic cell surface antigens. Bone 21:225^235 Bruder SP, Kurth AA, Shea M, Hayes WC, Jaiswal N, Kadiyala S 1998a Bone regeneration by implantation of puri¢ed, culture-expanded human mesenchymal stem cells. J Orthop Res 16:155^162 Bruder SP, Ricalton NS, Boynton RE et al 1998b Mesenchymal stem cell surface antigen SB-10 corresponds to activated leukocyte cell adhesion molecule and is involved in osteogenic di¡erentiation. J Bone Miner Res 13:655^663 Convery FR, Akeson WH, Amiel D, Meyers MH, Monosov A 1996 Long-term survival of chondrocytes in an osteochondral articular cartilage allograft. A case report. J Bone Joint Surg Am 78:1082^1088 Coutts RD, Healey RM, Ostrander R, Sah RL, Goomer R, Amiel D 2001 Matrices for cartilage repair. Clin Orthop 391:S271^S279 Dennis JE, Haynesworth SE et al 1992 Osteogenesis in marrow-derived mesenchymal cell porous ceramic composites transplanted subcutaneously: e¡ect of ¢bronectin and laminin on cell retention and rate of osteogenic expression. Cell Transplant 1:23^32 Friedenstein AJ 1976 Precursor cells of mechanocytes. Int Rev Cytol 47:327^359 Hangody L, Feczko P, Bartha L, Bodo G, Kish G 2001 Mosaicplasty for the treatment of articular defects of the knee and ankle. Clin Orthop 391:S328^S336 Haynesworth SE, Goshima J, Goldberg VM, Caplan AI 1992a Characterization of cells with osteogenic potential from human marrow. Bone 13:81^88 Haynesworth SE, Baber MA, Caplan AI 1992b Cell surface antigens on human marrow-derived mesenchymal cells are detected by monoclonal antibodies. Bone 13:69^80 Horwitz EM, Prockop DJ, Fitzpatrick LA et al 1999 Transplantability and therapeutic e¡ects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat Med 5:309^313 Horwitz EM, Prockop DJ, Gordon PL et al 2001 Clinical responses to bone marrow transplantation in children with severe osteogenesis imperfecta. Blood 97:1227^1231 Jaiswal N, Haynesworth SE, Caplan AI, Bruder SP 1997 Osteogenic di¡erentiation of puri¢ed, culture-expanded human mesenchymal stem cells in vitro. J Cell Biochem 64:295^312 Johnson LL 2001 Arthroscopic abrasion arthroplasty: a review. Clin Orthop 391:S306^S317 Johnstone B, Hering TM, Caplan AI, Goldberg VM, Yoo JU 1998 In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res 238:265^272 Kavalkovich KW, Boynton R, Murphy JM, Barry FP 2000 E¡ect of cell density on chondrogenic di¡erentiation of mesenchymal stem cells. Trans Orthop Res Soc 46:975 Koc ON, Gerson SL, Cooper BW et al 2000 Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J Clin Oncol 18:307^316 Mackay AM, Beck SC, Murphy JM, Barry FP, Chichester CO, Pittenger MF 1998 Chondrogenic di¡erentiation of cultured human mesenchymal stem cells from marrow. Tissue Eng 4:415^ 428 Majumdar MK, Wang E, Morris EA 2001 BMP-2 and BMP-9 promote chondrogenic di¡erentiation of human multipotential mesenchymal cells and overcome the inhibitory e¡ect of IL-1. J Cell Physiol 189:275^284 Minas T, Nehrer S 1997 Current concepts in the treatment of articular cartilage defects. Orthopedics 20:525^538

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Murphy JM, Barry FP 2000 Chondrogenic di¡erentiation of mesenchymal stem cells on matrices of hyaluronan derivatives. In Abatangelo G, Weigel PH (eds) New frontiers in medical sciences: rede¢ning hyaluronan. Elsevier, Amsterdam, p 247^254 Murphy JM, Heinegard R, McIntosh A, Sterchi D, Barry FP 1999a Distribution of cartilage molecules in the developing mouse joint. Matrix Biol 18:487^497 Murphy JM, Boynton RE, Kraus K, Cole JC, Hunziker EB, Barry FP 1999b An experimental model of osteoarthritis in goats. Trans Orthop Res Soc 45:435 Murphy M, Kavalkovich K, Fink D, Hunziker E, Barry F 2001 Injected mesenchymal stem cells stimulate meniscal repair and protection of articular cartilage. Trans Orthop Res Soc 47:193 Murphy JM, Dixon K, Beck S, Fabian D, Feldman A, Barry F 2002 Reduced chondrogenic and adipogenic activity of mesenchymal stem cells from patients with advanced osteoarthritis. Arthritis Rheum 46:704^713 Owen M, Friedenstein AJ 1988 Stromal stem cells: marrow-derived osteogenic precursors. In: Cells and molecular biology of vertebrate hard tissues. Wiley, Chichester (Ciba Found Symp 136) p 42^60 Pittenger MF, Mackay AM, Beck SC et al 1999 Multilineage potential of adult human mesenchymal stem cells. Science 284:143^147 Ponticiello MS, Schinagl RM, Kadiyala S, Barry FP 2000 Gelatin-based resorbable sponge as a carrier matrix for human mesenchymal stem cells in cartilage regeneration therapy. J Biomed Mater Res 52:246^255 Shake JG, Gruber PJ, Baumgartner WA et al 2002 Mesenchymal stem cell implantation in a swine myocardial infarct model: engraftment and functional e¡ects. Ann Thorac Surg 73:1919^1925 Solchaga LA, Yoo JU, Lundberg M et al 2000 Hyaluronan-based polymers in the treatment of osteochondral defects. J Orthop Res 18:773^780 Tri⁄tt JT, Ore¡o RO, Virdi AS, Xia Z 2001 Osteogenic stem-cell characterization and development: potentials for cytotherapy. Cytotherapy 3:413^416 Wakitani S, Goto T, Pineda SJ et al 1994 Mesenchymal cell-based repair of large, full-thickness defects of articular cartilage. J Bone Joint Surg Am 76:579^592 Wakitani S, Imoto K, Yamamoto T, Saito M, Murata N, Yoneda M 2002 Human autologous culture expanded bone marrow mesenchymal cell transplantation for repair of cartilage defects in osteoarthritic knees. Osteoarthritis Cartilage 10:199^206 Weber M, Steinert A, Jork A et al 2002 Formation of cartilage matrix proteins by BMPtransfected murine mesenchymal stem cells encapsulated in a novel class of alginates. Biomaterials 23:2003^2013 Weissman IL 2000 Translating stem and progenitor cell biology to the clinic: barriers and opportunities. Science 287:1442^1446 Young RG, Butler DL, Weber W, Caplan AI, Gordon SL, Fink DJ 1998 Use of mesenchymal stem cells in a collagen matrix for Achilles tendon repair. J Orthop Res 16:406^413

DISCUSSION Lohmander: As I interpret the information you gave us here, you are associating these bene¢cial e¡ects with the generation of the neomeniscal tissue. You trace cells in the neomeniscal tissue: have you found cells in any other locations in the joint? Barry: Yes. The cells are generally associated with all the soft tissues of the joint. We see cells associated with the newly formed meniscal tissue, the fat pad and the

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synovium. We don’t see cells directly associated with the articular cartilage. The change that we see in terms of a remodelling event seems to occur predominantly in the meniscus. Lohmander: What was the source of the autologous cells that you used in both the human studies and these studies? Barry: In all of the animal studies the cells are taken from the iliac crest. In the human studies they were taken from the same individual from the iliac crest and the site of surgery. Lohmander: What is the site of surgery? Barry: Either the knee or the hip. All of the data I showed involved cells derived from bone marrow samples taken from OA individuals. Roughly half were taken from the hip and half were from the knee joint. Caplan: One is an aspirate and one is a scoop. Pavesio: You said that there was no evidence of repair of the ligament. Can you speculate why? Barry: It didn’t work. It is probably because of the exercise regime that these animals have. Pavesio: Did you expect ligament repair? Barry: Because of the exercise regime there is a lot of movement in the joint, and it would be impossible to expect the ligament to repair under those conditions. Perhaps with some ¢xation of the resected ligament we might see the possibility of repair. Cancedda: I found this work very interesting, but I have a major concern. If I understood correctly, you have always injected cells very close to the time of the injury. This is very di¡erent from the case in real life. There is increasing evidence that these types of cells will behave di¡erently if they are exposed to a microenvironment where there is a fresh injury. Barry: I agree fully. The thinking behind these experiments at the start was that we wanted to evaluate whether we could develop a therapy that might be e¡ective in the chronic condition OA. We found that when we looked at the timing of delivery that in fact we were impacting the early repair response. What we ended up with, it seems, is potentially a therapy for repair of meniscal injury. We are not yet at the point where we can use these cells to treat OA. Caplan: Can you give us some guess as to the proportion of donor to host cells in the neomeniscal tissue? Barry: I would say about 90% host-derived cells, 10% implanted cells. Helms: It seemed to me that from the pictures you showed the only place that the stem cells were located was on the surface, implying that their participation in the regeneration of that meniscal tissue is really at the very end. If the stem cells did have an e¡ect in some way, perhaps it is through the release of something that they are making.

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Barry: It would be a mistake to think that these stem cells wholly give rise to this tissue. They contribute in its organization in some way. Helms: But only at the very end, it seems. They are only on the surface. Barry: That’s correct, but they are associating with a lot of other tissues as well. Caplan: Do you agree with Jill Helms’ interpretation that their location can help you discern whether they present themselves at the beginning or the end of the process? Barry: I’m not sure. Helms: Perhaps their e¡ect is simply from what they are releasing, not from the cells themselves. Lohmander: Coming back to the discussion we had yesterday about the possibility of widening indications for tissue regeneration and engineering, if you could show that this is applicable to meniscal surgery, or of these kinds of injuries, you have expanded the indications by two orders of magnitude as compared with the current limited indication for tissue engineering in human joints. Even if you don’t go into full-blown advanced OA, your market would be several hundred thousand cases per year just in the USA, as compared with a few thousand today. Barry: The ¢rst attempt at evaluation of this protocol in a human trial will involve delivery of cells one week following partial meniscectomy. The control group will have no treatment. Lohmander: Are your ¢ndings unique to the goat model, or have you looked at any other models? Barry: One of the limitations of our research is that we have not con¢rmed these observations in other species. This is something that we need to do. We have been very happy with the goat as the experimental model. The quality and consistency of MSC preparations from goat bone marrow is very high. This is not always the case with other species. van Blitterswijk: I wonder about one of your graphs, in which you compared healthy individuals with OA patients in which you took bone marrow biopsies from the femur joint and compared this with the iliac crest. We have done the same thing. In general, what we ¢nd is that the cells from the femur proliferate at a much lower rate and are less successful, compared with the iliac crest cells from the same patient. We get much better results using cells from the iliac crest with OA patients. This is not what you are showing. How do you take your cells? Barry: Our expectation was that we might see di¡erences between the cells taken from the site of surgery versus those taken from the iliac crest aspirate. In all of the measurements that we did, we could not see a di¡erence between the two sites. The quality of the cells was independent of the site from where the bone marrow was taken.

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Caplan: I would inject a note of caution when we are trying to compare proliferative rates between two di¡erent sites from the same patient. George Muschler and others have shown that the colony density on a plate, which is your initial seeding, a¡ects the eventual proliferative rate (Muschler et al 2001). Unless you can take and properly dilute out each of the samples in such a way that the colony-forming units of the two sources are comparable, you have to be careful in trying to evaluate the proliferative rate. There is a dependence on cell density. Goldstein: It seems that there are a lot of mechanical issues to consider. First of all, you said that all the repair occurred in the posterior lateral region of the meniscus. Considering the goat knee is probably sloped posterior, it suggests that during ambulation the motion probably wipes all of the material to the posterior corner, causing some growth in the meniscus and a stabilization of the joint. In fact, the protection may be coming from the fact that you have now got a meniscus that may be creating increased joint stability. Have you measured anything to do with the joint stability at sacri¢ce? Barry: We have done a Drawer test (for the measurement of anterior^posterior movement), and we don’t see any di¡erence, although this is only relevant to the ligament, and not the meniscus. Goldstein: So you didn’t see a di¡erence in the control versus the experimental group? Barry: No. The ligament is severed in all groups. Goldstein: Perhaps the test was not sensitive to whether there was a meniscus. Did you do any rotation? Barry: No. Goldstein: And you saw no repair occurring in the medial and anterior regions of the meniscus? Barry: It was variable. Generally the response was most evident in the posterior compartment, but sometimes we saw a tissue response in the anterior area. Goldstein: Did you do any studies where you tried to take the cells down a di¡erentiated pathway and then inject them? Are you planning to do this? Barry: We did some limited studies where we induced the cells to become chondrocytes and then delivered them. These were small experiments focused more on the repair of cartilage defects, but we didn’t see a particularly e⁄cacious result. There are several reasons in terms of regulatory aspects why we don’t want to change the phenotype of the cells by treating them with growth factors before we implant them into humans. Then you have to revalidate those cells. Ohgushi: You transduced GFP to autogenous cells as well as allogeneic cells. Do you see a di¡erent distribution of cells with autologous versus allogeneic cells in the same model? Barry: Not as far as we can see.

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Ohgushi: Do you detect the allogeneic cells? Barry: Yes. Ohgushi: With the occasional allogeneic cells is there any in£ammatory reaction? Barry: In those studies where we did three injections one week apart we found a dramatic response on the second and third delivery of the cells. There was an acute lameness that occurred very rapidly and then resolved three or four days later. It turned out that this was a secondary response due to the GFP. We were able to go back later and treat these animals in another study with untransduced versus transduced cells. We showed very clearly that the response is due to the GFP. On ¢rst exposure to GFP there is no response in these animals. But when they are treated a second time with GFP-expressing cells there is a dramatic sensitization response to the GFP. We showed that this is a GFP phenomenon and not an allogeneic cell phenomenon. Caplan: Were the data you showed looking at three injections versus one injection obtained with non-GFP transduced cells? Barry: Those were GFP-transduced cells. Even with this in£ammatory response to the GFP we still see formation of neomeniscal tissue in those joints. Caplan: So is your clinical protocol to produce a really good in£ammatory response? Will this be required for a good clinical outcome? Barry: I hope not! Ratcli¡e: If you take the data at face value, you seem to have no major safety issue with doing an injection with allogeneic cells. In this case, what source are you going to use in the clinic? This is critical: you are moving away from an autologous approach. The autologous approach is not something that can be delivered to 200 000 patients per year. This is a problem. If you go the allogeneic route then you have the opportunity of addressing a large patient population, because you can create one cell bank. What are you going to use as your source? I am interested in site as well as age. Barry: The idea is to use a universal cell: a single donor that gives rise to a very large number of cells that can be prepared in a large number of doses. The idea is to use a very young donor and a relatively large aspirate of bone marrow. Exactly what the numbers are I am not sure, in terms of how many doses can be obtained from a single donor. Ratcli¡e: While this work is exciting, it is not clear how far you can take these cells out in terms of doublings. Barry; We can’t passage the cells forever. Clearly they lose their capacity to di¡erentiate if they go through many passages. Given the dose we are working with 10 million cells per injection, a small number of cells from a single aspirate of bone marrow we can prepare many billions of cells. The economies of scale would be such that we can actually prepare a large number of doses from a single aspirate.

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Schwartz: Have you considered using fresh bone marrow aspirate? Would this be a possibility? Barry: We have never evaluated whole bone marrow in this context. The results we saw with OA patients suggested this wouldn’t work. We have never pursued this. Schwartz: You give three injections of cells: have you considered giving just one injection with cells followed by two injections of HA? The standard clinical protocol for some OA patients is three injections of HA. Barry: The studies that we have done so far have involved three identical injections of cells plus HA. There is an alternative approach which is to increase dramatically the number of cells per injection. The next experiment we do is going to look at dose ranging. Richardson: I had a question about the experiments using allogeneic cells. I wondered whether you have made any attempt to see how mismatched the donors and recipients were. Did you do a mixed lymphocyte reaction (MLR)? Some goat colonies tend to be inbred, and they might therefore not be as mismatched as you have assumed. Barry: These goat colonies were not inbred. They are all relatively mixed. We did do an MLR. There is a complete mismatch on the basis of the MLR between the host and the donor. That is a good point, though. Richardson: Another proposal might be if you wanted to get more cells to attach to the eroded articular surface, you could transfect them with integrins that would then cause binding to the exposed matrix. Barry: One of the points about evaluating whether these cells can have an e¡ect in OA means that we have to move away from the instability model. As long as the instability is maintained, the therapy is potentially compromised. The next model that we are going to use will involve the arthroscopic creation of a very large super¢cial lesion on the medial femoral condyle so that there is no instability. We will leave this to develop for several weeks and then start doing cell injections. Trippel: In your controls that didn’t receive the MSCs, how much repair occurred? I’m curious about where these host cells are coming from that comprise 90% of the neomeniscus when you did give the MSCs. Did you see any host cell population forming on this in controls, or are they only accumulating there in response to the MSC injection. Barry: In the controls there is a host cell repair response. At the earliest time we looked, six weeks after injection, when we compare cell treated versus controls there is a dramatic di¡erence. In the cell-treated joints there is neo tissue but there is none in the controls. At the next time point, 20 weeks, there is tissue response in both, but it is more organized in the cell-treated joints. The most dramatic thing is the attachment to the tibial surface. If you try to insert an instrument between the new tissue and the tibia it is possible in the cell-treated joints but not in the controls.

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Trippel: So some of the cells in the controls may be coming up from the tibial surface. Barry: I presume the cells are coming from the synovium. Trippel: Presumably, the meniscectomy is done strictly through the avascular part of the meniscus. Is the goat meniscus avascular in its inner third? Barry: It has a vascular zone just like the human meniscus. Caplan: Did the partial meniscectomy intrude into the vascular zone? Barry: Usually there is no bleeding since the partial meniscectomy does not intrude into the vascular zone. Reference Muschler GF, Nitto H, Boehm CA, Easley KA 2001 Age- and gender-related changes in the cellularity of human bone marrow and the prevalence of osteoblastic progenitors. J Orthop Res 19:117^125

Di¡erentiated chondrocytes for cartilage tissue engineering J. Huckle, G. Dootson, N. Medcalf, S. McTaggart, E. Wright, A. Carter, R. Schreiber*, B. Kirby*, N. Dunkelman*, S. Stevenson*, S. Riley*, T. Davisson* and A. Ratcli¡e* Smith & Nephew Group Research Centre, York Science Park, Heslington, York YO10 5DF, UK and *Advanced Tissue Sciences, Inc 10933 North Torrey Pines Road, La Jolla, CA 92037-1005, USA

Abstract. Trauma to the articular cartilage surface of the joint represents a challenging clinical problem due to the very limited ability of this tissue to self-repair. Moreover, repair techniques such as microfracture, which introduce cells into the joint, have unpredictable clinical outcomes as they produce a ¢brocartilage tissue that degenerates with time. Alternative treatments include tissue reconstruction with autograft and allograft tissue. However, these procedures are restricted by the availability of suitable donor tissue. These limitations have been the driving force behind the emerging ¢eld of articular cartilage tissue engineering. This paper will highlight and contrast the key challenges associated with the tissue engineering of this neo-tissue using di¡erentiated adult cells. The various components of the tissue engineering process will be described including the choice of donor cell/tissue type and the selection of sca¡olds that guide the formation of tissue. The ability of the tissue engineered implants to stimulate the repair of defects in vivo will also be discussed. Tissue engineering approaches may, in the future, provide an ideal alternative to the current surgical treatments for cartilage repair. 2003 Tissue engineering of cartilage and bone. Wiley, Chichester (Novartis Foundation Symposium 249) p 103^117

During the last decade there has been an exponential increase in research activity in the ¢eld of cartilage tissue engineering. Articular cartilage is seen as an ideal candidate for a tissue engineering approach to tissue repair it has a relatively simple structure and lacks a vascular supply. There is also a great clinical need for cartilage treatment due to the lack of ability to self-repair. The approach described here is to develop sca¡old materials which, when seeded with cells, can be cultured in vitro to produce implantable constructs which stimulate cartilage repair in vivo. This paper will compare and contrast the various options available for cartilage tissue engineering including cell source and sca¡old type. Identi¢cation of a suitable cell population for cartilage tissue engineering is the critical ¢rst step in the process. Articular chondrocytes are the most obvious choice 103

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as a cell source for cartilage generation, however obtaining su⁄cient quantities of these cells whilst retaining their ability to form cartilage is a particular challenge. Other cell types have been proposed including stem cells isolated from adipose tissue (Erikson et al 2001), from muscle (Deasy et al 2002), mesenchymal stem cells (Pittenger et al 1999, Caplan et al 1997) and adult dermal ¢broblasts (Nicoll et al 1998). These cell types have a greater proliferative capacity compared to articular chondrocytes although they lack the speci¢c ability to di¡erentiate into chondroctyes without the addition of speci¢c stimuli. Our approach has been to utilise mature chondrocytes that although having a limited proliferative capacity, have the advantage of being able to form the correct tissue type. The feasibility of using meniscal cells to produce a ‘hyaline-like’ tissue has also been studied. Data are included on the evaluation of ovine, lapine and human cell sources.

Ovine studies Meniscal cells There is an ongoing debate as to whether the meniscus contains one cell type, which expresses di¡erent phenotypes depending on the location within the tissue, or if the meniscus contains populations of cells with many phenotypes including ¢broblasts and chondroctyes (Hellio Le Graverand et al 2001). Preliminary experiments have been carried out to assess the potential of meniscal cells to form a ‘hyaline-like’ tissue. Meniscal cells were isolated from entire ovine menisci using pronase/collagenase (pronase 0.1% for 3 h, followed by collagenase 0.2% overnight) digestion and then cultured to passage 3 in monolayer. These cells were seeded at 4 million cells/construct into 102 mm felt discs (45 mg/cc) made of poly(ethylene terephthalate), (constructed as described in Carter & Huckle 2002). The seeded constructs were then cultured in a custom made bioreactor for 2 weeks with either dynamic hydrostatic pressure (30 min/day, 1 Hz frequency, 50 bar overpressure maximum) or without hydrostatic pressure. Histological analysis, using haematoxylin and eosin (H&E) stain of the resulting constructs is shown in Fig. 1. The constructs cultured under dynamic hydrostatic pressure contained cells with a more rounded appearance and had a matrix with a metachromatic staining as compared to the control, which had a ¢broblastic and ¢brous appearance. These data indicate that menisci contain a population of cells that can either: (a) respond to di¡erent environments forming tissues with di¡erent phenotypes, or (b) contain a mixed population of cells that can be selected for, under di¡erent conditions. Moreover, these data indicate the potential for the use of meniscal cells for articular cartilage tissue engineering.

CARTILAGE TISSUE ENGINEERING

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FIG. 1. Histological analysis of constructs cultured from ovine meniscal cells with or without dynamic hydraulic pressure (the arrow indicates a poly(ethylene terephthalate) sca¡old ¢bre).

Donor age The source of donor tissue utilized in most cartilage tissue engineering approaches in the literature to date is from young animals. The donors utilized are predominantly young calves or rabbits (Cao et al 1997, Freed et al 1994, Sims et al 1998, Schreiber et al 1998, Schreiber et al 1999). Experiments were performed using ovine tissue to assess the e¡ect of donor age on construct quality. Primary ovine chondrocytes were isolated, via 0.2% collagenase digestion overnight, from 4, 26 and 100 week old donors and cultured in poly(glycolic acid) (PGA) felts (102 mm felt discs, 45 mg/cc, constructed as described in Carter & Huckle 2002) at a density of 4 million cells/disc. The seeded felts were cultured for 4 weeks in an enclosed bioreactor system as described by Dunkelman et al (1995). Histological analysis (H&E stain) of representative constructs grown from the di¡erent age donors is shown in Fig. 2. These data indicate that the chondrocytes isolated from younger donors produce a ‘hyaline-like’ tissue, as indicated by the metachromatic staining and cells within lacunae, in our enclosed bioreactor system. The constructs become more ¢brous with the cells having a more ¢broblastic appearance with increased donor age. Moreover, biochemical analysis of the glycosaminoglycan (S-GAG) (Farndale et al 1986) and collagen (Woessner 1961) content indicated that the constructs cultured from younger donors contained signi¢cantly more S-GAG, although the collagen content was similar (data not shown).

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FIG. 2. Histological analysis of constructs cultured from various aged donors (the arrows indicate PGA sca¡old ¢bres).

Cell expansion In order to manufacture numbers of allogeneic chondrocytes or culture autologous cartilage constructs it is necessary to be able to expand the cells in culture to obtain enough cells for the subsequent construct culture. Experiments were carried out to assess whether ovine chondrocytes could be expanded in monolayer and retain their ability to produce cartilage in our culture system. Ovine articular chondrocytes were isolated from 4 week old donors via 0.2% collagenase digestion overnight and either expanded in monolayer to passage 1 (approximately 4 cell doublings) or seeded directly into PGA felt (10 2 mm felt discs, 45 mg/cc, constructed as described in Carter & Huckle 2002) at a density of 4 million cells/disc, as primary isolates. The resulting cell seeded PGA sca¡olds were cultured for 4 weeks in an enclosed bioreactor system as described by Dunkelman et al (1995). The passage 1 cells were also seeded into PGA felt and cultured in the same bioreactor system under the same conditions. Histological analysis (H&E stain) of the resulting constructs is shown in Fig. 3. These data indicate that after one passage of monolayer culture the ovine chondrocytes have lost their ability to produce a ‘hyaline-like’ tissue in our bioreactor system. The constructs cultured form passage 1 cells have a very ¢brous appearance and lack metachromatic staining observed in the constructs cultured from primary isolated cells.

Human studies In order to develop articular cartilage for clinical indications it is imperative to study the e¡ects of donor age and passage number on cells isolated from human tissues. Human tissue was obtained with the appropriate consent from various aged donors (10 months, 6, 9 and 25 years of age). Articular chondrocytes were


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FIG. 3. Histological analysis of constructs cultured from primary isolated and passage 1 ovine chondrocytes (the arrows indicate PGA sca¡old ¢bres).

isolated from the cartilage dissected from knee joints using collagenase (0.2%) digestion overnight. The cells were cryopreserved prior to construct culture studies. The cells from the various donors were then serially passaged in monolayer culture (seeded at 6103 cells/cm2 in T £asks, then cultured to 90^95% con£uence). At each passage (approximately 3 doublings/passage) a subpopulation of the cells from the di¡erent donors was seeded (4 million cell/ felt) into PGA felt (102 mm 45 mg/cc, constructed as described in Carter & Huckle 2002) and the resulting constructs maintained in static culture in petri dishes for a 4 week period. The resulting constructs were digested with papain and analysed for collagen and S-GAG content essentially as described by Woessner (1961) and Farndale et al (1986), respectively. The S-GAG and collagen content of constructs produced from successive cell generations of the di¡erent donor lots are shown in Fig 4. The S-GAG content was the highest with cells from younger donors, as compared to older donors. Constructs produced from cells propagated to passage 3 contained between 20 and 40% S-GAG. The levels of S-GAG were lower (15^30%), but still high in constructs produced from cells harvested between passage 4 and passage 6. The collagen content of constructs showed little variation with the passage number or the donor age. Histological analysis (data not shown) suggested that articular cartilage cells expanded from the various donors (2 months^25 years) to passage 3 synthesized a predominately hyaline-like cartilage matrix. Articular cartilage cells expanded from the two younger donors (10 months and 6 years), but not the two older donors (9 and 25 years) to passage 5 synthesized a predominately hyaline-like cartilage matrix.

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FIG. 4. Biochemical composition of human constructs cultured from various donor ages and passage numbers.

These studies indicate that cells isolated from younger human donors have a greater chondrogenic capacity compared to cells isolated from older donors. It is also possible to culture expand cells isolated from younger donors to at least passage 5 and still maintain, or be able to rescue, the phenotype of these cells when they are subsequently cultured on PGA felt. E¡ect of sca¡old materials The ideal sca¡old for cartilage tissue engineering should; (a) be reproducible to process, (b) be three dimensional, (c) have a highly porous structure that permits a spatially uniform cell distribution and minimises di¡usional constraints, (d) have a controlled degradation rate matching that of matrix deposition, and (e) be biocompatible (Freed & Vunjak-Novakovic 2002). Various sca¡olds were evaluated for their ability to support chondrogenesis using ovine articular chondrocytes isolated from 4 week old donors. The sca¡olds were seeded at a density of 120106 cells/cc and then cultured in static culture (in Petri dishes) for a period of 4 weeks. The PGA sca¡olds were 97% porous and had a density of 45 mg/cc. The PCL (poly(caprolactone)) sca¡olds were 90% porous foams (pore size: 106^150 mm). The PLGA [poly(lactic-co glycolic acid)] sca¡olds were 75:25


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FIG. 5. Histological analysis of constructs cultured on various sca¡old materials.

PLGA, with a 90% porosity and a pore size of 500^710 mm. The hydrogel sca¡olds were 40% polyethylene glycol dimethacrylate (3.4 kDa) with 60% polyethylene oxide (100 kDa) made into a total 20% w/w solution in PBS and polymerized using 0.01% 1-hydroxycyclohexyl phenyl ketone (HPK) under UV light (*3 mW/cm2) for 3 min. Histogical analysis (safranin O stain) of the resulting 4 week cultures is shown in Fig. 5. All the sca¡olds were able to support chondrogenesis, however using PGA resulted in the most uniform constructs with an even distribution of matrix. In vivo studies Tissue-engineered constructs have been cultured from passage 2 cells isolated from 1 year old lapine donors. These allogeneic constructs were subsequently utilized to repair osteochondral defects in the patellar groove of rabbits. The quality of the

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repair tissue was analysed histologically compared with untreated defects using a modi¢ed O’Driscol scale (O’Driscol et al 1986). This study (Schreiber et al 1999) concluded that the treatment of osteochondral lesions with allogeneic tissue engineered cartilage constructs lead to superior repair tissue than that generated in untreated defects. Further studies are in progress in large animal models.

Discussion Di¡erences have been observed between the chrondrogenic potential of articular chondrocytes isolated from di¡erent species. Our experiments with ovine donors have shown that the younger the donor the greater the chondrogenic potential. However, it appears that upon passage in monolayer, that ovine cells lose their ability to regenerate cartilage in our culture system regardless of donor age. Younger human donors also seem to have a greater chondrogenic potential compared to older donors, however human chondrocytes can be isolated from younger donors and culture expanded to at least passage 5 and retain their ability to regenerate cartilage in our culture system. Some of our previous studies were carried out with lapine cells, 1 year old lapine donor cells can be expanded to at least passage 3 without losing their ability to regenerate cartilage in our culture system (Schreiber et al 1999), indicating that lapine cells behave di¡erently to ovine cells. The e¡ect of donor age has also been reported in other studies including humans. Adkinsson et al (1998) and Hollander et al (2001) both report the inability of adult human donors to produce a chondrocytic tissue in their culture systems. Donor age is therefore an important variable for cartilage tissue engineering and may limit the use of autologous approaches to cartilage tissue engineering (depending on patient age). However, there have been some recent intriguing studies performed by Hollander et al (2001) and Ka¢enah et al (2002), which indicate that adult nasal chondrocytes have a greater chondrogenic potential compared to chondroctyes isolated from the articular surface. Studies to establish whether nasal cartilage constructs can stimulate joint repair have yet to be undertaken. Our initial studies using ovine meniscal cells have indicated that there is a potential to culture expand these cells and culture them in an environment such that they produce a ‘hyaline-like’ tissue indicating that they may be of use in cartilage tissue engineering. We have evaluated the ability of various sca¡olds (PGA, PCL, PLGA and a hydrogel) to support chondrogenesis using ovine articular chondrocytes. All of these candidate sca¡olds materials were able to support chondrogenesis to di¡erent degrees. However, PGA is our preferred material, as PGA-grown constructs are superior to constructs cultured on the other materials.


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Our initial implantation studies in a lapine model have indicated the potential to improve cartilage repair using a tissue-engineered approach. Further construct developments are in progress to develop tissue-engineered constructs to repair osteochondral defects. These constructs are currently being evaluated in large animal models. Acknowledgements This work was funded by a joint venture between Smith & Nephew and Advanced Tissue Sciences.

References Adkisson H, Maloney W, Zang J, Hruska K 1999 Sca¡old-independent neocartilage formation: a novel approach to cartilage engineering. Trans Ortho Res Soc 24:803 Caplan AI, Elyaderani M, Mochiziuki Y, Wakitani S, Goldberg VM 1997 Principles of cartilage repair and regeneration. Clin Orthop Relat Res 342:254^269 Cao Y, Vacanti J, Paige K, Upton, J, Vacanti C 1997 Transplantation of chondrocytes utilizing a polymer-cell construct to produce tissue-engineered cartilage in the shape of a human ear. Plast Reconstr Surg 100:297^302 Carter A, Huckle J 2002 Meniscus. In: Atala A, Lanza RP (eds) Methods of tissue engineering. Academic Press, San Diego, CA, vol 95:1049^1057 Deasy B, Qu-Peterson Z, Huard J 2002 Multipotentiality of muscle-derived stem cells. Trans Ortho Res Soc 27:658 Dunkelman N, Zimber M, LeBaron R, Pavelec R, Kwan M, Purchio A 1995 Cartilage production by rabbit articular chondrocytes on polyglycolic acid sca¡olds in a closed bioreactor system. Biotechnol Bioeng 46:299^305 Erickson G, Franklin D, Gimble J, Guilak F 2001 Adipose tissue-derived stem cells display a chondrogenic phenotype in culture. Trans Ortho Res Soc 26:198 Farndale RW, Buttle DJ, Barrett AJ 1986 Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochim Biophys Acta 883:173^177 Freed L, Vunjak-Novakovic G, Biron R 1994 Biodegradable polymer sca¡olds for tissue engineering. Biotechnology (NY)12:689^693 Freed L, Vunjak-Novakovic G 2002 Culture environments: cell-polymer-bioreactor systems. In: Atala A, Lanza RP (eds) Methods of tissue engineering. Academic Press, San Diego, CA, 6:97^111 Hellio Le Graverand M, Eggerer J, Rattner JP, Barclay L, Hart D, Rattner JB 2001 Identi¢cation of 4 major morphologically distinct cell types in rabbit menisci. Trans Ortho Res Soc 26:811 Hollander A, Ka¢enah W, Martin I, De¤ marteau O, Barker M 2001 Use of nasal chondrocytes in tissue engineering of articular cartilage. Trans Ortho Res Soc 26:641 Ka¢enah W, Jakob M, De¤marteau O et al 2002 Three-dimensional tissue engineering of hyaline cartilage: comparison of adult nasal and articular chondrocytes. Tissue Eng 8:817^826 Nicoll S, Wedrychowska A, Bhatnagar R 1998 Induction of a chondrocyte-like phenotype in human dermal ¢broblasts by high density micromass culture in a functionally hypoxic environment: regulation by protein kinase C. Trans Ortho Res Soc 23:36 O’Driscol S, Keeley F, Salter RB 1986 The chondrogenic potential of free autogenous periosteal grafts for biological resurfacing of major full-thickness defects in joint surfaces under the

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in£uence of continuous passive motion. An experimental investigation in the rabbit. J Bone Joint Surg Am 68:1017^1035 Pittenger M, Mackay A, Beck S, Jaiswal R, Douglas R, Mosca J, Moorman M, Simonetti D, Marshak C 1999 Multilineage potential of adult human mesenchymal stem cells. Science. 284:143^147 Schreiber R, Kirby B, Dunkelman N, Stevenson S, Ratcli¡e A 1998 Repair of osteochondral defects with tissue engineered cartilage allografts. Trans Ortho Res Soc 23:383 Schreiber R, Kirby B, Dunkelman N et al 1999 Repair of osteochondral defects by allogeneic tissue engineered cartilage implants. Clin Orthop Relat Res 367S:382^395 Sims C, Butler P, Cao Y et al 1998 Tissue engineered neocartilage using plasma derived polymer substrates and chondrocytes. Plast Reconstr Surg 101:1580^1585 Woessner J 1961 The determination of hydroxyproline in tissue and protein samples containing small proportions of this amino acid. Arch Biochem Biophys 93:440^447

DISCUSSION Barry: The progenitor cells that you have observed are on the super¢cial zone of the articular cartilage. Within the knee joint there are progenitor cells associated with the cartilage, fat pad, synovium and the bone marrow. In terms of the basic biology of these cells do these all represent subpopulations of the same pool, or are they tissue-speci¢c cells? Huckle: The experiments I showed were from cartilage tissues taken from calves we have not looked in the other tissues within the joint as yet. We know that those stem cells are present in calves, but we don’t know whether they are present in adult human tissue yet. Caplan: Did you look at older animals in this regard? Huckle: We have looked at bovine and they are still present, but we haven’t looked in human. Caplan: What were the oldest cows you looked at? Huckle: About 18 months old. Martin: You mentioned that using meniscal cells is a promising approach, but you have shown that you still need to apply some kind of hydrostatic pressure to induce formation of hyaline cartilage. Are you planning to use these physical forces in vitro? If so, why hydrostatic pressure and not deformation, for example? Huckle: This shows that there is potential to put those meniscal cells into a cartilage lesion, and that environment may stimulate them to form a ‘hyalinelike’ tissue. The experiment also shows that the cells in the meniscus have the ability to change phenotype and response to physical environment. This has other implications for meniscal tissue engineering as well, in that when we ¢rst looked at the meniscus we thought it was a very complex structure that went from a hyaline-like material to a ligament-like material. To grow that type of complex heterogeneous construct outside of the body would be di⁄cult. These


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experiments indicate that you might not have to grow a complex heterogeneous construct outside the body: a homogenous construct may remodel in vivo. Caplan: I wasn’t impressed by the chondrogenic character of the meniscal cells. Is it more conservative to say that those cells could provide ¢brocartilage but not necessarily hyaline cartilage? Huckle: I agree. From looking at the meniscal biochemistry in more detail, even the avascular region of the meniscus, which is quoted as hyaline-like by some individuals, is about 95% type I collagen and about 5% type II collagen, and contains limited glycosaminoglycans. The constructs that we have produced from meniscal cells grown under hydrostatic pressure have a phenotype more similar to the avascular region of the meniscus as compared to articular cartilage. Trippel: In the experiments you described early on in your paper in which you showed that neonatal or juvenile but not adult cartilage could produce a cartilage matrix, were you using serum-free cultures or serum-containing media? Huckle: The medium that we’ve tended to use is standard DMEM with 10% fetal calf serum. We have done some experiments in which we have taken away the serum and grown them in insulin, transferrin and selenium (ITS)-type media with and without growth factors. But we found that within the bioreactor systems that we use, it doesn’t make much di¡erence. Other individuals have found that for them to get chondrogenesis in their systems they have to wean the chondrocytes o¡ the serum. We don’t see this. Trippel: Have you ever tried using adult bovine chondrocytes? Huckle: No. Trippel: There is a curious disconnect here. If you take adult bovine chondrocytes and put them into culture in their native matrix, they will respond very nicely to a variety of growth factors. Something that you are doing seems to prevent the ability of the cells to respond to the growth factors. Have you tried doing these cultures with cells that are simply isolated from their matrix, without putting them in the PGA felt at all? For example, you could try pellet cultures or high-density monolayer cultures. It seems that there is something that you are doing to these cells that is inhibiting them. Caplan: First of all, James Huckle is not dealing with bovine cells. As was discussed yesterday, bovine chondrocytes in particular make huge amounts of extracellular matrix, whereas cells ‘isolated’ from other animals don’t do that in the same way under the same conditions. As well as the species, the location these cells were taken from is also important. I’d like to point out that if you take and analyse the aggrecan made by cells from young donors human or animal versus cells from old donors, the chemistries of the GAG chains are uniquely and distinctly di¡erent. Therefore, when someone says they are getting di¡erences between young and old cells, and they don’t look at the GAG chains, the impression one has is that when you take a cell out of a matrix, get it to divide

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and try to reform a chondrogenic matrix, it goes back to what it was doing. In older samples, this wasn’t such a good job in the ¢rst place. The issue is whether a cultureexpanded cell that is used in tissue engineering, especially in allograft cases, is going to make a young cartilage or a cartilage like the one from which the cells arose. There are several di¡erent variables here, which all need to be de¢ned. Huckle: Other groups have also reported a similar age-dependent e¡ect. We have put these cells onto other felts, matrices and culture systems and there is still an agedependent e¡ect. Buschmann: We have done very similar experiments in bovine with fetal cells all the way up to ¢ve-year old cows. If you normalize per cell there is a very signi¢cant age e¡ect, almost up to a factor of three. Collagen is the most sensitive, and the amount of ascorbate in the medium is critical. Lindahl: I don’t think that we should argue that younger cartilage is better than old. We don’t experience this in human cultures, but we use autologous human serum as additive. If you look at fetal calf serum, you have to select a very good batch. I don’t know whether this is done in these studies. If you have human autologous serum you can expand the cells, at least to ¢ve passages, and get good chondrogenesis in agarose cultures as well as in pellet mass. Huckle: What would be your response to Accio et al (2001)? They found that the ability to get good chondrogenesis in alginate does not relate to what forms in vivo. Intramuscular injection of freshly isolated adult human articular chondrocytes resulted in cartilage formation in a nude mouse model. These cells lost the ability to form cartilage in vivo after monolayer expansion. However, the same cells could still form cartilage in alginate. This indicates that alginate culture is not a good predictor of cartilage formation in vivo. Caplan: You have to be careful about this term ‘in vivo’. You said it was implanted intramuscularly in a nude mouse. You are dealing with mouse serum as your growth medium. Although nude mouse is awesome for mesenchymal stem cell (MSC) osteogenesis, chondrogenesis in a subcutaneous location depends on exactly the source of the MSCs and the species of nude mouse. Pavesio: In your in vivo experiments, did you use cryopreserved constructs? Huckle: No. Pavesio: What do you plan to use in clinical practice? Huckle: We have done some work on cryopreservation and fresh preservation. We have found with fresh preservation at 4 8C we can preserve the construct for several months, without having to cryopreserve. Martin: Coming back to the issue of ageing, from your data you showed a dramatic di¡erence between donors aged seven months to two years, and the rest. But you didn’t see much of a di¡erence between a 20 year old and a 45 year old donor. Is this correct?


