A Practical Manual for
Musculoskeletal Research
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A Practical Manual for Musculoskeletal Research
Editors
Kwok-Sui Leung The Chinese University of Hong Kong, Hong Kong
Yi-Xian Qin State University of New York at Stony Brook, USA
Wing-Hoi Cheung The Chinese University of Hong Kong, Hong Kong
Ling Qin The Chinese University of Hong Kong, Hong Kong
Associate Editors
Kwong-Man Lee The Chinese University of Hong Kong, Hong Kong
Jian-Quan Feng Texas A&M Health Science Center, USA
Chun-Wai Chan The Chinese University of Hong Kong, Hong Kong
World Scientific NEW JERSEY
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LONDON
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Published by World Scientific Publishing Co. Pte. Ltd. 5 Toh Tuck Link, Singapore 596224 USA office: 27 Warren Street, Suite 401-402, Hackensack, NJ 07601 UK office: 57 Shelton Street, Covent Garden, London WC2H 9HE
British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library.
A PRACTICAL MANUAL FOR MUSCULOSKELETAL RESEARCH Copyright © 2008 by World Scientific Publishing Co. Pte. Ltd. All rights reserved. This book, or parts thereof, may not be reproduced in any form or by any means, electronic or mechanical, including photocopying, recording or any information storage and retrieval system now known or to be invented, without written permission from the Publisher.
For photocopying of material in this volume, please pay a copying fee through the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA. In this case permission to photocopy is not required from the publisher.
ISBN-13 978-981-270-610-2 ISBN-10 981-270-610-0
Typeset by Stallion Press Email:
[email protected] Printed in Singapore.
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Foreword Qian Chen
Many textbooks have been published in the musculoskeletal research field in recent years. However, very few of them are practical manuals for laboratory techniques. This timely book contains step-by-step protocols for performing various experiments in the musculoskeletal field, ranging from cell and molecular biology to histology and microscopy, and from laboratory animal models to imaging and biomechanical testing. It is truly a valuable tool that fills a gap in musculoskeletal research. When I was an undergraduate student majoring in biochemistry in Fudan University, China, we relied on Molecular Cloning: A Laboratory Manual to perform molecular biology experiments in our senior honors theses. We affectionately called it “Maniatis’ book”, which refers to Dr Tom Maniatis, an author of the book. This was easily the most widely used book during our senior year, and a “bible” in the nascent molecular biology field. Twenty-five years and many editions later, Molecular Cloning still remains a useful book, although Dr Maniatis no longer serves as a coauthor. I hope that this book can achieve the same extent of usefulness as Maniatis’ book. It definitely has the potential. Unlike the research Qian Chen is Ehrlich Professor and Director, The Warren Alpert Medical School of Brown University; and Chairman of Board, International Chinese Hard Tissue Society.
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field 25 years ago, cutting-edge research nowadays is often derived from multidisciplinary collaborations; this is certainly true in the musculoskeletal research field. Very often, different types of techniques including cell, molecular biology, and mechanical testing are necessary to be used at the same time in order to assess the function of a tissue in the skeletal system. This poses a great challenge for researchers and students, who are usually technically proficient in only one of the disciplines. Therefore, this book will be very useful to researchers and students, since it covers most (if not all) of the cutting-edge techniques in this field. Each technique described in this book is presented in the form of a step-by-step protocol. It is intended to be used not only by researchers within the discipline but also by those outside of the discipline, especially students who are performing these experiments for the first time. I would like to congratulate the editors who have assembled a fine team of authors from different disciplines including cell biology, molecular biology, histology, veterinary science, radiology, and bioengineering. Another common characteristic of the authors is that most of them are researchers of Chinese heritage working in different areas of the world such as the United States, Europe, Australia, mainland China, Hong Kong, Taiwan, and other parts of Asia. Many of them are members of the International Chinese Hard Tissue Society (ICHTS). Founded in August 1994 at Sun Valley, Idaho, the International Chinese Hard Tissue Society is a nonprofit professional organization, striving to facilitate the exchange of ideas and to promote collaborative research among scientists in the field of hard tissue research. The ICHTS does not have, nor do we intend to establish, any political affiliation with any specific nation or region. The ICHTS is a worldwide organization. Its doors are open to all professionals, trainees, and students working in hard tissue research (and other related fields) or clinical practice. The ICHTS supports junior members to participate in ICHTS annual meetings, which are held concurrently with annual meetings of the American Society of Bone and Mineral Research (ASBMR) and the Orthopaedic Research Society (ORS), by awarding them the Webster Jee ICHTS travel awards.
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In recent years, the ICHTS has matured rapidly into a vibrant organization with more than 1000 members worldwide, expanding its influence within Chinese as well as international research communities. ICHTS members are making outstanding contributions to the research community through scientific publications and presentations. Many ICHTS members have achieved international recognition and been appointed as leaders at academic institutions and pharmaceutical companies. These members have made a book like this possible. The ICHTS has published two books in bone biology to serve as useful tools for educational purposes. In addition, the ICHTS has formed a good working relationship with various organizations in the field, including the Chinese Orthopaedic Research Society (CORS), Chinese Speaking Orthopaedics Society (CSOS), and Chinese Society of Osteoporosis and Bone and Mineral Research (CSOBMR). The ICHTS has successfully co-organized international conferences in China with these societies. This book is a coproduction of the ICHTS with the CORS. We hope such fruitful collaborations will be continued to benefit the whole research field. Finally, I hope that this practical manual can help our colleagues, especially young students who are new to the field and trying to make an experiment work for the first time. Molecular Cloning taught me how to perform gene cloning as a college student, and convinced me that an experiment done correctly should yield beautiful results whether we expect them or not. I hope this book can inspire young students to launch into careers in research, just like what the Molecular Cloning manual did for me 25 years ago.