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Huckle: Yes, we tend to see a cut-o¡ at about age 25. After this you can’t produce a hyaline-like material. Martin: But also between ages 20 and 45 I didn’t see a major di¡erence. Huckle: You would if you looked histologically. The material is ¢brous with 45 year old donors. The most hyaline-like material comes from younger donors. van Blitterswijk: How much e¡ort have you put into getting the cells running from the older patients? If you focus on it more you might get better results. Huckle: We have put a lot of e¡ort into getting older cells to make hyaline cartilage, both in expanding them up and bioreactor conditions. van Blitterswijk: It surprises me, because you are going for allogenic cells. Why would you put a lot of e¡ort in trying to culture autologous cells? Huckle: Because it is di⁄cult to get hold of younger donors. van Blitterswijk: So you think the number of cells that you can grow from younger donors is relatively limited? Huckle: It is more di⁄cult to get hold of younger donors. Ratcli¡e: It is much more convenient to harvest from an adult donor than from a two-year old donor. You can do it from an allogeneic child source. The data as presented would say focus on the younger donors. Hardingham: Do you think the di¡erence seen with age is simply related to the abundance of progenitors within the tissue you harvest? Huckle: That’s a good question. We haven’t looked at this. Hollander: Where we see the di¡erence between nasal and articular cartilage in either bovine or human, this may well be the explanation: there is a greater abundance of progenitors in the nasal septum. Caplan: James Huckle was conservative in the way he presented the chondroprogenitor information. The prevalence of these cells in human is unknown. Let’s be careful here. Vunjak-Novakovic: I am curious about the cell densities that you used: what was the starting cell density? Our experience is that the e¡ects of many factors that normally a¡ect chondrogenesis are not expressed at very low cell densities. The best result you showed was for a pellet, which may be an indication that the high cell density within the pellet made a di¡erence. Huckle: The only pellet I showed was from the chondroprogenitor cells. We are using felts all the time. We normalize seeding density all the way through the process, both when we plate them down at primary, right the way through individual passages, to when we put them on the cells. We have looked at the seeding density of the constructs upon seeding into the felts. They are normalized as well. We are putting 4 million cells into a construct 10 mm in diameter and 2 mm thick. Vunjak-Novakovic: Cell density is extremely important for chondrogenesis and should be taken into account.

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Huckle: We did a lot of work where we optimized the seeding density of the constructs, and it is critical. Caplan: James Huckle’s observation in calf also brings us back to a question raised yesterday. This is the issue of chondroprogenitor cells within the joint, the ultimate location of these cells, and whether there are markers that one would want to use in human. Does anyone want to comment on turnover dynamics in adult cartilage, and the source of the progenitor cells? Lohmander: The idea that I have, based on following the literature, is that there is very little turnover of cells in adult cartilage. If they do anything, they die. Sometimes in osteoarthritis we see nests of cells that appear to be proliferating to some extent, but I ¢nd it hard to see that a cell could migrate through the cartilage matrix. However, there might be cells at the surface of the ‘normal’ cartilage that could somehow replicate and keep that surface intact. If that surface then somehow is removed, you could add cells back to regenerate a new surface layer. This is something that is worth looking into. We are beginning to understand that there is not just one tissue called ‘cartilage’. There is a great deal of heterogeneity both between joints and locations within the joints in the phenotype of the cartilage cells. There is more for us to understand. Barry: You put your ¢nger on one of the critical points in this whole area: cell surface markers. The development of stem cell-speci¢c markers has not happened, and we currently lack an MSC-speci¢c marker that can be used in vivo to identify a stem cell. These markers simply do not exist. This greatly limits the experiments we can do looking at turnover of cells in vivo. The next step in this ¢eld has to be a signi¢cant e¡ort in identifying novel markers for stem cells. Caplan: The whole question of tissue turnover dynamics, particularly in experimental animals, has been based on the injection of tritiated thymidine or BrdU and looking at labelling kinetics of cells. If you seriously compare these older data with current probes for apoptosis, the numbers and impressions don’t ¢t. I would suggest not only is the issue of the identi¢cation of the normal chondroprogenitor cells in adult cartilage of some concern, but also the issue of turnover dynamics. The apoptosis-positive cell population in adult cartilage is surprisingly large in ‘normal’ joints. With regard to this issue of tissue turnover, I know of no case neural tissue included of tissues that don’t repopulate. Therefore the dogma from older experiments that cartilage matrix doesn’t turn over and that chondrocytes don’t turnover needs to be re-evaluated. Hollander: I think Stefan Lohmander’s point is a critical one, though. If cells are going to track down from the cell surface through the cartilage, collagen degradation would have to occur to allow the cells to migrate. There is no evidence that this occurs. In normal cartilage we have shown that there is minimal turnover of type II collagen. But turnover of the surface may be very important, because the surface cells may be signalling down to an older


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population of cells in the deeper zone. You may not need to repopulate through the depth of cartilage. Lindahl: This is a central question in the whole ¢eld of cartilage. If there is a cell turnover, the whole concept of tissue engineering will have to change. The same thing was said for the heart that the cardiomyocytes did not regenerate. With the number of apoptotic cells that can be found in the mouse heart, the mouse would be dead in three months. But the mouse lives much longer. I don’t think that we have done the correct experiments to address the question of cell regeneration or migration in cartilage. Cell migration can be quite large, so I would de¢nitely not rule out the possibility. Caplan: It is unlikely that progenitor cells are migrating through cartilage. But if the top of the cartilage is neocartilage and the bottom is non-neocartilage in this regard, you can imagine a gradient of age of the cells from top to bottom. This is a testable hypothesis. As far as I know, there are no data on this. The important point is that tissue engineering can be shifted to chemical management of host cells in regenerative logic, if tissue engineering can solve some of the other aspects. In some way, the turnover dynamics, the presence and location of progenitor cells and the disposition of the tissue at this turnover point, will be central questions at some stage down the road. Reference Dell’Accio FD, De Bari CD, Luyten FP 2001 Molecular markers predictive of the capacity of expanded human articular chondrocytes to form stable cartilage in vivo. Arthritis Rheum 44:1608^1619

Mesenchymal stem cells and bioceramics: strategies to regenerate the skeleton Hajime Ohgushi, Jun Miyake and Tetsuya Tateishi Tissue Engineering Research Center (TERC), National Institute of Advanced Industrial Science and Technology (AIST), 3-11-46 Nakouji, Amagasaki City, Hyogo 661-0794, Japan

Abstract. Bone is formed by cells called osteoblasts, which arise from mesenchymal stem cells (MSCs). The cells are known to exist in thin tissues surrounding bone (periosteum) and bone marrow, but the population is extremely small. The number of marrow-derived MSCs can be expanded using tissue culture techniques. These culture-expanded MSCs have the in vitro capacity to di¡erentiate into osteoblasts. Importantly, the cultured osteoblasts can form extracellular matrix in culture. This matrix consists of ¢ne crystals of hydroxyapatite comparable to natural bone mineral, as evidenced by X-ray di¡raction and Fourier-transform infrared spectroscopy. It is possible to fabricate the osteoblasts/ bone matrix on the surface of bioceramics. Thus in vitro cultured bone can show further bone-forming capability after in vivo implantation. We have begun studying this tissueengineering approach in patients with skeletal problems. This paper describes this and other approaches using MSCs to regenerate skeletal tissue. 2003 Tissue engineering of cartilage and bone. Wiley, Chichester (Novartis Foundation Symposium 249) p 118^132

Recent technology has developed various materials applicable for clinical use in orthopaedic, dental and maxillofacial surgery. When materials such as metals or their alloys are implanted at bone defect sites, encapsulation of the implants by scar tissue and/or a ¢brous membrane occurs (Black 1992). However, when bioinert materials such as alumina (Al2O3) ceramics or titanium devices are implanted into the bone defects, the scar tissue/¢brous membrane formation is minimal, and some areas show regenerated bone tissue in contact with the implanted materials without other types of ¢brous tissues intervening (Hulbert et al 1970, Linder et al 1988). Despite this, the interface between the bone and the implants is not strong and the interface detaches easily upon shear and distraction loads. In contrast, when bioactive materials are implanted, chemical bonding is established between the regenerated bone tissue and the surface of the material. This bone/material interface is very strong and stable and, upon loading, 118

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breakage usually occurs inside the bone or material, but not at the interface. Such interaction between bone and the bioactive material is referred to as bone bonding. Glass ceramics containing SiO2, Na2O, CaO and P2O5 in speci¢c proportions exhibit bioactivity and were later termed bioactive glasses (Hench 1988). More recently, calcium phosphate ceramics, such as hydroxyapatite (HA), were shown to be bioactive because the ceramics directly (chemically) bond to bone (Jarcho 1981). These bioactive ceramics are now used as arti¢cial bone substitutes and as coating substances on various kinds of arti¢cial joints to promote the stability of the joint. Bone is formed by cells called osteoblasts, which arise from mesenchymal stem cells (MSCs) in a multi-step lineage cascade (Friedenstein et al 1968, Caplan 1991, Caplan & Bruder 1996, Ohgushi & Caplan 1999). The stem cells reside in thin cell layers surrounding bone surface and a tissue inside the bone (periosteum and bone marrow, respectively). We have proposed that the surfaces of bioactive materials support the osteoblastic di¡erentiation of mesenchymal stem cells whereas nonbioactive materials cannot support this di¡erentiation (Ohgushi et al 1992, Takaoka et al 1996). The osteoblastic di¡erentiation of the stem cells can be found in subcutaneous implantation of the composites of marrow cells/porous ceramics (Ohgushi et al 1989a, Okumura et al 1997). The bone formation pattern re£ects the bioactivity of the implants. Di¡erentiation occurs directly on the surface of the bioactive materials whereas the di¡erentiation occurs away from the surface of non-bioactive materials (Takaoka et al 1996, Okumura et al 1997). Because of the surface-dependent osteoblastic di¡erentiation of the bioactive materials, bone bonding occurs on their surfaces. Although the bonding does not occur on the surface of non-bioactive materials, such as bioinert alumina ceramics and titanium implants, a number of areas show bone apposition without intervening ¢brous tissue (Ohgushi et al 1992, Takaoka et al 1996). The present paper describes the manipulation of the osteoblastic di¡erentiation of mesenchymal stem cells with a view to clinical application. Osteogenic di¡erentiation of MSCs in porous ceramics Bioactive materials can show bone bonding when implanted in bony defect sites. This phenomenon can be reproduced by subcutaneous implantation of composites of bioactive materials and marrow cells (Ohgushi et al 1992, Okumura et al 1997). The composites show attachment of MSCs followed by osteoblastic di¡erentiation (Ohgushi et al 1993). The di¡erentiation cascade involves the attachment of ¢broblastic cells (stem cells), which then develop into small round cells (preosteoblasts) and ¢nally become large round cells (osteoblasts) fabricating a thin layer of primary bone onto the ceramic (Okumura et al 1997). The primary bone is partially mineralized osteoid, and later this tissue becomes fully

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mineralized bone with no intervening ¢brous tissue at the interface, which implies bone bonding. After fully mineralized bone formation, appositional bone formation follows; thus, invariably, bone formation always starts on the surface of the HA ceramic and proceeds toward the centre of the pore. The cascade is called bonding osteogenesis, and indicates the importance of a bioactive ceramic surface to support the osteogenic di¡erentiation of marrow MSCs (Okumura et al 1991). By analysing the cell/material composite graft, we have found that other bioactive materials, such as glass ceramics (apatite wollastonite-containing glass ceramics), tricalcium phosphate (TCP) and biphasic HA/TCP ceramics, exhibit the same cascade of bonding osteogenesis and show the same material performance relative to the cell di¡erentiation sequence (Ohgushi et al 1990, 1992). These results show the usefulness of bioactive ceramics when combined with MSCs for bone reconstruction surgery (Ohgushi et al 1989b, Bruder et al 1998, Quarto et al 2001). Manipulation of mesenchymal stem cells Fresh marrow and cultured MSCs/ceramic composites The number of MSCs in freshly isolated bone marrow cells is very small and therefore, isolation or condensation of the MSC-rich cellular fraction is needed for extensive bone formation in the porous HA ceramics (Ohgushi et al 1989a). In vitro culture techniques are available for the expansion of MSCs from fresh bone marrow and these cultured MSCs are useful for making a composite with HA (Caplan & Bruder 1996, Ohgushi & Caplan 1999). Initially, we used the MSCs from rat marrow but later, we and others con¢rmed the osteogenic potential of human MSCs in the composites (Ohgushi & Okumura 1990, Haynesworth et al 1992). The fresh and cultured cells require a step towards osteoblastic di¡erentiation. Therefore, 3^4 weeks are needed to show the bone formation in HA combined with fresh marrow cells or cultured MSCs (Fig. 1). In this regard, if we can di¡erentiate the cultured MSCs into osteoblasts in vitro, such di¡erentiated osteoblasts as well as extracellular matrix (bone matrix) produced by the osteoblasts can be available for making composites. As seen in the following section, we have established this novel concept and found that in vitro generated osteoblasts/bone matrix show prominent osteogenic capacity (Yoshikawa et al 1996). Cultured bone in porous ceramics Bone-like tissue can be formed by rat marrow cell culture in the presence of vitamin C, b-glycerophosphate and dexamethasone (Dex), which seems to be crucial (Maniatopoulos et al 1988). However, an important question is whether the

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FIG. 1. Various methods for demonstrating new bone formation after in vivo implantation. Dotted arrows indicate the culture periods. Solid arrows indicate the in vivo implantation periods to show new bone formation. HA indicates the porous hydroxyapatite ceramics and BMP indicates the recombinant human bone morphogenetic protein 2.

bone-like tissue derived from marrow is actual bone tissue or not. In other words, characterization of the cells and extracellular matrices is needed. After the culture expansion of rat MSCs, the cells were further cultured (subcultured) in the presence of vitamin C, b-glycerophosphate and Dex. Phase contrast microscopy showed a cluster of many cuboidal cells surrounded by abundant extracellular matrices, which appeared at about 7 days post subculture. The central area of the cluster showed early mineralization (nodule). As shown in Fig. 2, the number of the clusters increased and numerous nodules could be seen macroscopically at 2 weeks post subculture. The nodules as well as cuboidal cells were positive for alkaline phosphatase (ALP). Northern blot analysis and in situ hybridization using an antisense probe for bone-speci¢c osteocalcin revealed that the cells produced abundant osteocalcin mRNA. Fourier transform infrared (FTIR) spectral and X-ray di¡raction patterns of mineralized area were similar to those of genuine bone tissue harvested from femoral bone (Ohgushi et al 1996). Both the in vitro mineralized area and bone tissue evidenced the poorly crystallized carbonated apatite formation. The characteristics of the crystal are typical of the crystal in real bone mineral, called biological apatite. These physicochemical and biochemical analyses indicated that the in vitro fabricated mineralized tissue from cultured MSCs consisted of active osteoblasts as well as normal bone matrix and therefore this tissue is comparable to genuine bone tissue (Ohgushi et al 1996). Importantly, the in vitro bone can be formed by MSCs derived from human marrow (Jaiswal et al 1997, Yoshikawa et al 1998).

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FIG. 2. Marrow mesenchymal cell culture in the presence of dexamethasone, glycerophosphate and vitamin C. Phase contrast microscopy reveals the mineralized area (black area) as well as osteoblastic cells (arrows). The cells show the presence of bone-speci¢c osteocalcin mRNA demonstrated by in situ hybridization and Northern blot analysis. Characteristic of the mineral is carbonate-containing biological apatite evidenced by X-ray di¡raction and FTIR analysis. Modi¢ed from the ¢gures in Ohgushi et al (1996).

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We also have succeeded in making in vitro cultured bone in pore areas of the ceramics and interestingly, the composite shows continued osteogenic potential after in vivo implantation. Therefore, this in vitro generated bone has regenerative capability for new bone formation (Yoshikawa et al 1996, 1997). We con¢rmed this new bone formation after in vivo implantation by using not only rat but also human cultured cells (Yoshikawa et al 1998, de Bruijn JD et al 1999) and showed the importance of the in vitro bone formation in culture conditions. The boneforming ability of the human cells can be seen from an aged population. We have cultured cells from more than 30 cases, including 60^80 year olds, and all of them showed evidence of osteogenic responses (Ohgushi & Caplan 1999). These results indicate the importance of the tissue-engineered regenerative bone formed in porous ceramics for bone tissue repair. These composites can be applied to patients with various chronic skeletal diseases such as osteoarthritis and rheumatoid arthritis (Ohgushi & Caplan 1999). However, the total period needed to show in vivo thick bone formation after harvesting fresh marrow is more than 4 weeks (Fig. 1, method C) and therefore, these composites can not be applied for acute cases such as fracture patients. In this regard, additional factors, which promote the osteoblastic di¡erentiation of MSCs, may be necessary. Composites of MSCs and bone morphogenetic protein To promote the bone formation capability of MSCs/ceramic composites, we added small amounts of recombinant human bone morphogenetic protein 2 (BMP2) to the composite (Noshi et al 2001, Ikeuchi et al 2002). Both HA and the BMP/HA composites did not show bone formation at any time after implantation. The MSC/BMP/HA composites showed early in vivo bone formation together with active osteoblasts at 1 week, obvious bone formation at 2 weeks and more bone hereafter. The MSCs/BMP/HA composites demonstrated high alkaline phosphatase and osteocalcin expression at both protein and gene expression levels compared with MSCs/HA composites (Noshi et al 2001). These results provide histological and biochemical evidence that the combination of MSCs, porous HA and BMP can synergistically enhance osteogenic potential. This method (Fig. 1, method D) shows in vivo bone formation at an earlier stage compared with the other graft methods using marrow cells. Towards clinical applications All these results indicate the usefulness of culture-expanded osteogenic cells for the repair of skeletal diseases. Various methods exist for this purpose, including the use of cultured MSCs (method B), cultured osteoblasts/bone matrix (cultured

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FIG. 3. Fabrication process of cultured regenerative bone from patient’s marrow at the Tissue Engineering Research Center.

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regenerative bone; method C) and cultured MSCs supplemented with BMP (method D). The last method can show rapid bone formation and therefore may be ideal for the treatment of traumas such as massive fractures. However, this method requires BMP and, in considering clinical applications, human recombinant BMP produced by expensive multi-step purifying cascades must be used. Our experience is that the MSC cell number necessary for bone formation is about 200 000 per HA disk (5 mm diameter and 2 mm thickness) for methods B and D. However, cultured osteoblasts/bone matrix in HA (method C) can show bone formation with only 20 000 MSCs (Yoshikawa et al 1996). Therefore, fewer fresh marrow cells are required compared with other methods, which may be more clinically relevant. Method C has an another advantage, because the reagents necessary for the method are culture medium, serum (not only calf serum but patient’s serum is available), vitamin C phosphate, glycerophosphate and dexamethasone: all these reagents are stable and easy to use and thus the method is convenient and less expensive. For these reasons we have begun to use the cultured regenerative bone method (C) for the treatment of the patients with chronic skeletal diseases (osteoarthritis and benign bone tumours). The method consists of two steps: primary culture of patient’s marrow (expansion of MSCs) and then subculture of the MSCs to fabricate osteoblasts/bone matrix in the pore region of the ceramics. This in vitro cultured regenerative bone is delivered to the hospital and used for the transplantation surgery. The approach requires a tissue culture procedure, which carries the risk of bacterial/fungal contamination. To avoid the risk, biological safe areas and careful/safe protocols are required. Our facility is located in an area where animal cells are not allowed and has two clean rooms (class 1000 and class 10 000). As shown in Fig. 3, during the culture of patients’ cells, we check for bacterial, fungal and mycoplasma contamination at least twice. Furthermore, endotoxin levels in the reagents/medium are also checked. As described, we are applying the cultured regenerative bone derived from patient’s bone marrow cells (method C) for the treatment of chronic skeletal diseases under strict and secure conditions. We believe this new tissue-engineered approach has signi¢cant advantages for treating severe problems in orthopaedic, dental and maxillofacial cases.

Acknowledgements We thank our colleagues at Nara Medical University and Tissue Engineering Research Center (TERC). We especially thank Dr Y. Takakura, Dr T. Yoshikawa, Dr Y. Dohi, Dr S. Tabata, Dr M. Hirose, Dr M. Ikeuchi, Ms H. Machida, Ms N. Kotobuki and Ms A. Matsushima. We also thank Dr E.C. Shors (Interpore International, CA) for providing ceramics and Yamanouchi Pharmaceutical Co. (Tokyo, Japan) for providing recombinant human BMP2. A special note

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of gratitude is extended to Professor A.I. Caplan (Case Western Reserve University) for encouraging us to attend the Novartis Foundation Symposium and prepare this paper.

References Black J 1992 The in£ammatory process. In: Black J (ed) Biological performance of materials. Marcel Dekker Inc, New York, p 125^145 Bruder SP, Kraus KH, Goldberg VM, Kadiyala S 1998 The e¡ect of implants loaded with autologous mesenchymal stem cells in the healing of canine segmental bone defects. J Bone Joint Surg Am 80:985^996 Caplan AI 1991 Mesenchymal stem cells. J Orthop Res 9:641^650 Caplan AI, Bruder SP 1996 Cell and molecular engineering of bone regeneration. In: Lanza RP, Chick WL, Langer R (eds) Principles of tissue engineering. RG Landes Company, SpringerVerlag, New York, p 599^618 de Bruijn JD, van den Brink I, Mendes S, Dekker R, Bovell YP, van Blitterswijk CA 1999 Bone induction by implants coated with cultured osteogenic bone marrow cells. Adv Dent Res 13:74^81 Friedenstein AJ, Petrakova KV, Kurolesova AI, Frolova GP 1968 Heterotopic transplants of bone marrow. Analysis of precursor cells for osteogenic and haemopoietic tissues. Transplanation 6:230^247 Haynesworth SE, Goshima J, Goldberg VM, Caplan AI 1992 Characterization of cells with osteogenic potential from human marrow. Bone 13:81^88 Hench LL 1988 Bioactive ceramics. Ann NY Acad Sci 523:54^71 Hulbert SF, Young FA, Mathews RS, Klawitter JJ, Talbert CD, Stelling FH 1970 Potential of ceramic materials as permanently implantable skeletal prostheses. J Biomed Mater Res 4:433^456 Ikeuchi M, Dohi Y, Horiuchi K et al 2002 Recombinant human bone morphogenetic protein-2 promotes osteogenesis within atelopeptide type I collagen solution by combination with rat cultured marrow cells. J Biomed Mater Res 60:61^69 Jaiswal N, Haynesworth SE, Caplan AI, Bruder SP 1997 Osteogenic di¡erentiation of puri¢ed, culture-expanded human mesenchymal stem cells in vitro. J Cell Biochem 64:295^312 Jarcho M 1981 Calcium phosphate ceramics as hard tissue prosthetics. Clin Orthop 157:259^278 Linder L, Carlsson A, Marsal L, Bjursten LM, Branemark PI 1988 Clinical aspects of osseointegration in joint replacement. A histological study of titanium implants. J Bone Joint Surg Br 70:550^555 Maniatopoulos C, Sodek J, Melcher AH 1988 Bone formation in vitro by stromal cells obtained from bone marrow of young adult rats. Cell Tissue Res 254:317^330 Noshi T, Yoshikawa T, Dohi Y et al 2001 Recombinant human bone morphogenetic protein-2 potentiates the in vivo osteogenic ability of marrow/hydroxyapatite composites. Artif Organs 25:201^208 Ohgushi H, Okumura M 1990 Osteogenic capacity of rat and human marrow cells in porous ceramics. Acta Orthop Scand 61:431^434 Ohgushi H, Caplan AI 1999 Stem cell technology and bioceramics: from cell to gene engineering. J Biomed Mater Res 48:913^927 Ohgushi H, Goldberg VM, Caplan AI 1989a Heterotopic osteogenesis in porous ceramics induced by marrow cells. J Orthop Res 7:568^578 Ohgushi H, Goldberg VM, Caplan AI 1989b Repair of bone defects with marrow and porous ceramic. Experiments in rats. Acta Orthop Scand 60:334^339

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Ohgushi H, Okumura M, Tamai S, Shors EC, Caplan AI 1990 Marrow cell induced osteogenesis in porous hydroxyapatite and tricalcium phosphate: a comparative histomorphometric study of ectopic bone formation. J Biomed Mater Res 24:1563^1570 Ohgushi H, Okumura M, Yoshikawa T, Tamai S, Tabata S, Dohi Y 1992 Regulation of bone development and the relationship to bioactivity. In: Ducheyne P, Kokubo T, van Blitterswijk (eds) Bone-bonding biomaterials. Reed Healthcare Communications Publishers, The Netherlands, p 47^56 Ohgushi H, Dohi Y, Tamai S, Tabata S 1993 Osteogenic di¡erentiation of marrow stromal stem cells in porous hydroxyapatite ceramics. J Biomed Mater Res 27:1401^1407 Ohgushi H, Dohi Y, Katuda T, Tamai S, Tabata S, Suwa Y 1996 In vitro bone formation by rat marrow cell culture. J Biomed Mater Res 32:333^340 Okumura M, Ohgushi H, Tamai S 1991 Bonding osteogenesis in coralline hydroxyapatite combined with bone marrow cells. Biomaterials 12:411^416 Okumura M, Ohgushi H, Dohi T et al 1997 Osteoblastic phenotype expression on the surface of hydroxyapatite ceramics. J Biomed Mater Res 37:122^129 Quarto R, Mastrogiacomo M, Cancedda R et al 2001 Repair of large bone defects with the use of autologous bone marrow stromal cells. N Engl J Med 344:385^386 Takaoka T, Okumura M, Ohgushi H, Inoue K, Takakura Y, Tamai S 1996 Histological and biochemical evaluation of osteogenic response in porous hydroxyapatite coated alumina ceramics. Biomaterials 17:1499^1505 Yoshikawa T, Ohgushi H, Tamai S 1996 Immediate bone forming capability of prefabricated osteogenic hydroxyapatite. J Biomed Mater Res 32:481^492 Yoshikawa T, Ohgushi H, Dohi Y, Davies JE 1997 Viable bone formation in porous hydroxyapatite: marrow cell-derived in vitro bone on the surface of ceramics. Biomed Mater Eng 7:49^58 Yoshikawa T, Ohgushi H, Uemura T et al 1998 Human marrow cell-derived cultured bone in porous ceramics. Biomed Mater Eng 8:311^320

DISCUSSION [Editor’s note: in his oral paper, Dr Ohgushi presented unpublished clinical data using cultured regenerative bone (osteoblasts/bone matrix) derived from patient’s marrow cells. These included total joint replacement cases and a benign bone tumour case.] Wozney: Have there been any controlled clinical studies comparing your porous implants with and without MSCs? Ohgushi: There was a criticism from our department about the technique we were using, because they thought that the lesions we were treating could be treated just by using ceramic implants. However, I talked to the patients, and they accepted my concept. Sometimes we might not need to use cells, but we have to do a randomized study to show whether we have to or not. Wozney: So will you be doing a randomized controlled clinical study? Ohgushi: We’d like to, but it will be di⁄cult because these sorts of cases aren’t very similar. Wozney: What is the advantage of this sort of cell-based therapy over just using BMP and the appropriate matrix?

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Ohgushi: As I showed, a small amount of BMP didn’t work. The amount we used was too small. One reason we haven’t used BMP more frequently is that it is very expensive. But it could work, and it does accelerate bone formation by MSCs. Wozney: I don’t doubt that, but my question was more a clinical one. We know that you can take BMP alone with a matrix and get bone formation in humans. The question is, cost aside, what is the advantage of a cell-based therapy over just a protein plus matrix-based one? Ohgushi: I am not so familiar with BMP matrices. But for a large bone defect the BMP matrix might not work in the deep zone, and thus this approach could be inappropriate for treating a large cavity. In this case, adding cells may help. For a small defect, there’s no problem with using a BMP matrix technique. Caplan: John Wozney, in your clinical experience is there an age-dependence to the responsiveness of implanted BMP? Wozney: No. We have looked across all of our clinical studies. In some cases it is di⁄cult to tell, for example, in the trauma studies most of the patients are young males. But in the alveolar ridge augmentation studies, for example, the patients’ ages range into the 70s and we see no dependence on age. This is perhaps surprising. Pavesio: Were there any di¡erences between the e¡ects of di¡erent dosages of BMP in patients belonging to di¡erent age groups? Wozney: The studies I am speaking of involve at least two doses of BMP. They were not set up to look at age di¡erences. Pavesio: Did you see di¡erences in e⁄cacy with the two doses? Wozney: Yes. The higher the dose, the faster bone formation occurred. Caplan: In trauma situations, and particularly in people aged around 65, do you think that there is a use for cell-based therapies in which you use small numbers of cells, expand them in vitro and then induce them into the right phenotype in vitro before implanting? Wozney: That’s basically my question. I haven’t seen any data that would suggest that adding MSCs to a BMP-based therapy will augment it. It could well be true, but I haven’t seen that information yet. Caplan: Healthcare delivery is somewhat de¢cient in dealing with patients above the age of 65, because of lack of experience and the now expanding patient population. In six years, 25% of the population of the USA will be above the age of 65. If someone aged 90 has a fracture, it is clinically more di⁄cult to deal with than in someone who is 50, in terms of their time of responsiveness. It may be that this patient population will be more amenable to cell based therapy, but this needs to be proven. Lohmander: It is not my experience that fractures necessarily heal less well in the elderly. They have problems for other reasons, such as osteoporosis and fragile health. But fracture healing as such is surprisingly good.

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Cancedda: My feeling is that these are two di¡erent approaches. Each one has its own advantages. For the small defect, perhaps BMP with matrix can work very well. But my sense is that it is very di⁄cult to control the concentration of the factor, and if you have too low a concentration then there is no bone formation. If you have excess, you can overshoot the target and have too much bone formation. Then the question is whether BMP can work with extreme situations, such as a long segment or in a situation where the area has been heavily radiated because of osteosarcoma removal, and the osteogenic cells have probably been damaged. In this case, can BMP still recruit the cells? Ohgushi: Are there any data from, for example, non-union cases treated by BMP? Wozney: Let me answer the questions one by one. It is clear that BMP can induce large amounts of bone. In sinus augmentation studies we are putting in, per side, up to 16 cc. We have to induce signi¢cant amounts of bone. The other question is whether it will work in compromised healing environments. There, I have to rely more on non-clinical studies. We have looked in glucocorticoid-compromised healing environments, and BMP will heal fractures here. We have done some reconstructions in radiated dog mandibles, and BMP has worked well there. There is less extensive clinical experience at this point. Caplan: So there are sites where you would expect to have less access to progenitor cells. For example, the distal tibia is a particularly di⁄cult fracture site. It is a nightmare because it doesn’t have all the muscle and vasculature on each side to supply cells and other nutrients. There will be some unfavourable sites that are going to be more challenging. It will be interesting to see what happens in these cases. Ranieri Cancedda’s point is a good one: there are two approaches, which could be complementary. What is interesting here is that if it turns out that in broad-based use of molecules such as BMP, the high doses that are currently necessary cause some distant side e¡ects, the exposure of these agents in vitro would eliminate most of these kinds of considerations. It is an alternative approach that may have some mechanistic value. van Blitterswijk: I believe in the e¡ects of BMP, but theoretically you would expect that the BMP would trigger the cell into an osteoblastic di¡erentiation, and this is something that takes time. After the implantation of your BMP, this process starts to take place. From Professor Ohgushi’s paper we see that having cells which are already di¡erentiated is better than having cells that are less di¡erentiated. Theoretically, we would expect from this that the bone healing using these di¡erentiated cells should be faster than with BMP alone. Wozney: I think there clearly could be a synergy. When BMP is implanted, there must be responsive cells there. Schwartz: As opposed to having a ceramic matrix, have you considered using a demineralized bone matrix that has BMPs? Then you could use fresh bone marrow and get it to selectively bind and attach to this matrix. Animal studies have shown a

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favourable response to this approach. The matrix can be optimized by using di¡erent forms of demineralized bone. You could have all of this at the time of surgery. Ohgushi: Bone matrix contains various kinds of factors including BMPs, and thus it works with or without marrow. However, most of the demineralized bone matrix is derived from calves, and therefore it elicits immunological responses and may cause infectious diseases. van Blitterswijk: What was the age of the patients that you treated? Ohgushi: In the joint replacement cases the patients were over 60. The tumour patient was a teenager. van Blitterswijk: I have a word of caution. In our hands we see that culturing cells of older patients is more di⁄cult and less reproducible than culturing cells of younger patients. We have thought about this clinical indication, but we decided not to go for it at this stage because with an implant integrated in the surrounding bone, the interface is very important. You are covering the interface not only with cells, but also with extracellular matrix. If your cells are not going to induce bone formation, you might interfere with the normal osteointegration process and expose the patient to risk. Do you have any ideas on that? Ohgushi: So far we have treated 30^40 patients, aged up to 80 years old. Even the bone marrow from 80 year old patients can show high alkaline phosphatase activity. We demonstrated osteoblast di¡erentiation from this elderly marrow. It is not a big problem, because even marrow from elderly patients can cover most surface areas of the implants with high alkaline phosphatase activity. van Blitterswijk: The issue is not so much that they make alkaline phosphatase almost all cells do but whether they will be able to continue bone formation after implantation. Ohgushi: We saw mineralization in every case. And we have experienced in vivo bone forming capability of the cultured human marrow cells, when we used athymic nude mice as the recipients. Caplan: The orthopaedically complicated issue here is the integration of the new tissue with the host tissue. This is the more di⁄cult part of these kinds of implantations. Ohgushi: To answer this question, we need many more cases treated by such tissue-engineering approaches, and therefore we should explain to the patients about the risk of the approaches. Hunziker: I have a comment on the question of the usefulness of having osteoprogenitor cells available besides BMP. BMP can do a very good job if there are enough progenitor cells available. If there is a lack, you need to provide them. There are clinical situations, such as segmental defects, in which the construct implant is exposed to other kinds of tissues at the surface. In our own experience, BMP stimulates the proliferation of ¢broblasts tremendously, which

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results in rapid contamination of the construct, inhibiting bone formation. This may be a reason for providing progenitor cells, or even taking additional measures to protect the construct from being contaminated by other cell pools. Wozney: What situation do you see this occur in? We don’t see that in periodontal regeneration. Hunziker: We have seen this in studies using calcium phosphate ceramics in rabbit. When we put them in the proximal tibia, the surgeon drilled the hole too far dorsally, and we saw contamination from the dorsal side. This is just an example of where it would be important to provide progenitor cells, or at least to take measures to prevent ingrowth of the cell types. Vunjak-Novakovic: Can anyone comment on the value of the subcutaneous implantation model, even for making preliminary decisions? Are there other models that would put engineered constructs into more realistic environments and expose them to actual signals that may lead to bone formation? van Blitterswijk: Generally it is more di⁄cult to show the e⁄cacy of your technology in a bone model as compared with the subcutaneous model. The subcutaneous model in nude mice is not ideal. Caplan: Let me restate the question. Has either of you taken the same size implant and in the same animal taken the autologous cells, loaded them into the implant, and put them in two places: one implanted subcutaneously, and the other placed into a bony defect? The question is, what is the relationship between ectopic bone formation and orthotopic bone formation, given the same carrier and cell loading? In Hajime Ohgushi’s case you would also compare this with a third plug, which would be cells that have been di¡erentiated by exposure to BMP or dexamethasone. Ohgushi: Our old data using fresh marrow cells, which can show ectopic bone formation, indicate that they can also work at orthotopic sites. However, in this regard, we did not do extensive studies using cultured cells. Caplan: You should also compare that with the porous material that you have implanted subcutaneously. The question is whether subcutaneous implantation is a good predictor of what you would see orthotopically. van Blitterswijk: We have done those studies. With intramuscular implants we see good bone formation with the sca¡old plus cells, and hardly any bone formation with the sca¡olds without cells. If you go into the bone defect, we see good bone healing with and without cells. What we are doing now is focusing more on spinal fusion. With spinal fusion you are at the bony site, but it is easier to discriminate between the bone formation caused by the sca¡olds and the cells, as opposed to the bone growing in from the bony sides. In our hands, we feel this is the best model to work with this type of technology. Martin: Within the context of this cellular approach, I’d like to understand the relevance of preculturing the cells on the ceramic. You are saying that you

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accelerate the formation of bone, but you lose time in vitro. Have you done studies on the kinetics of bone formation? Perhaps after one week you would have more formation of bone with precultured cells, but after one month the two groups may be comparable. Ohgushi: No, we haven’t.

Bone marrow stromal cells and their use in regenerating bone Ranieri Cancedda*{, Maddalena Mastrogiacomo*{, Giordano Bianchi*{, Anna Derubeis*{, Anita Muraglia* and Rodolfo Quarto* *Istituto Nazionale per la Ricerca sul Cancro, Centro Biotecnologie Avanzate and {Dipartimento di Oncologia, Biologia e Genetica, Universita' di Genova, Largo R. Benzi 10, 16132 Genova, Italy

Abstract. Tissue engineering approaches have recently been devised to repair large bone losses. Tissue engineering takes advantages of the combined use of cultured living cells and 3D sca¡olds to deliver vital cells to the damaged site of the patient. Cultured bone marrow stromal cells (BMSCs) can be regarded as a mesenchymal progenitor/precursor cell population derived from adult stem cells. When implanted in immunode¢cient mice, BMSCs combined with mineralized 3D sca¡olds to form a primary bone tissue that is highly vascularized. We have used autologous BMSC/bioceramic composites to treat full-thickness gaps of tibial diaphysis in sheep. The healing process has been investigated. The sequence of events is as follows: (1) bone formation on the outer surface of the implant; (2) bone formation in the inner cylinder canal; (3) formation of ¢ssures and cracks in the implant body; (4) bone formation in the bioceramic pores. Similar composites whose size and shape re£ected each bone defect have been implanted at the lesion sites of three patients. External ¢xation was used. Patients have been followed for more than three years. The results obtained are very promising and we propose the use of culture-expanded osteoprogenitor cells in conjunction with hydroxyapatite bioceramics as a signi¢cant improvement in the repair of critical size long bone defects. 2003 Tissue engineering of cartilage and bone. Wiley, Chichester (Novartis Foundation Symposium 249) p 133^147

Large bone loss represents a major problem in orthopaedics for two reasons: its frequency and di⁄culties in reconstruction. Surgeons often adopt solutions that do not allow a complete functional recovery and, in some cases, they have to take drastic measures such as arthrodesis or limb amputation. Therefore, a feasible strategy for repairing and reconstituting bone should aim to combine a relatively traditional approach, such as biomaterial implants, with recently acquired knowledge about the growth and di¡erentiation of osteogenic cells. 133

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Bone marrow stromal cells as a source of osteogenic cells A stem cell is a cell from the embryo, fetus, or adult that, under certain conditions, can reproduce for long periods. It can also give rise to specialized cells of the body tissues and organs. An adult stem cell is an undi¡erentiated (unspecialized) cell that exists in a di¡erentiated tissue, renews itself, and becomes specialized to yield all of the cell types of the tissue from which it originated. Bone marrow stromal cells (BMSCs) are responsible for the maintenance of bone turnover throughout life and can be regarded as a mesenchymal progenitor/ precursor cell population derived from adult stem cells (Beresford 1989, Bianco & Cossu 1999). In the case of mesenchymal tissues, self-renewal is less crucial than cell plasticity and phenotypic £exibility (Bianco & Gehron Robey 2000). Commitment of cells of mesenchymal origin is reversible, probably in response to environmental cues, and the same cells can interconvert between di¡erent cell types at a later stage than that of the multipotential stem cell (Bennett et al 1991, Beresford et al 1992, Park et al 1999). These properties appear necessary to permit growth and remodelling of connective tissues in the living organism. Since BMSCs are easily isolated from the patient’s bone marrow and expanded in vitro, they represent a promising source of multipotent mesenchymal cells for tissue engineering and clinical applications. Cultured BMSCs can di¡erentiate into di¡erent mesenchymal lineages such as osteoblasts, chondrocytes, adipocytes and myocytes and, under certain conditions, also into cell types derived from di¡erent germal layers. The colony-forming units ¢broblastic (CFU-f) derived from bone marrow cells, have been shown to clonally undergo osteogenic, chondrogenic and adipogenic di¡erentiation (Muraglia et al 2000, Pittenger et al 1999). We have observed a sequential loss of lineage potential in tripotent cloned cell populations, thus suggesting a model of predetermined BMSC di¡erentiation (Table 1) (Muraglia et al 2000). According to the model of di¡erentiation we have proposed, the osteogenic pathway is the default lineage of this population (Fig. 1) (Ban¢ et al 2002). In order to maintain a tissue or a function for a lifetime, cells should have a high regenerative potential. However, the stromal microenvironment can be damaged by circumstances such as high-dose chemo/radiotherapy in bone marrow transplant recipients (Galotto et al 1999) (Fig. 2). Extensive in vitro proliferation a¡ects BMSC di¡erentiation capability and replicative potential (Ban¢ et al 2000, Bruder et al 1997). Fibroblast growth factor (FGF)2 can promote the proliferation of cultured BMSCs maintaining their osteogenic potential, possibly by keeping cells in a more immature state (Martin et al 1997). This FGF2 e¡ect is also observed in BMSC culture supplemented with dexamethasone, a hormone known to induce an osteogenic phenotype in BMSCs (Cheng et al 1994, Peter et al 1998).

TISSUE ENGINEERING OF BONE

TABLE 1

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Analysis of human BMSC clones

Phenotypes

Clones

Osteogenic

Chondrogenic

Adipogenic

Frequency

%

+ + + + ^ ^ ^ ^

+ + ^ ^ + + ^ ^

+ ^ ^ + + ^ + ^

45 132 7 0 0 0 0 1

24 71.5 4 0 0 0 0 0.5

To evaluate their di¡erentiation potential, we stimulated 185 BMSC clones to di¡erentiate into osteogenic, chondrogenic and adipogenic lineages under the appropriate conditions. Osteogenic and chondrogenic di¡erentiation were revealed by immunostaining respectively with antibodies against osteocalcin and type II collagen. Adipogenic di¡erentiation was revealed by sudan black staining. Most of the analysed clones were able to undergo both osteogenesis and chondrogenesis. A signi¢cant percentage of clones were able to di¡erentiate along all three induced lineages, and a small percentage were only osteogenic. Associations of phenotypes such as chondro-adipogenic or osteo-adipogenic, and pure phenotypes such as chondrogenic or adipogenic were never observed.

Indeed FGF2 increases the colony size, but a¡ects the colony number (decreased by about 30%) with respect to the same BMSC primary culture, plated in the absence of the factor (Martin et al 1997). This might be due either to an impaired adhesion or to apoptosis, induced by the factor in a subset of cells, or to both. In all cases, in vitro, BMSCs undergo limited mitotic divisions. Despite the sensitivity of the PCR-based TRAP (telomeric repeat ampli¢cation protocol) assay employed, no telomerase activity was detected in the expanding BMSC population (Ban¢ et al 2002). No conclusion can be inferred from this result about the in vivo behaviour of bone marrow mesenchymal progenitors. Instead, it suggests that BMSCs do not have a way to prevent telomere erosion during expansion under current culture conditions, despite being cultured in the presence of FGF2. We have therefore investigated the telomere kinetics of long-term cultured BMSCs together with the extent of expansion which they can undergo before the occurrence of growth arrest due to replicative aging. As expected for normal cells that do not display telomerase activity, BMSC telomeres shortened in culture (Ban¢ et al 2002). However, by comparing each individual culture grown either with or without FGF2, we have observed that a population of cells that displays longer telomeres is selected in our cultures, when cells are plated and maintained in

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FIG. 1. Alkaline phosphatase (ALP) expression by cultured human BMSCs. In the presence of FGF2 a low frequency of ALP-positive BMSC (darker cells) is detected (A). Frequency of ALP-positive BMSC is signi¢cantly increased by the addition of dexamethasone in the culture medium (B). (C) ALP produced by the cells and measured as enzymatic activity.

the presence of FGF2 (G. Bianchi, A. Ban¢, M. Mastrogiacomo, R. Notaro, L. Luzatto, R. Cancedda, R. Quarto, unpublished data). Tissue engineering of bone: preclinical and clinical studies BoneformationcanbeassessedinsmallanimalsbyimplantingBMSCscombinedwith mineralized 3D sca¡olds subcutaneously in immunode¢cient mice (Fig. 3). Cells derived from di¡erent animal species have been used (Goshima et al 1991, Kadiyala et al 1997, Krebsbach et al 1997, Muraglia et al 1998). BMSCs obtained from rat, quail, transgenic mice, human or dog form a primary highly vascularized bone tissue within theporesofthebioceramics.Osteoprogenitorcellstransplantedintoceramicsca¡olds were also used to create vascularized bone £aps (Casabona et al 1998). Current therapeutic approaches for repairing large bone defects can be divided into two groups, one excluding (Ilizarov bone transport) and the other one including graft transplant (autologous, homologous or heterologous bone grafts). Osteotomy followed by bone distraction, the Ilizarov technique, is based on the innate regenerative potential of bone, thus avoiding all problems related to graft integration, but it is highly inconvenient for the patient. The relatively high rate of


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FIG. 2. CFU-f frequency in male patients treated with bone marrow transplant (BMT) vs. age. Marrow samples were obtained from normal donors and from patients treated with BMT for di¡erent myelo-lymphoproliferative diseases. A ¢xed volume of total bone marrow was plated, colony number determined and relative levels (¢lled circles) compared to normal donor values (empty circles). Patient CFU-f frequencies were signi¢cantly lower than those found in matching age/gender normal donors (P50.05)

success obtained by this technique is counterbalanced by the long recovery time and the high rate of complications. Autologous bone implants are used as either non-vascularized or vascularized grafts. Vascularized grafts are the most widely used, being considered as the most successful. The success rate is high, but complications, such as infections and nonunions, are frequent, especially in large shaft reconstructions. Furthermore, large reconstructions by autologous bone require large harvest of healthy tissue with important donor site morbidity. Experiments with animal models have been performed to test the tissue engineering approach for bone repair in a situation similar to the ones occurring in the clinical practice. We have used BMSC/bioceramic composites to treat full-thickness gaps of tibial diaphysis in adult sheep (Kon et al 2000). Autologous cells isolated from bone

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FIG. 3. Implants of di¡erent sca¡olds loaded with cells in immunode¢cient mouse. Four di¡erent biomaterials were used to deliver human BMSCs in vivo: cross-linked collagen sponge, polyglycolate/polylactate (PGA/PLA), cross-linked collagen sponge with bioceramic particles (HA/TCP) and hydroxyapatite sponge (HA). Implants were harvested 8 weeks after the transplant, formalin ¢xed and processed for histological analysis. Only loose connective tissue and no bone formation were observed on collagen sponge and PGA/PLA. When either bioceramic particles/collagen sca¡old or hydroxyapatite sponges were used to deliver BMSC, a signi¢cant bone matrix deposition was evident.

marrow and expanded in vitro were loaded onto highly porous ceramic cylinders (100% hydroxyapatite; 70^80% porosity; pore size distribution: 510 mm 3% vol.; 10^150 mm 11% vol.; 4150 mm 86% vol) and implanted in critical-sized segmental defects in the tibia of the animals. External ¢xation was used to stabilize the grafts. Gross morphology, X-ray, histology, microradiography and SEM studies showed complete integration of ceramic with the bone and good functional recovery. We have also reported the existence of complementary integration and disintegration mechanisms within hydroxyapatite (HA) ceramic implants used to replace the critical-sized segmental defects (Fig. 4). The healing process involves four main steps: (1) bone formation on the outer surface of the implant; (2) bone formation in the inner cylinder canal; (3) formation


139

FIG. 4 Bone-implant integration in the sheep model. (A) Evidence of bone growth within the macroporous sca¡old. (B^C) Newly formed bone was found to ¢ll the system of internal ¢ssures of the HAC sca¡old. (D) Integration of newly formed bone with the HA smallest particles. In collaboration with Professor Paolo Bianco (Rome, Italy) and Professor Alan Boyde (London, UK) (Boyde et al 1999).

of ¢ssures and cracks in the implant body; (4) bone formation in the bioceramic pores. Radiographically and tomographically bone formation was far more prominent over the external surface and within the inner canal of the implants. This might be due to a higher density of loaded cells and/or to a better survival of cells within the outermost portions of the HA bioceramics. Alternatively the implanted cells could stimulate, via a paracrine loop, resident osteoprogenitor cells, located within the skeletal tissues at the resection ends. Similar results were also obtained by Petite et al (2000) by implanting a combination of a coral sca¡old with cultured marrow stromal cells in a large segmental defect in sheep tibiae and by Bruder and colleagues who have treated an experimentally induced non-union defect in adult dog femora with autologous cells loaded on a hydroxyapatite: b-tricalcium phosphate (65:35) sca¡old (Bruder et al 1998, Kadiyala et al 1997). Given these results, similar composites, whose size and shape re£ected each bone defect, were implanted at the lesion sites of few patients for whom a therapeutical alternative was very di⁄cult (Fig. 5) (Quarto et al 2001). External ¢xation was used also in these cases. No major complications occurred in the early or late postoperative period. No evidence of pain, swelling and infection were observed at the implantation site. Callus formation was radiographically evident at the interface between the host bone and the HA cylinder after one month. At the moment a more complete follow-up is available for three patients who have been followed for more than three years. Peri-implant bone formation was still undetectable after one month, but it became detectable during the following months. Consolidation between the

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FIG. 5 Bone repair: clinical study. Repair of a large bone defect in the humerus of a 22 year old patient by autologous bone marrow stromal cells. (A) Films obtained before surgery. (B^C) X-ray and CT post operative control view 18 months after surgery. In collaboration with Professor Maurilio Marcacci and Dr Elisaveta Kon (Bologna, Italy) (Quarto et al 2001).

implant and the host bone was considered completed 5 to 6 months after surgery. A full functional recovery of the treated limb occurred within 6 to 7 months after surgery. Regardless of the low number of patients so far treated, we consider the results obtained very promising. Using a traditional approach, the expected recovery time would have been much longer, under the most favorable conditions and in the absence of complications. We propose the use of culture-expanded osteoprogenitor cells in conjunction with HA bioceramics as a real and signi¢cant improvement in the repair of critical-size long bone defects. Treatment of bone genetic disorders by systemic injection of osteogenic cells In principle, transplantation of mesenchymal progenitor cells either from a healthy donor or genetically modi¢ed would attenuate or possibly correct genetic disorders of bone. Allogeneic bone marrow transplantation was performed in three children with osteogenesis imperfecta. Three months after transplantation, all patients increased total body bone mineral content and new dense bone formation was observed in trabecular bone (Horwitz et al 1999, 2001). Horwitz et al have suggested that the improvement of the clinical conditions was due to engraftment of functional mesenchymal progenitor cells. The durability of the improvements observed remains under question. The engraftment of mesenchymal stem/early progenitor cells contained in the transplanted bone marrow is critical to ensure successful treatment of the disease. Some animal studies suggest the occurrence of mesenchymal stem/progenitor cell engraftment


141

following bone marrow transplantation, whereas results on patients who underwent bone marrow transplantation for cancer treatment were somewhat discouraging. We propose that only a recent tissue damage, as in the case of tissue injuries or wounds, or a tissue remodelling, as it occurs during fetal development and in some genetic disorders, provide a permissive milieu for the engraftment of adult stem cells. A common set of proteins and growth factors is expressed in tissues where active remodelling is taking place during development and in tissues characterized by an acute phase response due to pathological conditions (Cermelli et al 2000). These molecules may provide the unique microenvironment required for stem cell engraftment. To test the possibility of using BMSCs as a vehicle for gene therapy we have performed some preliminary experiments in immunode¢cient mice. In order to evaluate BMSC engraftment by monitoring haematocrit levels, human BMSCs, transduced with the human erythropoietin gene, were either locally injected with a 3D sca¡old or systemically infused. Subcutaneously transplanted mice underwent a hematocrit increase up to 70% whereas administration via intravenous infusion did not a¡ect the hematocrit levels. The most likely explanation is that the engraftment and survival of BMSCs after transplant strictly depend on the ¢nding of an adequate environment as it occurs when a 3D sca¡old is provided for cell delivery (Daga et al 2002).

Acknowledgements Data in Fig. 4 were obtained in collaboration with Professor Paolo Bianco (Rome, Italy) and Professor Alan Boyde (London, UK). Data in Fig. 5 were obtained in collaboration with Professor Maurilio Marcacci and Dr Elisaveta Kon (Bologna, Italy). Partially supported by the Italian ‘Ministero Salute’ and ‘Ministero Istruzione, Universita' e Ricerca’ (MIUR).

References Ban¢ A, Muraglia A, Dozin B, Mastrogiacomo M, Cancedda R, Quarto R 2000 Proliferation kinetics and di¡erentiation potential of ex vivo expanded human bone marrow stromal cells: Implications for their use in cell therapy. Exp Hematol 28:707^715 Ban¢ A, Bianchi G, Notaro R, Luzzatto L, Cancedda R, Quarto R 2002 Replicative aging and gene expression in long term cultures of human bone marrow stromal cells. Tissue Eng, in press Bennett JH, Joyner CJ, Tri⁄tt JT, Owen ME 1991 Adipocytic cells cultured from marrow have osteogenic potential. J Cell Sci 99:131^139 Beresford JN 1989 Osteogenic stem cells and the stromal system of bone and marrow. Clin Orthop 240:270^280

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Beresford JN, Bennett JH, Devlin C, Leboy PS, Owen ME 1992 Evidence for an inverse relationship between the di¡erentiation of adipocytic and osteogenic cells in rat marrow stromal cell cultures. J Cell Sci 102:341^351 Bianco P, Cossu G 1999 Uno, nessuno e centomila: searching for the identity of mesodermal progenitors. Exp Cell Res 251:257^263 Bianco P, Gehron Robey P 2000 Marrow stromal stem cells. J Clin Invest 105:1663^1668. Boyde A, Corsi A, Quarto R, Cancedda R, Bianco P 1999 Osteoconduction in large macroporous hydroxyapatite ceramic implants: evidence for a complementary integration and disintegration mechanism. Bone 24:579^589 Bruder SP, Jaiswal N, Haynesworth SE 1997 Growth kinetics, self-renewal, and the osteogenic potential of puri¢ed human mesenchymal stem cells during extensive subcultivation and following cryopreservation. J Cell Biochem 64:278^294 Bruder SP, Kraus KH, Goldberg VM, Kadiyala S 1998 The e¡ect of implants loaded with autologous mesenchymal stem cells on the healing of canine segmental bone defects. J Bone Joint Surg Am 80:985^996 Casabona F, Martin I, Muraglia A et al 1998 Prefabricated engineered bone £aps: an experimental model of tissue reconstruction in plastic surgery. Plast Reconstr Surg 101:577^581 Cermelli S, Zerega B, Carlevaro M et al 2000 Extracellular fatty acid binding protein (Ex-FABP) modulation by in£ammatory agents: ‘physiological’ acute phase response in endochondral bone formation. Eur J Cell Biol 79:155^164 Cheng SL, Yang JW, Rifas L, Zhang SF, Avioli LV 1994 Di¡erentiation of human bone marrow osteogenic stromal cells in vitro: induction of the osteoblast phenotype by dexamethasone. Endocrinology 134:277^286 Daga A, Muraglia A, Quarto R, Cancedda R, Corte G 2002 Enhanced engraftment of EPOtransduced human bone marrow stromal cells transplanted in a 3D matrix in nonconditioned NOD/SCID mice. Gene Ther 9:915^921 Galotto M, Berisso G, Del¢no L et al 1999 Stromal damage as consequence of high-dose chemo/ radiotherapy in bone marrow transplant recipients. Exp Hematol 27:1460^1466 Goshima J, Goldberg VM, Caplan AI 1991 The origin of bone formed in composite grafts of porous calcium phosphate ceramic loaded with marrow cells. Clin Orthop 268:274^283 Horwitz EM, Prockop DJ, Fitzpatrick LA et al 1999 Transplantability and therapeutic e¡ects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat Med 5:309^313 Horwitz EM, Prockop DJ, Gordon PL et al 2001 Clinical responses to bone marrow transplantation in children with severe osteogenesis imperfecta. Blood 97:1227^1231 Kadiyala S, Young RG, Thiede MA, Bruder SP 1997 Culture expanded canine mesenchymal stem cells possess osteochondrogenic potential in vivo and in vitro. Cell Transplant 6:125^134 Kon E, Muraglia A, Corsi A et al 2000 Autologous bone marrow stromal cells loaded onto porous hydroxyapatite ceramic accelerate bone repair in critical-size defects of sheep long bones. J Biomed Mater Res 49:328^337 Krebsbach PH, Kuznetsov SA, Satomura K, Emmons RV, Rowe DW, Robey PG 1997 Bone formation in vivo: comparison of osteogenesis by transplanted mouse and human marrow stromal ¢broblasts. Transplantation 63:1059^1069 Martin I, Muraglia A, Campanile G, Cancedda R, Quarto R 1997 Fibroblast growth factor-2 supports ex vivo expansion and maintenance of osteogenic precursors from human bone marrow. Endocrinology 138:4456^4462 Muraglia A, Martin I, Cancedda R, Quarto R 1998 A nude mouse model for human bone formation in unloaded conditions. Bone 22:S131^S134 Muraglia A, Cancedda R, Quarto R 2000 Clonal mesenchymal progenitors from human bone marrow di¡erentiate in vitro according to a hierarchical model. J Cell Sci 113:1161^1166


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Park SR, Ore¡o RO, Tri⁄tt JT 1999 Interconversion potential of cloned human marrow adipocytes in vitro. Bone 24:549^554 Peter SJ, Liang CR, Kim DJ, Widmer MS, Mikos AG 1998 Osteoblastic phenotype of rat marrow stromal cells cultured in the presence of dexamethasone, beta-glycerolphosphate, and L-ascorbic acid. J Cell Biochem 71:55^62 Petite H, Viateau V, Bensaid W et al 2000 Tissue-engineered bone regeneration. Nat Biotechnol 18:959^963 Pittenger MF, Mackay AM, Beck SC et al 1999 Multilineage potential of adult human mesenchymal stem cells. Science 284:143^147 Quarto R, Mastrogiacomo M, Cancedda R et al 2001 Repair of large bone defects with the use of autologous bone marrow stromal cells. N Engl J Med 344:385^386

DISCUSSION Hollander: At the beginning of your paper you asked whether the marrow population was a partially committed progenitor population or consisted of pluripotent stem cells. You sort of answered this. Then at the end you suggested that you need to look for a more pluripotent stem cell. In terms of implantation, what point in that line is the optimum time for implanting your cells? More di¡erentiated, or more pluripotent? Cancedda: We prefer to implant as soon as possible. We prefer to have as few passages as possible in vitro. Keep in mind that just the expansion of the population to have enough cells takes three or four weeks. Starting from a normal bone marrow aspirate it is almost impossible to have enough cells in a shorter time. Although there is some commitment of the cells for osteogenic di¡erentiation, the cells still have the capability to di¡erentiate into other types if the environment is changed in vitro or in vivo. Hardingham: You were extolling the virtues of FGF2 in expanding the cells. Is that against a serum background? Cancedda: In this particular case, we added FGF2 in addition to the serum. We have also developed a serum-free medium that still contains FGF2. Again, in this case the addition of FGF2 is also very important. Hardingham: So it is e¡ective in the presence of other growth factors. Lindahl: You had your bone marrow cell limited to the mesenchymal tissue. Is this just a matter of conditions? Are there other cells within the bone marrow of broader potential? Cancedda: This is a good question. In the bone marrow there are probably cells that have the capability to di¡erentiate into lineages other than mesenchymal. I can’t say what the percentages of these cells are. What I am trying to say is that the cells we are working with have been taken from the bone marrow and selected just by their adherence. Then they are expanded in certain culture conditions not superimposable to the conditions in the stroma microenvironment. This cell

144

DISCUSSION

population is certainly pluripotent. The behaviour of the same cells in the stroma microenvironment is something that should be investigated. Barry: You described an experiment in which you transduced the cells to express erythropoietin. You indicated very nice results where you saw haematopoietic recovery. But your results suggested that when the transduced cells are implanted on a matrix, you get a better result. Cancedda: Intravenously injected cells had no e¡ect. Barry: Is this because cells in the matrix are protected from being rapidly cleared? Cancedda: We don’t know. The most likely explanations are that it is either because of increased vascularization or the cells are better protected by the matrix. Caplan: We have taken puri¢ed marrow MSCs, labelled them with indium oxide and injected them. This labels their membranes, and it is possible to see where the cells are going using a gamma camera available in the clinic. They go quantitatively to the lung and liver. It is only subsequently that a small proportion eventually get back to the bone marrow. Estimates we have are that at best 1% get back to the marrow by this route. When we use vasodilators to help them through the lung and liver, we get this ¢gure up to 2^3%. The word used, of ‘homing’ back to the bone marrow, is a loose term. Cancedda: I think this is important. If you think of the data on bone marrow transplantation, no one is really sure that there is an engraftment of stromal cells from the donor and also from MSCs expanded in vivo. Trippel: Later on in your paper you raised a point that may have been insu⁄ciently addressed so far this meeting. This is the issue of ¢xation. If we are going to create tissue-engineered constructs and then put them to use in clinical applications, they have to be ¢xed. This was touched on during articular cartilage repair discussions, including some of the di⁄culties that can be encountered in trying to get constructs to remain in place. You pointed out that in order to get your constructs to work in vivo, the bones surrounding the constructs must be well stabilized. I don’t have the answer as to what is the best means of ¢xation. I was wondering whether anyone else did? Cancedda: Our experience with large animals and human patients is with external ¢xation. We were collaborating with orthopaedic surgeons who were mostly using a standard ¢xation. We have done this just because this was the standard in the clinic we collaborated with. Then, looking in the literature, we ¢gured that the problem with ¢xation was more critical than what we were expecting at the beginning. In particular, I think one should compare external ¢xation with ¢xation that does not allows some loading. If you use just one external ¢xation there is some possibility of loading of the bone. We think this is very important, and we are just now performing a clinical trial with sheep, comparing external ¢xation versus plates. It is too early to comment on the results.


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Lohmander: I would stick with external ¢xation. Cancedda: This is our feeling, but we want data. Goldstein: The important point is the physical and mechanical environment. We shouldn’t think that there is just one way to design these tissueengineering constructs. It is very important to consider: once you have a concept of what your tissue-engineered construct is going to look like, then managing its biological and physical environment in vivo is going to be an important part of your design. This includes the ¢xation. We have already discussed whether cells are needed for bone tissue engineering, or whether we can get the same result by delivering growth factors. Are these just alternative approaches, or do people believe that there is a critical need for cells in addition to growth factors? Cancedda: This has already been discussed in depth. At the moment the choice is probably largely down to the traditions of the group in question. Eventually, a clinical trial comparing the two approaches should be performed. For the time being, the group that has worked with bone marrow probably hasn’t got experience working with cells, and vice versa. I’m sure each group believes their approach has some advantage. van Blitterswijk: It will be a di⁄cult comparison because the sca¡olds won’t be the same. I’d argue that for BMP you need a completely di¡erent sca¡old, so it would be comparing di¡erent entities. What we will probably see is trials comparing cultured or tissue engineered cells with autografts and allografts, and trials comparing BMPs versus auto- and allograft. From this we will have to distil which works best. Caplan: I don’t want us to get stuck on this issue. John Wozney is very eloquent in his articulation of his experience based on clinical trials. An extensive database is going to be released from this. But the number of individuals who will be treated by autologous cell-based therapy is minuscule compared with the database that will be available for growth factor-related clinical trials. We should be cautious in making judgements about whether one approach is better or worse, because in the next ¢ve years there won’t be comparable numbers of patients treated by these two techniques. It is unfair in reality to the people doing autologous cell clinical trials, because they are at a distinct numbers disadvantage, not to mention the ¢nancial aspect needed to drive those trials. Without making a judgement about which is better or worse, those are facts. It will take a longer time to evaluate cell-based therapy for these practical issues. Jill Helms mentioned that osteogenesis was vascular driven. In patients implanted with sca¡olds loaded with cells, there will be always areas of the implant which, no matter how conscientious you are about introducing cells into the full depth of the implant, will be a millimetre or more from the closest vascular supply. They will have a hard time surviving. Are there other strategies

146

DISCUSSION

that one should employ to increase the vascularization, or provide growth factors and cytokines that can increase the survival of the cells furthest from the vascular supply? Cancedda: One can consider supplementing the sca¡old not only with growth factors but also angiogenic factors. We are just doing some experiments involving the use of modi¢ed implants that are ceramic particles combined with di¡erent types of extracellular matrix molecules. In this case the microenvironment might be better for vascular ingress. This is preliminary work, though. Caplan: I have a derivative question. From embryology we know that vascular progenitor cells are dispersed in mesenchymal tissues and later coalesce into capillaries. A potential approach to enhance vascular formation might be to seed these kinds of materials with vascular progenitor cells in combination with mesenchymal progenitors. In the end, it is these two tissues together that are the components important for bone formation. Cancedda: I’m not sure that this mechanism can also occur in adult tissues. The alternative way is that the new vessel is dividing from the ingrowth of a preexisting vessel. This should be investigated. Helms: In the adult it is by both angiogenesis and recruitment of angioblasts. van Blitterswijk: Most of us would agree that vascularization is crucial for success in tissue engineering. But let’s try to challenge this idea. All people working with regular tissue culture or bioreactors realize how tremendously di⁄cult it is in culture to get nutrients to the cells in the centre of a sca¡old. There we don’t have any vasculature to assist us. Now we implant a sca¡old that has already made tissue under these unfavourable conditions into a patient, where I would argue that the conditions are much better. But suddenly we need vascularization. Is there a logic in this? Caplan: The logic is as follows. We know when we implant blocks that there is a dimension requirement. van Blitterswijk: Ranieri Cancedda has just demonstrated implantation in a patient with a segmental defect several centimetres long. How big can we go? Helms: I don’t doubt that there is bone at the surface, but I doubt very much whether there are osteoblasts in the central region. Lindahl: Do you think that within these long constructs the bone grows over hypertrophic chondrocytes? Or is the transition always directly to bone? Perhaps another approach would be to have a construct callus to put into the patient, to help the vascularization. Cancedda: It is possible. In this patient there didn’t seem to be much callus formation. Lindahl: If you take the MSCs through the chondrogenic lineage and implant them into the ceramic sca¡old, would this be better? It might induce vascularization quicker.


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Cancedda: I can’t answer that. Hypertrophic cartilage should promote vascularization. Caplan: To my knowledge no one has taken porous implants, loaded them with cells, driven those cells into a chondrogenic phenotype, and implanted them in an orthotopic site. We have done this ectopically, but never orthotopically.

Studying the e¡ect of di¡erent macrostructures on in vitro cell behaviour and in vivo bone formation using a tissue engineering approach R. J. Dekker*{, C. A. van Blitterswijk1*{, I. Ho£and{, P. J. Engelberts{, J. Li{ and J. D. de Bruijn{{ *iBME, University of Twente, {Biomaterials Research Group, University of Leiden, and {IsoTis BV, Professor Bronkhorstlaan 10, 3723MB Bilthoven, The Netherlands

Abstract. In the present study, we tested the in vitro process of di¡erentiation and mineralization as well as the process of in vivo bone formation on substrates with di¡erent macrostructures. We used carbonated apatite-coated titanium discs that were respectively smooth, plasma sprayed with titanium or had a porous structure. Subcultured rat bone marrow cells were seeded on the substrates and after 7 days of culture, the tissue-coated substrates were subcutaneously implanted in nude mice for 4 weeks. After 1 week of culture in the presence of the osteogenic di¡erentiation promoter dexamethasone, the cells had formed a continuous layer of mineralized tissue on the smooth and titanium plasma-sprayed discs. In the case of the porous titanium discs, the bone-like tissue coverage was restricted to the outer surface and the peripheral pores. The in£uence of the macrostructure on the process of di¡erentiation of the cultured cells depended on the presence of dexamethasone. When dexamethasone was present, the highest ALP/DNA ratios were obtained with the smooth surfaces. In the absence of dexamethasone, the highest ALP/DNA values were obtained with the rough macrostructured discs. We postulate that these di¡erent patterns were due to the shielding of cells in pits or pores of rough structured substrates by dense overlying cell layers. These cell layers are suggested to increase the exposure of excreted osteoinductive proteins and decrease the exposure of dexamethasone to underlying cells. Four weeks postimplantation, abundant bone formation could be observed on all in vitro tissue-coated substrates. The percentage of direct bone contact on the porous discs (42.3 22.3) was signi¢cantly lower compared to the non-porous discs. This was related to the process of bone in¢ltration into the central oriented pores that predominantly occurred in a centrifugal manner. The percentage of direct bone contact on the smooth discs (96.3 2.3) was signi¢cantly higher compared to the titanium plasma-sprayed discs (81.5 10.7). This was not due to ¢brous tissue in¢ltration, but due to the extensive formation of bone marrow. Nevertheless, for practical reasons regarding protection of

1This

paper was presented at the symposium by Clemens van Blitterswijk to whom correspondence should be addressed. 148

THE EFFECT OF DIFFERENT MACROSTRUCTURES

149

the layer of cultured cells during the implantation procedure, the use of rough or porous surface structures is suspected to be advantageous in revision surgery. 2003 Tissue engineering of cartilage and bone. Wiley, Chichester (Novartis Foundation Symposium 249) p 148^169

The number of revision hip arthroplasties in orthopaedic surgery is increasing because of still rising numbers of primary arthroplasties. However, the success rates of revisions are unsatisfactory due to the inferior quality of periprosthetic bone. Schmale et al (2000) reported a failure rate of 23% using cemented hips after a mean follow-up of 4 years. It is furthermore generally agreed that the failure rates in young and active patients are even higher. Stromberg et al (1994) reported a failure rate of 35% for the cup and 39% for the stem of cemented revisions in patients younger than 55 years, after 10 years’ implantation. The use of osteoinductive revision implants in combination with osteoinductive bone ¢ller materials would o¡er great potential in revision surgery. These implants could actively restore the periprosthetic bone loss. It has been postulated that osteoinductivity can be obtained by coating an implant surface with a layer of autologous living bone tissue. In literature, many investigators have reported the osteoinductive capacity of either cell-coated (Muraglia et al 1998, Haynesworth et al 1992, Goshima et al 1991, Krebsbach et al 1997, Kuznetsov et al 1997) or tissuecoated (de Bruijn et al 1999, Yoshikawa et al 2000, Ishaug-Riley et al 1997, Mendes et al 1998) macroporous bone ¢ller materials at ectopic sites. Some even showed the ability of this cell-based approach to accelerate repair in bone defects (Kon et al 2000, Petite et al 2000, Takushima et al 1998, Bruder et al 1998, Kadiyala et al 1997). However, little is published about bone tissue engineering on non-porous substrates (Dekker et al 1998). In order to optimize this technique for application on prosthetic implants, the ¢rst requisite is an implant with optimal surface characteristics for this cell-based approach. It is known that besides the chemical composition (Teti et al 1991, de Ruijter et al 2001, Knabe et al 1997) and the crystallinity of calcium phosphate (Ca-P) substrates (Morgan et al 1996, Chou et al 1999), also the microstructure (Hatano et al 1999, Matsuzaka et al 2000, Lincks et al 1998) and macrostructure (Kuboki et al 1998, Suzuki et al 1997, Hayashi et al 1999) can have a signi¢cant in£uence on both the in vitro cellular response and the process of in vivo bone formation. With respect to the microstructure, it is known that a rougher microstructure stimulates the process of di¡erentiation and mineralization and decreases cell proliferation (Hatano et al 1999, Matsuzaka et al 2000, Lincks et al 1998). However, only a few systematic studies have dealt with the macrostructure by using a cell-culture based approach. Most research on substrate geometry was performed in vivo with

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prosthetic implants that were not coated with a layer of cultured cells. These data revealed that an increased surface topography ultimately increases bone-to-implant contact and increases the biomechanical interlock of the implant with bone (Cooper 2000). Suzuki et al (1997) reported an increased bone volume with higher direct bone^implant contact around implants with a rough plasmasprayed titanium surface as compared to smooth-surfaced titanium surfaces. They suggested that the process of remodelling was less active around the roughsurfaced implants during the early stage after implantation. Nevertheless, to our knowledge the in£uence of the macrostructure on both the in vitro cell behavior and in vivo bone formation by using implants with a layer of cultured bone-like tissue has not been reported. In the present study we compared the process of in vitro di¡erentiation and mineralization and in vivo bone formation on carbonated apatite-coated titanium discs that were respectively smooth, plasma-sprayed with titanium or porous. Cultured rat bone marrow cells were seeded on the substrates and cultured for 1 week in vitro to facilitate extracellular matrix production. The in£uence of the di¡erent macrostructures on the di¡erentiation of the cultured cells was evaluated by determining the alkaline phosphatase (ALP) activity per microgram of DNA in the presence and absence of dexamethasone. We hypothesize that the di¡erentiation pattern of the cells on the di¡erent materials depends on the presence of dexamethasone. Cell layers that seal pores and pits on the porous and rough discs, respectively, can actively isolate underlying cells. This is expected to enhance di¡erentiation in the absence of dexamethasone, due to increasing levels of excreted osteoinductive proteins in the sealed areas. In the presence of dexamethasone these barriers are expected to delay di¡erentiation due to exclusion of dexamethasone to the cells in the pores and pits. In order to evaluate the osteogenic capacity of the tissue-coated discs, we subcutaneously implanted the hybrid constructs in the back of nude mice for 4 weeks. Both the amount of direct bone contact to the di¡erent substrates and the amount of newly formed bone were determined. Materials and methods Three types of titanium (Ti6Al4V) discs with di¡erent macrostructures were used. The discs were respectively smooth (Ra ¼3 mm, 0.5 mm and 1 mm thick), plasma sprayed with Ti6Al4V (Cam implants, Leiden, The Netherlands, Ra ¼20 mm, 0.5 mm and 1 mm thick) or had a porous structure (mean pore size 500 mm, 0.5 mm and 3 mm thick). The porous discs were made by soaking polyurethane foam in titanium slurry, which was made by using Ti6Al4V powder. After removal of the excessive slurry, the sponges were dried and sintered at 1250 8C under vacuum. All substrates were biomimetically coated with a layer of


151

FIG. 1. Scanning electron micrograph of a carbonate apatite-coated smooth (A), Ti plasmasprayed (B) and porous Ti disc (C). Field width 2.5 mm.

carbonated apatite according to Barrere et al (1999) (see Fig. 1). In summary, the titanium discs were soaked in a simulated body £uid for 24 h. Additionally, the discs were immersed in a Ca-P saturated solution for 24 h. This resulted in a 70% crystalline coating of approximately 40 mm in thickness. All discs were sterilized by autoclaving for 20 min at 121 8C and placed in 25-well bacteriological culture plates. Cell isolation and culture Bone marrow cells were obtained from 10 femora of 100^120 g young adult male Wistar rats according to the method described previously (Mendes et al 1998). Brie£y, the femora were excised and washed in a-minimal essential medium (a-MEM-RNA/DNA, Gibco) that contained penicillin G (100 units/ml; Gibco) and streptomycin (100 mg/ml; Gibco). The epiphyses were removed and the marrow from each diaphysis was £ushed out with supplemented medium (a-MEM-RNA/DNA, Gibco) with 15% fetal bovine serum (FBS, Gibco), penicillin G (100 units/ml; Gibco), streptomycin (100 mg/ml; Gibco), and freshly-added 10 mM b-glycerophosphate (Gibco) and 0.1 mM L-ascorbic acid-2phosphate (Sigma). The bone marrow of all diaphyses were pooled and split into

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two fractions. One fraction was cultured in fully supplemented medium in the presence of 10 nM dexamethasone (stimulator of osteoblastic di¡erentiation, Sigma) and the other fraction in the absence of dexamethasone. The medium was changed after 24 h and 4 days, respectively, and maintained at 37 8C in a humidi¢ed atmosphere of 95% air and 5% CO2.

Cell seeding of subcultured cells After 7 days of culture the cells were trypsinized with a 0.25% trypsin solution in 1 mM EDTA (Sigma). The cell suspensions were then concentrated by centrifugation at 100 g for 10 minutes and the cell pellet was resuspended in supplemented medium with or without dexamethasone. Droplets of cell suspension were seeded on the Ca-P-coated discs, which were respectively smooth, plasma-sprayed with titanium or porous, at a density of approximately 500 000 cells/cm2 (see Table 1). Whereas the speci¢c surface area of the porous substrates was known, the surface areas of the dense discs were calculated by assuming a totally £at surface. In order to compensate for di¡erences in the seeding density, the excessive amount of cells ensured a total coverage of each substrate. The non-attached cells were washed away after the ¢rst medium refreshing, which resulted in a similar cell loading on each substrate per speci¢c surface area. For alkaline phosphatase analysis, the cells were seeded at a 10 times lower cell density in order to avoid cell clumps that would make the TABLE 1

Overview of the number of samples used in this study In vitro

In vivo

ALP/DNA analysis +/+ Dex / Dex Ca-P-coated 6 smooth discs 6 Ca-P-coated Ti plasma-sprayed discs Ca-P-coated 6 porous Ti discs

+ cells / Dex

cells +/+ Dex

+ cells +/+ Dex

cell +/+ Dex

6

4

4

6

4

6

4

4

6

4

6

4

4

6

4

The signs +/+ Dex, etc., represent the presence or absence of 10 nM dexamethasone during the primary culture and subculture, respectively. The signs + cells and cells indicate whether cells were or were not seeded on the substrates.


153

analysis impossible. After 3 h, 2 ml of supplemented medium with or without dexamethasone was added to each well containing a cell-seeded disc. Respectively 2 and 4 days after seeding the cells on the materials, the medium was refreshed. In order to check the degradation of the coating, 4 Ca-P-coated discs with the various macrostructures and without cultured cells were incubated in supplemented culture medium during the 7 d culture period. ALP/DNA analysis The alkaline phosphatase (ALP) activity per microgram DNA was determined using a colorimetric assay. After 7 days of culture in the presence or absence of dexamethasone, 6 cell-seeded constructs per substrate (see Table 1) were washed with phosphate-bu¡ered saline (PBS; Life Technologies) and stored at 80 8C for at least 24 h. After thawing, the cells were sonicated (Branson 250) in PBS with 0.2% Triton X-100 (Sigma). After adding the substrate p-nitrophenylphosphate (Sigma), the ALP activity was assayed by measuring the amount of released p-nitrophenol. A standard curve was generated using known concentrations of p-nitrophenol (Sigma). All samples and standards were measured on a BIO-TEK automated microplate reader (New York, USA) at 405 nm. To measure the DNA contents, we used a cyquant assay Kit (Molecular Probes) and a LS-50B £uorimeter (Perkin Elmer, Beacons¢eld, UK). The ALP activity was presented as micromoles p-nitrophenol per microgram DNA per minute. Implantation procedure Cell-seeded Ca-P-coated discs that were either smooth, plasma-sprayed with titanium or porous (n ¼6) were implanted in the backs of approximately 12 week old nude mice (Balb/cOla Hsd-nu mice; approximately 25 g) after 7 days of culture. Materials without cultured cells were also implanted after 7 days incubation in supplemented medium (see Table 1). Prior to implantation, the samples were soaked in serum-free culture medium and in PBS. The mice (n ¼8) were anaesthetized by an intramuscular injection of 0.04 ml of a ketamine (46.7 mg/ml), xylazine (8 mg/ml) and atropine (67 mg/ml) mix. The surgical sites were cleaned with 70% ethanol and 2 subcutaneous pockets were created on each lateral site of the spine. Each sample was inserted in a pocket, whereby each mouse contained the various types of substrates. Light microscopy Two samples of each substrate with or without cultured cells were ¢xed in 1.5% glutaraldehyde in 0.14 M cacodylate bu¡er after 7 days of culture. Furthermore, the

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in vivo samples that were implanted for 4 weeks were ¢xed after explantation and all samples were dehydrated through a graded series of ethanol. Subsequently, the samples were embedded in methyl methacrylate (MMA; Merck) and sectioned by using a Leica SP 1600 Saw Microtome (Nussloch, Germany). The undecalci¢ed sections were stained with methylene blue (Sigma) and basic fuchsine (Sigma) and examined with light microscopy. Histomorphometrical analysis All implanted tissue-coated discs were evaluated with histomorphometrical analysis. Histological sections through the centre of the samples were used to quantify the percentage of direct bone contact with the discs and the amount of in vivo formed bone and marrow by using a computerized image analysis system (VIDAS). Direct bone contact was calculated as the percentage of the implant surface length that was in direct contact with the bone. The bone and marrow volumes were calculated as the total bone and marrow area in the histological sections in relation to the implant surface length, including the surface length of pits and pores. These latter values equal the average bone and marrow volume in relation to the surface area of the discs. Per substrate we used 1 section of each sample (n ¼6) and calculated the mean values and standard deviations for direct bone contact and bone and marrow volume. Statistical analysis An ANOVA analysis with a Bonferroni correction was used to determine the statistical signi¢cance of the ALP/DNA values and the histomorphometrical values. The signi¢cance was de¢ned as P50.05. Scanning electron microscopy After 7 days of culture, two tissue-coated samples and two control substrates without cultured cells of each substrate were ¢xed in 1.5% glutaraldehyde in 0.14 M cacodylate bu¡er. Subsequently, the samples were dehydrated, critical point dried from carbon dioxide (Balzers model CPD 030 Critical Point Dryer) and sputter coated with a layer of gold (Balzers sputter coater model SCD 004). The samples were examined in a Philips XL 30 FEG environmental scanning electron microscope at an accelerating voltage of 10 kV. Backscatter electron imaging The blocks containing in vivo samples that had initially been prepared for light microscopy were polished with 4000 grit silicon carbide sandpaper (Stuers). The


155

blocks were cleaned with 70% ethanol and then carbon coated in a Bal-Tec CEA035 carbon evaporation supply installation. The samples were viewed in a Philips XL 30 FEG environmental scanning electron microscope at an accelerating voltage of 20 kV in backscatter mode. Results Cell growth and ALP analysis After one week of culture, a layer of cultured cells was present on the entire surface area of the Ca-P-coated discs that were either smooth or plasma sprayed with titanium. In case of the porous titanium discs, the cell ingrowth into the pores was fairly limited and was restricted only to the surface of the porous discs and the pores in the periphery. Light microscopy of cross-sections of porous discs revealed that the layer of cultured cells had bridged the pores, instead of following the surface towards the center of the discs. A similar process could be seen on the titanium plasma-sprayed coated discs, where cells bridged the extending plasma-sprayed projections. This resulted in locally loosely organized cell structures underneath the dense layer of cells that spanned pores and extending areas on porous discs and titanium plasma-sprayed discs, respectively. On all substrates, the cell layers were approximately two cells thick (see Fig. 2). The areas stained by basic fuchsine indicated that the amount of mineralized extracellular matrix was roughly comparable on all di¡erent substrates. Scanning electron microscopy revealed that the cultured cells had a £attened morphology. In between the layer of £attened cells and the substrates, abundant collagen-like extracellular matrix formation could be detected. Deposition of mineral on the excreted matrix resulted in fusion of the ¢bres, which was indicated by the large areas of extensively mineralized matrix (see Fig. 3). The control samples without cultured cells that had been immersed in culture medium did not show any morphological changes during the 1 week culture period. The perpendicularly orientated crystals were still visible on the outer surface of the discs as well as in the pits of the titanium plasma-sprayed discs and the pores of the porous discs. The ALP/DNA analysis of the cells cultured in both the presence and the absence of dexamethasone revealed signi¢cant di¡erences between the ALP expressions per microgram DNA on the di¡erent substrates after 7 days. The cells cultured on the Ca-P-coated smooth discs and the porous titanium discs did not reveal any signi¢cant di¡erences when cultured in the presence of dexamethasone. Nevertheless, both had a signi¢cantly higher speci¢c ALP expression in the presence of dexamethasone as compared to the Ca-P-coated titanium plasma-sprayed discs. The ALP/DNA expression of the cells cultured in the absence of dexamethasone was signi¢cantly higher on the Ca-P-coated porous

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FIG. 2. Light micrograph of respectively a smooth, Ti plasma-sprayed and porous Ti disc, coated with a 70% crystalline biomimetic coating after 1 week of culture. Note the layer of cells on the discs and the abundant mineralized spots between the layer of cultured cells and the discs. Field width 315 mm.