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Foreword Shu-Xun Hou
As chairman of the Chinese Orthopaedic Research Society (CORS), I would like to express my sincere congratulations on the publication of this book. It is a useful manual of laboratory techniques developed and adopted for musculoskeletal research, with many contributions from CORS board members. I would like to show my appreciation to the editorial team for their endeavors in the design, coordination, and preparation of this manual book, which bridges laboratory research and clinical studies. It will definitively benefit our dynamic orthopedic research and the WHO-designated “bone joint decade” (BJD). The development of cutting-edge technologies is not only for the sake of basic sciences, but is also employed in translational research in the treatment of fracture healing, osteoporosis, osteoporotic fracture repair, osteonecrosis, osteoarthritis, scoliosis, and many other musculoskeletal conditions. The remarkable achievements in orthopedic surgery in recent years are attributable to innovations in basic science, especially in biomechanics, bioengineering, and material sciences. It is exciting that more and more orthopedic surgeons have recognized the importance of basic science and have devoted themselves to this field. However, not every researcher can reach their expected goals Shu-Xun Hou is Chairman, Chinese Orthopaedic Research Society (CORS); and Vice Chairman, Chinese Orthopaedic Association (COA).
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when exploring the secrets of nature. In scientific studies, there are many components in an experiment that may determine its success, such as study objectives, methods, accuracy in every step of the whole procedure, and data analysis. This book supplements several previous handbooks in related research areas, and is the first one with major contributions from Chinese scholars. It encourages multidisciplinary research and collaboration, which current science promotes. Therefore, I strongly recommend this cutting-edge volume to all students, research scientists, and personnel working in the field of musculoskeletal research and development.
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Foreword Stephan M. Perren
Over the past decade, we have experienced a fascinating evolution of scientific knowledge of the musculoskeletal system. If we take the treatment of bone fractures as an example, we find that, on the one hand, mechanobiology and biological research have resulted in a basic change of the clinical approach to the treatment of bone fractures with a move away from the temporary replacement of function toward taking advantage of biology; on the other hand, the scientist is digging deeper into a new world of molecular biology where knowledge is exploding and awaits practical application. Another area of concern in relation to the musculoskeletal system is the literally exploding area of osteoporosis, where our practical approaches still only skim the surface and we need help from basic science and the support of up-to-date research technology. New areas are currently evolving at a rapid pace. Furthermore, the areas tend to evolve differently in different parts of the world. Thus, the divide between the frontier of scientific technology and guidance for the newcomer or the specialist changing the scope of his/her working area tends to vary widely. Stephan M. Perren is Co-founder and Senior Scientific Advisor, AO Foundation, Davos, Switzerland; and Professor, Orthopaedics and Trauma Research Group, Queensland University of Technology, Brisbane, Australia.
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This manual book addresses the lack of concise practical guidance. It spans from immunohistochemistry via stem cells to cartilage- and bone-specific tissue culture. Improved conventional technologies and new technologies like micro-CT and PET are techniques that we need to understand with regard to their opportunities, limitations, and methodologies. A large part of this book is dedicated to essential laboratory animal techniques. The extension of laboratory technology involving small rodents for molecular genetic studies as well as osteoporosis and osteonecrosis models has come at the right time. Additional information concerns the ligament and tendon areas, where major clinical problems still await scientific progress. Diagnostic methods for osteoporosis, like DXA and the clinically and scientifically superior high-resolution QCT, deserve special interest and are expounded in proper detail. The chapter on biomechanics and motion analysis covers micromechanical techniques down to the nanoworld. The editors, collaborators, and authors have invested a great deal of work in providing us with invaluable knowledge and practical guidance. We are grateful for their effort and accomplishment.
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Foreword L. K. Hung
Musculoskeletal research has gained great momentum over the past decade with the availability of advanced techniques for assessment of bone (classically described as “hard” tissue), gross imaging and histological studies, and functional assessment in terms of bone mineral density and radionuclide uptake measurements. Molecular biology as well as cell and tissue culture techniques have also found wider applications in the musculoskeletal system with special adaptations. The Department of Orthopaedics & Traumatology of The Chinese University of Hong Kong has, over the past few years, studied different aspects of the musculoskeletal system and has established some useful models and techniques either de novo or with our collaborators, especially with contributors from the International Chinese Hard Tissue Society (ICHTS) and other orthopedic and bioengineering societies devoted to basic and clinical research in the musculoskeletal scheme. It is timely that these models and protocols are compiled into this manual for the benefit of the wider scientific community. Also included are some protocols developed by centers which we are in regular communication with. The techniques described are not comprehensive and not without our own bias, but they have definitely been well tried out and readers may find them L. K. Hung is Professor and Chairman, Department of Orthopaedics & Traumatology, The Chinese University of Hong Kong, Hong Kong.
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as useful stepping stones for further scientific exploration and improvements. Congratulations go to Professors K. S. Leung, X. Y. Qin, W. H. Cheung, and L. Qin, who have led the editorial team so successfully in putting together such a valuable publication.
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Preface
The preparation of this manual book started 3 years ago as a systemic introduction to general laboratory protocols from the molecular level to biomechanical testing. It is designed to be an experimental guide for personnel who work in the areas of basic and clinical musculoskeletal research. During the period of preparation, feedback obtained from musculoskeletal research scientists urged editors and authors to emphasize more on the translational aspect, i.e. towards problem- or disease-oriented approaches, instead of regurgitating conventional systemic descriptions of common laboratory methods already partially covered in several earlier published handbooks. Current orthopedic practice requires extensive, multidisciplinary knowledge on musculoskeletal and related research, e.g. from molecular biology to bioengineering, from the application of new techniques and methods to the scientific evaluation of both operative and nonoperative or minimally invasive treatment outcomes. Orthopedic clinics and research have developed rapidly in China, Asia, and other regions in recent years, and will continue to do so in years to come. The authors of this book supported the editorial team in our endeavors to assemble this manual book, which addresses in detail the practical, step-by-step application of advanced technologies, their applications, and their limitations in musculoskeletal research. The book supplements previously published handbooks on specific aspects of musculoskeletal tissues, including (1) Handbook of Histology Methods for Bone and Cartilage by Y. H. An and K. L. Martin (Humana Press, 2003); (2) Mechanical Testing of Bone and the Bone–Implant Interface xv
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by Y. H. An and R. A. Draughn (CRC Press, 1999); (3) Biomechanics and Biomaterials in Orthopedics by D. Poitout (Springer, 2004); (4) Handbook of Biomaterials Evaluation: Scientific, Technical and Clinical Testing of Implant Materials by A. F. von Recum (Taylor & Francis Inc., 1998); and (5) Animal Models in Orthopaedic Research by Y. H. An and R. J. Freidman (CRC Press, 1998), to name a few. This book is also an accompaniment to a recently published book — Advanced Bioimaging Technologies in Assessment of Quality of Bone and Scaffold Biomaterials by L. Qin, H. K. Genant, J. F. Griffith, and K. S. Leung (Springer, 2007) — that introduces cutting-edge bioimaging technologies for assessing the quality of musculoskeletal tissues with an emphasis on bone and cartilage, and for evaluating the quality of scaffold biomaterials developed for enhancement of the repair of musculoskeletal tissues. This manual book is categorized into the following parts: cell culture and molecular biology (microarray, primary cell culture technique, mechanotransduction), histology and histomorphometry (general and undecalcified histology, ultrasonic acceleration of decalcification), microscopy and bioimaging (micro-CT, PET), laboratory animal models (bone-, tendon-, and cartilage-oriented defect models), CT- and MRI-based densitometry (DXA, pQCT, XtremeCT, MRI), and biomechanics and motion analysis (cell traction force microscopy, nanoindentation, motion analysis). More practical than theoretical, the text is simple and straightforward, with many illustrations for easy reference to establish and/or modify the described technical protocols for researchers’ own studies. Full bibliographies at the end of each chapter also guide the reader to additional detailed information. We hope that this manual book will help those engaging in musculoskeletal research to establish new techniques in their laboratories for extended applications. For those already experienced in related research, we hope that they will benefit from the detailed description of the methods, in particular the many pearls and pitfalls which the authors were especially asked to discuss. Thus, this book provides a unique platform for multidisciplinary collaborations among various professions, including orthopedics, biomedical engineering, biomaterials,
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and basic and clinical medicine. More importantly, this book provides information on how to make an independent research design and perform analysis for the results, offering readers general guidelines to initiate and achieve unique research goals in the musculoskeletal research field and other areas. Finally, the editors would like to cite a comment recently published in Nature that was primarily addressed to research universities in the USA, but is also generally applicable globally: “The university of the future will be inclusive of broad swaths of the population, actively engaged in issues that concern them, relatively open to commercial influence, and fundamentally interdisciplinary in its approach to both teaching and research” (Nature 446(7139): 949, 2007). We sincerely hope that this manuscript can contribute to this notion in basic and clinical musculoskeletal research. Editors Kwok-Sui Leung Yi-Xian Qin Wing-Hoi Cheung Ling Qin May 2008
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Contents
Foreword Qian Chen Shu-Xun Hou Stephan M. Perren L. K. Hung
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Preface
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List of Contributors Part I
Cell Culture and Molecular Biology
1
Chapter 1
DNA Microarray Xiao-Ling Zhang and Yan-Zhi Du
3
Chapter 2
Visualizing Gene Products: Immunohistochemistry, in situ Hybridization, and Staining for β-Galactosidase Activity Yong-Bo Lu, Yi-Xia Xie and Jian-Quan Feng
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Chapter 3
Mesenchymal Stem Cell Culture, Expansion, and Osteogenic and Adipogenic Differentiation Hui Sheng, Ling Qin, Ge Zhang, Wei-Fang Jin, Jian-Jun Cao, Hong-Fu Wang, Yi-Xiang Wang and Wen-Song Tan
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Chapter 4
Stem Cells and Their Role in Bone Formation and Regeneration Yi-Zhi Meng and Yi-Xian Qin
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Chapter 5
Stem Cells and Tissue Engineering Applications in the Musculoskeletal System Chang-Hun Lee, Gregory Yourek, Eduardo Moioli, Paul A. Clark and Jeremy Jian Mao
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Chapter 6
Osteoblast Culture and Pharmacological Evaluation in vitro Hong-Fu Wang, Wei-Fang Jin, Jian-Jun Cao and Hui Sheng
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Chapter 7
Osteoclast Culture and Pharmacological Evaluation in vitro Jian-Jun Cao, Wei-Fang Jin, Hong-Fu Wang and Hui Sheng
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Chapter 8
Primary Cultures of Human Periosteal Cells Wing-Hoi Cheung, Wing-Sze Lee, C. Zhang and Kwok-Sui Leung
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Chapter 9
Visualization of Osteocytes and Mineralization Yi-Xia Xie, Ling Ye, Shu-Bin Zhang, Vladimir Dusevich and Jian-Quan Feng