FIG. 3. Scanning electron micrograph of a cell-seeded smooth biomimetic coated disc after 1 week of culture. Note the abundant collagen ¢bres, which are extensively mineralized. Field width 10 mm.


157

FIG. 4. Chart of the ALP activity that was assayed as the release of p-nitrophenol from p-nitrophenylphosphate per microgram DNA per minute. The cells cultured in the presence of dexamethasone on respectively the smooth discs and the porous titanium discs had a signi¢cantly higher speci¢c ALP expression as compared to the titanium plasma-sprayed discs. The ALP/ DNA expression of the cells cultured in the absence of dexamethasone was signi¢cantly higher on the porous discs as compared to the smooth discs. The relative ALP expression on the titanium plasma-sprayed discs was signi¢cantly higher than both the smooth discs and the porous titanium discs.

discs as compared to the Ca-P-coated smooth discs. In contrast to the results obtained in culture medium with dexamethasone, the relative ALP expression on the Ca-P-coated titanium plasma-sprayed discs was signi¢cantly higher than both the Ca-P-coated smooth discs and the porous titanium discs (see Fig. 4). Finally, the ALP/DNA ratios of the cells that were cultured in the presence of dexamethasone were signi¢cantly higher on all substrates as compared to the cells cultured in the absence of dexamethasone. Evaluation of in vivo samples In the control samples without cultured cells, no bone formation was seen after 4 weeks of implantation. Light microscopy showed no visible changes to the biomimetic Ca-P coating on the di¡erent macro-structured substrates after the in vivo period. The histological ¢ndings with the ex vivo tissue-coated discs did reveal de novo bone formation on all implanted samples. The layer of bone fully covered the surface of both the Ca-P-coated smooth and titanium plasma-sprayed discs and could only be detected on the in vitro tissue-coated site of the discs. In the porous Ca-P-coated discs, vascularized ¢brous tissue invasion into the porous regions of the discs could be detected. Clear de novo bone formation was present at the periphery and in the pores of the porous discs. It appeared that the bone had in¢ltrated the central pores from the peripheral pores, which resulted in the presence of bone throughout the whole porous disc. This process of bone in¢ltration did not only proceed via the surface of the substrate, but also proceeded through the middle of the pores (see Figs 5 and 6). This resulted in a lower amount of direct bone contact in the central regions of the porous discs as compared to the more peripheral regions.

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FIG. 5. Light micrograph of respectively a Ca-P-coated smooth (A), Ti plasma-sprayed (B) and porous Ti disc (C, D), exhibiting de novo bone formation, after 4 weeks’ subcutaneous implantation. Note the bone marrow formation in all the presented samples and the process of bone in¢ltration into the pores of the Ti disc. Field width 630 mm (A, B), 1260 mm (C) and 315 mm (D).

The average percentage of direct bone contact on the porous discs was therefore signi¢cantly lower than on both types of non-porous discs. The percentage of direct bone contact on the Ca-P-coated smooth discs was signi¢cantly higher compared to the Ca-P-coated titanium plasma-sprayed discs (Table 2). This could be due to the interference of bone marrow with the contact of the newly formed bone to the Ca-P-coated titanium plasma-sprayed discs (see Fig. 6). The bone marrow formation was more extensive and present on more discs on the titanium plasma-sprayed discs as compared to the Ca-P-coated smooth discs (see Table 2). The impact of the bone marrow interference with respect to the percentage of bone contact was, however, limited, because a thin layer of bone was often present between the surface of the substrates and the cavities ¢lled with bone marrow. The bone marrow formation in the porous samples was the most pronounced and ¢lled large areas of the pores in all samples. Finally, the bone volume on the


159

FIG. 6. Back-scattered electron images of respectively a smooth (A), Ti plasma-sprayed (B) and porous Ti disc (C, D), coated with a 70% crystalline biomimetic coating and a layer of cultured tissue after 4 weeks’ subcutaneous implantation. Although all di¡erent substrates revealed direct bone contact (A, B, C), the amount of direct bone contact with the porous discs was the lowest (D). Note the bone marrow cavity in the layer of bone on the smooth and Ti plasma-sprayed disc. Field width 350 mm (A, B, C) and 2500 mm (D).

di¡erent macro-structured discs did not reveal any signi¢cant di¡erences in relation to the surface area of the discs. Neither could any signi¢cant di¡erences be seen when the volume of the bone marrow was added to the volume of the bone and subsequently related to the surface area (see Table 2). Discussion Surface topography varies widely among commercially available femoral prostheses and is known to in£uence the biological ¢xation (Lauer et al 2001, McAuley et al 1998). Animal models reveal increases in bone-to-implant contact and increases in the biomechanical interlock of the implant with bone for implants of increased surface topography (Cooper 2000). Nevertheless, despite these developments in implant topography, the results obtained in revision hip arthroplasty are still sub-optimal. Bone tissue engineering could provide an additional tool to increase the success rates and survival periods of revision hips.

160

TABLE 2

DEKKER ET AL

Overview of the results obtained in this study

Bone formation ( cells) Ca-P-coated 0/4 smooth discs 0/4 Ca-P-coated Ti plasma-sprayed discs 0/4 Ca-P-coated porous Ti discs

Bone formation (+ cells)

Bone marrow formation

% Direct bone contact

Bone + marrow Bone volume volume (mm3/mm2) (mm3/mm2)

6/6

3/6

96.3 2.3

9.2 1.1

6/6

6/6

81.5 10.7 11.0 2.6

16.2 7.1

6/6

6/6

42.3 22.3 7.6 3.0

20.4 10.7

10.9 3.2

The numbers with regard to bone and bone marrow formation represent the number of samples with de novo bone and bone marrow formation in vivo versus the total number of implanted cell-seeded substrates. Direct bone contact was calculated as the length of newly formed bone that was in direct contact with the surface of the discs as a percentage of the total surface length of the discs. Bone (and marrow) volume was calculated as the total bone (and marrow) volume on the discs in relation to the implant surface. The percentage of direct bone contact on the porous discs was signi¢cantly lower than on the non-porous discs. The percentage of direct bone contact on the smooth discs was signi¢cantly higher compared to the titanium plasma sprayed discs. No signi¢cant di¡erences were obtained with respect to the bone (and marrow) volumes.

The topography of the implant is expected to in£uence the in vitro cell behaviour and the process of in vivo bone formation on tissue-engineered implants. Several reports related to the in£uence of the macrostructure on in vitro cell proliferation and di¡erentiation have been published (Perizzolo et al 2001, Kieswetter et al 1996, Boyan et al 1998, Batzer et al 1998, McCarthy et al 1991, Vehof et al 2000). However, to our knowledge these ¢ndings were never correlated with in vivo outcomes. In the present study, we tested the in vitro process of di¡erentiation and mineralization as well as the process of in vivo bone formation on substrates with di¡erent macrostructures. We used Ca-P-coated titanium discs that were smooth, plasma-sprayed with titanium or had a porous structure. The Ca-P coating was applied to ensure identical microstructures of the surface of all substrates and to promote proper cell attachment in vitro. After 1 week of culture, the cells had formed a continuous layer that covered the total surface area of the Ca-P-coated discs that were either smooth or plasmasprayed with titanium. In the case of the porous titanium discs the cell ingrowth into the pores was fairly limited and was restricted only to the surface of the porous discs and the pores in the periphery. It is therefore di⁄cult to compare the results obtained with the porous discs and the results obtained with the non-porous discs with regard to cell proliferation and cell coverage. Nevertheless, the cell layers that


161

could be detected were approximately 2 cells thick on all substrates after 7 days of culture. It thus appeared that the cells had not proliferated extensively, since a multi-layer of cells was already seeded onto the samples. It can be concluded that the di¡erent macrostructures did not result in di¡erences in cell coverage after 7 days of culture, with the exception of the central oriented pores of the porous discs. Lauer et al (2001) reported that cultured human maxillar osteoblasts had identical growth patterns on polished, sandblasted and plasma-sprayed titanium surfaces. However, this was not in accordance with other in vitro ¢ndings, in which a decrease in proliferation was observed with increasing macrostructures (Batzer et al 1998). After 7 days of culture we could detect di¡erences in the amount of ALP expression per DNA content. When the cells were cultured in the presence of dexamethasone, a higher average ALP/DNA value was obtained with the Ca-Pcoated smooth discs as compared to the Ca-P-coated porous discs. Furthermore, they both had a signi¢cantly higher speci¢c ALP/DNA expression than the Ca-Pcoated titanium plasma-sprayed discs. The opposite pattern was observed when the cells were cultured in the absence of dexamethasone. The ALP/DNA expression was signi¢cantly higher on the titanium plasma-sprayed discs compared to the porous discs, which was again signi¢cantly higher than the ALP/DNA expression obtained with the smooth discs. We hypothesize that these patterns are the result of an increased supply of dexamethasone and a decreased disposal of excreted stimulatory factors for di¡erentiation. When the cells are cultured in the presence of dexamethasone, the most signi¢cant factor for di¡erentiation is expected to be the e¡ect of dexamethasone. This is con¢rmed by our results, because cells cultured in the absence of dexamethasone always exhibited signi¢cantly lower ALP/DNA values than the cells cultured in the presence of dexamethasone. When dexamethasone is easily accessible for all cells, the average ALP/DNA values can therefore expected to be high. However, we hypothesize that the cells present in the peripheral pores of the porous discs and in the coves on the titanium plasma-sprayed discs are partly excluded from the stimulatory e¡ect of dexamethasone, due to the barrier e¡ect of the layer of cultured cells above. This results in the creation of a microenvironment in the peripheral pores and the coves, in which cells experience a lower concentration of dexamethasone as compared to the cells cultured on the Ca-Pcoated smooth discs. The creation of this microenvironment is, however, also believed to be the reason for the faster di¡erentiation of the cultured cells in the absence of dexamethasone. In the absence of dexamethasone, the expected signi¢cant factors for di¡erentiation are the autocrine and paracrine factors that are excreted by the cells themselves. In the created microenvironments, the elevated concentrations of excreted factors are suspected to increase the stimulatory e¡ect on the process of di¡erentiation. This theory also indicates,

162

DEKKER ET AL

that the smaller microenvironments created on the Ca-P-coated titanium plasmasprayed discs are more e¡ective regarding di¡erentiation compared to the larger sealed areas in the porous discs. Perizzolo et al (2001) also suggested the stimulatory e¡ect of a microenvironment on the process of osteogenic di¡erentiation. They showed that the process of di¡erentiation and mineralization were increased on grooved substrates compared to smooth surfaces and that the e¡ects depended on the dimensions of the grooves. They hypothesized that grooved substrata might limit free exchange of ions released from the Ca-P surface, as well as cell secretions with the bulk culture medium that could favour di¡erentiation and mineralization. Furthermore, not only a decreased disposal, but also an increased secretion of autocrine and paracrine mediators in response to the macrostructure, such as prostaglandin E2 (PGE2), could be an explanation for the enhanced ALP expression. Boyan and colleagues showed that an increase in macrostructure resulted in an increased production of PGE2 (Kieswetter et al 1996, Boyan et al 1998, Batzer et al 1998). The actions of PGE2 may stimulate the production of cytokines, such as insulin-like growth factors, which are associated with cellular di¡erentiation (McCarthy et al 1991). This indicates that surface roughness has a direct e¡ect on cell activity and may have an indirect e¡ect mediated through local factor production. Groessner-Schreiber & Tuan (1992) also demonstrated the enhanced expression of a di¡erentiated phenotype in response to the macrostructure. They showed that the synthesis of extracellular matrix and subsequent mineralization of chick embryonic calvarial osteoblast cultures were both enhanced on rough-textured and porous-coated titanium compared to smooth titanium surfaces. Four weeks after implantation, all ex vivo tissue-coated substrates exhibited de novo bone formation. The layer of bone covered the total surface of both the CaP-coated smooth and titanium plasma-sprayed discs. In the porous discs, the bone was present in both the peripheral pores and the central pores, although the percentage of bone contact and bone volume was visibly lower in the centrally orientated pores. This was most likely due to the fact that cultured osteogenic cells with excreted extracellular matrix were absent in the central pores at the moment of implantation. It seemed that the bone formation in the peripheral pores had started at the ex vivo tissue-coated implant surface and proceeded away in a so-called centripetal direction. The bone in¢ltration towards the central orientated pores occurred by two types of bone formation. The bone migrated both via the surface of the substrate and within the three-dimensional soft biological matrix. The bone formation via the surface was due to osteoconduction and resulted in bonding osteogenesis as indicated by the direct bone contact with the substrate. However, the process of bone formation through the lumen of the pores proceeded in a centrifugal manner, as indicated by the cone


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shape of the in¢ltrating bone. This bone formation towards the implant surface was not always completed in the central pores and resulted in a lower percentage of direct bone contact as compared to the peripheral pores. Vehof et al (2000) also observed this predominant bone deposition in a centrifugal manner, by implanting cell loaded porous Ca-P-coated titanium ¢bre mesh substrates and injecting £uorochrome markers at di¡erent time points. The areas with low direct bone contact in the porous discs resulted in an overall signi¢cantly lower percentage of direct bone contact compared to the non-porous discs. There was also a di¡erence in the percentage of direct bone contact between the smooth discs and titanium plasma-sprayed discs. The percentage of direct bone contact on the smooth discs was signi¢cantly higher compared to the titanium plasma sprayed discs. This is not in accordance with most reports about direct bone contact around implants with di¡erent surface structures. Several investigators (Suzuki et al 1997, Hayashi et al 1999, Cooper 2000, Ericsson et al 1994, Feighan et al 1995) concluded that the percentage of direct bone contact is positively correlated with the increasing roughness of the implant surface. These implants were however not coated with a layer of cultured tissue and were implanted in a bony environment. The samples were thus completely dependent on the attraction, attachment and di¡erentiation of osteogenic cells on the surface of the implants. An explanation for an enhanced bone contact on rough structured implants might be that an enhanced surface area leads to greater binding of attachment proteins and regulatory factors. El-Ghannam et al (1999) showed that adsorption of serum ¢bronectin to the surface of calcium phosphate results in an enhanced osteoblast adhesion, whereas Bowers et al (1992) showed that signi¢cantly higher levels of cell attachment of osteoblast-like cells were found on rough surfaces rather than smooth surfaces. Moreover, other biologically active molecules present in solution, such as bone morphogenetic proteins, may also adsorb at higher quantities on rougher surfaces and a¡ect bone apposition. In our study, however, the osteogenic cells were already present on the surface and did not require the in vivo attachment of proteins. The lower direct bone contact on the rough titanium plasma-sprayed discs versus the smooth discs in our study most probably was due to the increased production of bone marrow on the rough discs. This bone marrow formation interfered with the bone contact on the substrates, although a thin layer of bone was often present between the surface of the substrates and the cavities ¢lled with bone marrow. It should be mentioned that the highest degree of direct bone contact obtained with the smooth implants does not guarantee optimal results in a clinical application. When this tissue-engineered technique would be applied in revision hip surgery, the relatively tough implantation procedure may cause damaging and detachment of the layer of cultured cells. When the layer of cells is partly protected by a rough structure and mainly present within the outer diameter of the prosthesis, the chance of damaging

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the biological coating is much smaller. Furthermore, bone ingrowth rather than bone contact, is expected to be the critical factor for the ¢xation of prosthesis with a rough or porous surface. The volume of the newly formed bone, including or excluding the volume of the formed bone marrow, was not signi¢cantly di¡erent on the di¡erent macrostructured discs. It can, however, be expected that the bone volume per unit surface area would be the highest in the porous discs after a longer implantation period. The process of bone in¢ltration could continue in the central pores and could therefore enhance the total bone volume per unit surface area in time. Nevertheless, the absence of di¡erences in bone volume contrasts with the results of Suzuki et al (1997), who reported an increased bone volume around non tissuecoated implants with a rough plasma-sprayed titanium surface as compared to smooth-surfaced titanium surfaces. They suggested that the process of remodelling was less active around the rough-surfaced implants during the early stage after implantation. This is in contrast with our results, since the higher frequency of bone marrow around the rough and in the porous discs indicates an increased process of remodelling. It is, however, still questionable whether these di¡erent observations are due to the presence of a layer of cultured mineralized bone-like tissue. In conclusion, this study has shown that a rough macrostructure can both enhance and lower the process of in vitro osteogenic di¡erentiation, depending on the presence of di¡erentiation-stimulating agents in the culture medium. Nevertheless, when cultured in the presence of dexamethasone, a layer of mineralized bone-like tissue can be formed on smooth, rough and porous macrostructures. These tissue-coated substrates can, regardless of the macrostructure, initiate bone formation in vivo. For practical reasons connected with the protection of the layer of cultured cells during the implantation procedure, the use of rough or porous surface structures is expected to be advantageous in revision surgery. Acknowledgements The authors would like to acknowledge the European Community Brite-Euram project BE97-4612 and the Dutch Department of Economic A¡airs for ¢nancial support.

References Barrere F, Layrolle P, van Blitterswijk CA, de Groot K 1999 Biomimetic calcium phosphate coatings on Ti6AI4V: a crystal growth study of octacalcium phosphate and inhibition by Mg2+ and HCO3. Bone 25:107S^111S Batzer R, Liu Y, Cochran DL et al 1998 Prostaglandins mediate the e¡ects of titanium surface roughness on MG63 osteoblast-like cells and alter cell responsiveness to 1a,25-(OH)2D3. J Biomed Mater Res 41:489^496


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Bowers KT, Keller JC, Randolph BA, Wick DG, Michaels CM 1992 Optimization of surface micromorphology for enhanced osteoblast responses in vitro. Int J Oral Maxillofac Implants 7:302^310 Boyan BD, Batzer R, Kieswetter K et al 1998 Titanium surface roughness alters responsiveness of MG63 osteoblast-like cells to 1a,25-(OH)2D3. J Biomed Mater Res 39:77^85 Bruder SP, Kraus KH, Goldberg VM, Kadiyala S 1998 The e¡ect of implants loaded with autologous mesenchymal stem cells on the healing of canine segmental bone defects. J Bone Joint Surg Am 80:985^996 Chou L, Marek B, Wagner WR 1999 E¡ects of hydroxylapatite coating crystallinity on biosolubility, cell attachment e⁄ciency and proliferation in vitro. Biomaterials 20:977^985 Cooper LF 2000 A role for surface topography in creating and maintaining bone at titanium endosseous implants. J Prosthet Dent 84:522^534 de Bruijn JD, van den Brink I, Mendes S, Dekker R, Bovell YP, van Blitterswijk CA 1999 Bone induction by implants coated with cultured osteogenic bone marrow cells. Adv Dent Res 13:74^81 de Ruijter JE, ter Brugge PJ, Dieudonne SC, van Vliet SJ, Torensma R, Jansen JA 2001 Analysis of integrin expression in U2OS cells cultured on various calcium phosphate ceramic substrates. Tissue Eng 7:279^289 Dekker RJ, de Bruijn JD, van den Brink I, Bovell YP, Layrolle P, van Blitterswijk CA 1998 Bone tissue engineering on calcium phosphate-coated titanium plates utilizing cultured rat bone marrow cells: a preliminary study. J Mater Sci Mater Med 9:859^863 El-Ghannam A, Ducheyne P, Shapiro IM 1999 E¡ect of serum proteins on osteoblast adhesion to surface-modi¢ed bioactive glass and hydroxyapatite. J Orthop Res 17:340^345 Ericsson I, Johansson CB, Bystedt H, Norton MR 1994 A histomorphometric evaluation of bone-to-implant contact on machine-prepared and roughened titanium dental implants. A pilot study in the dog. Clin Oral Implants Res 5:202^206 Feighan JE, Goldberg VM, Davy D, Parr JA, Stevenson S 1995 The in£uence of surfaceblasting on the incorporation of titanium-alloy implants in a rabbit intramedullary model. J Bone Joint Surg Am 77:1380^1395 Goshima J, Goldberg VM, Caplan AI 1991 Osteogenic potential of culture-expanded rat marrow cells as assayed in vivo with porous calcium phosphate ceramic. Biomaterials 12:253^258 Groessner-Schreiber B, Tuan RS 1992 Enhanced extracellular matrix production and mineralization by osteoblasts cultured on titanium surfaces in vitro. J Cell Sci 101:209^217 Hatano K, Inoue H, Kojo T et al 1999 E¡ect of surface roughness on proliferation and alkaline phosphatase expression of rat calvarial cells cultured on polystyrene. Bone 25: 439^445 Hayashi K, Mashima T, Uenoyama K 1999 The e¡ect of hydroxyapatite coating on bony ingrowth into grooved titanium implants. Biomaterials 20:111^119 Haynesworth SE, Goshima J, Goldberg VM, Caplan AI 1992 Characterization of cells with osteogenic potential from human marrow. Bone 13:81^88 Ishaug-Riley SL, Crane GM, Gurlek A et al 1997 Ectopic bone formation by marrow stromal osteoblast transplantation using poly(DL-lactic-co-glycolic acid) foams implanted into the rat mesentery. J Biomed Mater Res 36:1^8 Kadiyala S, Jaiswal N, Bruder SP 1997 Culture-expanded, bone marrow-derived mesenchymal stem cells can regenerate a critical-sized segmental bone defect. Tissue Eng 3:173^185 Kieswetter K, Schwartz Z, Hummert TW et al 1996 Surface roughness modulates the local production of growth factors and cytokines by osteoblast-like MG-63 cells. J Biomed Mater Res 32:55^63 Knabe C, Gildenhaar R, Berger G et al 1997 Morphological evaluation of osteoblasts cultured on di¡erent calcium phosphate ceramics. Biomaterials 18:1339^1347

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Kon E, Muraglia A, Corsi A et al 2000 Autologous bone marrow stromal cells loaded onto porous hydroxyapatite ceramic accelerate bone repair in critical-size defects of sheep long bones. J Biomed Mater Res 49:328^337 Krebsbach PH, Kuznetsov SA, Satomura K, Emmons RV, Rowe DW, Robey PG 1997 Bone formation in vivo: comparison of osteogenesis by transplanted mouse and human marrow stromal ¢broblasts. Transplantation 63:1059^1069 Kuboki Y, Takita H, Kobayashi D et al 1998 BMP-induced osteogenesis on the surface of hydroxyapatite with geometrically feasible and nonfeasible structures: topology of osteogenesis. J Biomed Mater Res 39:190^199 Kuznetsov SA, Krebsbach PH, Satomura K et al 1997 Single-colony derived strains of human marrow stromal ¢broblasts form bone after transplantation in vivo. J Bone Miner Res 12:1335^1347 Lauer G, Wiedmann-Al-Ahmad M, Otten JE, Hubner U, Schmelzeisen R, Schilli W 2001 The titanium surface texture e¡ects adherence and growth of human gingival keratinocytes and human maxillar osteoblast-like cells in vitro. Biomaterials 22:2799^2809 Lincks J, Boyan BD, Blanchard CR et al 1998 Response of MG63 osteoblast-like cells to titanium and titanium alloy is dependent on surface roughness and composition. Biomaterials 19:2219^2232 Matsuzaka K, Walboomers F, de Ruijter A, Jansen JA 2000 E¡ect of microgrooved poly-l-lactic (PLA) surfaces on proliferation, cytoskeletal organization, and mineralized matrix formation of rat bone marrow cells. Clin Oral Implants Res. 11:325^333 McAuley JP, Culpepper WJ, Engh CA 1998 Total hip arthroplasty. Concerns with extensively porous coated femoral components. Clin Orthop 355:182^188 McCarthy TL, Centrella M, Raisz LG, Canalis E 1991 Prostaglandin E2 stimulates insulin-like growth factor I synthesis in osteoblast-enriched cultures from fetal rat bone. Endocrinology 128:2895^2900 Mendes SC, van den Brink I, de Bruijn JD, van Blitterswijk CA 1998 In vivo bone formation by human bone marrow cells: e¡ect of osteogenic culture supplements and cell densities. J Mater Sci Mater Med 9:855^858 Morgan J, Holtman KR, Keller JC, Stanford CM 1996 In vitro mineralization and implant calcium phosphate-hydroxyapatite crystallinity. Implant Dent 5:264^271 Muraglia A, Martin I, Cancedda R, Quarto R 1998 A nude mouse model for human bone formation in unloaded conditions. Bone 22:131S^134S Perizzolo D, Lace¢eld WR, Brunette DM 2001 Interaction between topography and coating in the formation of bone nodules in culture for hydroxyapatite- and titanium-coated micromachined surfaces. J Biomed Mater Res 56:494^503 Petite H, Viateau V, Bensaid W et al 2000 Tissue-engineered bone regeneration. Nat Biotechnol 18:959^963 Schmale GA, Lachiewicz PF, Kelley SS 2000 Early failure of revision total hip arthroplasty with cemented precoated femoral components: comparison with uncemented components at 2 to 8 years. J Arthroplasty 15:718^729 Stromberg CN, Herberts P 1994 A multicenter 10-year study of cemented revision total hip arthroplasty in patients younger than 55 years old. A follow-up report. J Arthroplasty 9:595^601 Suzuki K, Aoki K, Ohya K 1997 E¡ects of surface roughness of titanium implants on bone remodeling activity of femur in rabbits. Bone 21:507^514 Takushima A, Kitano Y, Harii K 1998 Osteogenic potential of cultured periosteal cells in a distracted bone gap in rabbits. J Surg Res. 78:68^77 Teti A, Tarquilio A, Grano M et al 1991 E¡ects of calcium-phosphate-based materials on proliferation and alkaline phosphatase activity of newborn rat periosteal cells in vitro. J Dent Res 70:997^1001


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Vehof JW, Spauwen PH, Jansen JA 2000 Bone formation in calcium-phosphate-coated titanium mesh. Biomaterials 21:2003^2009 Yoshikawa T, Ohgushi H, Nakajima H et al 2000 In vivo osteogenic durability of cultured bone in porous ceramics: a novel method for autogenous bone graft substitution. Transplantation 69:128^134

DISCUSSION Hunziker: I don’t fully understand the di¡erence between a non-osteoinductive particle and an osteoinductive particle. Could you explain this? van Blitterswijk: At the moment people are not absolutely sure. Many of the early studies wrongly assumed that it had to do with the shape of the pores. We have checked this, and have found that instead it is actually porosity that is important: a microporous surface is needed for osteoinduction. For a long time we thought that osteoinduction had to do with calcium phosphate. This is not the case, because aluminium oxide also works, provided that it is microporous. I wouldn’t be surprised if a whole range of other materials also worked. It seems to be a physical process, and it’s not yet fully understood. Hardingham: On the same issue, hydroxyapatite is renowned for protein adsorption. As soon as you put it in contact with any biological £uid it will be coated with proteins. Have you looked at this as a variable in your di¡erent surface area presentation studies? van Blitterswijk: That’s true. We had these discussions about 15 years ago on bone-bonding materials. People still think that this has to do with protein adsorption. But I don’t believe this at all, because we see bone bonding occur with a large variety of materials with completely di¡erent surface chemistries. They are certainly not going to bind the same proteins, but they still show bonebonding behaviour. This mostly has to do with mineralizaton. Caplan: Is it the time-dependent residency of inductive versus non-inductive materials that gives them this property, or is it contact with wound £uid in your surgical implantation or subcutaneous site that is the key element? The test of this would be various precoatings that could be applied to these surfaces. van Blitterswijk: We truly do not know what the crystal induction mechanism is. What we can imagine is that it might have to do with spontaneous liberation of calcium phosphate ions from the surface. We have seen in the past that even with aluminium ¢lters with appropriately sized indentations that you will see the growth of calcium phosphate crystals on the surface. Perhaps this is what these materials have in common: they are all able to induce some type of calcium phosphate precipitation. Ohgushi: You have demonstrated the osteoinductive property of the ceramics using the goat and dog models. Does this phenomenon also occur in rats and mice?

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van Blitterswijk: We have seen it in dog, goat, rabbit, rat and mouse. In the dog we saw 30% e¡ective bone formation and it goes down in the rabbit, goat rat and mouse. Ohgushi: What is the incidence of osteoinduction using subcutaneous implantation in the rat? van Blitterswijk: I don’t know the exact ¢gure, but it is in the region of 5%. Ohgushi: I have implanted more than 500 ceramics (Interpore ceramics) in rat subcutaneous sites but I have never seen this sort of spontaneous bone formation. However, the same ceramics showed the spontaneous bone formation when implanted at dog intramuscular sites. van Blitterswijk: In our mouse models we don’t see any spontaneous bone formation, but intramuscularly in the goat we do. Helms: When you say ‘microporous’, do you mean ‘micro’ or ‘nano’? If it is micro, how does the cell perceive this, when it is much smaller than the pore? van Blitterswijk: The pore is about 300 mm in diameter, and the surface of the pore is microporous. There we are talking about pores with a diameter of microns or even lower. Cancedda: You say that in many cases your inductive biomaterial does not induce bone formation in nude mice. van Blitterswijk: It occurs at a very low frequency: we hardly ever see it. Cancedda: Is this because of an in£ammatory response? van Blitterswijk: Because we do not understand the underlying mechanisms, it is very hard to say what accounts for the di¡erences between species. Cancedda: I’m not fully convinced by the data from one of your experiments. In this experiment you are comparing the implantation of a ceramic that has been preincubated in vitro with the implantation of a ceramic that has not been preincubated. You showed a quicker response in the case of the in vitro preincubated implant, but you don’t take into account the time of the in vitro preincubation. So I am not convinced that you can make this kind of comparison. In both cases for you time zero is the time of implantation in the animal. In the reality, in the case of the preincubated implant there should be a shift of ¢ve days corresponding to the preincubation time. Time zero should correspond in both cases to the loading of the cells. If you did this, there would be no major di¡erence between the two conditions. van Blitterswijk: That’s not true. We compared 100 000 cells with 5 d culture, 100 000 cells with 16 d culture and an implant with no cells. Cancedda: If you shift the line of the graph for the 5 d in vitro preincubated implant by those 5 d, the two graphs are almost superimposable. van Blitterswijk: It would be impossible to make this experiment completely ideal. We have done our best. We end up with roughly the same amount of cells. Only the cultivation period is di¡erent.


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Hunziker: I have a technical question: There are products on the market for use in orthopaedic surgery, which typically consist of hydroxyapatite and are available with various porosities. Are these products generally formed under hightemperature conditions? Was the material you talked about formed biomimetically? van Blitterswijk: This is not a biomimetic calcium phosphate in the sense that it was made at ambient temperatures. What is important is the sintering temperature, because the microporosity of the surface depends on this. The sintering temperature we use here is relatively low, in the region of 1100 8C. Ka¢enah: In terms of organized sca¡olds, could it be argued that a bit of heterogeneity is needed to establish the roughness you are seeking? van Blitterswijk: It might be that di¡erent biological phenomena are involved in the success of tissue engineering, and it might be possible that surface roughness is involved. But we don’t know. The main point I want to make is that the sca¡olds we use are not reproducible, so they are hard to compare within the group. I am a little bit surprised that many groups focus on the biology of the process and then just use one particular sca¡old, drawing a lot of conclusions from this. The sca¡old itself is so essential in the outcome of the experiment that we really have to work with it and spend a lot of e¡ort on it. Vunjak-Novakovic: Do you have any insight into the progression of bone formation, and the spatial and temporal distribution of the new bone? van Blitterswijk: In general we see continuing bone formation. Most studies haven’t gone further than six months, but it is a continuous increase out till then. Lindahl: I have a question concerning the oxygen tension measurement of your constructs. If I recall correctly you had an oxygen partial pressure of about 5 kPa in the centre. If you implant this construct, do you see a di¡erence between the centre and the outside in terms of bone formation? van Blitterswijk: I can’t answer that. We see a di¡erence in the amount of cartilage formation in sca¡olds in vitro. Typically this is ¢rst seen at the periphery and in the centre only at a later stage. Lindahl: Tissue oxygen partial pressure is around 5^7 kPa. Is high oxygen tension a goal in the culture? van Blitterswijk: We have played with oxygen tension in culture, and high tension can be good for cartilage. Hollander: What is the best measure of functionality of your engineered bone. Is it the volume, or the average width of the trabeculae? van Blitterswijk: It depends on the application. A good general measurement is the amount of bone. However, it is more complicated than that. In our spinal fusion model we take particles. We might get beautiful bone formation in each particle, but if we have no interconnection between the particles it will still be a useless process. This is one of the challenges we are facing. We need to ¢nd a way to interconnect those particles.

General discussion I Goldstein: What is the fate of the cells that are used to seed constructs, and how many of them actually become part of the extracellular matrix in a bone construct? Or is their role simply to pump out growth factors or to elicit a response from the surrounding milieu causing factors to be expressed that then stimulate bone formation? Cancedda: I can only speculate. I think they are doing both things. They are participating in bone formation and also making the right microenvironment for other cells to join in. Martin: I think it is more than a speculation. You demonstrated that the osteoblasts, at least in the nude mouse model, are of human origin. So the cells are not only producing factors that recruit host cells but also are participating themselves. Caplan: Steve Goldstein is asking a good question. For small distances and small pores that can be evenly coated, and in which you can get good vascularization, the ¢rst bone that is made is made by donor cells. But eventually, since osteoblasts have short half lives, the layer of osteoblasts that are making bone will contain host as well as donor cells. For well vascularized, good continuous pore structure, there is a sequence of events that goes from donor to host. The point I tried to make earlier is that I suggest that only the outer edge of the solid implant actually has host cells that survive. Although there’s bone throughout the thickness of this implanted material, it may be that some proportion of the outer circumference has bone that is derived from donor, and the rest is from the host. Steve is asking the question of whether the input donor cells somehow stimulate the production of cytokines and growth factors that keep the momentum of that bone formation going so that it ¢lls the whole volume of that implant material. Is this another function of cellbased therapy? If it is, and we can identify what agents are made by these cells, could we then mimic that with a growth factor or cytokine-based approach? It’s a tissue engineering logic that is quite di¡erent from just saying that we are putting in a collagen gel with some BMP2, and we are going to get bone there. My suggestion to John Wozney is that this process is also quite complicated. The end result is bone, but what BMP is doing and how it is functioning within that milieu is food for speculation. The question is, is there a growth factor approach complementary to or founded on a cell-based approach that we can start to think about? 170

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van Blitterswijk: Theoretically, yes, but in practice, no. There is a range of factors involved, and these factors have to be released at di¡erent times. If you want to do this you need a biomaterial in which you’d have to build in as many as 50 di¡erent factors, which have to be released at speci¢c times. Caplan: How can you say that? Puri¢ed recombinant BMP2 gets the same result that other people have obtained using cells. van Blitterswijk: That’s a good point. What we see in tissue engineering is that if we only take the matrix, nothing happens. If we add frozen dead cells, nothing happens, but if we use living cells, something starts to happen. We also see that these cells, at least in culture, express a multitude of di¡erent factors. Everyone doing basic biological research on bone regeneration also sees that a multitude of factors are involved. I take it as logical that in order to do optimal bone regeneration, you would want to mimic the natural situation as closely as possible. This is too complicated to do in practice. Caplan: But we don’t know what the rate-limiting and key factors are in this cascade. This is the issue. You can paint whatever picture you want. You documented that osteogenesis was a multistep cascade, and we don’t know the exact identity of each of those factors. I suspect that BMP is just a wonderful chemoattractant for mesenchymal stem cells (MSCs), and they are the most important part of this regeneration. It is this multifactor cascade that Ranieri Cancedda is kicking o¡ by putting in bone marrow cells from the beginning, so he doesn’t need BMP2 to bring those cells in. van Blitterswijk: To make a parallel with gene therapy, in the early days people thought gene therapy would create miracles, and yet it has only worked with a very limited number of diseases involving just one particular gene. In tissue engineering, perhaps BMP is an equivalent example. We are lucky that we have this speci¢c protein that can show a major e¡ect, but it may be too optimistic to think that this will keep happening in tissue engineering. Cancedda: At this point we have to distinguish between what works in the orthotopic nude mice assays and the real clinical situation. If the orthotopic mouse model is correct, bone is formed only because we are putting in some cells that are osteogenic. My explanation is that in this area there are no osteogenic cells that can be recruited with more ‘soft’ approaches. I don’t consider addition of BMP a very ‘soft’ approach. For sure, BMP is able to recruit cells to the osteogenic lineage which cannot be recruited just by adding osteogenic cells. The situation is probably di¡erent in the clinic. In the real clinical situation we have shown that a good biomaterial by itself can be osteoconductive, if not osteoinductive. In this case, the bone that is formed is just a combination of bone that is made by cells that are implanted and by other cells that can be recruited by the speci¢c microenvironment that the implanted cells are organizing.

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Goldstein: It’s clear that there have already been a number of di¡erent avenues, all of which have created the same end. That is, that bone has formed. It is a balance between what exogenous factor (cells or biomaterial) you can put in that will then couple with existing cells and factors to regenerate tissue. It will be a matter of optimization of the di¡erent approaches to strike the right balance. Ratcli¡e: We have discussed that there are multiple ways that we can now make bone. What I’m not hearing is what the true outcome measure is that you are trying to achieve. Someone mentioned functionality and then went into a histology analysis. Histology isn’t function. To really pull this out we need a clear de¢nition of what the true objective is. Caplan: Could you amplify that by giving the experimental example that you have worked on in which you assess whether cartilage provides an appropriate source for bone? Ratcli¡e: This morning’s discussion addressed whether it is possible to throw in a cartilage-type matrix as a piece of a pathway along which you can move to make bone. We asked the same question using tissue-engineered cartilage constructs. In collaboration with Dr Robert Guldberg a bone chamber was designed which could be inserted in the rabbit tibia and used to follow bone formation (Case et al 2002). We put cartilage constructs in that chamber, and compared viable or non-viable constructs. The non-viable constructs didn’t generate much bone formation. The viable constructs generated much more apparent bone. We also did an experiment in which we applied a load during the incubation period, and this generated even more bone formation. So cartilage constructs using viable cells can generate a bone repair pathway. Grodzinsky: What sort of load was this? Ratcli¡e: It was a compressive load. Caplan: The point that Tony Ratcli¡e is making is that the cartilage construct that he made in a bioreactor with adult rabbit chondrocytes, when put in a bone chamber, eventually went hypertrophic and became the substrate for eventual bone formation. The preloading for a certain time enhanced the amount of bone seen at the end of the experiment. Vunjak-Novakovic: We had a similar result with an in vivo experiment in adult (8 month old) rabbits. Large osteochondral defects in (755 mm, femoropatellar groove) were repaired using in vitro engineered cartilage constructs (Schaefer et al 2002). We expanded articular chondrocytes, cultured them for 4^6 weeks on polyglycolic acid sca¡olds, and sutured to an osteoconductive support. Cell-free sca¡olds and untreated defects served as controls. Repair tissue was evaluated histologically, biochemically and biomechanically (by indentation, in the whole joint). Over 6 months in vivo, defects implanted with engineered composites underwent orderly repair and yielded cartilage that had normal thickness, columnar cells and tidemark at an appropriate depth, and new subchondral bone.