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Chapter 10 Tissue Culture of Giant Cell Tumor of Bone Lin Huang and Ming-Hao Zheng
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Chapter 11 Chondrocyte Mechanotransduction in Three-Dimensional Cell Culture Xu Yang, Riaz Gillani and Qian Chen
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Chapter 12 Chondrocyte-Pellet Culture for Cartilage Repair Research Wing-Hoi Cheung, Kwoon-Ho Chow, Kwong-Man Lee and Kwok-Sui Leung
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Part II
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Histology and Histomorphometry
Chapter 13 Tissue Preparations Yong-Bo Lu, Yi-Xia Xie and Jian-Quan Feng
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Chapter 14 Acceleration of Bone Decalcification by Ultrasound Xia Guo and Wai-Ling Lam
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Chapter 15 Stains of Bone and Cartilage Yong-Bo Lu, Yi-Xia Xie and Jian-Quan Feng
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Chapter 16 Contact Microradiography for Studying the Degree of Bone Mineralization Yong-Ping Cao, Tasuku Mashiba, Xin Yang, Chao Liu and Satoshi Mori
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Chapter 17 Undecalcified Histology in Studying Hard Tissue Implanted with Calcium Phosphate–based Ceramics Chun-Wai Chan and Ling Qin
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Part III
Microscopy and Bioimaging
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Chapter 18 Protocols of Micro-Computed Tomographic Analysis Established for Musculoskeletal Applications Hiu-Yan Yeung, Kwok-Sui Leung, Jack Chun-Yiu Cheng, Po-Yee Lui, Ge Zhang and Ling Qin
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Chapter 19 Microangiography for Studying Neovascularization During Long Bone Fracture Repair in a Rat Model Xiao-Zhong Zhou, Ge Zhang, Qi-Rong Dong and Ling Qin
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Chapter 20 High-Resolution Imaging of Organs and Tissues by in vivo Micro-Computed Tomography Engin Ozcivici, Yen-Kim Luu, Clinton Rubin and Stefan Judex
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Chapter 21 Positron Emission Tomography of Bone in Small Animals Erik Mittra, Shahriar S. Yaghoubi and Yi-Xian Qin
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Part IV
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Laboratory Animal Models
Chapter 22 Surgical Anesthesia and Analgesia for Animals in Musculoskeletal Research Dewi K. Rowlands and Anthony E. James
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Chapter 23 Mouse Model of Calvarial Osteolysis Chao Zhang and Ting-Ting Tang
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Chapter 24 Distraction Osteogenesis Model Chun-Wai Chan and Kwok-Sui Leung
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Chapter 25 Fracture Nonunion Animal Model Xia Guo, Mu-Qing Liu, Chi-Cheung Hui and Zheng Guo
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Chapter 26 Establishment of Osteoporosis Model in Goats Wing-Sum Siu, Ling Qin and Kwok-Sui Leung
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Chapter 27 Posterior Spinal Fusion Model Chun-Wai Chan and Jack Chun-Yiu Cheng
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Chapter 28 Functional Disuse Model for Musculoskeletal Adaptation Ho-Yan Lam and Yi-Xian Qin
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Chapter 29 Neurogenic Limb Disuse Animal Models Xia Guo, Xiao-Yun Wang and Wai-Ling Lam
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Chapter 30 Establishment of Steroid-Associated Osteonecrosis Rabbit Model Ge Zhang, Ling Qin and Hui Sheng
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Chapter 31 Establishment of Anterior Cruciate Ligament Reconstruction Model in Rabbit Chun-Yi Wen and Kai-Ming Chan
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Chapter 32 Establishment of Normal and Delayed Bone–Tendon Junction Repair Models Kwok-Sui Leung and Ling Qin
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Chapter 33 Anterior Cruciate Ligament Transection (ACLT)-Induced Osteoarthritis in Rats Ya-Feng Zhang, Jun-Fei Wang and Ge Zhang
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Chapter 34 Establishment of Rabbit Partial Growth Plate Defect Model Kwoon-Ho Chow, Ngai-Man Cheung, Wing-Hoi Cheung and Kwok-Sui Leung
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Part V
X-Ray- and MRI-based Densitometry
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Chapter 35 Micro-CT 3D Image Analysis Techniques for Orthopedic Applications: Metal Implant-to-Bone Contact Surface and Porosity of Biomaterials Phil Salmon
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Chapter 36 Application of DXA to Assess Orthopedic Implants Tom V. Sanchez and Jing-Mei Wang
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Chapter 37 Clinical Monitoring of Bone Mineralization in Distraction Osteogenesis Using DXA Vivian Wing-Yin Hung, Bobby Kin-Wah Ng and Jack Chun-Yiu Cheng
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Chapter 38 In vivo and ex vivo Bone Mineral Density and Structure Measurements Using XtremeCTR — A High-Resolution pQCT (HRpQCT) Maurus Neff, Helmut R. Radspieler, Ling Qin and Maximilian A. Dambacher
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Chapter 39 Advanced 3D Image Processing Methods for Quantifying Proximal Femur and Vertebra Structures from QCT Images Wen-Jun Li, Ying Lu and Thomas Lang
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Chapter 40 Micro-Finite Element Analysis of Bone He Gong, Ming Zhang and Ling Qin
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Chapter 41 The Characterization of Cortical Bone Water 691 Distribution and Structure Changes on Age, Microdamage, and Disuse by Nuclear Magnetic Resonance Qing-Wen Ni, Daniel P. Nicolella, Xiao-Du Wang, Jeffry S. Nyman and Yi-Xian Qin
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Chapter 42 Dynamic Contrast-Enhanced Magnetic Resonance 729 Imaging of the Musculoskeletal System: Basic Principles and Clinical Applications in Bone Sarcomas and Rheumatoid Arthritis Yi-Xiang Wang Chapter 43 Noninvasive Evaluation of Knee Cartilage Morphology by Magnetic Resonance Imaging Yi-Xiang Wang
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Part VI
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Biomechanics and Motion Analysis
Chapter 44 Cell Traction Force Microscopy for Musculoskeletal Research James Hui-Cong Wang, Bin Li and Jeen-Shang Lin
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Chapter 45 Nanoindentation: Techniques and Technical Considerations for Musculoskeletal Research Suzanne Ferreri, Stefan Judex and Yi-Xian Qin
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Chapter 46 Micromechanical Testing of Bone Tissues in Tension Xiao-Du Wang, Michael Reyes, Xuan-Liang Dong and Hui-Jie Leng
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Chapter 47 Technical Manual for Biomechanical Testing of Musculoskeletal Tissues Daniel Hung-Kay Chow, Andrew D. Holmes, Ling Qin, Wing-Sum Siu and Alon Lai
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Chapter 48 Motion Analysis in Musculoskeletal Research Zong-Ming Li
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Index
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List of Contributors
Jian-Jun Cao Department of Bone Metabolism Institute of Radiation Medicine Fudan University, Shanghai P. R. China Yong-Ping Cao Department of Orthopedic Surgery Peking University, First Hospital, Beijing P. R. China Department of Orthopedic Surgery, Medical Faculty Kagawa University, Kagawa Japan Chun-Wai Chan Musculoskeletal Research Laboratory Department of Orthopaedics & Traumatology The Chinese University of Hong Kong, Hong Kong SAR China Kai-Ming Chan Musculoskeletal Research Laboratory Department of Orthopaedics & Traumatology The Chinese University of Hong Kong, Hong Kong SAR China xxvii
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Qian Chen Cell and Molecular Biology Laboratories Department of Orthopaedics Rhode Island Hospital and The Warren Alpert Medical School of Brown University Providence, RI USA Jack Chun-Yiu Cheng Musculoskeletal Research Laboratory Department of Orthopaedics & Traumatology The Chinese University of Hong Kong, Hong Kong SAR China Ngai-Man Cheung Musculoskeletal Research Laboratory Department of Orthopaedics & Traumatology The Chinese University of Hong Kong, Hong Kong SAR China Wing-Hoi Cheung Musculoskeletal Research Laboratory Department of Orthopaedics & Traumatology The Chinese University of Hong Kong, Hong Kong SAR China Daniel Hung-Kay Chow Department of Health Technology & Informatics The Hong Kong Polytechnic University, Hong Kong SAR China Kwoon-Ho Chow Musculoskeletal Research Laboratory Department of Orthopaedics & Traumatology The Chinese University of Hong Kong, Hong Kong SAR China
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List of Contributors
Paul A. Clark Columbia University College of Dental Medicine The Fu Foundation School of Engineering and Applied Science Department of Biomedical Engineering New York USA Maximilian A. Dambacher Zurich Osteoporosis Research Group Zurich–Munich–Hong Kong, Zurich Switzerland Qi-Rong Dong Department of Orthopaedics The Second Affiliated Hospital Suzhou University, Suzhou 215004 P. R. China Xuan-Liang Dong Department of Mechanical Engineering, Engineering Division The University of Texas at San Antonio San Antonio, TX 78229 USA Yan-Zhi Du Institute of Health Sciences Shanghai Institutes for Biological Sciences Chinese Academy of Sciences; and Shanghai Jiao Tong University School of Medicine P. R. China Vladimir Dusevich Department of Oral Biology, School of Dentistry University of Missouri–Kansas City, Kansas City USA
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Jian-Quan Feng Department of Biomedical Sciences Baylor College of Dentistry Texas A&M Health Science Center, Dallas USA Suzanne Ferreri Department of Biomedical Engineering State University of New York at Stony Brook Stony Brook, NY 11794 USA Riaz Gillani Cell and Molecular Biology Laboratories Department of Orthopaedics Rhode Island Hospital and The Warren Alpert Medical School of Brown University Providence, RI USA He Gong Department of Health Technology & Informatics The Hong Kong Polytechnic University, Hong Kong SAR China Department of Mechanics Jilin University, Changchun P. R. China Xia Guo Department of Rehabilitation Sciences The Hong Kong Polytechnic University, Hong Kong SAR China Zheng Guo Department of Orthopaedic Surgery Fourth Military Medical University, Xi’an P. R. China
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Andrew D. Holmes Department of Health Technology & Informatics The Hong Kong Polytechnic University, Hong Kong SAR China Lin Huang Division of Plastic and Reconstructive Surgery Department of Surgery The Chinese University of Hong Kong Prince of Wales Hospital Hong Kong SAR China Chi-Cheung Hui Department of Rehabilitation Sciences The Hong Kong Polytechnic University, Hong Kong SAR China Vivian Wing-Yin Hung Musculoskeletal Research Laboratory Department of Orthopaedics & Traumatology The Chinese University of Hong Kong, Hong Kong SAR China Anthony E. James Laboratory Animal Services Centre The Chinese University of Hong Kong, Hong Kong SAR China Wei-Fang Jin Department of Bone Metabolism Institute of Radiation Medicine Fudan University, Shanghai P. R. China
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Stefan Judex Department of Biomedical Engineering State University of New York at Stony Brook Stony Brook, NY 11794 USA Alon Lai Department of Health Technology & Informatics The Hong Kong Polytechnic University, Hong Kong SAR China Ho-Yan Lam Department of Biomedical Engineering State University of New York at Stony Brook Stony Brook, NY 11794 USA Wai-Ling Lam Department of Rehabilitation Sciences The Hong Kong Polytechnic University, Hong Kong SAR China Thomas Lang Department of Radiology University of California, San Francisco San Francisco, CA 94143 USA Chang-Hun Lee Columbia University College of Dental Medicine The Fu Foundation School of Engineering and Applied Science Department of Biomedical Engineering New York USA
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Kwong-Man Lee Lee Hysan Clinical Research Laboratories and Musculoskeletal Research Laboratory Department of Orthopaedics & Traumatology The Chinese University of Hong Kong, Hong Kong SAR China Wing-Sze Lee Department of Orthopaedics & Traumatology The Chinese University of Hong Kong, Hong Kong SAR China Hui-Jie Leng Department of Mechanical Engineering, Engineering Division The University of Texas at San Antonio San Antonio, TX 78229 USA Kwok-Sui Leung Musculoskeletal Research Laboratory Department of Orthopaedics & Traumatology The Chinese University of Hong Kong, Hong Kong SAR China Bin Li MechanoBiology Laboratory Department of Orthopaedic Surgery University of Pittsburgh Pittsburgh, PA 15213 USA Wen-Jun Li Department of Radiology University of California, San Francisco San Francisco, CA 94143 USA
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Zong-Ming Li Hand Research Laboratory Department of Orthopaedic Surgery University of Pittsburgh Pittsburgh, PA 15213 USA Jeen-Shang Lin Department of Civil and Environmental Engineering School of Engineering, University of Pittsburgh Pittsburgh, PA 15260 USA Chao Liu Department of Orthopedic Surgery Peking University, First Hospital, Beijing P. R. China Mu-Qing Liu Department of Rehabilitation Sciences The Hong Kong Polytechnic University, Hong Kong SAR China Ying Lu Department of Radiology University of California, San Francisco San Francisco, CA 94143 USA Yong-Bo Lu Department of Oral Biology, School of Dentistry University of Missouri–Kansas City, Kansas City USA Po-Yee Lui Musculoskeletal Research Laboratory Department of Orthopaedics & Traumatology The Chinese University of Hong Kong, Hong Kong SAR China