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In contrast, controls repaired with ¢brocartilage. In particular, the tissueengineered cartilage had normal Young’s moduli. Integration with subchondral bone was complete in most cases, whereas integration with cartilage was not consistently good. Functional cartilage engineered in vitro can thus provide a mechanically stable template that can yield osteochondral tissue with physiologically thick and sti¡ cartilage. Hollander: I’d like to bring the discussion back to the question of the functionality of bone, and whether there is an issue here. There are various approaches to bone formation, and at some point we have to ask whether these are all producing a similar tissue. On what basis should we compare them? Goldstein: The outcome is a little more simple than with cartilage. First of all, there is a geometric requirement that is related to the anatomic site. Secondly, there are required mechanical properties that are relatively well characterized according to the site. The third criterion is that it has to be a remodellable material: it has to participate in normal bone turnover and metabolic remodelling. These are all measurable in animal models. In humans, we have to have surrogate measures. The geometric measures can be done by computed tomography scans, but mechanical properties can only be inferred, perhaps by densitometry measures. These are the targets. Hollander: Are these sorts of data coming through from the studies we are hearing about? Goldstein: In the ¢eld of bone tissue engineering in general, biomechanics is a critical endpoint. So the answer is yes. Vunjak-Novakovic: Integration is also a factor in graft functionality. Trippel: Using the third criterion of remodellability, Clemens van Blitterswijk, is the hydroxyapatite that you are using resorbed so that the bone is remodellable? van Blitterswijk: Any type of hydroxyapatite will ultimately resorb, but it is a very slow process. It is so slow that I would say hydroxyapatite is non-resorbable. This is a good thing, because we are using it in acetabular reconstruction, and you wouldn’t want the sca¡old to go away before the bone has taken over. Trippel: Would you disagree that remodellability is important? van Blitterswijk: No, because for other indications I would go for sca¡olds which go away. In some cases though, non-degradable sca¡olds are called for. Lohmander: I would agree that in some circumstances the ability of the matrix to be removed is not relevant, or at least it comes far down on the list. Goldstein: In those cases you would create a composite, and often the bone surface of those non-degradable materials can still be remodelled. We have seen this happen. Caplan: One variable hasn’t been discussed yet, but it is the key variable in de¢ning our success for metal implants, which is how long is the implant going to last? This a¡ects the logic of implantations of knees and hips. Is there a time

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component to successful functional outcome, particularly for materials that don’t degrade or degrade only very slowly? Lohmander: It depends on the use. If you are thinking about hip revisions, you would like it to last as long as the patient does. Caplan: What about a tumour resection for example, which is a huge defect? You want to replace it with something other than a dead allograft and a metal plate with 25 screws. You would like to put functional living bone in there. The current standard of care is to take a piece of lyophilized allograft, and a plate with 25 screws. We know that 10 years later a revision of this will be needed because of osteoclastic activity. The issue is whether tissue engineering will provide a better standard of care. Lohmander: That is not necessarily the standard. We can do free transplants of the ¢bula to other sites. Caplan: Not in the femur. Lohmander: These cases are getting increasingly rare, due to the advances in oncology. Unless you get substitution by host-built bone to substitute for the arti¢cial material, it will fail. Cancedda: I would be cautious about expanding bone marrow stromal cells from osteosarcoma patients. Goldstein: Large segmental allografts are becoming rare. They are being replaced by other means, including distraction osteogenesis. References Case ND, Duty AO, Ratcli¡e A, Muller R, Guldberg RE 2002 Bone formation on tissueengineered cartilage constructs in vivo: e¡ects of chondrocyte viability and mechanical loading. Tissue Eng, in press Schaefer D, Martin I, Jundt G et al 2002 Tissue-engineered composites for the repair of large osteochondral defects. Arthritis Rheum 46:2524^2534

Cartilage repair with chondrocytes: clinical and cellular aspects Anders Lindahl, Mats Brittberg* and Lars Peterson* Institute of Laboratory Medicine, Department of Clinical Chemistry and Transfusion Medicine, *Department of Orthopaedic Surgery, Sahlgrenska Academy at Gothenburg University, Sahlgrenska University Hospital, S-413 G˛teborg, Sweden

Abstract. Articular cartilage has a limited potential to repair. Unsatisfactory results with current treatment methods (e.g. osteochondral autografts, drilling or microfracturing) has triggered the development of new cartilage restoration techniques including autologous cell transplantation (mesenchymal stem cells or chondrocytes) with or without supporting sca¡olds. Autologous chondrocyte transplantation (ACT) was ¢rst used in humans in 1987 and the ¢rst pilot was published in 1994. Two years after transplantation, 14 of the 16 patients with femoral condyle transplants had a restored joint function and 11 of 15 femoral transplants demonstrated a hyaline repair tissue. Results from patellar transplants were less encouraging. To date, we have treated over 1000 and other groups over 6000 patients. The technique gives stable long-term results with a high percentage of good to excellent results (84^90%) in patients with di¡erent types of single femoral condyle lesions, whereas in patients with other types of lesions in the knee it is less successful (average 74%). A better understanding of the repair mechanism induced by the cultured chondrocytes and the regulatory mechanisms controlling chondrogenic di¡erentiation combined with identi¢cation and culture of stem cells with chondrogenic potential will be the key to new cartilage treatments. 2003 Tissue engineering of cartilage and bone. Wiley, Chichester (Novartis Foundation Symposium 249) p 175^189

Chondrocytes are cells of mesenchymal origin producing di¡erent cartilaginous tissues (hyaline, elastic and ¢brous) where the cartilage phenotype is dependent on the composition of the extracellular matrix. In fetal life cartilage is crucial for the formation of the skeletal system through endochondral ossi¢cation. In adult life hyaline cartilage provides a smooth surface at the end of long bones, functions as a cushion between bones and allows frictionless movements between joints. Hyaline cartilage is considered a permanently established tissue and the repair response to injury is limited due to the lack of vascularization, lymphatic drainage and inervation (Shapiro et al 1993, Hunziker & Rosenberg 1996). Epidemiological studies have demonstrated a relation between knee injuries and later development of osteoarthritis (OA). Chondral and osteochondral injuries are 175

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common in trauma (Noyes et al 1980) and cartilage damages give rise to pain and joint dysfunction (Johnson-Nurse & Dandy 1985) with a potential risk of generalized destruction of the cartilage in the joint. Cartilage defects with a diameter over 2^4 mm rarely heal and will, under certain conditions, progress to OA (Mankin 1966). The aim for all clinical interventions is thus to prevent the expansion of the cartilage injury and to regenerate hyaline cartilage in order to prevent further progress and to save the joint. Cartilage repair potential Cell division in hyaline cartilage can be found in the growing rat and rabbit and with long term labelling it is possible to identify a population of label-retaining cells found in hyaline cartilage, perichondrium and the growth plate (Ohlsson 1992, Fujimoto 2000). In the skeletally matured rabbit no cell division can be detected in hyaline joint cartilage and label-retaining cells can only be found in the perichondreal ring also called the groove of Ranvier, a potential stem cell source for repair (Robinson et al 2000). The aim for a cartilage repair treatment should be the induction of biological healing and regeneration of cartilage, a goal that could be reached either by enhancement of the intrinsic repair potential of articular cartilage or by the introduction of cells or tissue with a potential of regenerating new cartilage. Increasing the intrinsic repair has traditionally been focused on the recruitment of pluripotent cells from the bone marrow by penetration of the subchondral bone by drilling or microfracturing (Johnson 1991, Aglietti et al 1994, Insall 1974, Ficat et al 1979). However, the treatments usually result in a ¢brocartilaginous repair tissue lacking the biomechanical properties of the hyaline cartilage and with a poor long term clinical outcome (44 years). Periosteal/perichondral transplantations combined with opening of the bone marrow has resulted in a hyaline-like tissue (Homminga et al 1989, Rubak 1982) generated by the transplant or pluripotent stem cells from the bone marrow. The clinical long term results from these treatments are encouraging in a well selected group of patients with small cartilage injuries but for the majority of patients with cartilage defects the long term outcome is poor manly due to calci¢cation of the transplant (Angermann et al 1998, Bouwmeester et al 1997). An extensive literature search was performed by The National Coordinating Centre for Health Technology Assessment in UK (Jobanputra et al 2001) in conjunction with evaluation of the autologous chondrocyte transplantation (ACT) technique. The report concluded that the e¡ectiveness of other treatments mostly have been reported as case studies and no other systematic review of surgical techniques for cartilage defects were found. Only one study with a control group could be found (Hubbard 1996).

CHONDROCYTE CARTILAGE REPAIR

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FIG. 1. Current human cellular therapies in clinical and experimental use.

The poor outcome of current treatments have encouraged the development of alternative techniques of cartilage replacement either with a series of small osteochondral plugs mosaicplasty (Kish et al 1999) or completely with a matched osteochondral transplant (Bugbee & Convery 1999). Both techniques have shown good clinical results in the time frame between 1 and 5 years. An alternative treatment method would be transplantation of cultured chondrocytes, since the cause of the inadequate cellular response of hyaline cartilage to injury could be due to the entrapment of chondrocytes in the cartilaginous matrix resulting in a limited number of cells capable of a proper cartilage repair. In rabbit studies where autologous cultured chondrocytes were transplanted to a de¢ned defect in the patella a restoration of the defect volume to 85% with a hyaline like tissue was seen after one year (Brittberg et al 1996). Biological and synthetic sca¡olds have also been used for repair of cartilage defects but our own results with ¢brine glue and carbon ¢bre pads as sca¡olds for chondrocyte implantation has been discouraging (Brittberg 1996). Cell therapy for treatment of tissue injuries in humans has been in limited clinical use over the last two decades (Fig. 1) and for cartilage the ¢rst clinical treatment attempt was made in Sweden almost 15 years ago. Since the ¢rst pilot report (Brittberg et al 1994), the use of transplanted autologous chondrocytes has been regarded as an alternative approach to regenerate hyaline cartilage, and the method has been widespread during the last 5 years (Minas 1998, L˛hnert et al 1999, Richardson et al 1999).

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The principal methodology of ACT The chondrocytes are isolated from a biopsy specimen from the upper medial femoral condyle taken at the time of the clinical examination. At the same time 1010 ml autologous venous blood samples are collected for preparation of serum additive to the culture medium. Individual chondrocytes are isolated by collagenase digestion overnight and cultured in DMEM/F12 with 10% autologous human serum supplement. Primary cultures are performed in 25 cm2 culture £asks and after one week the cells are trypsinized and passaged to 75 cm2 culture £ask at a cell density of 8000 cells/cm2. After two weeks of culture the cells are trypsinized, washed and resuspended to a treatment density of 30 million cells/ml. The cells are subsequently transferred to the operating room were the knee has been opened by a parapatellar incision and the cartilage injury debrided to healthy cartilage. A sterile template is made from the defect and a piece of periosteum, isolated from the upper part of the tibia through a skin incision, is trimmed to the correct size and sutured over the defect. The cells are injected under the £ap with a treatment dose of 1106 cells/cm2 defect area. The patient is subsequently put on a 24 h passive motion machine and is allowed to walk on crutches with a gradual increase in weight bearing until full load 8^12 weeks after surgery. Several modi¢cations of the treatment model exist e.g. the replacement of the periosteum with a collagen membrane or seeding the chondrocytes on a collagen membrane that is then implanted (MACI technique). Results from the pilot study The pilot study involving 23 patients with cartilage injuries treated with ACT was published in 1994 (Brittberg et al 1994). Fourteen of 16 patients with cartilage injuries on the femoral condyle had restored joint function and 11/15 biopsy specimens demonstrated a hyaline repair tissue that all correlated with good clinical outcome. Treatment of patellar lesions had a less successful clinical result with only 2 of 7 patients demonstrating a restored joint function. Long term clinical results The clinical use of ACT is over 14 years old and we have followed all treated patients from the start with questionnaires and directed clinical examinations. We recently published a long-term clinical follow up in 102 patients 2^9 years after ACT (Peterson et al 2000). In the treatment groups (i) isolated femoral condyles, (ii) osteochondritis dissecans (OCD) and (iii) isolated patellar lesions with correction of malalignment the good to excellent clinical results ranged from 83^92% and biopsy specimens demonstrated hyaline cartilage repair tissue in 80% of the cases.


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FIG. 2. Clinical outcome from ACT in 61 patients treated with ACT after 0, 2 and 11 years’ follow up. Clinical grading ‘Excellent’ means no clinical symptoms from the knee. Excellent, no pain, swelling or locking with strenuous activities; Good, mild aching with strenuous activity but no swelling or locking.

In cartilage repair treatments using microfracturing or drilling the clinical outcome is usually satisfactory for the ¢rst two years but gradually the clinical result declines and long-term results are usually poor. In a recent study from our group the long-term durability of ACT treatment was evaluated. When treatment results after 2 years (50/61 patients treated were graded good to excellent) were compared to the clinical outcome 7^11 years later, 100% clinical durability was demonstrated. Of the 50 patients with good to excellent clinical results, all had the same or better clinical results, and one patient was improved from fair to good during the observation period (Fig. 2) (Peterson et al 2002). Other clinical applications for ACT are the treatment of multiple and patellar lesions where the clinical results in later studies are much better than initially published (Peterson et al 2002). We have also extended the treatment indication to other joints, i.e. ankle, shoulder, elbow and hip. The clinical results in the ankle are very promising (14 examined patients), but results from the other joints are too early to be elucidated. The successful clinical outcome from the treatment is dependent on strict clinical indications. When deciding if a patient is eligible for ACT treatment we use the following clinical algorithm (Fig. 3). We conclude that for smaller juvenile injuries microfracturing, osteochondral grafts and ACT give the most socioeconomic bene¢ts. In larger defects and in revision surgery ACT is the best treatment option.

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FIG. 3. Treatment algorithm for patient with cartilage injuries.

Independent clinical results collected from over 19 centres in the USA now demonstrate a 3 year follow up similar to our clinical result (Micheli et al 2001) as well as results from other surgeons (Minas 2001).

Health economic implications of ACT The treatment with cultured cells is an expensive method with prices for a single treatment in the USA costing in the range of US$20 000 and in Europe E6000^ 9000, where the cost for cells represents approximately 50% of the treatment cost. Many studies have examined the clinical e¡ect of treatment but few if any papers have correlated the outcome with the e¡ect on workplace disability, absenteeism and the economic burden to society. We followed 57 patients with full thickness cartilage defects treated between 1987 and 1996 and evaluated the e¡ect of ACT on clinical outcome, absenteeism, disability status and total economic burden (Lindahl et al 2001). Pre ACT 57/57 patients were disabled; post ACT 44/57 had no disability, 10/57 had minor disability and 1/57 was disabled. In the 10 years prior to ACT the average cost of absenteeism and surgery was SEK982 457 and SEK47 000, respectively compared to the post-ACT period where both absenteeism and medical costs were dramatically reduced to SEK9508 and SEK7050 respectively. Our estimate was that ACT is cost saving when more


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FIG. 4. Biopsy specimen T117 in 26 year old male patient 3.9 years after ACT treatment. Indenter force 3.7 N on grafted area. Indenter force 3.8 N on surrounding normal cartilage. Lower section: alcian blue^van Gieson staining. Upper section: Same specimen in polarized light. Articular surface (left): observe ¢brous appearance of periosteal tissue.

than 50% of the patients fail a surgical procedure within a 10 year period. What types of patients are suitable for ACT when considering the cost saving e¡ect? Dzioba (1988) found that 100% of patients with large chronic and or full thickness defects failed to respond to treatment with debridement or drilling. A large portion of the cost savings were due to the reduction in absenteeism a fact even more important when considering the young age of the study group (mean 31 years, range 15^49 years). The economic calculation is relevant to a socialized healthcare system but also in a private insurance system a net margin e¡ect was demonstrated for ACT treatment (Minas 1998).

Graft repair tissue: biomechanical function and histology When evaluating the histological structure of the graft tissue generated by ACT it is important to evaluate the defect location carefully since cartilage formation is dependent on the defect location as well as the biomechanical loading (Peterson

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FIG. 5. Hypothetical cellular mechanism for cartilage repair induced by ACT: (1) cellular adhesion; (2) cellular adhesion followed by clonal growth; (3) cellular condensation; (4) paracrine stimulation from periosteum; (5) inhibitory e¡ects on apoptosis.

et al 2002). Therefore, when a biopsy specimen is taken from a central portion of the grafted area it should be compared with a reference biopsy from surrounding cartilage. The grafted area undergoes a signi¢cant remodelling process during the ¢rst 18^24 months after surgery and we recommend that biopsies should not be taken earlier than three years after operation. Examination of cartilage components in the repair tissue has been focused on the following components: cartilage oligomeric matrix protein (COMP), type I and II collagen and aggrecan. Repair tissue formed after ACT has a positive staining for COMP, aggrecan and type II collagen, although 10^20% of the repair tissue facing the joint cavity had a more ¢brous appearance with a type I collagen staining, probably a sign of a remaining periosteal tissue. Polarized light microscopy demonstrates an opalescent tissue structure similar to normal hyaline cartilage (Fig. 4). When testing the hardness of the grafted area we use an indenter probe and the hardness is similar to normal hyaline cartilage (Peterson et al 2002). Similar results have been found by other investigators (Richardson et al 1999). The same authors recently studied biopsy samples from the grafted area in order to examine the cellular mechanisms and determine whether remodelling of the matrix occurred, by using antibodies directed against C-propeptide of type II collagen as an indicator of anabolism and Col2-3/4m as marker of catabolism. The authors conclude that ACT is capable of repair and regeneration of the cartilage tissue (Roberts et al 2001). Biological repair mechanisms induced by ACT are similar to mechanisms used in embryological cartilage development Although ACT has been used in treatment of cartilage defects in over 6000 patients world-wide, the biological mechanism behind the repair process is poorly understood. The development of a superior treatment process for cartilage repair will be dependent upon the understanding of the cellular processes involved in fetal cartilage development. Nature uses a limited toolbox of genes for the


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development of organs and similar genes are used for fetal development and regeneration in di¡erent organs. The development of the skeletal systems and joints starts with cells from the mesenchyme that condense into the limb bud structure, a process called mesenchymal condensation. There is no reason to believe that tissue repair is not restricted to the same cellular processes. We have addressed the hypothesis that the expansion of chondrocytes in vitro involves the selection of a limited number of chondrocyte progenitor clones and that the culture process allows the cells to revert to a fetal stage. Then the repair process initiated by the injection of the chondrocytes in the cartilage defect initiates cell condensation and cartilage formation in a similar way as the mesenchymal condensation in the limb formation. To investigate this we have designed primers for a limited number of key genes expressed during limb and cartilage development: collagen type IIA and IIB (Coll types 2A+B), osteocalcin, SRY-related HMG box gene (Sox9), cartilage-derived morphogenetic protein 1 (cdmp1), wingless-type MMTV integration site family member 14 (Wnt14) and core binding factor a1(cbfa1). We have found that all genes were expressed by the cultured cells. Especially interesting are SOX9 expression, since it is critical for mesenchymal condensation (Bi et al 1999), WNT14, critical for the joint development (Hartman & Tabin 2001) and FGFR3, expressed on chondrogenic progenitor cells (Robinson et al 2000). The ACT procedure not only induces the formation of a new hyaline cartilage in the repair area by the activation of cellular mechanisms used during fetal development, but also appears to reduce the number of apoptotic cells in the surrounding cartilage in a rabbit model (Fig. 5) (Kato et al 2000). Role of periosteum The e¡ect of transplantation of cultured chondrocytes has been reproduced in various animal models with variable results. The conclusion that can be drawn is that a good animal model for studies of cartilage repair does not exist. The live periosteum is crucial to the repair process in a rabbit model and the periosteum is able to support the new cartilage formation via a paracrine action (Brittberg 1996). The nature of such agents is currently unknown but potential candidates could be transforming growth factor b, ¢broblast growth factor 2, interleukin 6 and others. Quality standards for ACT treatments The use of autologous tissue to repair cartilage avoids the potential of graft reduction and it reduces the potential of viral infection. However, the laboratory handling of the tissue outside the body introduces a number of potential risks which have to be handled properly. Such risks include cross contamination of cells, errors in labelling, failure in the transportation process and cultivation

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failure in isolation, propagation and freezing. ACT is regulated in the USA but not in the European Union. Due to the lack of regulatory structure an emerging ‘spot market’ for cheap chondrocytes exists in many European countries. Producers culturing cells without subjecting themselves to the very rigid quality system of GMP can produce the cells at a lower cost but to a higher risk for the patients. The low price in combination with an unregulated environment for cell therapies hamper the future possibility of developing new techniques. The use of ACT is regulated by FDA in the USA and Genzyme Biosurgery received a biological license for ACT procedures in 1997. Although ACT is not regulated in Europe, the Gothenburg group have, for patient safety, decided on a voluntary regulation supervised by the Swedish Medical Product Agency. We have worked with two quality standards for the production facility in Gothenburg and since 2000 the laboratory is regulated both according to the ISO 17025 for medical laboratories and according to the GMP regulations. There are issues not addressed in the current regulatory system. One of the key ones concerns establishing the purity of the biopsy obtained prior to ACT, since it is hard to distinguish the cartilage from possible contaminating tissues such as periosteum, synovium or bone: it is not possible to identify contaminating cells morphologically in cell cultures. When a panel of selected genes were tested we found that a combination of collagen types 2A+B and osteocalcin primers were able to positively identify the chondrocyte phenotype and exclude a potential contamination of periosteum synovium or osteoblast cells. We have also started a development project for replacing the current culture media, not intended for human use, with de¢ned culture media according to the European Pharmacopoeia. Future development of chondrocyte transplantation technology The use of cultured cells for repair of cartilage is promising and several investigators have been able to reproduce the clinical results. Several modi¢cations for the method have been suggested, such as the use of matrix and variations employing cultured cells on membranes and injected in gels. As the original founder of the technology we have been cautious in changing the method since the basic understanding of the repair mechanisms are unclear. More important for patients is expanding the surgical indication to joints other than the knee, and our experience is that ACT can be used for cartilage repair in almost any joint. We are also focusing on the long-term follow up of patients since we now have the possibility to follow 5 and 10 year cohorts of the patients. When these aspects have been properly addressed the improvement of the method can start and we believe that an ultimate cartilage regeneration procedure has to be based on the understanding of developmental biology and the genes


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involved. The identi¢cation of chondrocyte progenitor cells and the use of cartilaginous cells derived from embryonic stem (ES) cells are other options. An interesting option for ES cell therapy was prompted by the observation that chimeric bone marrow is formed by ES cells in mice, abolishing the immunological issues of ES cell therapies (Fandrich et al 2002).

References Aglietti P, Buzzi R, Bassi PB, Fioriti M 1994 Arthroscopic drilling in juvenile osteochondritis dissecans of the medial femoral condyle. Arthroscopy 10:286^291 Angermann P, Riegels-Nielsen P, Pedersen H 1998 Osteochondritis dissecans of the femoral condyle treated with periosteal transplantation. Poor outcome in 14 patients followed for 6^9 years. Acta Orthop Scand 69:595^597 Bi W, Deng JM, Zhang Z, Behringer RR, de Crombrugghe B 1999 Sox9 is required for cartilage formation. Nat Genet 22:85^89 Brittberg M 1996 Cartilage repair. PhD thesis Gothenburg University, Gothenburg, Sweden Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peterson L 1994 Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. New Engl J Med 331:889^895 Brittberg M, Nilsson A, Lindahl A, Ohlsson C, Peterson L 1996 Rabbit articular cartilage defects treated with autologous cultured chondrocytes. Clin Orthop 326:270^283 Bouwmeester SJ, Beckers JM, Kuijer R, van der Linden AJ, Bulstra SK 1997 Long-term results of rib perichondrial grafts for repair of cartilage defects in the human knee. Int Orthop 21:313^317 Bugbee WD, Convery FR 1999 Osteochondral allograft transplantation. Clin Sports Med 18:67^ 75 Dzioba RB 1988 The classi¢cation and treatment of acute articular cartilage lesions. Arthroscopy 4:72^80 Fandrich F, Lin X, Chai GX et al 2002 Preimplantation-stage stem cells induce long-term allogeneic graft acceptance without supplementary host conditioning. Nat Med 8:171^178 Fujimoto E, Kato H, Lindahl A 2000 Proliferating capacity and identi¢cation of stem cells in articular cartilage. 3rd International Cartilage Repair Society meeting, April 2000, Gothenburg, Sweden, Abstract 37 Ficat RP, Ficat C, Gedeon P, Toussaint JB 1979 Spongialization: a new treatment for diseased patellae. Clin Orthop 144:74^83 Hartmann C, Tabin CJ 2001 Wnt-14 plays a pivotal role in inducing synovial joint formation in the developing appendicular skeleton. Cell 104:341^351 Homminga G, van der Linden TJ, Terwindt-Rouwenhorst W, Drukker J 1989 Repair of articular defects by perichondral grafts. Acta Orthop Scand 60:326^329 Hubbard MJ 1996 Articular debridement versus washout for degeneration of the medial femoral condyle. J Bone Joint Surg Br 78:217^219 Hunziker EB, Rosenberg LC 1996 Repair of partial thickness defects in articular cartilage: cellrecruitment from synovial membrane. J Bone Joint Surg Am 78:721^733 Insall J 1974 The Pridie debridement operation for osteoarthritis of the knee. Clin Orthop 101:61^67 Jobanputra P, Parry D, Fry-Smith A, Burls A 2001 E¡ectiveness of autologous chondrocyte transplantation for hyaline cartilage defects in knees: a rapid and systemic review. Health Technol Assess 5:1^57

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Johnson LL 1991 Arthroscopic abrasion arthroplasty. In: McGinty JB (ed) Operative arthroscopy. New York, Raven Press, p 341^359 Johnson-Nurse C, Dandy DJ 1985 Fracture separation of articular cartilage in the adult knee. J Bone Joint Surg Br 67:42^43 Kato H, Lindahl A, Sterner H, Fujimoto E, Sjogren-Jansson E 2000 Chondrocytes implantation is e¡ective to inhibit the appearance of chondrocyte apoptosis around injury cartilage. 3rd International Cartilage Repair Society meeting, April 2000, Gothenburg, Sweden, Abstract 12 Kish G, Modis L, Hangody L 1999 Osteochondral mosaicplasty for the treatment of focal chondral and osteochondral lesions of the knee and talus in the athlete. Clin Sports Med 18:45^66 Lindahl A, Brittberg M, Peterson L 2001 Health economics bene¢ts following autologous chondrocyte transplantation for patients with focal chondral lesions of the knee. Knee Surg Sport Traumatol Arthrosc 9:358^363 L˛hnert J, Ruhnau K, Gossen A, Bernsmann K, Weise M 1999 Autologie Chondrozytentransplantation (ACT) im Kneigelenk. Arthroscopie 12:34^42 Mankin HJ 1966 Current concept reviews. The response of articular cartilage to mechanical injury. J Bone and Joint Surg Am 64:460^466 Micheli LJ, Browne JE, Erggelet C et al 2001 Autologous chondrocyte implantation of the knee: multicenter experience and minimum 3-year follow-up. Clin J Sport Med 11:223^228 Minas T 1998 Chondrocyte implantation in the repair of chondral lesions of the knee: economics and quality of life. Am J Orthop 27:739^744 Minas T 2001 Autologous chondrocyte implantation for focal chondral defects of the knee. Clin Orthop 391:S349^S361 Noyes FR, Bassett RW, Grood ES, Butler DL 1980 Arthroscopy in acute traumatic hemarthrosis of the knee. Incidence of anterior cruciate tears and other injuries. J Bone Joint Surg Am 62:687^695 Ohlsson C, Nilsson A, Isaksson O, Lindahl A 1992 Growth hormone induces multiplication of the slowly cycling germinal cells of the rat tibial growth plate. Proc Natl Acad Sci USA 89:9826^9830 Peterson L, Minas T, Brittberg M, Nilsson A, Sj˛gren-Jansson E, Lindahl A 2000 Two- to 9 year outcome after autologous chondrocyte transplantation of the knee. Clin Orthop 374:212^234 Peterson L, Brittberg M, Kiviranta I, —kerlund EL, Lindahl A 2002 Autologous chondrocyte transplantation. Biomechanics and long-term durability. Am J Sports Med 30:2^12 Richardsson JB, Caterson B, Evans EH, Ashton BA, Roberts S 1999 Repair of human articular cartilage after implantation of autologous chondrocytes. J Bone Joint Surg Br 81:1064^1068 Roberts S, Hollander AP, Caterson B, Menage J, Richardson JB 2001 Matrix turnover in human cartilage repair tissue in autologous chondrocyte implantation. Arthritis Rheum 44:2586^2598 Robinson D, Hasharoni A, Evron Z, Segal M, Nevo Z 2000 Synovial chondromatosis:the possible role of FGF9 and FGF receptor 3 in its pathology. Int J Exp Pathol 81:183^189 Rubak JM 1982 Reconstruction of articular cartilage defects with free periosteal grafts. Acta Orthop Scand 53:175^180 Shapiro F, Koide S, Glimcher MJ 1993 Cell origin and di¡erentiation in the repair of fullthickness defects of articular cartilage. J Bone Joint Surg Am 75:532^553

DISCUSSION Grodzinsky: You mentioned the other surgical approaches that are used, such as drilling and microfracture. Is the repair tissue that you ¢nd with ACT fundamentally di¡erent from the repair tissue seen with these other surgical approaches?


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Lindahl: There are very few studies presenting histological results after drilling and microfracture, but the data presented usually demonstrate the production of a ¢brous repair tissue. We also see this in a frequency of around 15% in our patient groups. But we see a higher frequency of hyaline repair tissue compared to the other techniques. Lohmander: You indicated that the success rate varies among di¡erent locations in the joint. Is this because of technical di⁄culties, or the di¡erent biomechanics and loading patterns? Or could this be because of the biology of the cartilage in these di¡erent locations? Lindahl: I think that it is a combination of all of these factors. It is di⁄cult to repair tibial cartilage. Even in cases where we are able to get a perfect repair with hyaline tissue, the patient still has pain. Then there are technical problems. In the patella, isolated defects are the best candidates for repair. A contributing factor could be that the individual patient has di¡erent sensitivity to pain in di¡erent areas of the joint. Buschmann: I thought that OCD was primarily a bone problem that resulted in delamination of articular cartilage from subchondral bone. How is this an indication for ACT? Lindahl: You are correct that it is a bone disease. There is a plug of bone and cartilage which is loosening. We were hesitant to begin with because we are only treating full-thickness cartilage defects. If the defect is too deep, we have to pack the bone ¢rst and then go back later with chondrocytes. But it looks like we are able to form a cartilage plug in the defect and over time the bone remodels and builds up underneath. Caplan: In these cases, do you add more chondrocytes, or do you keep the procedure standardized to just the chondral defect standards? Lindahl: If you look at the optimum dose, in the beginning we used everything we had, and we could ¢nd no correlation with what we inserted and the result. Now we use 1 million cells per cm2. This is independent of the nature of the defect. Caplan: The other question of importance is the percentage of delivered cells which survive the initial 72 h post implantation, and then what percentage mature into repair tissue. Can you estimate this? Lindahl: No. There are others who have tried to label cells and come up with a ¢gure of 20^50% survival. Cells attach to the surrounding cartilage and bone. The repair tissue is later formed over a period of several months, and the question is whether later on it is still the same cells that are involved, or whether cells from surrounding cartilage have migrated in as a part of a remodelling process. Caplan: Especially with OCD, we now have other sources of cells that can come into the repair structure. Has anyone attempted quantitation in these larger OCD defects? Lindahl: No.

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Barry: Would it be feasible to use chondrocytes derived from another type of cartilage, for example nasal septum, in this procedure? Lindahl: It’s an interesting approach. We have used knee cartilage to treat the ankle and hip. It looks like it is working ¢ne. The nasal cartilage is a little too di¡erent from joint cartilage to be useful, but I might be wrong. Barry: Could you use cells from an allogeneic source such as neonatal cartilage? Lindahl: In principal, if you are able to make a construct producing enough matrix to cover the cells, they can be implanted. Osteochondral allografts are well accepted by the patients. Hollander: We have promoted the concept of using nasal chondrocytes for cartilage repair. I fully accept that nasal and articular chondrocytes have di¡erent ‘addresses’, but it may be that you can move address, and that we don’t have to have an identical cartilage being formed to that that was already there, as long as it is performing a function. The two questions I’d want answered about the nasal chondrocytes is whether they can survive the low oxygen tension in the joint, and whether they will respond to load in a way that would allow them to remodel the cartilage matrix. Lindahl: When we culture articular chondrocytes it looks as if they re-express a fetal programme. If you go back that far, are you adaptive to a new environment? This has not been shown yet. Caplan: I have reviewed a manuscript (not yet in print) in which horse nasal septum chondrocytes were used in a knee environment. The long-term results weren’t very positive. Huckle: Did the knee cells work in the same model? Caplan: This group is experienced with cultured chondrocytes, and the hyaline cartilage chondrocytes gave a positive result. Helms: One thing is clear is that the nasal cartilage is derived from the cranial neural crest, whereas the rest is from the mesoderm. Is there a di¡erence because of the way that the cranial neural crest di¡erentiates to form cartilage? They could be formed using di¡erent molecular mechanisms. Caplan: They look the same, but they have di¡erent molecular constituents. They have lots of aggrecan and type II collagen, and other di¡erences in their minor collagen components. They also have di¡erent mechanical properties. Helms: Do you think tissues derived from cranial crest are di¡erent from those derived from the mesoderm? Caplan: This is a discussion that has been going on for many years. The problem is that with the single exception of tooth pulp, all the other structures are piped to the rest of the body. There are circulating progenitor cells and there are turnover dynamics in all these tissues. By the time you are an adult the embryological origin may or may not play an important role in the actual tissue dynamics. The pragmatic issue is whether cartilage from a di¡erent address in the body, which has di¡erent


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minor molecular constituents and subtly di¡erent mechanical properties, can modulate to the new site of implantation. This is something we will have to determine empirically. The advantage of nasal and auricular chondrocytes is that these cells are easier to culture and make wonderful cartilage. But they make the cartilage from which they originated. Whether this happens in the joint under ACT conditions needs to be experimentally determined. Hollander: Is that passage-number dependent? Do they lose their address with multiple passages? Caplan: In our lab we try to keep all cells at low passage. That’s what we believe will be clinically relevant in terms of keeping problems down to a minimum. Martin: Apart from OCD, are you also using this technique for osteochondral lesions, or just chondral lesions? Has anyone ever used your approach for osteochondral lesions? Lindahl: Not really. There are others who are using it for osteochondral lesions. We have tried to limit the treatment to the classical OCD group. Martin: Which fraction of patients injured at the cartilage site have an osteochondral injury as opposed to a purely chondral one? Lindahl: If you look at the fraction of our treated patients with OCD, it is around 15%. If you look at this other group, where there are osteochondral injuries, most of them are due to an osteochondral defect. Caplan: There’s a fundamental question, particularly for those who are interested in both bone and cartilage: since there are 7000 procedures being done, I’m sure that osteochondral lesions have been subjected to this technology. Is there some general information you could provide us with regarding this more complicated lesion, and whether this technology is perceived to work in this case? Lindahl: What you end up with is a cartilage plug including the bone defect. If you look long term in these patients, the bone is remodelled and the cartilage is reduced. We treat the defect for bone and cartilage with cartilage, which might remodel over the years to bone. Caplan: So do you estimate the total volume and adjust the cell load appropriately? Lindahl: No, the dose is 1 million cells per cm2. This is what we have used irrespective of whether there is also a bond defect. Hunziker: You mentioned the use of di¡erent types of periosteal £aps. Some companies now have marketed acellular £aps for use in autologous chondrocyte transplantation approaches. How do the clinical results compare between these di¡erent types of £aps? Lindahl: The clinical data for the acellular £aps are quite sparse. It looks like they work, though.

Qualitative and quantitative in vivo assessment of articular cartilage using magnetic resonance imaging Elizabeth O’Byrne, Theodore Pellas and Didier Laurent Department of Arthritis Biology, Novartis Institute for Biomedical Research, 556 Morris Avenue, Summit, NJ 07901-1398, USA

Abstract. The molecular organization and biochemical composition that give cartilage the viscoelasticity necessary for load distribution also convey unique magnetic resonance (MR) properties. In that context, MR imaging has the potential to detect cartilage degeneration and regeneration. Magnetization transfer (MT) imaging probes the exchange of magnetization between the bulk water pool and the water pool bound to macromolecules such as collagen and hence MT may be applied for evaluation of collagen integrity. In addition, Gd(DTPA)2-induced T1 changes have been proposed as a surrogate marker of proteoglycan (PG) loss based on the principle that the paramagnetic agent Gd(DTPA)2 penetrates cartilage to an equilibrium concentration inversely proportional to the negative charge density (i.e. the PG concentration). Results obtained in vivo from MT and Gd(DTPA)2-enhanced MRI acquisitions on the goat knee showed early signs of biochemical changes in response to a papain injection. A dose-dependent e¡ect of papain was observed with both approaches over a wide range of PG depletion (i.e. T1 measurement) and collagen damage (i.e. MT measurement) as con¢rmed with post-mortem biochemistry and histology. Development of MRI protocols for non-invasive assessment of cartilage will facilitate diagnosis and monitoring of treatment e⁄cacy in the clinic. 2003 Tissue engineering of cartilage and bone. Wiley, Chichester (Novartis Foundation Symposium 249) p190^202

Magnetic resonance properties of cartilage Magnetic resonance imaging (MRI) is most frequently based on atomic nuclei with spin properties, such as the hydrogen proton of water molecules in tissues and £uids. Hence the high water content, or proton density, of articular cartilage forms the basis for the MRI signal in this tissue. Since the environment of water in the extracellular matrix dictates both biomechanical and MR properties of cartilage, proton MRI has also the potential to monitor non-invasively the 190

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physical properties of tissue-engineered cartilage in clinical trials. In search of surrogate markers that would better assess cartilage degradation and regeneration, we have developed special MRI techniques for improving signal quality and speci¢city for the macromolecular content of cartilage. This overview is aimed at introducing the reader to such new approaches. In addition to protons, several other nuclei (i.e. 13C, 23Na) in cartilage possess MR spin properties which are altered when the matrix is degraded (Jelicks et al 1993). However hardware limitations and inherent di⁄culties to the spin physics make them less suitable for biological applications. When placed into a magnetic ¢eld of the MR scanner these nuclei align in the direction of the magnetic ¢eld (B0). To produce a change in the energy state of the spin population, an oscillating magnetic ¢eld or radio frequency (RF) pulse (B1) is applied to the sample from a transmitter/receiver coil oriented perpendicular to B0. Following activation, while the RF pulse is turned o¡, the protons radiate electromagnetic energy that can then be observed as the nuclear magnetic resonance (NMR) signal by using the same antenna close to the tissue. Varying the RF pulses can enhance the contrast between tissues. The rate at which the energized spins lose their excess energy to their immediate environment is called spin-lattice or T1 relaxation. The di¡erence in frequency in the spins of neighbouring protons that are out of alignment with one another is called spin^spin or T2 relaxation. Based on relaxation characteristics of water protons, tissue contrast may be best displayed from T1- or T2-weighted images depending on the purpose of the examination. However, the measurement of T1/T2 relaxation and other MR variables by applying appropriate MRI techniques may o¡er additional new information about the physiology and molecular structure of tissues such as cartilage.

MRI measurement of cartilage thickness and volume In two-dimensional radiographs, cartilage thickness measured as joint space width has been used as a parameter indicating pathology and disease progression (Buckland-Wright et al 1995). In addition to its non-invasiveness, MRI is capable of acquiring cross-sections in any plane detecting all the tissues in the joint. Rapid pulse sequences that involve fat saturation usually meet large agreement among radiologist since a good cartilage/bone contrast can be obtained (Peterfy 1999). Speci¢c contrast agents are also widely used especially when looking at focal damages. Meanwhile MRI-determined cartilage volume provides a direct measure of tissue growth, adaptation and tissue loss (Eckstein et al 1998). Threedimensional MRI may indeed be less prone to errors resulting from malpositioning and more speci¢c information can be obtained on disease status such as di¡erentiation between femoral versus tibial loss (Burgkart et al 2001).

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However, volume assessments are prone to inaccuracies with respect to the actual cartilage loss as both weight-bearing and non-weight-bearing regions are often taken into consideration. This is why cartilage thickness maps may provide better insights into the location of cartilage lesions and progression (Peterfy 1999). Since the ultimate goal for a MRI method is to quantify the extent and severity of cartilage abnormality, alternative ways of imaging far more sensitively for early diagnosis are necessary. Potentially, animal research is more demanding than clinical applications; for instance, animals require MR images to be obtained with a high in-plane resolution since structures such as articular cartilage rarely exceed 1 mm thickness (e.g. dog, goat). Especially for investigations on the early stage of osteoarthritis (OA), conventional MRI is of limited value as macroscopic signs of cartilage degeneration may not yet be visible. As one illustration of these new developments, high-resolution MRI was applied to the rabbit knee to measure in vivo cartilage swelling at the early stage of experimental OA (Calvo et al 2001). However, cartilage thickness in the articular region of the rabbit knee typically is around 0.5 mm. The accurate determination of a focal increase in cartilage volume less than 10% of the whole volume therefore is, of course, limited by the pixel size in the crosssection plane. A rather large pixel size as used in the study above (195 mm2 or 3 pixels across thickness) most likely is beyond the detection limit; hence, it is di⁄cult to use this imaging technique to demonstrate subtle changes of cartilage thickness as well as potential bene¢cial e¡ect of novel therapies. These limitations emphasize the need for methods not restricted to inspection of the cartilage morphology, but to provide non-invasive assessment of biomechanical/ biochemical functional properties.

Utilization of magnetization transfer imaging to assess cartilage collagen The use of magnetization transfer (MT) has been suggested to measure cartilage collagen from the ratio of free water protons and restricted motion protons in the macromolecule (Kim et al 1993, Tyler et al 1999). From a practical point of view, MT data are acquired by applying a weak source of RF energy o¡-resonance from the frequency of freely mobile water; the signal of water in contact with macromolecules is then selectively saturated and suppressed as a result of the constant exchange between both pools of water. In theory, the degree of MT saturation achieved by a polymer is proportional to its concentration, a⁄nity to water, and degree o¡ cross-linking. Articular cartilage exhibits very high saturation of signal (Kim et al 1993). This e¡ect is caused in part by the crosslinked collagen network, which supports the hypothesis that MT measurements may be used to identify the initial stages of cross-link disruption and later loss of


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FIG. 1. MT image from a goat knee using 50 ms saturation pulse, Ms/Mo ¼ 0.63, MTR ¼ 0.37.

collagen from cartilage during degeneration or, alternatively, to monitor the formation of new type II collagen network during repair (Tyler et al 1999). In our laboratory, MT imaging was applied to investigate (1) the role of collagen concentration on the saturation transfer, (2) the in£uence of collagen type and the exact contribution of proteoglycans (PG) to the MT e¡ect, (3) the e¡ect of enzyme treatments on articular cartilage explants and (4) its in vivo applicability to the goat knee (Laurent et al 2001a). The goat was chosen because of its similarity to human anatomy and its relatively thick articular cartilage (1^2 mm) in the knee. Sensitivity of the MT measurement was assessed in goats that were injected with various amounts of papain (30^390 units/goat) into the knee the day prior to MRI. MRI experiments were conducted in a 3-T, 60 cm magnet (Bruker Medical Inc., Billerica, MA). Two-millimetre thick slices were obtained using a 2D gradientrecalled standard sequence with an in-plane resolution of 312 mm2. Pulse parameters were optimized for a good contrast between cartilage and surrounding structures in the knee along within a reasonable time of acquisition. The same pulse parameter set was applied to all three steps of the study, e.g. in vitro, ex vivo and in vivo measurements. For measurements on the live goat, the position of the coronal section was adjusted from a previous series of sagittal and transverse scout images to depict the weight-bearing region of cartilage at the medial condyle. Typically, a reference proton image was ¢rst obtained with the MT pulse sequence but zero amplitude of irradiation RF power for the saturation pulse (Fig. 1). Then the measurement was repeated with e¡ective RF power for the saturation pulse. The amount of MT was quanti¢ed by calculating the Ms/M0 ratio or MTR¼(M0^Ms)/M0 which, refers to signal intensities before (M0) and after (Ms) saturation. With a MT pulse applied for a total of 50 ms at 1 kHz o¡-resonance, approximately 30% signal attenuation (MTR¼0.30+0.02, n ¼6) was measured

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FIG. 2. MT dose^response as measured 24 h after intra-articular injection of papain in the goat knee. Reprinted with permission from Laurent et al (2001a).

in articular cartilage on the femoral condyles. When applied to the papain-injected knee, a dose^responsive decrease in the MTR was observed (Fig. 2). Our in vitro data obtained in articular cartilage explants indicated that the in£uence of other macromolecule structures is minimal since trypsin-induced PG depletion did not alter the MT e¡ect. This is in agreement with a previous study (Kim et al 1993) where the authors observed that cartilage depleted of PG exhibited the same degree of MT e¡ect as untreated samples. On the other hand, the MTR value showed a high variability in control goats (0.24^0.37). This may be a consequence of the pressure applied in the weight-bearing area of articular cartilage as would an inverse MTR^body weight correlation suggest. These data were found consistent with recent data obtained on normal volunteers whose lowest values of MT coe⁄cient were measured in the medial cartilage where compression forces are the greatest as opposed to the lateral and patellar cartilage (Hohe et al 2000). This led to the hypothesis that, as a result of pressure, free water is extruded from cartilage, which in turn may lower the relaxation time T1. Since MT not only causes a decrease in net magnetization but also shortening in the T1 relaxation time of water (Wolf & Balaban 1989), the ¢nal MTR measurement may then be a¡ected as well. To circumvent this and prevent misinterpretation of MT data, although it is slightly more time-consuming (15 min versus 5 min) we suggest that a determination of the magnetization exchange rate be added to the MTR measurement as it may re£ect collagen integrity in a more reliable manner. Indeed, by minimizing the T1 e¡ect, the determination of the rate constant k may show more sensitive in terms of measuring changes of collagen content. At this point it should be emphasized that, while no more than 5% change of collagen


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content is typically encountered in osteoarthritic tissue, the variability of MTR observed in vivo may be due to structural changes in collagen rather a change in collagen concentration. Focal lesions in cartilage may be di⁄cult to detect using conventional MRI techniques due to limitation in spatial resolution leading to partial volume e¡ects. This is of importance considering that in some cases, OA is diagnosed as cartilage defects at the surface (Hodler et al 1992). In this respect, MT imaging can also be employed for improved contrast and consequent better delineation of articular structures (Balaban & Ceckler 1992, Vahlensieck et al 1994). Speci¢cally the di¡erentiation of cartilage and adjacent meniscus may be improved following o¡-resonance irradiation (Peterfy et al 1994, Adler et al 1996). Utilization of contrast MRI to assess proteoglycan and related charge density of cartilage Relaxation enhancing agents with paramagnetic properties are widely used as a clinical modality to provide adequate discrimination between tissue structures. However, other applications of such contrast agents recently emerged. As one of them, the gadolinium-enhanced MRI technique is now viewed as a possible way to provide a surrogate marker of PG depletion in cartilage (Bashir et al 1996). When administered at the right dose, usually through an i.v. injection, gadolinium diethylenetriamine penta-acetic acid [Gd(DTPA)2, Magnevist1, Berlex Imaging] is particularly e¡ective at accelerating T1 relaxation of tissues where it accumulates. In those regions, a bright signal appears if using the adequate pulse sequence (e.g. T1-weighted MRI). The application of gadolinium to monitor PG is based on the principle that negatively charged Gd(DTPA)2 penetrates cartilage to an equilibrium concentration inversely proportional to the ¢xed charge density (FCD) of cartilage that depends on PG concentration (Allen et al 1999). Promising results have shown that this technique can be used in a clinical setting as a diagnostic tool with the ability to monitor cartilage molecular composition, and hence to visualize early stages of degeneration (Gray et al 2001). In vivo clinical results have demonstrated a good correlation between cartilage T1 in the presence of Gd(DTPA)2 and ex vivo histological data (Bashir et al 1999). However, while all the patients in the previous study underwent knee replacement surgery, the question still remains as to whether, based on such an approach, OA can be diagnosed at an earlier stage of the disease. In routine clinical use, nondestructive biochemical analysis of cartilage may provide in the future surrogate markers of cartilage degeneration that should help develop e¡ective therapy. In our laboratory, we used the same MRI approach to look at whether the enzymatic treatment of the goat knee with varying amounts of papain would result in a Gd(DTPA)2-induced T1 decrease of articular cartilage in a graded

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FIG 3. Gadolinium-enhanced T1 images of control and papain-injected goat knees.

fashion (Laurent et al 2001b). Partial to total PG depletion was induced by an intraarticular injection of 30^390 units of papain the day prior to MRI. T1 calculated maps were obtained by using an inversion-recovery turbo-spin echo pulse sequence. After baseline measurement of the T1 value in the weight-bearing cartilage area, each goat received a 0.2 ml/kg dose of Gd(DTPA)2 as an i.v. infusion. Time-course variation of T1 within the cartilage was then followed for the next 3^4 h. In untreated goats, normal cartilage T1 at baseline was 1884245 ms (n ¼4). In the same goats, there was Gd(DTPA)2 penetration visible near the cruciate ligaments, but the T1 of the weight-bearing cartilage was only slightly a¡ected, with a maximum 20% reduction (1468182 ms, n ¼4). In the goat knees treated with 30, 100 and 390 units of papain, Gd(DTPA)2 penetration involving nearly the whole cartilage after 2 h (Fig. 3) resulted in a T1 decrease to 1346150 (n ¼4), 888157 (n ¼3) and 480117 ms (n ¼2), respectively. An excellent correlation was observed between the percentage PG depletion as determined biochemically post-mortem and the relative T1 decrease (r ¼0.85). Evidence for partial-to-complete PG depletion in this area could also be demonstrated from histology with safranin O and toluidine blue staining. Our results have shown for the ¢rst time in an in vivo model of cartilage degeneration the relationship between the extent of PG loss and the Gd(DTPA)2-induced T1 decrease. A dose-dependent e¡ect of papain was observed from minimal to almost 100% PG depletion as con¢rmed with necropsy. Based on these preliminary data, we now intend to monitor gadolinium uptake during the development of focal surgically created cartilage lesions in goat knees.


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Future MRI approaches to monitor cartilage degeneration and regeneration Because of the pivotal role of proteoglycan for cartilage function, new applications of MRI are focusing on proteoglycan. In addition to reducing FCD, another consequence associated with the loss of PG is a concomitant loss of Na+ counterions. Signal intensity in 23Na MRI can be used to measure the Na+ concentration in articular cartilage. Subsequently, the Na+ concentration can be used to calculate FCD. 23Na MRI protocols have been applied to quantify FCD of articular cartilage of human volunteers in vivo (Shapiro et al 2002). However, hardware limitations especially still make this technique di⁄cult to apply for routine applications. Another approach to monitor PG using proton MRI is based on spin lattice relaxation in a rotating frame of spins under the in£uence of a radio-frequency ¢eld T1r(T1-rho). PG depletion induces changes in T1r-relaxation and dispersion in articular cartilage (Akella et al 2001). T1rimages have been collected using a clinical 1.5 T MRI scanner (Duvvuri et al 2001). In preliminary studies, T1rweighted images had better signal di¡erence:noise ratios than T2-weighted images of the knee joint. T1r relaxation and dispersion show promise for detecting cartilage abnormalities. Re¢nement of proton MRI techniques will increase the sensitivity of measurements of disruption collagen matrix and PG content. MRI is exquisitely suited to follow biophysical properties of tissue engineered cartilage in a speci¢c articular lesion. Animal studies that compare MRI with biochemical and histological data are still required to validate MRI as a surrogate to follow cartilage regeneration in clinical trials. Application of MRI will allow development of standard imaging protocols to set benchmarks for cartilage regeneration.

References Adler RS, Swanson SD, Doi K, Craig JG, Aisen AM 1996 The e¡ect of magnetization transfer in meniscal ¢brocartilage. Magn Reson Med 35:591^595 Akella SV, Regatte RR, Gougoutas A et al 2001 Proteoglycan-induced changes in T1 rhorelaxation of articular cartilage at 4T. Magn Reson Med 46:419^423 Allen RG, Burnstein D, Gray ML 1999 Monitoring glycosaminoglycan replenishment in cartilage explants with gadolinium-enhanced magnetic resonance imaging. J Orthop Res 17:430^436 Balaban RS, Ceckler TL 1992 Magnetization transfer contrast in magnetic resonance imaging. Mag Reson Q 8:116^137 Bashir A, Gray ML, Burstein D 1996 Gd-DTPA2 as a measure of cartilage degradation. Magn Reson Med 36:665^673 Bashir A, Gray ML, Hartke J, Burstein D 1999 Nondestructive imaging of human cartilage glycosaminoglycan concentration by MRI. Magn Reson Med 41:857^865

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Buckland-Wright JC, MacFarlane DG, Lynch JA, Jasani MK, Bradshaw CR 1995 Joint space width measures cartilage thickness in osteoarthritis of the knee: high resolution plain ¢lm double contrast macroradiographic investigation. Ann Rheum Dis 54:263^268 Burgkart R, Glaser C, Hyhlik-Dˇrr A, Englmeier KH, Reiser M, Eckstein F 2001 Magnetic resonance imaging-based assessment of cartilage loss in severe osteoarthritis: accuracy, precision, and diagnostic value. Arthritis Rheum 44:2072^2077 Calvo E, Palacios I, Delgado E et al 2001 High-resolution MRI detects cartilage swelling at the early stages of experimental osteoarthritis. Osteoarthritis Cartilage 9:463^472 Duvvuri U, Charagundla SR, Kudchodkar SB et al 2001 Human knee: in vivo T1(rho)- weighted MR imaging at 1.5 T preliminary experience. Radiology 220:822^826 Eckstein F, Winzheimer M, Westho¡ J et al 1998 Quantitative relationships of normal cartilage volumes of the human knee joint assessment by magnetic resonance imaging. Anat Embryol (Berl) 197:383^390 Gray ML, Burstein D, Xia Y 2001 Biochemical (and functional) imaging of articular cartilage. Semin Musculoskelet Radiol 5:329^343 Hodler J, Berthiaume MJ, Schweitzer ME, Resnick D 1992 Knee joint hyaline cartilage defects: a comparative study of MR and anatomic sections. J Comput Assist Tomogr 16:597^603 Hohe J, Faber S, Stammberger T, Reiser M, Englmeier K, Eckstein F 2000 A technique for 3D in vivo quanti¢cation of proton density and magnetization transfer coe⁄cients of knee joint cartilage. Osteoarthritis Cartilage 8:426^433 Jelicks LA, Paul PK, O’Byrne E, Gupta RK 1993 Hydrogen-1, sodium-23, and carbon-13 MR spectroscopy of cartilage degradation in vitro. J Magn Reson Imaging 3:565^568 Kim DK, Ceckler T, Hascall VC, Calabro A, Balaban RS 1993 Analysis of water-macromolecule proton magnetization transfer in articular cartilage. Magn Reson Med 29:211^215 Laurent D, Wasvary J, Yin J, Rudin M, Pellas T, O’Byrne E 2001a Quantitative and qualitative assessment of articular cartilage in the goat knee with magnetization transfer. Magn Reson Imaging 19:1279^1286 Laurent D, Wasvary J, Yin J et al 2001b In vivo assessment of proteoglycan loss in the articular cartilage of the goat knee with gadolinium-enhanced MRI after papain injection. Proc 9th Intl Soc Magn Reson Med, Glasgow, Scotland, p 37 Peterfy CG 1999 Applications of MRI for evaluating osteoarthritis. In: Tanaka S, Hamanishi C (eds) Advances in osteoarthritis. Springer-Verlag, Tokyo, p 74^92 Peterfy CG, Majumdar S, Lang P, Van Dijke CF, Sack K, Genant HK 1994 MR imaging of the arthritic knee: improved discrimination of cartilage, synovium, and e¡usion with pulsed saturation transfer and fat-suppressed T1 weighted sequences. Radiology 191:413^419 Shapiro EM, Borthakur A, Gougoutas A, Reddy R 2002 23Na MRI accurately measures ¢xed charge density in articular cartilage. Magn Reson Med 47:284^291 Tyler JA, Hall LD, Watson PJ 1999 Development of quantitative magnetic resonance imaging for assessment of cartilage damage and repair in vivo. In: Tanaka S, Hamanishi C (eds) Advances in osteoarthritis. Springer-Verlag, Tokyo, p 93^106 Vahlensieck M, Dombrowski F, Leutner C, Wagner U, Reiser M 1994 Magnetization transfer contrast (MTC) and MTC-subtraction: enhancement of cartilage lesions and intracartilaginous degeneration in vitro. Skeletal Radiol 23:535^539 Wol¡ SD, Balaban RS 1989 Magnetization transfer contrast (MTC) and tissue water proton relaxation in vivo. Magn Reson Med 10:135^144

DISCUSSION Barry: Thank you for sharing some outstanding data. The correlation between the proteoglycan depletion and the decrease in the T1 signal is very useful. As we have


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devised cell therapy applications for treating joint injury, we have always wondered about the potential e¡ects that Gd might have on cells. Can you point us to any in vitro studies that have looked at the potential e¡ects of Gd on the viability or function of chondrocytes or mesenchymal stem cells? O’Byrne: We haven’t really done that in vitro. Gd has been used for many years clinically. It was ¢rst used to look at stroke and brain applications. It has been used multiple times in the same patients. We haven’t looked at chondrocytes or MSCs, but that is a good idea. Martha Gray and colleagues at MIT have used Gd for imaging tissue-engineered cartilage in vitro. Barry: Would direct intra-articular delivery of the Gd be more e¡ective than infusion two hours earlier which you currently carry out? O’Byrne: When we let Gd di¡use against a negative charge gradient in cartilage we can measure di¡erences in the rate of T1 change due to proteoglycan retarding Gd. When we do the intra-articular injection of Gd, there is such a bolus it is hard to look at it as a rate change. Instead, we have to look at the total change. We could see the in£ammation, Gd given intravenously goes to an in£amed joint where the joint capsule is permeable; the T1 changes are faster in synovial £uid than in non-in£amed joint. With the intra-articular injection you overpower that di¡usion. Huckle: Is the instrument you are using a clinically relevant one in terms of resolution, or is this higher resolution than we get in the clinic? O’Byrne: It is clinically relevant because it is used for brain injury, but it is 3 T. Most clinical trials will be with 1.5 T machines. As we develop methods, we need to translate them into the lower powered machines. The 3 T imagers are now available. Hunziker: I have a question relating to the Gd di¡usion into the articular cartilage in the control groups, following the intravenous injection of this material. Were you able to estimate what proportion of Gd di¡used from the subchondral bone plate into the articular cartilage, and what proportion di¡used into it from the synovial side? O’Byrne: We cannot calculate the proportion. We very rarely observe it coming from the subchondral bone plate soon after i.v. injection. After Gd is given intravenously we observe changes in MR images near the insertion point of the cruciate ligaments. The access of the Gd into the knee joint does not appear to be even. Hunziker: If I recall correctly Martha Gray estimated that about 10% of the Gd di¡uses into the articular cartilage from the subchondral bone plate. There is thus an additional di¡usional contribution from this side. O’Byrne: We look from an early point, and at ¢rst we don’t see it come from the subchondral bone, although it does eventually. We de¢nitely do see the Gd around that ligament insertion point before it comes evenly into the joint

200

DISCUSSION

£uid. Note that the goat knee is constrained in an extended position during imaging. Caplan: I’m taken aback by the comment you just threw out about ligament insertion points being dominant with regard to the di¡usion of small molecules. Are there some measurements of the relative contribution of this to the synovial environment when we compare subchondral plate, synovial membrane, and ligament insertion sites? Lohmander: Not that I am aware of. The assumption has been that most of it comes from the synovial tissue. Perhaps a small portion of the nutrition to the cartilage might come from the subchondral plate. But ligament insertions have largely been ignored. O’Byrne: This is a qualitative phenomenon. When we looked at joint levels of pharmaceuticals it was usually proportional to the plasma levels which supports the idea that molecules come via synovial tissue. Grodzinsky: Could you share some hunches with us? From your description it sounds like the timing is critical. If I try to imagine the Gd di¡using into the cartilage, the longer you wait the bigger the di¡erence is between the region with high density of aggrecan and regions where it is missing. But if you wait too long, there will probably be some washout. There will be an optimal time period. So in designing a clinical trial, how does one try to standardize with patient weight? O’Byrne: It is going to be di⁄cult. Now that we are going to start applying this imaging protocol to focal cartilage lesions changes, there might be something like two hours for optimal change. A lot of information will come when Gd imaging is applied to cartilage repair, when we look at the same area of interest over time. If we standardize the conditions, we can compare the results from the ¢rst day, two weeks later and then four weeks later, for example. This might be the more important issue. Caplan: Why is it disadvantageous to introduce very small doses of Gd into the joint to do acute imaging, as opposed to systemic introduction of Gd? If you do small doses then the toxicology you mentioned is probably not relevant. O’Byrne: Toxicology is not a problem. One of the reasons we chose Gd is because it has been used in the clinic for a long time. The problem is that if you give the bolus, it is uneven wherever you give it. We haven’t tried it much in the goats, but when we did it in rabbits it caused a massive change of T1 in the whole joint, and it was hard to see any subtle changes. This is because it was a fast di¡usion. The problem with Gd is kinetics. It is a surrogate that is based on a kinetic change, not an absolute change. Human cartilage is much thicker than rabbit cartilage; so clinically intra-articular injection of contrast agents may be useful. Caplan: Has MR been used for any repair circumstances? I know it has been used diagnostically, but has it been used in clinical follow-up?


201

Lohmander: I am aware of two pilot-scale studies where this Gd technique has been used for this purpose, with divergent interpretations of the data. Listening to your presentation, it seems to me that at any given variation of the technique you used, the dynamic range of your read-out is somewhat limited. This makes me think that if the dynamic range of the changes in the joints of di¡erent patients exceeds that, you would have to adjust the window by time for di¡usion, or exercise or not exercise, for example. This would complicate the interpretation. O’Byrne: This is a problem we will have with almost any contrast agent. This is why if we can develop techniques where we are actually looking at the physical and MR properties of cartilage, it will be much better. Trippel: What is preventing you from doing what you described as being the more desirable approach? You mentioned resolution of sodium as being a problem. Is this not resolvable in the foreseeable future? What would be even better than sodium imaging? O’Byrne: Sodium was ¢rst applied within a clinical situation within the last year. Sodium signal is proportional to ¢xed charge density due to negative charges on proteoglycan. One of the most promising new proton approaches out there now is T1r (T1rho) from Ravinder Reddy’s lab at University of Pennsylvania. It will be up to the physicists to ¢nd some set of gradients and pulses where we can have something that is dependent on proteoglycan. Clinical MRI is based on protons in water (1H2O). Since proteoglycan changes the physical properties of bound water there is optimism that proton-based imaging will someday re£ect proteoglycan in cartilage. Trippel: In 10 years do we still want to be looking at protons, or should we be looking at something else? O’Byrne: I think we still will be looking at protons, because this is the most abundant of the MR signals in tissues due to water. Hardingham: In your model system you are depleting the matrix in various ways. In many of the applications we are thinking of, they will have been MR imaged using standard techniques. We obviously see defects in normal MRI. How does that image compare with your procedure as a follow-up? How much more sensitive do methods need to be than those currently used? O’Byrne: With current techniques we can see joint morphology. MRI can detect, for example, whether surgery is successful in reattaching a ligament; to follow whether an implant stayed in we ¢rst need more MRI data on the implant in vitro to compare with adjacent cartilage. If you want to assess whether or not you have a functional collagen matrix then you need high resolution and MT data. Hardingham: Anders Lindahl, if your patients are looked at with MRI a year after the procedure, can you see any evidence of the procedure? Lindahl: We have done MRI on a number of our patients. What we see is that there is some type of tissue at the repair area. But we cannot judge whether it is

202

DISCUSSION

hyaline or ¢brous. We do the studies at the patients’ home clinics, so the MRI quality varies. But there are skilled radiologists who focus on cartilage and are able to produce very nice images. Hardingham: So if Liz O’Byrne’s approach could discriminate between ¢brous and hyaline tissue, this would be very desirable.

Hyaluronan-based sca¡olds (Hyalograft1 C) in the treatment of knee cartilage defects: preliminary clinical ¢ndings Alessandra Pavesio, Giovanni Abatangelo*, Anna Borrione, Domenico Brocchetta{, Anthony P. Hollander}, Elizaveta Kon{, Francesca Torasso, Stefano Zanasi} and Maurilio Marcacci{ Fidia Advanced Biopolymers srl, Via Ponte della Fabbrica 3/B, 35031 Abano Terme (PD), *Institute of Histology and Embriology, Faculty of Medicine, University of Padova, Viale G. Colombo 3, 35121 Padova, {Istituto Clinico S. Ambrogio, Reparto di Ortopedia, Via Faravelli 16, 20149 Milano, {Istituti Ortopedici Rizzoli, Laboratorio di Biomeccanica, Via Di Barbiano 1/10, 40136 Bologna, }Policlinico di Monza, Reparto di Ortopedia, Via Amati 111, 20052 Monza, Italy and }University of Bristol Academic Rheumatology, Avon Orthopaedic Centre, Southmead Hospital, Bristol BS10 5NB, UK

Abstract. Hyalograft1 C is an innovative tissue-engineering approach for the treatment of knee cartilage defects involving the implantation of laboratory expanded autologous chondrocytes grown on a three-dimensional hyaluronan-based sca¡old. This technique has recently been introduced into clinical practice, with more than 600 patients treated so far. Because no periosteal coverage is required to keep the graft in place, surgical time and morbidity are reduced, and handling of the graft is much simpler than currently available autologous chondrocyte implantation techniques. The safety pro¢le of the treatment appears positive, with a limited number of adverse events reported. Here we discuss the clinical, arthroscopic and histological results from a cohort of 67 patients treated with Hyalograft1 C (mean follow-up time from implantation of 17.5 months). Results are reported based on four endpoints: patients’ subjective evaluation of knee conditions (97% of patients improved) and quality of life (94% improved), surgeons’ knee functional test (87% of patients with the best scores), arthroscopic evaluation of cartilage repair (96.7% biologically acceptable) and histological assessment of the grafted site (majority of specimens hyaline-like). The positive clinical results obtained indicate that Hyalograft1 C may be a viable therapeutic option for the treatment of acute cartilage lesions. 2003 Tissue engineering of cartilage and bone. Wiley, Chichester (Novartis Foundation Symposium 249) p 203^217

Full-thickness lesions in knee articular cartilage remain a signi¢cant problem in medical practice, with no treatment method providing consistent acceptable 203

204

PAVESIO ET AL

long-term clinical results to date. At present, there are no e¡ective pharmacological alternatives to orthopaedic surgery. Arthroscopic lavage and debridement may o¡er temporary relief of pain, but no prospect of long-term results (Messner & Maletius 1996). Bone marrow stimulation techniques, including abrasion arthroplasty, subchondral drilling, microfracture and spongialization typically promote the development of a repair tissue of ¢brocartilage or ¢brous nature, lacking the mechanical characteristics of articular hyaline cartilage (Bert & Maschka 1989, Ogilvie-Harris & Fitsialos 1991, Johnson 1991, Dandy 1991, Rodrigo et al 1994). Osteochondral autograft transplantation (Hangody et al 1998) is usually restricted to small defects (52 cm2) due to donor site morbidity when lesions are larger. Autologous periosteum or perichondrium grafts can produce hyaline cartilage, but they are limited by the amount of tissue available for grafting and the tendency towards endochondral ossi¢cation of the newly formed tissue (Homminga 1997, Hoikka et al 1990, Sandelin 1997, Bouwmeester et al 1997). Autologous chondrocyte implantation (ACI) is a novel biological approach, introduced in Sweden in 1987, for the treatment of large full thickness chondral defects of the knee. It is based on the implantation of a suspension of dedi¡erentiated cultured autologous chondrocytes beneath a tightly sealed periosteal £ap (Brittberg et al 1994). The clinical experience with this technique now exceeds 10 years, with more than 3900 patients treated. However, despite the promising clinical results (Peterson et al 2000) the use of ACI carries a number of limitations, essentially correlated with the complexity of the surgical procedure. Additionally, as the cells are grown in a liquid suspension, lack of chondrocyte di¡erentiation and limited in vitro matrix production are a concern. Recently, the use of three-dimensional sca¡olds has been shown to favour the maintenance of a chondrocyte di¡erentiated phenotype. Consequently, e¡orts are now focused towards a tissue-engineered approach, which combines laboratory grown cells with appropriate three-dimensional biocompatible sca¡olds for the purpose of generating new tissues or tissue equivalents (Hendrickson et al 1994, Freed et al 1993, Stanton et al 1995, van Susante et al 1995). Hyaluronan in cartilage reconstruction Hyaluronan is a naturally occurring, widely distributed and highly conserved glycosaminoglycan, which plays an important role in many biological processes such as hydration, proteoglycan organization and cell di¡erentiation; it is an important matrix component of native cartilage tissue (Chen & Abatangelo 1999). Hyaluronan, in a highly puri¢ed form, has found extensive applications in clinical practice (Abatangelo & O’Regan 1995, Goa & Ben¢eld 1994). However,

HYALURONAN SCAFFOLDS IN CARTILAGE REPAIR

205

its water solubility and short residence time in the tissue along with its poor ductility limit the opportunities for further applications. HYAFF111 is a semi-synthetic derivative of hyaluronan, obtained by chemical esteri¢cation of the native molecule with benzyl alcohol. It may be processed to obtain a variety of devices with di¡erent physical forms such as microspheres, membranes, ¢bres, and knitted and non-woven materials. Extensive biocompatibility studies have demonstrated that HYAFF111-based biomaterials are intrinsically safe (Campoccia et al 1998). A three-dimensional HYAFF111 non-woven ¢brous mesh (¢bre diameter¼10 mm, ¢bre density¼120 g/m2), in in vitro and in vivo experimental settings, was proven to be e¡ective in supporting the ingrowth of chondrocytes, which remain highly viable and capable of di¡erentiation. De-di¡erentiated chondrocytes seeded into the HYAFF1 sca¡old produce a characteristic extracellular matrix, rich in proteoglycans, and re-express markers typical of hyaline cartilage, such as collagen II and aggrecan (Aigner et al 1998, Brun et al 1999, Grigolo et al 2002). When implanted in full-thickness defects of the femoral condyle in rabbits, chondrocytes cultured in the HYAFF1 matrix regenerated a cartilage-like tissue (Grigolo et al 2001, Solchaga et al 2000). Moreover, it has been hypothesized that HYAFF111, when used in conjunction with mesenchymal progenitor cells, can be both ‘inductive and informational’ with respect to the transplanted cells, leading to an in situ chondrocyte di¡erentation of mesenchymal cells followed by later repair processes such as angiogenesis, endochondral bone formation and neo-cartilage integration promoted by the release of hyaluronan fragments (Solchaga et al 2000). These preclinical ¢ndings have supported the use of HYAFF111 sca¡old for autologous chondrocyte implantation in a clinical setting.

Clinical experience with Hyalograft1C Hyalograft1 C (Fidia Advanced Biopolymers, Abano Terme, Italy), a tissueengineered graft consisting of autologous chondrocytes grown on a threedimensional HYAFF111 sca¡old in a non woven con¢guration, was introduced into clinical practice in 1999. Autologous chondrocytes are isolated from a patient’s cartilage biopsy harvested arthroscopically from a non weight-bearing area in the knee. Chondrocytes are expanded in vitro as previously described (Brittberg et al 1994). Subsequently the cells are cultivated into the HYAFF111 sca¡old for 14 days (Scapinelli et al 2002). Re-implantation at the lesion site occurs via a limited arthrotomy under regional or general anaesthesia, in a tourniquet controlled bloodless ¢eld.

206

PAVESIO ET AL

Articular cartilage lesions are prepared with curettage debriding to the best cartilage available. Border stabilization is performed and subchondral perforation is limited in order to minimize bleeding at the site of implantation. Hyalograft1 C, trimmed if necessary to ¢t the defect, is placed into the lesion. In the majority of cases, no graft ¢xation is required; however, depending on the size and location of the defect, ¢brin glue and/or sutures may be used to keep the graft in place (Zorzi et al 2000). After tourniquet release, the knee is mobilized to con¢rm adherence and stability of the graft prior to standard closure of the arthrotomy. Hyalograft1 C may also be implanted arthroscopically using an appropriate cannula, as proposed by Marcacci et al (2002). Post-surgical joint drainage and compressive bandaging are applied for a minimum of 24 h postoperatively. Immobilization is recommended for 24 h postgrafting, thereafter a postoperative rehabilitation programme begins with passive motion and isometric quadriceps exercises. Articular weight bearing can be applied from the third week and gradually increased thereafter. As of December 2001, 751 cartilage biopsies have been collected from over 40 clinical centres in Italy and in Austria, and 622 patients have been grafted with Hyalograft1 C. The population, 61.4% male and 38.6% female, with a mean age of 37.4 years, was treated for a single lesion in 68.5% of cases and for multiple lesions in the remaining percentage. The majority of the lesions were graded as Outerbridge IV (72.5%) and were localized on the femoral condyle (71%). The lesions were smaller than 4 cm2 in 57.1% of the cases, 4^8 cm2 in 37.6% of cases and greater than 8 cm2 in the remaining 5.3%. Hyalograft1 C safety According to the quality system implemented for the ISO 9002 Certi¢ed Service which supplies Hyalograft1 C, adverse events which are reported from the clinical use of the product are ¢led and evaluated by Fidia Advanced Biopolymers s.r.l. As of December 2001 (n ¼622), no relevant or serious adverse events related to the product had been reported. A total of eight adverse events had been recorded. Three cases of slight postoperative fever were seen within seven days after surgery, but resolved in few days with negative laboratory ¢ndings and no further consequences. One of these cases was also associated with pain and swelling. One case of postoperative haematoma, which resolved spontaneously, was reported. Intra-articular adhesions were seen in 2 patients 12 months after surgery. One patient with a lesion at the medial femoral condyle underwent medial meniscus surgery and tibial osteotomy at the time of implantation. A marked ¢broarthrosis


207

was seen on arthroscopic examination. The second case, treated for a patellar lesion, had patellar alignment concomitant to the biopsy for chondrocyte procurement. The patient did not comply with the recommended post-operative rehabilitation programme and reported a marked sti¡ness. Symptoms resolved after arthroscopic adhesiolysis in both cases. Periosteal hypertrophy was seen in two cases where a periosteal £ap was used to secure Hyalograft1 C. One patient was symptomatic with signi¢cant catching and locking of the knee. In the other, the hypertrophy was seen incidentally at the time of a reoperation for a traumatic anterior cruciate ligament rupture. Both patients experienced symptomatic relief after arthroscopic excision performed 12 and 24 months after implantation respectively. No graft failures, de¢ned as requiring a re-operation to remove the implant or re-implant it, or a procedure that violates the subchondral bone to treat the defect, have been reported.

Pilot clinical studies Initial clinical reports focused on the safety pro¢le and symptomatology of patients treated with Hyalograft1 C and are hereby brie£y reviewed. A normal postoperative period with no serious adverse events related to the product has been reported on the ¢rst series of cases treated with Hyalograft1 C, consisting of more than 100 patients a¡ected by symptomatic chondral lesions of femoral condyle or patella or tibial plateau (Zorzi et al 2000, Schatz 2001a,b, Scapinelli et al 2002). In this cohort of patients, there were no cases of joint infections, necrosis or oedema. Also, no intra-articular adhesions or hypertrophy occurred. In the experience reported by Scapinelli et al (2002), within 21 months of implantation no graft failures were noted. Clinical symptoms such as pain, locking and swelling decreased, as reported by Zorzi et al (2000), starting from one month after grafting and joint functionality improved constantly. Immediate pain relief with good range of motion was also observed in the Austrian clinical experience (Schatz 2001b).

Patient registry: preliminary ¢ndings A patient registry has recently been set up with the aim of establishing an extensive database of patients treated with Hyalograft1 C. The objective is to monitor the progress of the patients and to collect relevant clinical information for evaluating the outcome of the treated lesions. To this end, patients will be monitored for at least 4 years from implantation.

208

PAVESIO ET AL

Data collection is based on a Case Report Form which has been designed taking into account the most recent guidelines of the International Cartilage Repair Society (ICRS) discussed at the 3rd ICRS meeting of 28 April, 2000 in Gothenburg (ICRS 2000). We present here the preliminary ¢ndings on a cohort of 67 patients, treated in 4 Italian clinical centres.

Patient characteristics and evaluations performed This ¢rst series of 67 cases refer to 39 male and 28 females, with a mean age of 36.5 years, a¡ected by chondral defects of the knee caused by trauma (39.4%), osteochondritis disseccans (16.7%) and lesions that are degenerative and/or microtraumatic in nature (43.9%). 70% of the subjects had a single defect. The remaining 30% had a multifocal lesion, with defects localized at di¡erent sites within the knee. The total number of defects treated in this population was 85. The most common defect location was the femoral condyle (86%). The remainder of the defects was localized on the patella (7%) and on the tibial plateau (7%). The defects treated were graded Outerbridge IV in 86% of cases and grade III in the remaining 14%. The mean surface area implanted was 3.8 cm2 (SD¼2.5), with a range of 1.0^12.2 cm2. Most of the patients had undergone previous surgery on the a¡ected knee. Remarkably, 52% of the total population had been previously subjected to cartilage surgery, including debridement, lavage and bone marrow stimulation techniques, which eventually proved unsuccessful. 34% of patients underwent one or more concomitant procedures at the time of Hyalograft1 C grafting. These included high tibial valgus osteotomy and/or patellar realignment osteotomy (in 8.9% of the 67 patients), meniscus and/or ligament surgery (29.8%), and synovectomy (12%). Hyalograft1 C was implanted without any coverage or ¢xation system in 62.7% of cases, whilst ¢brin glue and/or sutures were used in 31.3% of cases. A periosteal £ap was used in only 4 cases, at the very beginning of the clinical experience with this novel approach. A minimally invasive arthroscopic technique for positioning the graft (Marcacci et al 2002) was used in 13.4% of the patients. The average follow-up time for this series of patients was 17.5 months after implantation (SD¼6.3). Patients were asked for a subjective evaluation of the knee symptoms and physical function using the IKDC Subjective Knee Evaluation Form. According to this questionnaire, a higher score represents higher levels of function and lower


209

levels of symptoms. Therefore, a score of 100 is interpreted to mean no limitations on daily living activities or sports and the absence of symptoms. Patients were also asked to evaluate their quality of life using the EuroQol EQ-5D questionnaire (Rabin & de Charro 2001). This is a recognized assessment of health-related quality of life based on self-care, mobility, usual activities, pain/ discomfort and anxiety/depression dimensions. It includes a 0^100 Visual Analogue Scale (EQ-VAS) for a self-rating of the global health state, in which the 100 value represents the best imaginable health state. Baseline conditions data were obtained by asking patients to answer these questionnaires retrospectively for the conditions of their knee and their quality of life after injury but just before Hyalograft1 C implantation, and by a review of their medical records. A knee functional test was performed by the surgeon according to the IKDC Knee Examination Form. The lowest ratings in e¡usion, passive motion de¢cit and ligament examination were used to determine the ¢nal functional grade of the knee (normal, nearly normal, abnormal or severely abnormal). Thirty patients underwent second-look arthroscopy: this was performed as a consequence of an adverse event in four cases and for investigative purposes subsequent to patient’s consent in the remaining cases. Quality of the repair tissue was classi¢ed according to the integration of the graft into the surrounding cartilage (0^4 points), the degree of defect ¢ll (0^4 points), and the macroscopic appearance (0^4 points), giving the best possible defect-repair score of 12 points (Brittberg score, Peterson et al 2000). Twenty-two patients out of the 30 who underwent second-look arthroscopy consented for a biopsy harvesting in the area of graft implantation. Two patients consented to two biopsy harvests at di¡erent time-points after surgery. Processing and analysis of the biopsy specimens obtained were performed by two independent investigators blinded to treatment outcomes using standard histological techniques. Based on criteria of cellularity, cell distribution, matrix composition and collagen type I and II immunolocalization, regenerated cartilage was rated as hyaline-like, ¢brocartilage or mixed tissue. Quantitative biochemical analysis was carried out on some of the specimens as reported elsewhere in this volume (Hollander et al 2003, this volume). Moreover, any adverse event which occurred in this patient population (n ¼67) was recorded. Results Patient’s assessment ofknee conditions and quality of life. The mean IKDC score obtained (n ¼67) was 37.0 (SD¼9.2) at baseline and 78.1 (SD¼17.7) at the follow-up control (P50.01, Student’s t-test, Fig. 1A).

210

PAVESIO ET AL

FIG. 1. Patient’s subjective assessment (n ¼ 67). (A) IKDC Subjective Knee Evaluation: mean score before surgery and at last follow-up. Patients were asked to rate their knee symptomatology, functionality and activity level. High scores represent high level of function and low level of symptoms. Therefore, a score of 100 is interpreted to mean no limitations with daily living activities or sports and the absence of symptoms. Di¡erence between baseline and post-treatment values proved statistically signi¢cant (P50.01, Student’s t-test). (B) EuroQol EQ-5D Visual Analogue Scale (EQ VAS): mean score before surgery and at last follow-up. EQ VAS was used for patient self-rating of the global health state. A value of 100 represents the best imaginable health state. The di¡erence between baseline and post-treatment is statistically signi¢cant (P50.05, Student’s t-test).

Remarkably, 97% of the patients experienced a subjective improvement in knee function and symptoms. Only two patients (3%) experienced a worsening of their knee conditions. 94% of patients experienced an improvement in their quality of life, as assessed by the EQ-VAS. The situation was found to be unchanged in two patients and worsened in two patients (the same subjects for whom a worsening in the subjective knee evaluation scores was also observed). The di¡erence in the mean EQ-VAS score (Fig. 1B), which changed from 59.1 (SD¼17) to 88.1 (SD¼14.1) was statistically signi¢cant (P5 0.05, Student’s t-test). The majority of the improvements was related to mobility (66.7% of patients improved), usual activities (74.2%) and reduced pain/discomfort (86.4%); no patients showed a worsening of their status, except for the pain/discomfort parameter (1.5%). As expected, the majority of patients registered no changes with respect to their baseline conditions when assessed for self-care and anxiety/ depression.


211

FIG. 2. IKDC Knee Examination performed by the surgeon (n ¼ 39). Patient distribution among the four knee functional group grades (normal, nearly normal, abnormal or severely abnormal). The ¢nal functional grade was determined by the lowest ratings in e¡usion, passive motion de¢cit and ligament examination sections of the IKDC Knee Examination Form.

Surgeon’s knee examination. Figure 2 shows the distribution of the percentage of patients displaying the four possible IKDC knee group grades (n ¼ 39). Final grade was normal in 46.2% of patients and nearly normal in 41.0%, resulting in a proportion of 87.2% of patients displaying knee conditions within the two best categories. Only one patient had the worst possible score (severely abnormal); this could have been related to the occurrence of a marked ¢broarthrosis. Macroscopicassessmentofthe repair tissue. The mean repair score for this set of patients (n ¼ 30, mean arthroscopy time from surgery¼12.5 months) was 10.7 (range 3^12). The maximum value of the Brittberg scale was reported in 14 cases (46.7%). Nine patients scored 11, six patients had scores between 8 and 10.Thus, in 29/30 cases the cartilage repair was rated as biologically acceptable (Fig. 3). Only one case, associated to periosteal hypertrophy, was rated as severely abnormal. This graft had a biologically unacceptable appearance with signi¢cant ¢ssuring of the repair tissue and no integration with the surrounding cartilage. Biopsy analysis. Table 1 reports the results of histological analysis, cartilage repair assessment by arthroscopy, clinical patient’s and physician’s scores and the occurrence of adverse events in this group of patients (n ¼22). Average secondlook arthroscopy time post-surgery was 13.9 months.

212

PAVESIO ET AL

FIG. 3. Macroscopic evaluation of cartilage defect repair (n ¼ 30). Quality of cartilage repair: distribution according to the Brittberg score system (Peterson 2000), based on the assessment of the degree of defect ¢ll (0^4 points), graft integration into the surrounding cartilage (0^4 points) and the macroscopic appearance (0^4 points). The overall repair assessment was then classi¢ed as follows: 12 points ¼ Grade I ¼ Normal; 11^8 points ¼ Grade II ¼ Nearly Normal; 7^4 points ¼ Grade III ¼ Abnormal; 3^1 points ¼ Grade IV ¼ Severely Abnormal. For a classi¢cation as biologically acceptable appearance a minimal score of 7 points with at least 3 in Group 1 (degree of defect ¢ll) and 2 in the other groups is necessary. The cartilage repair was scored as biologically acceptable in 29/30 cases (96.7%).

The majority of the biopsies (n ¼14) was rated as hyaline-like, four cases were rated as mixed tissue and four as ¢brocartilage. For reference, Fig. 4 is representative of a typical hyaline-like case, while Fig. 5 shows a typical ¢brocartilage case. According to the Brittberg score, the repair was evaluated as biologically acceptable in all these cases. 18/22 patients scored very high for cartilage repair (i.e. 11 or 12). Correlation between cartilage repair grade and histological appearance was found in 77% of cases (17/22). The data also suggest that longer time points from implantation may be correlated to better histological appearance (see time-dependent improvement in the two patients with both a second- and third-look biopsy; Table 1). Discussion In this cohort of 67 patients, evaluated at a mean follow-up period of 17.5 months, 97% of subjects had improved when assessed for subjective knee symptoms and functionality, 94% when assessed for quality of life and, according to the surgeon’s evaluation, almost 90% of patients showed a normal or nearly normal knee.


213

FIG. 4 Example of a hyaline-like rated biopsy specimen. (A) Safranin O staining. Intense glycosaminoglycan (GAG) deposition is observed, starting from the deeper area of the implant. Here, chondrocyte-like cells are surrounded by lacunae while, on the external layers of the tissue, cells are still undergoing di¡erentiation and cartilage matrix is still remodelling (Case C 405 IT, original magni¢cation 25). (B) Immunolocalization of collagen type II (horse radish peroxidase [HRP] detection). Intense staining was found starting from the inner part of the biopsy, cellularity is moderate or rare in the core while cells are more concentrated on the surface of the specimen (Case C 405, original magni¢cation 25).