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Yen-Kim Luu Department of Biomedical Engineering State University of New York at Stony Brook Stony Brook, NY 11794 USA Jeremy Jian Mao Columbia University College of Dental Medicine The Fu Foundation School of Engineering and Applied Science Department of Biomedical Engineering New York USA Tasuku Mashiba Department of Orthopedic Surgery, Medical Faculty Kagawa University, Kagawa Japan Yi-Zhi Meng Department of Biomedical Engineering State University of New York at Stony Brook Stony Brook, NY 11794 USA Erik Mittra Division of Nuclear Medicine Stanford University Medical Center, Stanford, CA USA Eduardo Moioli Columbia University College of Dental Medicine The Fu Foundation School of Engineering and Applied Science Department of Biomedical Engineering New York USA
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Satoshi Mori Department of Orthopedic Surgery, Medical Faculty Kagawa University, Kagawa Japan Maurus Neff Zurich Osteoporosis Research Group Zurich–Munich–Hong Kong, Zurich Switzerland Bobby Kin-Wah Ng Musculoskeletal Research Laboratory Department of Orthopaedics & Traumatology The Chinese University of Hong Kong, Hong Kong SAR China Qing-Wen Ni Southwest Research Institute San Antonio, TX 78238 USA Department of Mathematical & Physical Sciences Texas A&M International University Laredo, TX 78041 USA Daniel P. Nicolella Southwest Research Institute San Antonio, TX 78238 USA Jeffry S. Nyman Department of Orthopaedics & Rehabilitation Vanderbilt University Medical Center Nashville, TN 37215 USA
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Engin Ozcivici Department of Biomedical Engineering State University of New York at Stony Brook Stony Brook, NY 11794 USA Ling Qin Musculoskeletal Research Laboratory Department of Orthopaedics & Traumatology The Chinese University of Hong Kong, Hong Kong SAR China Zurich Osteoporosis Research Group Zurich–Munich–Hong Kong, Zurich Switzerland Yi-Xian Qin Department of Biomedical Engineering State University of New York at Stony Brook Stony Brook, NY 11794 USA Helmut R. Radspieler Zurich Osteoporosis Research Group Zurich–Munich–Hong Kong, Zurich Switzerland Michael Reyes Department of Biomedical Engineering The University of Texas at San Antonio San Antonio, TX USA Dewi K. Rowlands Laboratory Animal Services Centre The Chinese University of Hong Kong, Hong Kong SAR China
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Clinton Rubin Department of Biomedical Engineering State University of New York at Stony Brook Stony Brook, NY 11794 USA Phil Salmon SkyScan N.V., Kontich Belgium Tom V. Sanchez Norland — a CooperSurgical Company Socorro, NM 87801 USA Hui Sheng Department of Orthopaedics & Traumatology The Chinese University of Hong Kong, Hong Kong SAR China Department of Bone Metabolism Institute of Radiation Medicine Fudan University, Shanghai P. R. China Wing-Sum Siu Department of Health Technology & Informatics The Hong Kong Polytechnic University, Hong Kong SAR China Department of Orthopaedics & Traumatology The Chinese University of Hong Kong, Hong Kong SAR China Wen-Song Tan State Key Laboratory of Bioreactor Engineering East China University of Science and Technology, Shanghai P. R. China
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Ting-Ting Tang Department of Orthopaedic Surgery Shanghai Ninth People’s Hospital Shanghai Jiao Tong University School of Medicine, Shanghai P. R. China Hong-Fu Wang Department of Bone Metabolism Institute of Radiation Medicine Fudan University, Shanghai P. R. China James Hui-Cong Wang MechanoBiology Laboratory Department of Orthopaedic Surgery Department of Bioengineering Department of Mechanical Engineering Department of Materials Science and Engineering Department of Physical Medicine & Rehabilitation University of Pittsburgh Pittsburgh, PA 15213 USA Jing-Mei Wang Norland — a CooperSurgical Company Beijing 100032 P. R. China Jun-Fei Wang The Center of Diagnosis and Treatment for Joint Disease Drum Tower Hospital Affiliated to the Medical School of Nanjing University, Nanjing P. R. China Xiao-Du Wang Department of Mechanical Engineering, Engineering Division The University of Texas at San Antonio San Antonio, TX 78229 USA
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Xiao-Yun Wang Department of Rehabilitation Sciences The Hong Kong Polytechnic University, Hong Kong SAR China Yi-Xiang Wang Department of Diagnostic Radiology & Organ Imaging The Chinese University of Hong Kong Prince of Wales Hospital Hong Kong SAR China Chun-Yi Wen Musculoskeletal Research Laboratory Department of Orthopaedics & Traumatology The Chinese University of Hong Kong, Hong Kong SAR China Yi-Xia Xie Department of Biomedical Sciences Baylor College of Dentistry Texas A&M Health Science Center, Dallas USA Shahriar S. Yaghoubi Molecular Imaging Program at Stanford Department of Radiology Stanford University, Stanford, CA USA Xin Yang Department of Orthopedic Surgery Peking University, First Hospital, Beijing P. R. China
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Xu Yang Cell and Molecular Biology Laboratories Department of Orthopaedics Rhode Island Hospital and The Warren Alpert Medical School of Brown University Providence, RI USA Ling Ye Department of Oral Biology, School of Dentistry University of Missouri–Kansas City, Kansas City USA Hiu-Yan Yeung Musculoskeletal Research Laboratory Department of Orthopaedics & Traumatology The Chinese University of Hong Kong, Hong Kong SAR China Gregory Yourek Columbia University College of Dental Medicine The Fu Foundation School of Engineering and Applied Science Department of Biomedical Engineering New York USA C. Zhang Department of Orthopaedics Navy General Hospital of Chinese PLA, Beijing P. R. China Chao Zhang Department of Orthopaedic Surgery Shanghai Ninth People’s Hospital Shanghai Jiao Tong University School of Medicine, Shanghai P. R. China
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Ge Zhang Musculoskeletal Research Laboratory Department of Orthopaedics & Traumatology The Chinese University of Hong Kong, Hong Kong SAR China Ming Zhang Department of Health Technology & Informatics The Hong Kong Polytechnic University, Hong Kong SAR China Shu-Bin Zhang Department of Oral Biology, School of Dentistry University of Missouri–Kansas City, Kansas City USA Xiao-Ling Zhang Institute of Health Sciences Shanghai Institutes for Biological Sciences Chinese Academy of Sciences; and Shanghai Jiao Tong University School of Medicine P. R. China Ya-Feng Zhang The Center of Diagnosis and Treatment for Joint Disease Drum Tower Hospital Affiliated to the Medical School of Nanjing University, Nanjing P. R. China Department of Orthopaedics & Traumatology The Chinese University of Hong Kong, Hong Kong SAR China