The patient’s improvement was further analysed taking into account the following possible predictive factors: concomitant surgery, size of lesions and surgical procedure for implantation. Interestingly, the majority of the patients (66%) did not undergo any other surgical procedures associated with Hyalograft1 C implantation. Associated

FIG. 5. Example of a ¢brocartilage biopsy specimen. (A) Safranin O staining. Weak and uniform GAG staining; at higher magni¢cation ¢brotic organization of the matrix is recognizable (Case C 468, original magni¢cation 25). (B) Collagen type I immunolocalization (HRP detection). Strong reactivity throughout the specimen, high cellularity, cells not encased in the matrix nor surrounded by lacunae (Case C 468, original magni¢cation 100).

3.14 4.91

3.92

2.36

C 218 42 C 323 29

C 384 14

C 076 31

tibial plateau

patella

patella patella

medial femoral condyle medial femoral condyle medial femoral condyle medial femoral condyle medial femoral condyle lateral femoral condyle medial femoral condyle medial femoral condyle lateral femoral condyle medial femoral condyle lateral femoral condyle medial femoral condyle medial femoral condyle medial femoral condyle lateral femoral condyle lateral femoral condyle medial femoral condyle medial femoral condyle

Location

13.9

13

18

12 12

8 12 12 12 12 12 12 12 12 12 13 14 14 15 15 17 17 30

HL/FC

HL

FC FC

HL FC HL/FC HL/FC HL HL HL HL HL HL HL FC HL HL HL HL HL/FC HL

Histological appearance

11.1

12 (BA)

12 (BA)

8 (BA) 8 (BA)

11 (BA) 10 (BA) 11 (BA) 11 (BA) 11 (BA) 12 (BA) 11 (BA) 12 (BA) 12 (BA) 12 (BA) 11 (BA) 12 (BA) 12 (BA) 11 (BA) 12 (BA) 12 (BA) 10 (BA) 12 (BA)

78.58

98.9

96.6

40.8 79.3

50.6 61.5 92.0 70.1 46.0 100.0 92.0 100.0 60.9 81.6 85.1 98.3 75.9 85.8 79.3 85.1 54.0 95.4

Cartilage IKDC repair score at assessment follow-up

43.73

56.3

54.0

8.0 41.4

13.8 45.4 50.6 37.9 6.9 62.6 NA 56.7 35.6 44.8 47.1 54.0 43.1 60.5 56.3 58.1 18.4 66.7

IKDC improvement baseline/ follow-up

*Patients with two biopsies. C 253*: Second-look biopsy taken at 9 months, evaluated as HL/FC and the cartilage repair was graded 9 (BA). C 013*: Second-look biopsy taken at 12 months, evaluated as HL and the cartilage repair was graded 11 (BA). **Third-look biopsy for patients C253 and C013. HL: Hyaline-like tissue; HL/FC: Mixed tissue; FC: Fibrocartilage tissue; BA: biologically acceptable.

mean 38.9 3.5

2.00 2.35 1.18 3.00 1.50 7.85 4.71 5.50 2.00 3.92 1.50 2.36 3.53 3.53 2.00 4.71 2.00 8.83

C 035 38 C 468 56 C 033 47 C 456 34 C 038 47 C 077 28 C 234 49 C 252 30 C 405 28 C 572 53 C 226 25 C 065 37 C 253* 46 C 032 47 C 450 22 C 425 56 C 436 45 C 013* 51

Code

2nd-look biopsy time from surgery (months)**

85.12

97.5

95.0

45.0 82.5

70.0 75.0 87.5 90.0 70.0 95.0 NA 97.5 80.0 90.0 95.0 92.5 75.0 80.0 90.0 95.0 85.0 100.0

37.17

42.5

38.0

10.0 42.5

20.0 50.0 42.5 20.0 5.0 47.5 NA 40.0 40.0 45.0 35.0 47.5 40.0 40.0 40.0 65.0 20.0 50.0

EQ-VAS EQ-VAS improvement baseline/ score at follow-up follow-up

Patient’s subjective evaluation

¢broarthrosis

Adverse events

nearly normal

severely abnormal ¢broarthrosis normal periosteal hypertrophy normal

NA abnormal nearly normal nearly normal NA nearly normal nearly normal nearly normal nearly normal nearly normal NA nearly normal nearly normal abnormal normal nearly normal NA normal

IKDC at follow-up

Surgeon’s evaluation

Histological, arthroscopical, clinical and quality of life outcomes of patients with second-look biopsies (n ¼ 22)

Size of principal defect Age (cm2)

TABLE 1


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surgery was indeed used in 34% of the patients and in all these cases lesions were 42 cm2. When the improvement (de¢ned as the mean di¡erence between the scores obtained at baseline and at follow-up) was analysed by lesion size, a positive trend was observed (the larger the size, the greater the improvement). Moreover, data obtained so far suggest no di¡erence in the results considering the technique used for implantation. Comparison of the clinical outcomes with arthroscopic cartilage assessment and histological ¢ndings showed that a hyaline-like regenerated tissue correlated with the most favourable clinical results. Conclusions ACI shows promise for the treatment of full-thickness cartilage defects and is ¢nding growing interest in the orthopaedic community. An improved methodology based on the use of Hyalograft1 C, a hyaluronan-based sca¡old for delivery of cultured autologous chondrocytes, has recently been introduced in clinical practice. Advantages of Hyalograft1 C over the currently available ACI technique include ease of handling, the possibility to be tailored to ¢t the defect and to be delivered by a minimally invasive technique. With this novel approach, no coverage is required to keep the graft in place. Hyalograft1 C is applied via a miniarthrotomy which signi¢cantly reduces surgical time and morbidity. Positive clinical outcomes following arthroscopic insertion of Hyalograft1 C have also been reported here and elsewhere (Marcacci et al 2002). A limited number of adverse events were registered with the use of Hyalograft1 C. Post-operative adverse reactions included fever, haematoma and swelling. Preliminary clinical ¢ndings have been reviewed and suggest that the vast majority of patients treated improved when assessed for relief of symptoms, improvement of mobility and quality of repaired cartilage tissue. The authors acknowledge that the patient’s series analysed here has no control group and represents a retrospective case series. Thus, it lacks the methodological strength of a clinical trial. However, clinical follow-up data are being prospectively collected, and will represent a valuable starting point for the design of future clinical studies. Additional clinical investigation is also required to con¢rm the preliminary ¢ndings reported here at longer time points. With the above limitations in mind, the authors believe that Hyalograft1 C may be an e¡ective treatment option for large defects (42 cm2), particularly for patients who have high physical demands and in those cases in which, regardless of the size and the patient’s demand, lesions did not respond to alternative cartilage repair techniques.

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References Abatangelo G, O’Regan M 1995 Hyaluronan: biological role and function in articular joints. Eur J Rheumatol In£amm 15:9^16 Aigner J, Tegeler J, Hutzler P et al 1998 Cartilage tissue engineering with novel nonwoven structured biomaterial based on hyaluronic acid benzyl ester. J Biomed Mater Res 42:172^181 Bert JM, Maschka K 1989 The arthroscopic treatment of unicompartmental gonarthrosis: a ¢veyear follow-up study of abrasion arthroplasty plus arthroscopic debridement and arthroscopic debridement alone. Arthroscopy 5:25^32 Bouwmeester SJ, Beckers JM, Kuijer R, van der Linden AJ, Bulstra SK 1997 Long-term results of rib perichondral grafts for repair of cartilage defects in the human knee. Int Orthop 21:313^317 Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peterson L 1994 Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med 331:889^895 Brun P, Abatangelo G, Radice M et al 1999 Chondrocyte aggregation and reorganization into three-dimensional sca¡olds. J Biomed Mater Res 46:337^346 Campoccia D, Doherty P, Radice M, Brun P, Abatangelo G, Williams DF 1998 Semisynthetic resorbable materials from hyaluronan esteri¢cation. Biomaterials 19:2101^2127 Chen WY, Abatangelo G 1999 Functions of hyaluronan in wound repair. Wound Repair Regen 7:79^89 Dandy DJ 1991 Arthroscopic debridement of the knee for osteoarthritis. J Bone Joint Surg Br 73:877^888 Freed LE, Marquis JC, Nohria A, Emmanual J, Mikos AG, Langer R 1993 Neocartilage formation in vitro and in vivo using cells cultured on synthetic biodegradable polymers. J Biomed Mater Res 27:11^23 Goa KL, Ben¢eld P 1994 Hyaluronic acid. A review of its pharmacology and use as a surgical aid in ophthalmology, and its therapeutic potential in joint disease and wound healing. Drugs 47:536^566 Grigolo B, Roseti L, Fiorini M et al 2001 Transplantation of chondrocytes seeded on a hyaluronan derivative (HYAFF11) into cartilage defects in rabbits. Biomaterials 22: 2417^2424 Grigolo B, Lisignoli G, Piacentini A et al 2002 Evidence for redi¡erentiation of human chondrocytes grown on a hyaluronan-based biomaterial (HYAFF11): molecular, immunohistochemical and ultrastructural analysis. Biomaterials 23:1187^1195 Hangody L, Kish G, Ka'rpa' ti Z, Udvarhelyi I, Szigeti I, Be' ly M 1998 Mosaicplasty for the treatment of articular cartilage defects: application in clinical practice. Orthopedics 21:751^ 756 Hendrickson DA, Nixon AJ, Grande DA et al 1994 Chondrocyte-¢brin matrix transplants for resurfacing extensive articular cartilage defects. J Orthop Res 12:484^497 Hoikka VEJ, Jaroma HJ, Ritsila VA 1990 Reconstruction of the patellar articulation with periosteal grafts. A 4-year follow-up of 13 cases. Acta Orthop Scand 61:36^39 Hollander AP, Dickinson SC, Sims TJ, Soranzo C, Pavesio A 2003 Quantitative analysis of repair tissue biopsies following chondrocyte implantation. In: Tissue engineering of cartilage and bone. Wiley, Chichester (Novartis Found Symp 249) p 218^233 Homminga GN 1997 Long-term follow-up of perichondral grafting for cartilage lesions of the knee. Cartilage Repair Symposium, Bermuda ICRS Cartilage Injury Evaluation Package 2000 http://www.cartilage.org/Evaluation ___ Package/ ICRS ____ Evaluation.pdf Johnson LL 1991 Arthroscopic abrasion arthroplasty. In: McGinty JB (ed) Operative arthroscopy. Raven Press, New York, p 341^359


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Marcacci M, Za¡agnini S, Kon E, Visani A, Iacono F, Loreti I 2002 Arthroscopic autologous chondrocyte transplantation: technical note. Knee Surg Sports Traumatol Arthrosc 10:154^ 159 Messner K, Maletius W 1996 The long-term prognosis for severe damage to weight-bearing cartilage in the knee: a 14-year clinical and radiographic follow-up in 28 young athletes. Acta Orthop Scand 67:165^168 Ogilvie-Harris DJ, Fitsialos DP 1991 Arthroscopic management of the degenerative knee. Arthroscopy 7:151^157 Peterson L, Minas T, Brittberg M, Nilsson A, Sj˛gren-Jansson E, Lindahl A 2000 Two to 9years outcome after autologous chondrocyte transplantation of the knee. Clin Orthop 374:212^234 Rabin R, de Charro F 2001 EQ-5D: a measure of health status from the EuroQol Group. Ann Med 33:337^343 Rodrigo JJ, Steadman JR, Silliman JF, Fulstone HA 1994 Improvement of full-thickness chondral defect healing in the human knee after debridement and microfracture using continuous passive motion. Am J Knee Surg 7:109^116 Sandelin J 1997 Long-term results of reconstruction of the patellar articulation with periosteal grafts. Cartilage Repair Symposium, Bermuda Scapinelli R, Aglietti P, Baldovin M, Giron F, Teitge R 2002 Biological resurfacing of the patella. Current Status. Clinics Sports Med 21:547^573 Schatz KD 2001a Erste Erfahrungen Mit Matrixassistierter Knorpel-Zelltransplantation (Summary experiences with autologous chondrocyte implantation). Arthritis Rheum (Munch ) 5:262^268 Schatz KD 2001b Treatment of cartilage lesions using expanded cartilage cells seeded on a three dimensional ¢brous sca¡old based on benzylic ester of hyaluronic acid preliminary clinical results. Osteoarthritis Cartilage 9 suppl B PA32 S29 Solchaga LA, Yoo JU, Lundberg M et al 2000 Hyaluronan-based polymers in the treatment of osteochondral defects. J Orthop Res 18:773^780 Stanton JS, Salih V, Bentley G, Downes S 1995. The growth of chondrocytes using Gelfoam as a biodegradable sca¡old. J Mater Sci Mater Med 6:739^744 van Susante JL, Buma P, van Osch GJ et al 1995 Culture of chondrocytes in alginate and collagen carriers gel. Acta Orthop Scand 66:549^556 Zorzi C, Hyalograft C Clinical Study Group 2000 Tissue engineered cartilage grafting: preliminary clinical data. ICRS Symposium, April 2000, Gothenburg, Sweden, p 124

[Editor’s note: Dr Pavesio’s paper was discussed together with the paper by Hollander et al; this discussion can be found on pages 229^233.]

Quantitative analysis of repair tissue biopsies following chondrocyte implantation Anthony P. Hollander, Sally C. Dickinson, Trevor J. Sims, Carlo Soranzo* and Alessandra Pavesio* University of Bristol Academic Rheumatology, Avon Orthopaedic Centre, Southmead Hospital, Bristol BS10 5NB, UK and *Fidia Advanced Biopolymers srl, Via Ponte della Fabbrica, 3/B, 35031 Abano Terme (PD), Italy

Abstract. Outcome measures for cartilage repair techniques include clinical assessment of functional status, magnetic resonance imaging, mechanical indentation in situ and secondlook biopsies, which are used for detailed ex vivo histological and immunohistochemical assessment. Biopsy analysis is considered an important outcome measure, despite being highly invasive, since it provides a visual record of the spatial organization of matrix proteins and cells. We propose that the value of second-look biopsies would be signi¢cantly enhanced if accurate quanti¢cation of cartilage matrix molecules could also be obtained. The goal of our work has been to develop a combined method for histological and biochemical analysis of a single biopsy. We have developed a method of cutting frozen sections of cartilage and recovering the uncut tissue for subsequent biochemical analysis. We have also developed a range of miniaturized assays that can be performed after cartilage digestion with trypsin. In this way we are now able to analyse biopsies with a wet weight as low as 5 mg using both histological and biochemical methods, so obtaining the maximum amount of information from the minimum volume of tissue. This new approach will allow a more accurate assessment of the quality of cartilage repair tissue than histological analysis alone. 2003 Tissue engineering of cartilage and bone. Wiley, Chichester (Novartis Foundation Symposium 249) p 218^233

Cell-based cartilage repair techniques are now in widespread use. Autologous chondrocyte implantation (ACI) was developed in Sweden (Brittberg et al 1994) and has been used there in clinical practice for over 10 years (Peterson et al 2000). The potential use of stem cells for chondrocyte formation and cell-based therapy is being actively investigated by a number of groups (Gao et al 2001, Johnstone et al 1998, Martin et al 2001). Biodegradable sca¡olds have also been developed to act as carriers for cells or for complete tissue engineering in vitro (Campoccia et al 1998, Freed et al 1998, Langer & Vacanti 1993). Therefore there are many competing 218

QUANTITATIVE ANALYSIS OF BIOPSIES

219

techniques, several of which are likely to be used in patients for the treatment of otherwise intractable problems of knee articular cartilage (Langer 1995). Assessment of the e⁄cacy of these new technologies is dependent on the availability of reliable outcome measures. At present the range of assessments includes clinical improvement scores, magnetic resonance imaging and the histological analysis of second-look biopsy (Peterson et al 2000, Roberts et al 2001). The function of the biopsy is to provide evidence of hyaline cartilage formation and the assumption is that formation of hyaline-like cartilage is essential for the long-term survival of the repair tissue. But to date the only way of measuring this outcome has been by immunohistochemical analysis, allowing comparison of the extent of staining of types I and II collagen (Roberts et al 2001). This approach has two major problems: Each tissue cryosection represents less than 0.5% of the total volume of the biopsy (A. P. Hollander, unpublished results) and is therefore only a very minor fraction of the original repair tissue site. Data obtained by semi-quantitative analysis of immunolocalization is unreliable and therefore potentially misleading. Di¡erent antibodies have di¡erent a⁄nities for their epitopes as well as di¡erent speci¢c activities of enzyme or £uorescent label, making any comparison between di¡erent antibodies tenuous. Furthermore, although it is reasonable to assume that there is a linear relationship between the amount of bound antibody and the amount of immunostaining observed, it is also expected that this relationship will reach a plateau, above which there will be minimal increase in staining even with increased antibody binding. Similarly, below a minimal threshold of bound antibody there may be limited association with staining intensity. Therefore, unless a dose^response curve is carried out using di¡erent amounts of antibody for each biopsy, it is almost impossible to interpret a quantitative relationship in this way. We have therefore attempted to develop a protocol for processing cartilage biopsies that would allow us to prepare cryosections for histochemical analysis and at the same time to measure the concentration of speci¢c analytes in the repair tissue. For each biopsy we would like to be able to determine (a) the histological organization, (b) the relative proportion of hyaline and ¢brocartilage, (c) the relative rate at which its extracellular matrix is turning over and (d) the abundance of extracellular matrix. Type II collagen is the characteristic collagen of cartilage. It accounts for 90% of hyaline cartilage collagen but it is also found in ¢brocartilage (Kucharz 1992). Type I collagen is absent from hyaline cartilage but abundant in ¢brocartilage (Kucharz 1992). Therefore the relative proportion of these collagens in a repair tissue biopsy will provide a strong indication of the extent to which hyaline cartilage has formed.

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Cross-linking of collagen is mediated by the enzyme lysyl hydroxylase, which drives the formation of immature cross-links through hydroxylysine residues at four speci¢c sites in each collagen molecule (Eyre et al 1984). The immature, reducible cross-link is hydroxylysylketonorleucine (HLKNL) which matures spontaneously into the mature, non-reducible hydroxypyridinium cross-link. Formation of the mature cross-link is directly related to time after HLKNL formation (Uchiyama et al 1981) and therefore is dependent on the rate of collagen turnover. Mature hyaline cartilage undergoes very limited collagen turnover and therefore almost all the cross-links are mature. However cartilage in younger individuals is richer in the immature cross-link because of relatively rapid degradation of the collagenous network and replacement with newly synthesized collagen. All forms of reducible, immature cross-links fall in concentration with tissue maturation (Bailey & Peach 1971, Bailey et al 1974, Fujii & Tanzer 1974). Conversely, there is good evidence that removal of the mature cross-links is indicative of collagen degradation (Robins 1982). Therefore the relative proportion of mature and immature collagen cross-links in a repair tissue will provide an index of the degree of maturity of the extracellular matrix. In addition to these important markers, we would like to be able to measure total collagen as hydroxyproline and proteoglycan as glycosaminoglycans (GAGs), as indicators of abundance of the extracellular matrix. The aim of this project was to develop assays with enough sensitivity for detection of each of the marker analytes in a small volume of extracted cartilage. For this purpose it was important to identify a single proteinase with which to extract all the marker molecules. We describe here an e¡ective protocol which provides us with histological and quantitative data from a single small biopsy of repair tissue. Methods Patients We evaluated the quality of repair tissue, generated in vivo following implantation in humans of autologous chondrocytes seeded onto a hyaluronic acid-based sca¡old (Hyalograft1 C). This study is part of a follow-up project which involves a systematic and standardized collection of qualitative and quantitative clinical data concerning the treatment of cartilage lesions of the knee with Hyalograft1 C. Chondrocytes isolated from the low weight-bearing cartilage of patients were expanded in monolayer culture, seeded onto Hyalograft1 C sca¡olds and cultured for a further 14 days, as previously described (Grigolo et al 2002). The cell-loaded sca¡old was implanted into traumatic cartilage lesions in the knee (1^9 cm2 in area) which were ¢rst debrided if necessary. Most were at the


221

medial femoral condyles though some were lateral condyles, tibial plateau or patella. The second-look biopsies analysed here were harvested at arthroscopy 12^30 months after implantation. Full-depth cartilage and bone plugs were removed and immersed in saline prior to transportation. Preparation of cryosections from biopsies and recovery of uncut tissue For each second-look biopsy, cartilage was separated from the subchondral bone using a scalpel and the cartilage pieces were frozen in OCT embedding medium (B.D.H. Laboratory Supplies, Poole, Dorset, UK). Sections (7 mm) were cut using a cryostat for use in histochemical studies. The remaining, uncut cartilage was thawed and the OCT removed by exhaustive washing in distilled water. Development of a new type I collagen assay An anti-peptide antibody to an 8 amino acid sequence in the C-terminal telopeptide of the a1 chain of type I collagen, was raised in rabbits using a conventional immunization scheme. An N-terminal cysteine residue was added for coupling to keyhole limpet haemocyanin. Type I collagen levels were measured using an inhibition ELISA similar to that we have previously described for type II collagen (Hollander et al 1994), with the following modi¢cations. The 8 amino acid peptide against which the antibody was raised was used as a standard (5^1000 ng/ml) in all assays. Standards and samples were dissolved in 0.8% (w/v) sodium dodecyl sulfate (SDS; Bio-Rad) in 50 mM Tris-HCl, pH 7.6, where the SDS was required to solubilize the type I collagen peptides and dissociate any interactions with other proteins present in the sample. The standards and samples, assayed in triplicate, were mixed in a preincubation plate with an equal volume of the rabbit anti-peptide antiserum, diluted 1:1000 in Tris-HCl, pH 7.6, containing 4% (v/v) Triton X-100 (BioRad). After an overnight incubation at 37 8C, samples were transferred to an Immulon-2 96-well ELISA plate (Dynex) which had been coated with 40 mg/ml peptide, dissolved in carbonate bu¡er, pH 9.2, for 3 days at 4 8C and blocked with 1% (w/v) BSA, in PBS, to minimize non-speci¢c binding. After 30 min, plates were washed with PBS containing 0.1% (v/v) Tween-20 (Sigma) and alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin (Southern Biotechnology) was added for a further 2 h to detect bound antibodies. Following further washing, the enzyme substrate p-nitrophenyl phosphate (Sigma), dissolved at 0.5 mg/ml in 1 M diethanolamine (Sigma), pH 9.8, containing 0.5 mM MgCl2, was added to each well and the absorbance read at 405 nm. A standard curve was included on each plate from which the concentration of type I collagen in each sample was derived.

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Other analytical assays Type II collagen, GAG and hydroxyproline were all assayed as previously described (Fenwick et al 2001, Handley & Buttle 1995, Hollander et al 1994). However, the type II collagen ELISA was modi¢ed for use with a 384-well plate allowing analysis of 10 ml samples rather than the 50 l volumes required in 96-well plate assays. Collagen cross-links were determined by amino acid analysis, as previously described (Sims et al 2000, Sims & Bailey 1992). Proteolytic extraction of tissues It was essential to develop a means of extracting all the analytes from cartilage in a single proteolytic step. Various proteolytic enzymes were tested, including proteinase K, chymotrypsin, papain and elastase, but each destroyed either or both of the type I or type II collagen recognition epitopes. However, trypsin was found to cleave both collagen types into small, undetectable peptides (as judged by SDS-PAGE, data not shown) without the loss of either epitope. In order to solubilize intact tissues by trypsin digestion, samples were ¢rst milled in liquid nitrogen to obtain a ¢ne particulate. An initial incubation for 15 h at 37 8C with 250 ml 2 mg/ml TPCK-treated trypsin in 50 mM Tris-HCl, pH 7.6, containing 1 mM iodoacetamide, 1 mM EDTA and 10 mg/ml pepstatin A (all from Sigma), was followed by a further 2 h incubation at 65 8C after the addition of a further 250 ml fresh trypsin. Samples were then boiled for 15 min to inactivate the enzyme. Results and discussion Natural bovine tissues were used as controls for all our studies to provide an index with which to compare patent biopsies. Protein analysis of natural bovine hyaline and ¢bro-cartilage There were clear di¡erences in the abundance of di¡erent proteins in hyaline and ¢brous tissues (Fig. 1). Both types of cartilage were rich in collagen, measured as hydroxyproline, determined by colorimetric assay (Fig. 1A). Our hydroxyproline assay standard has been calibrated by amino acid analysis to ensure accuracy of the data. Proteoglycan content, measured by colorimetric assay of GAG, was greater than 10% of dry weight in all hyaline cartilage specimens but less than 10% of dry weight in all ¢brocartilage specimens. Speci¢c immunoassay of collagens type I and II revealed, as expected, that hyaline cartilage contained a high concentration of type II collagen but negligible amounts of type I collagen (Fig. 2A), whereas in ¢brocartilage both these collagens


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FIG. 1. Abundance of extracellular matrix proteins in di¡erent types of cartilage. Adult bovine articular cartilage (hyaline) and meniscus (¢bro-cartilage) were freeze-dried, weighed and then processed for analysis of total collagen (panel A) and proteoglycan (panel B) as described in the methods section. Each point is the result for one animal.

were detectable with a signi¢cant preponderance of type I (Fig. 2B). The mean ratio of collagen II to I was 4340 for hyaline and 0.13 for ¢brocartilage. Turnover of natural bovine hyaline cartilage The total cross-link content relative to total collagen was similar in cartilage obtained from all ages of animal (Fig. 3A). However there was a signi¢cant variation in the proportion of mature to immature cross-link with age (Fig. 3B). Tissue from skeletally young animals had far more immature cross-links than mature, indicating rapid turnover of the tissue. However, animals which had

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FIG. 2. Abundance of speci¢c collagens in di¡erent types of cartilage. Types I and II collagen were determined in adult bovine cartilage by immunoassay. Extracts of freeze-dried cartilage were prepared as described in the methods section. Panel A is articular cartilage (hyaline) and panel B is meniscal cartilage (¢brocartilage). Each line shows the results for one animal.

reached skeletal maturity had a marked excess of mature cross-links over immature, indicating much slower turnover of their hyaline cartilage. Clinical observations More than 700 patients have been implanted with Hyalograft1 C and to date a total of 22 second-look biopsies have been analysed after removal from the patient 12^30 months following cell implantation. All patients showed clinical improvement and


225

FIG. 3. Collagen cross-links in articular cartilage at di¡erent ages. Bovine articular cartilage was obtained from animals at the ages shown, extracted and assayed for total collagen cross-links per collagen (Panel A) and the ratio of mature to immature cross-links (Panel B) as described in the methods section. Each bar is the result for one animal.

the macroscopic appearance of repair tissue at arthroscopy was normal or nearly normal in all cases, as is described by Dr Alessandra Pavesio elsewhere in this volume (Pavesio et al 2003, this volume). Analysis of biopsies from patients treated using Hyalograft1 C In all cases we were able to cut frozen sections for histological analysis, recover uncut tissue and analyse each specimen after extraction with trypsin. In this way

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FIG. 4. Abundance of extracellular matrix proteins in second-look biopsies from patients treated with chondrocytes seeded onto Hyalograft1 C. Each biopsy was freeze-dried, extracted and assayed for total collagen and proteoglycan (panel A) as well as for speci¢c collagens (panel B) as described in the methods section. Each point (A) or line (B) is the result for one patient.

we could obtain a full range of quantitative data from specimens with dry weights in the range of 0.4^3.2 mg. All biopsies contained abundant collagen and proteoglycan, with content similar to that of natural hyaline cartilage (Fig. 4A). However there was marked variation in the type of collagen. Three biopsies had a clear excess of type I collagen but some type II collagen, indicative of ¢brocartilage, whilst three had more type II collagen than type I indicating a more hyaline phenotype (Fig. 4B). In previous studies (Brittberg et al 2001, Roberts et al 2001), tissue sections sometimes showed evidence of both ¢brous and hyaline regions, though this may be in part a result of the periosteal £ap used to contain the cells (Brittberg et al 2001). The biopsies analysed here encompassed a much larger volume of tissue than any one histological section. Except in one case, the collagen content appeared to be either predominantly type I or type II rather than a mixture of the two, suggesting that for the biopsy as a whole one type of tissue structure is dominant. Total collagen cross-link content was in the normal range for hyaline and ¢brocartilage (Fig. 5A). However the ratio of mature to immature cross-links was low in all cases (Fig. 5B), indicating that the repair tissues are all turning over as rapidly as cartilage from skeletally immature joints. In a previous study of ACI (Roberts et al 2001), we found that repair tissue showed immunohistochemical signs of turnover as judged by collagen and aggrecan neoepitope exposure. The cross-link analyses we have described here extend


227

FIG. 5. Collagen cross-links in second-look biopsies from patients treated with chondrocytes seeded onto Hyalograft1 C. Each biopsy was freeze-dried, extracted and assayed for total collagen cross-links per collagen (panel A) and the ratio of mature to immature cross-links (panel B) as described in the methods section. Each bar is the result for one patient.

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our earlier data by providing the ¢rst quantitative evaluation of the turnover rate. Conclusion These results demonstrate for the ¢rst time that it is possible to analyse small second-look biopsies using quantitative biochemical techniques in addition to standard histological analysis. We have found that all biopsies so far studied have an abundant extracellular matrix which is still turning over as fast as immature natural cartilage, 12^30 months after cell implantation. This approach to the analysis of biopsies provides an e¡ective means of comparing di¡erent cartilage repair techniques in an objective, quantitative way. Acknowledgement This work was funded in part by the European Union framework ¢ve ‘SCAFCART’ consortium. References Bailey AJ, Peach CM 1971 The chemistry of the collagen cross-links. The absence of reduction of dehydrolysinonorleucine and dehydrohydroxylysinonorleucine in vivo. Biochem J 121:257^259 Bailey AJ, Robins SP, Balian G 1974 Biological signi¢cance of the intermolecular crosslinks of collagen. Nature 251:105^109 Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peterson L 1994 Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med 331:889^895 Brittberg M, Tallheden T, Sjogren-Jansson B, Lindahl A, Peterson L 2001 Autologous chondrocytes used for articular cartilage repair: an update. Clin Orthop S337^S348 Campoccia D, Doherty P, Radice M, Brun P, Abatangelo G, Williams DF 1998 Semisynthetic resorbable materials from hyaluronan esteri¢cation. Biomaterials 19:2101^2127 Eyre DR, Paz MA, Gallop PM 1984 Cross-linking in collagen and elastin. Ann Rev Biochem 53:717^748 Fenwick SA, Curry V, Clements S, Hazleman BL, Riley GP 2001 96-well plate-based method for total collagen analysis of cell cultures. Biotechniques 30:1010^1014 Freed LE, Hollander AP, Martin I, Barry JR, Langer R, Vunjak-Novakovic G 1998 Chondrogenesis in a cell-polymer bioreactor system. Exp Cell Res 240:58^65 Fujii K, Tanzer ML 1974 Age-related changes in the reducible crosslinks of human tendon collagen. FEBS Lett 43:300^302 Gao J, Dennis JE, Muzic RF, Lundberg M, Caplan AI 2001 The dynamic in vivo distribution of bone marrow-derived mesenchymal stem cells after infusion. Cells Tissues Organs 169:12^20 Grigolo B, Lisignoli G, Piacentini A et al 2002 Evidence for redi¡erentiation of human chondrocytes grown on a hyaluronan-based biomaterial (HYA¡ 11): molecular, immunohistochemical and ultrastructural analysis. Biomaterials 23:1187^1195 Handley CJ, Buttle DJ 1995 Assay of proteoglycan degradation. Methods Enzymol 248:47^58


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Hollander AP, Heath¢eld TF, Webber C et al 1994 Increased damage to type II collagen in osteoarthritic articular cartilage detected by a new immunoassay. J Clin Invest 93:1722^1732 Johnstone B, Hering TM, Caplan AI, Goldberg VM, Yoo JU 1998 In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res 238:265^272 Kucharz EJ 1992 The collagens: biochemistry and pathophysiology. Springer-Verlag, Berlin Langer R 1995 1994 Whitaker lecture: polymers for drug delivery and tissue engineering. Ann Biomed Eng 23:101^111 Langer R, Vacanti JP 1993 Tissue engineering. Science 260:920^926 Martin I, Shastri VP, Padera RF et al 2001 Selective di¡erentiation of mammalian bone marrow stromal cells cultured on three-dimensional polymer foams. J Biomed Mater Res 55:229^235 Pavesio A, Abatangelo G, Borrione A et al 2003 Hyaluronan-based sca¡olds (Hyalograft1 C) in the treatment of knee cartilage defects: preliminary clinical ¢ndings. In: Tissue engineering of cartilage and bone. Wiley, Chichester (Novartis Found Symp 249) p 203^217 Peterson L, Minas T, Brittberg M, Nilsson A, Sjogren-Jansson E, Lindahl A 2000 Two- to 9year outcome after autologous chondrocyte transplantation of the knee. Clin Orthop 374:212^234 Roberts S, Hollander AP, Caterson B, Menage J, Richardson JB 2001 Matrix turnover in human cartilage repair tissue in autologous chondrocyte implantation. Arthritis Rheum 44:2586^ 2598 Robins SP 1982 An enzyme-linked immunoassay for the collagen cross-link pyridinoline. Biochem J 207:617^620 Sims TJ, Bailey AJ 1992 Quantitative analysis of collagen and elastin cross-links using a singlecolumn system. J Chromatogr 582:49^55 Sims TJ, Avery NC, Bailey AJ 2000 Quantitative determination of collagen crosslinks. Methods Mol Biol 139:11^26 Uchiyama A, Inoue T, Fujimoto D 1981 Synthesis of pyridinoline during in vitro aging of bone collagen. J Biochem (Tokyo) 90:1795^1798

DISCUSSION Barry: What time after implantation were these biopsies produced? Hollander: Between 12 and 15 months. Barry: Do you intend to look using indentation testing at any of the material properties of the tissue? Pavesio: No, we did not look at indentation testing because the equipment was not available at the sites. That is work that can only be done in a prospective controlled clinical evaluation. Trippel: One of the biggest limitations of evaluating these patients is the small quantity of tissue that you can legitimately go back and take out, after you have tried so hard to ¢ll these defects in! One of the things that was unclear from your technique development was just how much variability there is in the histology from one part of the biopsy to another. Is it fairly uniform? Hollander: I can’t tell you, because if we used up all our biopsy for multiple sections we wouldn’t have any left for biochemistry. My prediction is that there will be variation, otherwise I would have expected a better match-up between the biochemistry and the histochemistry.

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DISCUSSION

Caplan: I think that question can be handled in a more controlled setting by having access to cartilage where you take two or three biopsies from the same patient and compare them quantitatively and qualitatively side-by-side. The micromethods are technically complicated. The question is, what is the reproducibility like between cartilage samples from the same region? The only way to answer this is with multiple biopsies from the same area, which can probably be done with discarded material or human material that has nothing to do with the clinical study. You should do this retrospectively, because otherwise this question of the reproducibility of your measurements will surface time and time again. Hollander: We have con¢dence in the measurements because we can take the same sample and assay it two or three times. Trippel: Alessandra Pavesio, what is holding your implants in place? Do they adhere to the underlying bone? Do the little trabeculae grab onto it like velcro? Pavesio: Normally the surgeons apply the material and then release the tourniquet to allow some blood into this region. It is this blood that ¢xes the device in place. Hyaluronan is also mucoadhesive and therefore has some intrinsic stickiness. The combination of these factors holds the material in place. In the Swedish technique, the recommendation to minimize bleeding, is related to the concern about blood building up beneath the periosteal £ap and rupturing it. On the other hand, given the porous nature of the HYAFF sca¡old, the blood tends to impregnate the sca¡old with the cells, and contributes to keeping the device in place. Ratcli¡e: Carrying on from there, have you done any labelling of cells in animal studies to see how many remain from the donor? Pavesio: No. I should point out that the cells are of autologous origin. Ratcli¡e: In control experiments have you tried just putting the hyaluronan there and going through the same process, comparing it to the with-cells approach? Pavesio: This was one of the objectives of our preclinical investigation. Publications by Dr Caplan’s group have shown that in osteochondral lesions created in young rabbits, cellular and acellular HYAFF implants were not statistically di¡erent. In fact, there have been a number of reports showing that the presence of a sca¡old encourages a natural repair in the absence of cells in a preclinical setting. Ratcli¡e: If you go to the literature and look at analysis of just creating a defect and then looking at the repair tissue 12 months later, there are data similar to the type of data you are showing here (Furukawa et al 1980). Pavesio: I’m not sure I agree with you. In my views, the literature reports the formation of hyaline-like cartilage using the Swedish technique. In this surgery, bleeding is kept to a minimum, given the constraints that we discussed about the use of the periosteal £ap. Note, however, that a completely bloodless ¢eld in


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cartilage surgery is an ideal, and that therefore some bleeding is always associated with this type of surgery. The literature also reports formation of ¢brous-like cartilage after microfracture and drilling in this case, bleeding is deliberately created. Our data suggest that hyaline-like cartilage can be obtained by delivering cells on a HYAFF sca¡old, in the absence of a periosteal £ap, and in the presence of some bleeding. The question of the contribution of the blood components versus the matrix versus the laboratory cultured cells remains open for debate. Hollander: I would add that to my knowledge, no one has quanti¢ed what is going on in the matrix in a microfracture situation. Ratcli¡e: Let me rephrase the point. The literature describes animal studies where the repair tissue contains type II collagen (as well as type I) and proteoglycans, and is a mixture of hyaline and ¢brous cartilage (Furukawa et al 1980). Hollander: The point here is that by measuring it in the biopsies you can begin to compare techniques on a fair basis. Ratcli¡e: Absolutely. You have done a nice job of being able to extract an immense amount of information out of those tiny biopsies. Pavesio: I think we need to exercise caution in looking at preclinical data and making clinical conclusions or even hypotheses. Lindahl: Alessandra Pavesio, you showed a promising clinical picture, which mirrors what we have seen from the beginning with our technique. There remains the question of the relative contributions of the hyaluronan and the cells. This needs to be addressed, as does the long-term result. In addition, just a comment on the analysis. We saw that the patient variability is very high. If you are going to do quanti¢cation, make sure that there is a biopsy from a surrounding untreated area. This is the best comparison of your quantitative data. Lohmander: If you were able to prove that the results of the Italian technique are as good clinically as the Swedish technique, this would be a step forward in the ease of use. One of the great problems in dealing with the Gothenburg technique is the issue of periosteum, and suturing this to the cartilage. This is what takes most of the time and requires the most technical skill. It also requires open surgery. I wonder about the question of whether the matrix as such could be enough on its own. What if you were to, in your patients, take the original cartilage and biopsy not by just shaving a sliver o¡ but by punch biopsy? At that time you can ¢ll the punched-out with your matrix only but without cells, and when you go back 30 days later for the transplant you can retrieve that piece, and look for the presence of a cell repopulation of this originally empty matrix. This is a possible way of getting some early human data of whether these matrices can be repopulated ‘spontaneously’ by endogenous cells. Pavesio: That’s a brilliant suggestion. Thank you. Caplan: But in your punch biopsies for tissue acquisition, is this a bleeding or non-bleeding wound?

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DISCUSSION

Pavesio: It’s a non-bleeding wound. Caplan: That’s a problem. Pavesio: I would like to point out that our preliminary ¢ndings suggest that the same dose of cells produces clinical outcomes that are similar to those reported by the Swedish group. We now have a delivery system that will improve the current surgical technique, making it much simpler. But, there are quite a lot of similarities between our work and the ¢rst reports by Peterson we should not forget that the cell doses in these two approaches are identical. Caplan: Victor Goldberg went and monitored a couple of these operations in Italy. I asked him what he thought. He said it was great because it is a combination of the Swedish and Steadman techniques, and it takes the best of both. For those of us who are interested in other cell types that di¡erentiate into chondrocytes, this aspect of a bleeding wound provides some food for thought. Also, the porosity of the matrix provides a di¡erent kind of tissue engineering logic. Helms: Alessandra, you mentioned that during limb development, hyaluronan is higher and then goes down. My recollection of old data is that hyaluronan is antiangiogenic. It is the breakdown of hyaluronan that allows the formation of bone. Do you have a perception that it is anti-angiogenic until it is broken down? Pavesio: I need to clarify two technical issues. First, the hyaluronan used in the HYAFF sca¡old is a low molecular weight fraction, in the region of 200 000 Da. The fractions reported in the literature to be anti-angiogenic are fractions beyond 1.5 million Da. Second, the fragments proven to be angiogenic belong to very low molecular weight fractions: essentially the hexa- and pentasaccharide fragments that are released after hyaluronan cleavage. The hypothesis we are putting forward is that the in vivo degradation of the HYAFF molecule releases these fragments, which are in turn promoting angiogenesis in speci¢c settings. Schwartz: When you went back to look at these grafts by arthroscopy, how did they look grossly? Did it look like the graft was integrating with the native tissue? Pavesio: We have recorded these and we are currently attempting to correlate the histology with the arthroscopy ¢ndings. We are using the Brittberg scoring system, which takes into account integration, degree of defect ¢ll and mechanical properties. Schwartz: What is your ‘feel’ regarding this technology from what you have seen? Pavesio: Our results show a majority of cartilage repairs graded as ‘normal’ or ‘nearly normal’ at a mean follow-up time of 18 months. These observations correlated well with the clinical scorings. Lindahl: The reason we avoid bleeding is that our cells are implanted into a closed compartment. Red blood cells haemolyse, and haemoglobin has been shown to be toxic to chondrocytes. The clinical observation is that when we cover the cartilage defect, independent of what it is covered with, in the early phase the patients are improved.


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Lohmander: I admit to being guilty as charged for introducing the term ‘garage trial’ during this meeting. With regards to the Peterson technique it is currently being argued that it is unethical to use anything else, because it is so much better than the alternatives. But I don’t see that there would be an ethical problem in trying to set up a trial that would compare the technique discussed here with the Peterson technique. Pavesio: I am aware of some work currently being performed spontaneously by clinical users. In my opinion, however, I feel that there is more clinical value in identifying the clinical indications of ACI, regardless of the surgical technique used for cell implantation. Now that we have better protocols and methods for analysing clinical data, I see great value in setting out to demonstrate whether there is an advantage of tissue engineering over techniques that don’t require a two-step procedure. Lindahl: The comparison would be interesting. It is probably inevitable in Europe since we will have a regulatory standard that a new technique has to be compared with something else. Probably our ‘garage technique’ will be the golden standard. Pavesio: There are always objections. Surgeons ask how this technique compares with mosaicplasty, subchondral bone perforation or other cartilage surgeries. Hunziker: I have a question that relates to the sca¡old. You used HYAFF111. My impression was in the video that the surgeon, who applied the HYAFF111, under¢lled the defect site (so that it can swell up later to surface levels). Isn’t the fact that this material has a slight swelling tendency an additional factor that contributes to its mechanical ¢xation in place? Pavesio: This is true for the arthroscopical application, as, the instrumentation is designed to create a small groove, so the graft is placed inside this space. In open surgery this is not the case. Hunziker: What happens to the sca¡old when it is placed in a physiological ionic strength solution? Does the volume of the construct change? Pavesio: It comes in a transport medium that is at physiological ionic strength; therefore it is already swollen. Hunziker: You have introduced a chemically hydrophobic group into your HYAFF111 construct. Is this group immunogenic? Pavesio: No, it isn’t. This has been demonstrated in preclinical settings. Also, note that the HYAFF biomaterial has been approved for use, both in Europe and in the USA, for a variety of medical indications, and we have to date received no report of immunological reactions in humans. Reference Furukawa T, Eyre DR, Koide S, Glimcher MJ 1980 Biochemical studies on repair cartilage resurfacing experimental defects in the rabbit knee. J Bone Joint Surg Am 62:79^89

General discussion II Tissue engineering using recombinant human BMP2 Wozney: I am going to describe some of our clinical work with recombinant human BMP2 (rhBMP2). Several presentations here have outlined the elements of tissue engineering. These include cells, some kind of matrix or sca¡old, perhaps bioactive factors, and then placing these constructs into the appropriate mechanical environment. Our approach with rhBMP2 is somewhat di¡erent. We are supplying a single bioactive factorrhBMP2with a matrix material. This is implanted, and the host provides the responsive cells, whether they be mesenchymal stem cells (MSCs) or other cells. The concept is to cause the host to induce its own bone. I would also argue that mechanical load conditions are irrelevant to the initial bone-induction process. Once bone is induced, the bone then responds physiologically depending on its appropriate biomechanical environment. Clearly rhBMP2 is osteoinductive, but when it is placed in an in vivo environment it leads to a very complicated series of biochemical and cellular events. The implants become hypercellular, from which one would infer that proliferation and chemotactic events are taking place. The primary mode of action of rhBMP2 is probably to di¡erentiate mesenchymal cells into cartilage and bone forming cells. This results in both endochondral and intramembranous bone formation. rhBMP2 ¢rst induces immature woven trabecular bone, which then remodels depending on the site it is in and its biomechanical environment. There are also large numbers of blood vessels induced concurrent with bone formation. While rhBMP2 itself is gone several weeks after implantation, preclinically and clinically we can still see bone induction or bone formation occurring several months after the application of rhBMP2. Thus, this is a multifactorial process, and we are adding an initiator. In order to implant BMP, we use a carrier system, not necessarily a sca¡old. The major mode of action of the carrier is to keep a high local level of rhBMP2; this appears to be necessary for optimal bone induction. The matrix may also provide an environment for bone induction. Most of the matrix materials that we have evaluated are resorbed, so after the bone induction process all that is left is normal native bone. The ¢rst implantable product that we have used in the clinic is rhBMP2 with an absorbable collagen sponge (ACS). This is a haemostatic collagen sponge that has been on the market for decades. We reconstitute lyophilized rhBMP2 in a vial, take the liquid out, express it onto the sponge and this then makes a mouldable implant material: it is like wet tissue paper in 234

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consistency and can be cut or rolled. It has high tensile strength so does not tear; it is also adhesive, so it stays at the site of implantation. I will use one preclinical model to illustrate some important aspects of bone induction by rhBMP2. A segmental defect model clearly shows what rhBMP2 can do. In this study, a 3 cm defect in a dog mandible was created and then implanted with rhBMP2. After 12 weeks we see full bone induction throughout the defect radiographically. There is also functional restoration, because the ¢xation plate can be removed and the dogs can eat. Over time, the induced bone density tends to increase and it remodels very nicely into the surrounding boneit is hard to see where the defect margins are nine months later. Two years later the bone has further remodelled and increased in density. In our clinical studies we have tried to take a broad approach, and looked at the possibility of using rhBMP2/ACS in a variety of applications (Valentin-Opran et al 2002). I will describe one clinical study from dental/craniofacial reconstruction, one from orthopaedic trauma (fracture repair), and one from spinal fusion. In the dental/craniofacial area, the ¢rst indication we have looked at is augmentation of the alveolar ridge. Concentrating on maxillary sinus £oor augmentation, we have run a series of studies starting with a small feasibility study, a dose-ranging study and then a pivotal study. The latter is still ongoing. In this procedure, a lateral incision is made into the maxillary sinus, and graft material is placed to augment the quantity the maxillary bone, in order to have enough bone to place dental implants. This procedure allows one to use the patient as their own control, as no bone is present at the beginning of the procedure. From computed tomography (CT) scans we can see an induction of radiodense material relative to baseline, six months post-operatively, which one would presume to be bone (Boyne et al 1997). At the time of dental implant placement we have the opportunity to take a biopsy and evaluate the bone histologically. From these biopsy specimens, it is evident that rhBMP2 does induce bone in humans. At this time point the bone is relatively sparse trabecular bone, but the overall density is higher than the existing bone. Unfortunately, bone induction as a clinical outcome measure; a functional outcome must be demonstrated. In this case it is dental restoration: the placement and loading of dental implants. At the di¡erent doses of rhBMP2 relative to the allograft control, we have about the same proportion of dental implant success. We do CT scans with an internal standard so we can measure the actual bone density, and after the bone is loaded the density increases dramatically with time. Conversely, in some cases where the bone was not loaded, it goes away. The safety assessment was more than satisfactory: there were no serious adverse events associated with the device in any case. In summary, rhBMP2/ACS is osteoinductive, and induces normal physiological bone that behaves as you would expect the host bone to behave. In terms of the concentration of rhBMP2, we have chosen 1.5 mg/ml as the most e¡ective dose. While the amount of bone

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formed at di¡erent concentrations of rhBMP2 is the same, the density of the bone at a set time point is higher at higher concentrations. My assumption is that the bone induction process occurs more rapidly. In orthopaedic trauma, we have done a large study looking at rhBMP2/ACS and acceleration of healing of open tibial shaft fractures (BESTT Study Group 2002). We chose this indication because it is one of the most di⁄cult healing environments, which results in signi¢cant numbers of treatment failures. Our primary e⁄cacy outcome was the reoperation rate for failed union. We also evaluated the time to union and adverse events. This was a prospective, randomized, controlled, single-blind study. It was conducted at 49 centres worldwide and included 450 patients. Even in this trauma patient population, we were able to obtain 96% follow-up at the 12 month outcome point. There were three treatment groups with 150 patients per group. The ¢rst was the standard of care, including ¢xation with an intramedullary nail. The second group was standard of care plus 0.75 mg/ml rhBMP2/ACS wrapped around the fracture. The third group was a higher concentration of rhBMP2/ACS wrapped around the fracture (1.5 mg/ml). Results from the 12 month time point show that, in the control group, about half of the patients had to have an additional subsequent operation to heal their fracture, for example receive an additional bone graft or have the nail exchanged. In the high dose rhBMP2/ACS group there was a 45% reduction in the re-operation rate. The rate of fracture healing was also evaluated both clinically and by a blinded radiographic panel. As early as 10 weeks post surgery, at the high concentration of rhBMP2 there was an increase in the number of patients that were scored as healed, and this continued throughout the study. While overall there was no di¡erence in infection rate between groups, in the most severe fractures there was a signi¢cant reduction in the infection rate in the patients treated with rhBMP2. It is likely an indirect e¡ect. Caplan: Did the BMP a¡ect wound closure? Wozney: There was a di¡erence in the soft tissue healing rate in the BMP-treated patients. The reduction in infections could be secondary to closure and maturation of the soft tissue around the fracture site, or due to the increase in vascularity commonly seen in conjunction with bone induction by BMP. Caplan: Is it dose dependent? Wozney: We do not have enough data to answer that yet. rhBMP2/ACS treatment reduced the reintervention rate due to delayed union or non-union. Even in the patients who did have secondary interventions, the invasiveness of the operation was reduced in the BMP-treated patients. I will ¢nish by describing an interbody spinal fusion clinical study, conducted by our corporate collaborators, Medtronic Sofamor Danek, which will form the basis of the ¢rst approval of rhBMP2/ACS in the USA. In this procedure, titanium threaded interbody cages are placed in the disc space to create spinal arthrodesis.


237

Currently, the procedure involves the use of autogenous bone graft harvested from the iliac crest to provide a bony bridge between the vertebral bodies. Again, both pilot and pivotal clinical studies have been performed (Boden et al 2000). In both cases, the treatment groups were rhBMP2/ACS at 1.5 mg/ml compared with autogenous bone graft. Bone induction is evident in the metal cage by CT scan, and this material becomes increasingly dense with time: even out at two years it is signi¢cantly denser than it was at the one year time point. The fusion rate is equivalent to autograft, approaching 100%. This completely eliminates the need for an iliac crest autograft harvest. Some of the patients in the control group were still complaining of pain two years after the surgery. In addition, there is decreased operating-room time and blood loss when rhBMP2/ACS was used. Finally, I want to comment on where I think we are going with these sorts of technologies in the future. I think we can expand the indications for rhBMP2 by looking at injectable formulations of BMP and other implantable matrices or sca¡olds, in a variety of clinical scenarios. Looking to other tissue engineering areas, we can think about using BMPs in cartilage repair and tendon repair. van Blitterswijk: How did you de¢ne success in the dental trial? Wozney: The ability to place and load dental implants. These implants fail at a variety of times. They can fail when you put them in, or when they are uncovered and loaded. Success is de¢ned as the ability to load the implant and for it to stay loaded for 12 months. It is a long procedure: typically the bone induction phase is six months, then implants are placed and allowed to osseointegrate for another six months, and then loading is begun. Ohgushi: For tibial fracture you use two di¡erent concentrations of BMP2. What is the average volume of the implant, and therefore the total amount of BMP used? Wozney: We used one sponge, which is about 8 ml. This equates to 6 or 12 mg of BMP. Ohgushi: How much did you use in the spinal fusion? Wozney: The concentration is the important parameter. 1.5 mg/ml is the most e¡ective concentration in all these applications. Helms: What I know about BMPs has come in part from their role in embryonic development. Thinking about what happens when you put BMPs into a site at the time of surgery, have you ever evaluated the e¡ect on programmed cell death? This is a well documented role of BMPs. Wozney: This has been best characterized during embryonic development. Helms: I have never seen anything published on this. Have they shown that BMPs do not induce programmed cell death? Wozney: There are several published reports of BMPs inducing apoptosis of cancer cell lines, and anectodal reports of BMPs inducing, or inhibiting, apoptosis in other cell types. We have not done a comprehensive study of the e¡ect of rhBMP2 on apoptosis. However, we have conducted extensive

238


toxicology studies following implantation of rhBMP2/ACS. No local cell death or other toxicological consequences were seen histologically. Quintavalla: You used a sponge to carry the BMP. Have you tried using sponges that the BMP would adhere to, where the sponge could break down, releasing the BMP? Wozney: We have tried a variety of matrix materials. It’s a bit of a trade-o¡. You need a material that will bind the BMP and keep it there long enough, but if it binds it too strongly there won’t be su⁄cient bioavailability. There has to be some continuous release. We have evaluated both hyaluronic acids and hydroxyapatites, which bind BMP more strongly than collagen. In the right format both work well. van Blitterswijk: What would happen if you used just a sca¡old that was known to be an e¡ective bone forming matrix such as hydroxyapatite ceramic, without BMP? How would this compare with BMP2/collagen? Wozney: In spinal fusion surgeons don’t use these sorts of void bone ¢llers because they don’t work at all. Autograft is needed. I think this is also true in sinus £oor augmentation. The standard of care is either autograft alone or autograft plus allograft extender. van Blitterswijk: I’m surprised that no one has tried using much more osteoconductive carriers. Caplan: In all fairness, there is a phenomenological base for this which goes back to the old Urist and Reddi studies in which BMP-loaded material is placed into a particular site, and there is a cascade of events. van Blitterswijk: I am convinced the BMP is doing something; I was just wondering whether it could be even more e¡ective using a more osteoconductive sca¡old. References Govender S, Csimma C, Genant HK et al (BESTT Study Group) 2002 Recombinant human bone morphogenetic protein 2 for open tibial fractures: a prospective, controlled, randomized study of four hundred and ¢fty patients. J Bone Joint Surg Am 84-A:2123^2134 Boden SD, Zdeblick TA, Sandhu HS, Heim SE 2000 The use of rhBMP-2 in interbody fusion cages. De¢nitive evidence of osteoinduction in humans: a preliminary report. Spine 25:376^ 381 Boyne PJ, Marx RE, Nevins M et al 1997 A feasibility study evaluating rhBMP-2/absorbable collagen sponge for maxillary sinus £oor augmentation. Int J Periodontics Resorative Dent 17:11^25 Valentin-Opran A, Wozney J, Csimma C, Lilly L, Riedel GE 2002 Clinical evaluation of recombinant human bone morphogenetic protein 2. Clin Orthop 395:110^120

Final discussion and summing-up Caplan: I’d like to end this meeting by asking a couple of people to summarize what we have discussed, and suggest where this technology is going to go in the future. First I’d like to call upon Steve Goldstein. Goldstein: I’d like to highlight some of the critical issues that came out of our discussions at this meeting. All of us are convinced that some combination of four components seems to be critical for tissue engineering success: cells, biofactors, matrices and mechanical/environmental factors. In those areas, while there will be a multitude of approaches, there are a number of issues we need to continue to concentrate on. One of these is the biofactors. Whether they are given exogenously or endogenous expression is stimulated, but which factors will be the more critical ones? In particular, is there some sequence or temporal aspect to when they should be made available or stimulated? There is an implication that there have been innumerable sca¡olds and matrices utilized. There was also an inference that each of them has a variety of characteristics that may be of bene¢t in one condition versus another. Perhaps what we are missing is a clear articulation of the attributes of these matrices such that a tissue engineer could strategically decide on which one might be more appropriate for their chosen use and location. Then there is the role of the cells. We have had a lot of discussion about this. First, there seems to be insu⁄cient information about the relationship between the level of commitment of the cells to a particular fate and their role in promoting tissue formation. Are the cells in their pluripotential state, or are they already down the pathway of di¡erentiation towards chondrocytes and osteoblasts? How might it be bene¢cial to take those cells at any one of those stages? Another issue is that we all have professional opinions, and yet the experiments aren’t there: that is, trying to follow the fate of these cells. What role do they play in becoming part of the matrix-incorporated cells in the tissueengineered construct, and what role might they be playing in either expressing or pumping out factors that are stimulating a response? We need to try to understand the fate of these cells better so that we might use that information. The next area is that of vascularization. Arnold Caplan promoted this at the beginning of the meeting. We need a better understanding of whether it is just vascularity itself, or whether it is about angiogenic or anti-angiogenic factors, and how this leads to the evolving incorporation of the variety of tissues that we are interested in. In some respects it may be more advantageous to have more 239

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FINAL DISCUSSION AND SUMMING-UP

anti-angiogenic activity. In other tissues the opposite might be true. Another issue discussed was the need for a more quantitative understanding of how physical forces are going to play of role. In a bioreactor, if you are trying to create a tissue with certain characteristics, what are the mechanical conditions that will be valuable? In vivo, what are the parameters associated with how much local strain or stress is critical for promoting the formation and remodelling of these tissues? There were some comments about the need for clear de¢nitions of the targeted functional properties we should have for these constructs. These might be di¡erent from the normal properties of the tissues that we are trying to replace. In some cases our initial targets may not be absolutely normal. How good does engineered cartilage have to be? We have to think of our ¢eld as an evolving one. There seems to be some need for understanding the surgical usability of these devices. You may have a great construct, but if it can’t be held into a defect and easily handled by the physician, this is a limitation. Then we had a lot of discussion about the design of the clinical studies. How should they be designed? What is the feasibility of controls and prospective randomized procedures? What comes with this is the development of outcome measures, which are very di⁄cult. There is hope that through imaging technologies and others we will have more quantitative measures, but this is an area we need to pay attention to. Ratcli¡e: We have had an interesting set of presentations at this meeting, covering a variety of aspects of tissue engineering. Perhaps what we haven’t done is to describe the di¡erent ways that tissue engineering can be used to generate a clinical result. The ¢rst way is simply to facilitate the body to repair itself. If you put in a tissue-engineered material the body is allowed and helped to repair itself. What we did see a lot of is the next level, in terms of complication and di⁄culty, where the tissue-engineered products induce the body to repair itself. Finally, the next stage is where a tissue-engineered construct is put in and expected to function at or near the time of implantation. An example would be if we made perfect articular cartilage and it had a good compressive sti¡ness: the patient would have a functional joint immediately after surgery. This is very di⁄cult, but it is where people tend to think that tissue engineering should be. We are not there yet. We are still at the facilitating and inducing repair processes stage. These are therefore the gradations that exist within the applications of tissue engineering in clinical treatments. Steve Goldstein mentioned a couple of things that deserve reiteration. One was that in terms of our animal study designs, it is critical that these studies are designed correctly. At the present time it is unclear in animal studies what the objectives should be, and it is also unclear what we should regard as successful outcome. Secondly, we are using these animal studies to feed into the clinical trials, and we are uncertain of how to design those clinical trials and what the true outcome

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measures should be. There needs to be work done to generate useful outlines for clinical trials. Caplan: I’d like to take a few minutes to provide my summary. First I’d like to congratulate the participants here and others in our industry who have e¡ectively translated tissue engineering ideas into clinically relevant protocols. In the early stages of this industry, we have some notable examples of success. It is interesting that starting at 1850 and continuing to today, it has taken 20 years on average for individual discoveries to translate to clinically relevant protocols or to functionally relevant materials. This stands true for innovations as diverse as BMPs, light bulbs and radios. If any of you have developed something really exciting last week before you came here, it will take 20 years to translate to therapy! I see a huge horizon: there are a variety of technologies for which we now have imperfect solutions. These need to be made perfect. We have a variety of medical and practical issues that have to do with our lifestyles and demands on our bodies. These will require complex and multicomponent solutions. There is an entire area that is underdeveloped and under-served, which is the delivery of adequate and useful healthcare management for people above the age of 65. This is one of the last, and more challenging frontiers of medicine. I see tissue engineering as a complicated, multicomponent management of biological processes that will tax the basic science information base and will require innovative combinations of basic information in order to proceed. The cleverness and creativity of clinical and basic scientists are the key factors for success here. The thoughtful criticism of each other’s work is the cornerstone of scienti¢c enquiry. You can see at this small meeting that there were insightful suggestions made by the audience, which some people will take on board and use to change their protocols. I thank you all for donating so much of your time to participate in this meeting.

Index of contributors Non-participating co-authors are indicated by asterisks. Entries in bold indicatepapers; other entries refer to discussion contributions.

A

E

*Abatangelo, G. 203

*Engelberts, P. J. 148

B

G

Barry, F. P. 29, 49, 86, 96, 97, 98, 99, 100, 101, 102, 112, 116, 144, 188, 198, 199, 229 *Bianchi, G. 133 *Borrione, A. 203 *Brittberg, M. 175 *Brocchetta, D. 203 Buschmann, M. D. 67, 80, 114, 187

Goldstein, S. 15, 31, 64, 67, 84, 99, 145, 170, 172, 173, 174, 239 Grodzinsky, A. J. 52, 64, 65, 66, 67, 68, 172, 186, 200 H Hardingham, T. 26, 30, 32, 66, 68, 115, 143, 167, 201, 202 Helms, J. 25, 28, 97, 98, 146, 168, 188, 232, 237 *Ho£and, I. 148 Hollander, A. P. 13, 32, 50, 115, 116, 143, 169, 173, 188, 189, 203, 218, 229, 230, 231 Huckle, J. 32, 103, 112, 113, 114, 115, 116, 188, 199 Hunziker, E. B. 11, 12, 70, 78, 79, 80, 130, 131, 167, 169, 189, 199, 233

C Cancedda, R. 12, 14, 27, 97, 129, 133, 143, 144, 145, 146, 147, 168, 170, 171, 174 Caplan, A. I. 1, 10, 11, 13, 14, 15, 17, 25, 26, 27, 28, 29, 30, 31, 32, 46, 47, 48, 65, 67, 68, 69, 78, 79, 80, 81, 82, 83, 84, 85, 97, 98, 99, 100, 102, 112, 113, 114, 115, 116, 117, 128, 129, 130, 131, 144, 145, 146, 147, 167, 170, 171, 172, 173, 174, 187, 188, 189, 200, 230, 231, 232, 236, 238, 239, 241 *Carter, A. 103

K Ka¢enah 65, 169 *Kirby, B. 103 *Kisiday, J. 52 *Kon, E. 203

D *Davisson, T. 103 *de Bruijn, J. D. 148 *Dekker, R. J. 148 *Derubeis, A. 133 *Dickinson, S. C. 218 *Dootson, G. 103 *Dunkelman, N. 103

L *Laurent, D. 190 *Lee, J. H. 52 *Li, J. 148 242

INDEX OF CONTRIBUTORS

Lindahl, A. 10, 25, 31, 48, 69, 78, 114, 117, 143, 146, 169, 175, 187, 188, 189, 201, 231, 232, 233 Lohmander, L. S. 2, 10, 11, 12, 13, 14, 15, 47, 48, 49, 82, 83, 85, 96, 97, 98, 116, 128, 145, 173, 174, 187, 200, 201, 231, 233 M *McTaggart, S. 103 *Marcacci, M. 203 Martin, I. 48, 112, 114, 115, 131, 170, 189 *Mastrogiacomo, M. 133 *Medcalf, N. 103 *Miyake, J. 118 *Muraglia, A. 133 O O’Byrne, E. 190, 199, 200, 201 Ohgushi, H. 26, 99, 100, 118, 127, 128, 129, 130, 131, 132, 167, 168, 237 P Pavesio, A. 14, 15, 49, 68, 69, 84, 97, 114, 128, 203, 218, 229, 230, 231, 232, 233 *Pellas, T. 190 *Peterson, L. 175 Q *Quarto, R. 133 Quintavalla, J. 11, 50, 238

243

Richardson, B. 101 *Riley, S. 103 S *Schreiber, R. 103 Schwartz, H. 80, 101, 129, 232 *Sims, T. J. 218 *Soranzo, C. 218 *Stevenson, S. 103 T *Tateishi, T. 118 *Torasso, F. 203 Trippel, S. 28, 29, 48, 81, 84, 101, 102, 113, 144, 173, 201, 229, 230 V van Blitterswijk, C. A. 14, 15, 48, 68, 82, 84, 98, 115, 129, 130, 131, 145, 146, 148, 167, 168, 169, 171, 173, 237, 238 Vunjak-Novakovic, G. 30, 34, 46, 47, 48, 49, 50, 66, 115, 131, 169, 172, 173 W Wozney, J. 29, 46, 127, 128, 129, 131, 234, 236, 237, 238 *Wright, E. 103

R Ratcli¡e, A. 12, 13, 50, 83, 85, 100, 103, 115, 172, 230, 231, 240

Z *Zanasi, S. 203

Subject index angiogenic factor supplementation 146 animal models 84 cell-based cartilage repair 5 ideal 70 limitations 1 outcome measures 84^85 variability 80^81 anlagen 21 anterior cruciate ligament tears 4, 10^11 apatite 121 apoptosis autologous chondrocyte transplantation 183 BMP2 stimulation 20, 237^238 Wnt14 25^26 arthritic joint environment 16 arthroscopic lavage and debridement 204 arthroscopic probes 7 articular cartilage biomechanical environment 52^54 magnetic resonance imaging 190^198 mesenchymal stem cell therapy 86 arti¢cial joint failures 83 ascorbic acid-2-phosphate 88 autogenic perichondrium/periosteum transplantation 5 autologous bone implants 137 autologous cells 90 donor age 110 genetic modi¢cation 8 sources 97 autologous chondrocyte transplantation 92, 176, 177, 204, 218 biological repair mechanisms 182^183 biopsies 182, 218^229 economics 180^181 future developments 184^185 long term results 178^180 methodology 178 patient eligibility 179^180 pilot studies 178 quality standards 183^184 regulation 184

A abrasion arthroplasty 92, 204 absorbable collagen sponge 234^235 acellular £aps 189 activated leukocyte cell adhesion molecule (ALCAM) 87 active-MMP9 60 adipocytes 89 adipose tissue, stem cells 89, 104 agarose gels 37 age and ageing aggrecan synthesis 90 alkaline phosphatase activity 130 bone morphogenetic protein responsiveness 128 cartilage repair 15 cartilage turnover 223^224 cell cultures 130 compression response 68 fracture healing 128 mesenchymal stem cells 3 tissue donors see donor age aggrecan ageing and synthesis 90 autologous chondrocyte transplantation 182 biological half-life 3 biomechanics 53^54 collagen network 66, 68 expression 88 extracellular matrix 21 alginate culture 62, 87, 114 alkaline phosphatase age 130 ceramic cultured bone 121 DNA content 153, 155, 157, 161 monolayer mesenchymal stem cells 88 prostaglandin E2 162 allogenic cell sources 100 allografts 92 alumina ceramics 118, 119 alveolar ridge augmentation 235 244

SUBJECT INDEX

repair tissue 79, 181^182, 186^187 success rate 187 autologous grafts, donor age 110 autologous perichondrium/periosteum grafts 204 B back-yard trials see garage trials BESTT Study Group 236 b-sheets 55 b1 integrin 62 bias 6 bioactive glasses 119 bioceramics 118^127, 136 bone marrow stromal cell composites 137^139 biocompatibility 71 biodegradability 71 biofactors 239 biomechanics 76 autologous chondrocyte transplantation 181^182 cartilage 54^55 chondrocytes 52^54, 68 engineered cartilage 64^65 biopsies 7, 182, 218^229 bioreactors 35, 75, 104 cultivation duration 42^45 culture vessels 37^39 functions 37 biphasic HA/TCP ceramics 120 blinded trials 6^7, 13, 14, 15 blood-borne cells 73 Bmp5 knockout mice 26 bonding osteogenesis 120 bone bonding 119^120 chamber 172 demineralized matrix 129^130 di¡erent types 26 formation 22, 119 functionality 169, 173 genetic disorders, osteogenic cell injection 140^141 implants 137 large bone loss 133, 136 porous ceramic cultures 120^123 regeneration 118^127, 133^143 tumours, bone culture 125 vascularization 73

245

bone marrow aspirate, fresh 101 donors 100 stimulation 204 transplant recipients 134 bone marrow stromal cells 133^143 bioceramic composites 137^139 gene therapy 141 osteogenic cell source 134^135 recovery 87 bone morphogenetic protein (BMP) bone apposition 163 carrier system 234 cell-based therapy compared 127^128 chondrocyte cultures 62 mesenchymal stem cell attractant 171 bone morphogenetic protein 2 (BMP2) 234^238 apoptosis 20, 237^238 carrier system 234 dental implants 235, 237 mandible restoration 235 mesenchymal stem cell composites 123 spinal fusion 236^237 tibial fractures 236, 237 bone morphogenetic protein 7 (BMP7) 62 BrdU 116 C Ca2+/calmodulin-dependent cascades 59 calcium phosphate 22, 167 calcium phosphate ceramics 119, 131 cAMP 59, 60 cartilage anisotropia 3 biomechanical environment 52^54 biomechanical properties 54^55 charge density 195^196 collagen quality 67 composition 2^3 con¢ned-compression equilibrium moduli 49 di¡erent types 23 explants 57^59 full/partial thickness injuries 4 heterogeneity 65, 116 magnetic resonance imaging 190^198 nourishment 3 rods 21^22 surgical protocols 92

246

cartilage (cont.) thickness and volume 191^192 turnover 27, 31, 116^117, 223^224, 226^228 cartilage-derived morphogenetic protein 1 (cdmp1) 183 cartilage matrix protein 88 cartilage oligomeric matrix protein (COMP) 88 autologous chondrocyte transplantation 182 cartilage repair 3^8 ageing and 15 bene¢ts 6 chondrocytes 175^186 clinical trials 7, 14^15 global perspective 12 in vitro culture and in vivo implantation 34^46 mesenchymal stem cells 8, 90, 92^93, 104 osteoarthritis 3^4 patient-base 7^8 potential 176^177 CD73 87 CD105 87 CD166 87 cell cultures 19^20 elderly 130 cell density 115^116 cell di¡erentiation 20 cell expansion 106 cell markers, chondroprogenitor cells 32, 183 cell^sca¡old^bioreactor system 35 cell-surface markers, mesenchymal stem cells 87, 116 cell tra⁄cking 31 cell turnover 116^117 ceramics see bioceramics chondrocytes 2, 3 biomechanical environment 52^54, 68 cartilage repair 175^186 di¡erentiated 103^112 expansion 106 gene expression 57^59 high-density monolayer 59^60 hypertrophic 27^28, 29 immature versus adult 29 metabolism, mechanical regulation 57^60 nasal 110, 188

SUBJECT INDEX

chondrogenesis 35^36 cell density 115^116 in vitro 39^41 mesenchymal stem cells 87^88 chondroprogenitor cell marker 32, 183 clinical algorithm 179^180 clinical trials 7, 14^15 collagen cross-linking amino-acid analysis 222 Hyalograft1 C biopsies 226^228 lysyl hydroxylase 220 collagen ¢brils 3, 53 collagen sponge 234^235 collagen type I assay 221 autologous chondrocyte transplantation 182 cartilage content 219 extracellular matrix 22 osteoid 22 repair tissue biopsies 222^223 collagen type II 88 assay 222 autologous chondrocyte transplantation 182 cartilage content 219 chondrocyte cultures 62 compression sensitivity 61 extracellular matrix 21 gels 61 magnetization transfer imaging 193 repair tissue biopsies 222^223 collagen type IIA 183 collagen type IIB 183 colony-forming units ¢broblastic 134 compliance 48 compression 54, 61, 65, 68 coral sca¡olds 139 cryopreservation 114 cyclic stretching 59^60 cytokines 90, 170

D degenerative disease, stem cell depletion 89^90 demineralized bone matrix 129^130 dental implants 235, 237 dermal ¢broblasts 104

SUBJECT INDEX

dexamethasone 88, 89, 120^121, 134, 152, 153 alkaline phosphatase expression 155, 157, 161 DNA synthesis 60 donor age 110 construct quality 105 dynamic compression response 68 GAG chains 105, 107^108, 113 hyaline-like material 107, 115 Drawer test 99 dynamic compression 54, 65, 68 E economics, autologous chondrocyte transplantation 180^181 elderly cell culturing 130 fractures 128 see also age and ageing embryonic development 3, 17^25 embryonic stem cell therapy 185 endoglin 87 environmental factors arthritic joint 16 developing tissue 30^31 healing outcome 72^73 normal articular cartilage 52^54 EQ-5D (EuroQol questionnaire) 209 equilibrium compressive modulus 54 equilibrium shear modulus 54 equilibrium tensile modulus 54 ethical issues 6, 83 evaluator blinding 6, 14 external ¢xation 144^145 externally regulated kinase (ERK) 59 extracellular matrix 21, 22^23, 53^54 F fatty acid synthetase 89 femoral condyles 178 femur cell proliferation 98 ¢brin glue 73^74 ¢broblast growth factor 2 (FGF2) 59, 134^135, 143, 183 ¢broblast growth factor receptor 3 (FGFR3) 32, 183 ¢brocartilage 48, 82, 92, 176 ¢bromodulin 88

247

¢bronectin 22, 163 ¢brous meshes 37 ¢xation 144^145 £uid £ow 54 see also water management £uid shear forces 59 follow-up 6 MRI studies 200^201 fractures bone culture 123, 125 distal tibia 129 in elderly 128 tibial shaft 236, 237 functionality 169, 173 G gadolinium-enhanced MRI 195^196, 199^201 garage trials 82, 84, 233 gel sca¡olds 36^37, 61 Gelfoam 93 gene expression chondrocytes 57^59 hydrostatic pressure 60 limb and cartilage development 183 gene knockouts 23 gene therapy, bone marrow stromal cells 141 genetic factors 4, 11 genetically modi¢ed cells 8 Genzyme 78, 82 glass ceramics 119, 120 global perspective, cartilage repair 12 b-glycerophosphate 88, 120^121 glycosaminoglycans assay 222 chondrogenesis marker 41 compression 61, 65 cyclic stretching 60 donor age 105, 107^108, 113 £uid £ow resistance 54, 67 synthesis rate and tissue composition 66 goat model 80, 98, 99, 102 green £uorescent protein 94, 99^100 groove of Ranvier 176 growth factors 4, 72, 170 H haematopoietic stem cells 29 healing outcome 72^73

248

healing process 81 heart muscle embryology 31 mesenchymal stem cell therapy 86^87 heat shock protein 70 (hsp70) 59, 60 hip implants 82 revision surgery 149, 163, 164 histology autologous chondrocyte transplantation 181^182 unreliabiity 7 HYAFF1 11 sponge 49, 93, 205, 232, 233 hyaline cartilage 175 di¡erentiation 21 turnover 223^224 ‘hyaline-like’ tissue 104, 105, 106, 107, 110, 115 Hyalograft1 C 203^217 arthroscopic implantation 206, 208, 215 biopsies 220^221, 224^228 patient registry 207^208 pilot studies 207 safety 206^207 hyaluronan anti-angiogenicity 232 cartilage reconstruction 204^205 extracellular matrix 22 hyaluronan sca¡olds 23^24, 230, 232 see also Hyalograft1 C hyaluronidase 23 hydrogel sca¡olds 109 hydrostatic pressure 59, 60, 104, 112 hydroxyapatite 101, 119, 167, 173 hydroxyapatite; b-tricalcium phosphate sca¡old 139 hydroxylysylketonorleucine 220 hydroxyproline assay 222 hydroxypyridinium cross-link 220 I iliac crest cell proliferation 98 Ilizarov technique 136 immobilization 47, 48 implant lifetime 173^174 topography 160 indium oxide labelling 144 in£ammatory cytokines 90 inositol-1,4,5-triphosphate 59

SUBJECT INDEX

insulin 89 insulin-like growth factor 1 (IGF1) 36 integrins 62, 101 interbody spinal fusion 236^237 intercellular matrix 2^3 interleukin 1 (IL1) 60 interleukin 1a (IL1a) 90 interleukin 1b (IL1b) 61 interleukin 6 183 intramuscular implants 131 J joint replacement criterion 13 K keratan sulfate 88 knee injuries, osteoarthritis development 3, 175^176 L lateral plate mesenchyme 18 ligament repair 97 loading protocol 76 loss to follow-up 6 lysyl hydroxylase 220 M MACI technique 178 magnetic resonance imaging, cartilage 190^198 follow-up studies 200^201 gadolinium-enhanced 195^196, 199^201 sodium imaging 197, 201 magnetization exchange rate 194 magnetization transfer imaging 192^195 mandible bone induction 235 matrix metalloproteinases (MMPs) 59 MMP9 60 pro-MMP9 60 matrix proteins 2 maxillary sinus £oor augmentation 235 mechanical stimulation 37 mechanics chondrocyte metabolism 57^60 fetal development 31 sca¡old cultures 60^62 see also biomechanics medium perfusion 62

SUBJECT INDEX

meniscus cell types 104 injuries, osteoarthritis 3, 4, 12 remodelling 94, 96^97 removal 12 mesenchymal progenitor cells 18, 30, 32, 33, 140^141 mesenchymal stem cells 86^96 ageing 3 bioceramics and 118^127 bone morphogenetic protein as chemoattractant for 171 bone morphogenetic protein composites 123 cartilage repair 8, 90, 92^93, 104 cell surface markers 87, 116 chondroprotection 94 compression sensitivity 61 degenerative disease 89^90 di¡erentiation 29, 87^89 in vitro culture from fresh bone marrow 120 manipulation 120^123 multipotency 86 osteoarthritis 93^94 osteoblasts 119 osteogenic di¡erentiation in ceramics 119^120 mesh sca¡olds 37 methyl-isobutylxanthine 89 microenvironment arthritic joints 16 healing outcome 72^73 microfracture 92, 204 mineralized matrix 88 mitogens 19^20 mixed lymphocyte reaction 101 monoclonal antibodies 87 morphogenesis 19 mosaicplasty 92, 177 multiple techniques 81 muscle myosin isoforms 31 stem cells 104 myocardium embryology 31 mesenchymal stem cell therapy 86^87 myosin isoforms 31

249

N Na+ concentration 197, 201 nasal cartilage 115, 188 nasal chondrocytes 110, 188 nuclear factor of activated T cells ‘p’ (NFATp) 27 nuclear transfer experiments 30

O oligomers 23 osteoarthritis 2 anterior cruciate ligament tears 4, 10^11 bone culture 123, 125 cartilage repair 3^4 clinical trial design 7 knee injuries 3, 175^176 magnetic resonance imaging 192 magnetization transfer imaging 195 meniscal injuries 3, 4, 12 progress 6 stem cell depletion 89^90 stem cell therapy 93^94 osteoblasts 21, 22, 119 de¢nition 28, 29 osteocalcin 183 osteocalcin mRNA 121 osteochondral transplant 177, 204 osteochondritis dissecans (OCD) 178, 187, 189 osteogenesis imperfecta allogeneic bone marrow transplantation 140 stem cell therapy 87 osteogenic cells bone marrow stromal cell source 134^135 genetic disorders 140^141 osteoid 22 osteoinductivity 149, 167^168 osteosarcoma 129 outcome, surgeon versus patient assessment 14, 209^212 outcome measures animal models 84^85 biopsy 7, 219 choice 7 de¢nition 172 oxygen tension 62, 169

250

SUBJECT INDEX

P

R

pain-killers 13 papain, contrast MRI 195^196 patellar lesions 178, 179 patient immobilization 47, 48 patient registry, Hyalograft1 C 207^208 PCL sca¡olds 108 pellet cultures 87, 88 peptide hydrogels 55, 61 perichondreal ring 176 perichondrium grafts 204 inappropriate use of term 21 transplantation 4, 5, 176 periosteal £ap 72^73, 78, 79 periosteum di¡erentiation 21 grafts 204 role 183 transplantation 4, 5, 176 peroxisome proliferator activated receptor g (PPARg) 89 PGA sca¡olds 49, 61, 108, 109, 110 pharmacology 71^72 phosphoinositol 59 placebo e¡ect 6 plasticity 20, 27, 28, 29, 30 PLGA (polylactic-coglycolic acid) sca¡olds 108^109 PLGA-PEG sponge 49 pre-osteoarthritis 8 progenitor cells 3, 27 prostaglandin E2 162 protein synthesis 60, 61 proteoglycans 2, 66 cyclic stretching 60 extracellular matrix 22 magnetic resonance imaging 195^196, 197, 198^199 magnetization transfer e¡ect 193, 194 water management 64 proteolytic tissue extraction 222 punch biopsy 231

rabbit model, variability 80 randomized, controlled trials 6^7, 14, 81 recombinant bone morphogenetic protein (rhBMP) 62 recombinant bone morphogenetic protein 2 (rhBMP2) 234^238 apoptosis 237^238 carrier system 234 dental implants 235, 237 mandible restoration 235 mesenchymal stem cell composites 123 spinal fusion 236^237 tibial fractures 236, 237 regulation, autologous chondrocyte transplantation 184 revision hip surgery 149, 163, 164 rheumatoid arthritis, in vitro bone culture 123

Q quality standards, autologous chondrocyte transplantation 183^184

S safety Hyalograft1 C 206^207 recombinant bone morphogenetic protein 235 SB 10 (Sleeping Beauty) 87 sca¡olds 35, 36^37, 55, 93, 108^109, 110, 136 angiogenic factor supplementation 146 chemical properties 49 ideal 108 mechanobiology 60^62 scar tissue 18 second-look biopsies 214, 221, 224^228 self-assembling peptides 55, 61 serum-containing media 113 SH (serum hepatitis domains) 2/3/4 87 ‘short ears’ 26 sodium imaging 197, 201 somitic mesenchyme 18 SOX9 183 Sox9 183 SOX9 31 Sox9 61 spin-lattice 191 spin^spin 191 spinal fusion 131, 236^237 sponges 37, 49 spongialization 204

SUBJECT INDEX

251

spontaneous repair 73, 80 stack cell 25 stair climbing 53 stem cells 3, 104, 134 assay 28^29 cell markers 116 mechanical loading 61 see also mesenchymal stem cells Stro-1 87 structure^function 64 subchondral bone cysts 80 subchondral bone tissue 79 subchondral drilling 204 subcutaneous implantation model 131 surrogate measures 85 synovial £uid 3

tissue repair speed of 18 stem cells 89^90 titanium structures 118, 119, 150^151, 155 transforming growth factor b (TGFb) 60, 72, 87, 183 transforming growth factor b1 (TGFb1) 62 treatment algorithm 179^180 tricalcium phosphate 120 trypsin 222 tumour necrosis factor a (TNFa) 90 tumours bone culture 125 resection 174 type I /type II collagen see collagen type I; collagen type II

T

V

T1 relaxation 191 T2 relaxation 191 telomerase activity 135 tendons, mesenchymal stem cell therapy 86 thymidine 116 tibial fractures bone morphogenetic protein 236, 237 distal 129 tissue di¡erentiation 21^23 tissue inhibitors of metalloproteinases 59 tissue regeneration 18

vascular endothelial progenitor cells 18 vascularization 3, 73, 137, 145^147, 239 ‘virtual’ partial-thickness defects 73^74 vitamin C 120^121 W water management 64^65, 67 WNT14 183 Wnt14 183 WNT14 31 Wnt14, apoptosis 25^26

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