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Ming-Hao Zheng Department of Orthopaedics, School of Pathology and Surgery University of Western Australia, Perth Australia Xiao-Zhong Zhou Department of Orthopaedics The Second Affiliated Hospital Suzhou University, Suzhou 215004 P. R. China
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Part I Cell Culture and Molecular Biology
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Chapter 1
DNA Microarray Xiao-Ling Zhang and Yan-Zhi Du
DNA microarray, also known as DNA chip or gene chip, is a powerful tool that allows the measurement of tens of thousands of genes in parallel for gene expression and many other aspects of genome research. With the availability of increasing numbers of completely sequenced organisms, genomewide microarrays are becoming more and more popular in various biological areas. DNA microarray, like other hybridization-based techniques such as Southern and Northern blots, is based on the principle that every nucleic acid strand carries the capacity to recognize its complementary sequences through base pairing. DNA microarray has been intensively used in various areas of human disease studies. It has also been recently applied by a number of investigators to elucidate molecular programs that define osteoblast differentiation. Several cellular models have been used, including committed osteogenic precursors of murine and human origin, immortalized human cells at various stages of differentiation, and uncommitted mesodermal progenitor cells. We believe that the potential of DNA microarray in human bone studies has yet to be explored, and may dramatically expand our scope of understanding molecular programs underlying the physiological and pathological conditions of human bone. This chapter will focus primarily on detailed protocols of DNA microarrays, in particular expression arrays. Keywords:
DNA microarray; probe; hybridization; washing; image scanning; data analysis.
Corresponding author: Xiao-Ling Zhang. Tel: +86-21-63855434; fax: +86-21-63855434; E-mail:
[email protected] 3
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1. Introduction DNA microarray, also known as DNA chip or gene chip, is a powerful tool that allows the measurement of tens of thousands of genes in parallel for gene expression and many other aspects of genome research. With the availability of increasing numbers of completely sequenced organisms, genome-wide microarrays are becoming more and more popular in various biological areas. In addition to conventional cDNA arrays, many emerging types of arrays, such as single nucleotide polymorphism (SNP) arrays and comparative genomic hybridization (CGH) arrays, have facilitated genome-wide detection of single nucleotide polymorphisms and genetic alterations; more recently, promoter arrays have offered tremendous opportunities for gene transcriptional studies. Obviously, DNA microarray now goes beyond gene expression profiling to cover many other important aspects of biological research, and may therefore lead to significant advances in our understanding of the underlying mechanisms of diseases and their effective treatment (Sensen 2005; Calvano et al. 2005). Like many other hybridization-based techniques such as Southern and Northern blots, DNA microarray is based on the principle that every nucleic acid strand carries the capacity to recognize its complementary sequences through base pairing. Two important innovations have provided the foundation for DNA microarray technology. The first one is the use of a rigid and optically flat surface, which facilitates miniaturization of DNA arrays and fluorescence-based signal detection. cDNA microarrays contain discrete cDNA sequences at high spatial resolution in precise locations on a small surface such as a microscope slide. Fluorescencebased detection provides a sensitive, high-resolution measurement of molecular binding events on arrays. The second key innovation is the simultaneous hybridization on one slide with two pools of fluorescence-labeled cDNA, representing total RNA from test and reference samples. In addition to providing information on the expression pattern in each sample, the ratio of these measurements allows a direct and quantitative comparison of message abundance
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Fig. 1. Process of differential expression measurements using cDNA microarrays. DNA clones are first amplified and printed out to form a microarray. Test and reference RNA samples are then reverse-transcribed and labeled with different fluorodyes (Cy5 and Cy3), which fluorescence in different (red and green, respectively) wavelength bands; these are hybridized to the microarray. The fluorescence of each dye is then measured for each feature (gene) using laser excitation, and converted to relative expression levels in the two samples.
in the test and reference samples (Starkey 2001; Du et al. 2006; Zheng et al. 2005). The most widely used method of array fabrication is the robotic spotting of individual DNA clones onto a coated glass slide. Such spotted DNA arrays can have a density of up to 5000 features per cm2. The features comprise double-stranded DNA molecules (genomic clones or cDNAs) that must be denatured prior to hybridization (Ignacimuthu 2005) (Fig. 1). Originally, the DNA spots were largely incompletely characterized pieces of cloned DNAs, cDNAs, or expressed sequence tags (ESTs), representing known or unknown genes. Currently, it is possible to obtain arrays comprising synthetic
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oligonucleotides which represent well-characterized genes. These have the advantage that all of the oligos in the spots are of the same size, thus ensuring a uniform hybridization process to a large extent. DNA microarray has been intensively used in various areas of human disease studies. It has also been recently applied by a number of investigators to elucidate molecular programs that define osteoblast differentiation, osteoarthritis, and osteoporosis. Several cellular models have been used, including committed osteogenic precursors of murine and human origin (Raouf and Seth 2002; Seth et al. 2000; Beck et al. 2001; Doi et al. 2002), immortalized human cells at various stages of differentiation (Billiard et al. 2003), and uncommitted mesodermal progenitor cells (Qi et al. 2003; Locklin et al. 2001; Vaes et al. 2002; De et al. 2002; Balint et al. 2003). We believe that the potential of DNA microarray in human bone studies has yet to be explored, especially in musculoskeletal research, and may dramatically expand our scope of understanding molecular programs underlying the physiological and pathological conditions of musculoskeletal systems. This chapter will focus primarily on detailed protocols of DNA microarrays, in particular expression arrays.
2. Materials For the following materials, alternative vendors can be used, but pay special attention to the selection of microscope slides, reverse transcriptase, and Cy3- and Cy5-labeled oligonucleotides. • • • •
•
PCR primers modified with a 5′-amino-modifier C6 (Glen Research, Sterling, VA, USA) 96-well thermal cycler (Perkin-Elmer, Norwalk, CT, USA) 96-well PCR plates (Perkin-Elmer) Taq DNA polymerase and 10× PCR buffer: 500 mM KCl, 100 mM Tris-HCl, pH 8.3, 15 mM MgCl2, 0.1% (w/v) gelatin (Stratagene, La Jolla, CA, USA) PCR Purification Kit (TeleChem, Sunnyvale, CA, USA)
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Flat-bottomed 384-well plates (Nunc, Naperville, IL, USA) Robotic arrayer (built in-house, based on a Synteni design) ChipMaker microspotting device (TeleChem) Microspotting solution (TeleChem) Microscope slides coated with amine-reactive groups (e.g. silylated slides from CEL, Houston, TX, USA) Sodium borohydride (98%) (J.T. Baker, Philipsburg, NJ, USA) TRIZOL Reagent (Gibco-BRL, Grand Island, NY, USA) Oligotex mRNA Midi Kit (Qiagen, Valencia, CA, USA) RNA transcription kit (Stratagene) Oligo-dT 21mer (treated with 0.1% [w/v] diethyl pyrocarbonate to inactivate ribonucleases) 100 mM dATP, dCTP, dGTP, dTTP (Gibco-BRL) 1 mM Cy3-dCTP (Amersham, Arlington Heights, IL, USA) 1 mM Cy5-dCTP (Amersham) SuperScript II RNase H-Reverse Transcriptase (Gibco-BRL) RNase inhibitor (Gibco-BRL) Chromaspin-TE-30LC (Clontech, Mountain View, CA, USA) Hybridization cassettes (TeleChem) Staining dishes (Wheaton, Millville, NJ, USA) or microarray wash station (TeleChem) SpeedVac (Savant, Farmingdale, NY, USA) ScanArray 3000 microarray scanner (General Scanning, Watertown, MA, USA) Component plane presentation integrated self-organizing map (CPP-SOM) DNA array data mining and analysis software (http://www.arraylab.com/) Excel software (Microsoft, Seattle, WA, USA) TE buffer: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0
3. Methods The objective of the DNA array procedure is to compare mRNA populations of control (untreated) cells or tissues with corresponding mRNAs from treated cells or tissues, both qualitatively and quantitatively. In other words, we analyze and compare the
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transcription programs in terms of patterns or profiles. The controls usually comprise normal cells, or tissues obtained from normal animals. Treated cells/tissues refer to cells that have been exposed to a test material (e.g. a drug, infectious organism, chemical, etc.), or to selected tissues obtained from animals which have been inoculated or exposed to the test materials. The technique is also used to compare transcriptional programs of cells or tissues at different stages of development and differentiation (Hudson and Altamirano 2006). The implementation of a DNA microarray experiment, in essence, involves four steps (Fig. 2)a: (1) Total RNA (including all of the mRNAs) from the test cells or tissues are extracted and purified. (2) These RNAs are converted to labeled probes, e.g. cDNAs. (3) The probes are then hybridized to representative sequences of specific cellular genes embedded on the array slides. (4) The resulting arrays, one for control and one for each treated preparation, are then analyzed and compared in commercial scanners, and the data are interpreted and displayed by means of various software programs (including appropriate statistical analysis). A more recent modification involves comparison of both treated and control preparations with a reference preparation to overcome some technical problems inherent in the earlier analysis.
a
Most of the steps have now been commercialized (and some even robotized). This has resulted in some advantages, such as the use of commercial kits in several steps, giving greater consistency. However, one unfortunate disadvantage is that trainees entering this field may not be aware of the respective roles of all the components of the system, and consequently may not know how to fix or adjust the system when things go wrong. In other words, they may not know what they are doing if they simply follow the kit instructions. Of course, the easiest remedy for such a problem is to contact the local company technical representative(s); unfortunately, these individuals are usually young graduates who are equally unaware of the basis for most of the techniques, and who will likely suggest a replacement kit to “fix” the problem. At the very least, one collaborative research group should have one expert with the needed background, experience, and understanding.
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Fig. 2. Steps in the implementation of a DNA microarray experiment. Cy3 and Cy5 are the cyanine dyes (green and red, respectively) used to label the cDNAs.
3.1. Preparation of total RNA from cultured cells b Isolate total RNA using the TRIZOL Reagent one-step guanidinium thiocyanate acid-phenol extraction method (e.g. mammalian cultured cells grown in a monolayer): •
b
Rinse the cell monolayer with ice-cold phosphate buffered saline (PBS) three times.
There are several well-validated commercial kits for extracting intact mRNA populations from cells in such a way as to give us consistent reliable preparations free from contaminating DNA and protein. The overall integrity of the preparations can be checked (and should be checked) by denaturing gel electrophoresis techniques and measuring relative amounts of intact ribosomal RNAs.
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•
Lyse cells directly in a culture dish by adding 1 mL of TRIZOL Reagent per 3.5-cm-diameter dish and scraping with cell scraper. Pass the cell lysate several times through a pipette. Vortex thoroughly. Incubate the homogenized sample for 5 minutes at room temperature to permit the complete dissociation of nucleoprotein complexes. Centrifuge to remove cell debris. Transfer the supernatant to a new tube. Add 0.2 mL of chloroform per 1 mL of TRIZOL Reagent. Cap sample tubes securely. Vortex samples vigorously for 15 seconds and incubate them at room temperature for 2 to 3 minutes. Centrifuge the samples at 12 000 × g for 15 minutes at 2°C to 8°C. Transfer the upper aqueous phase carefully, without disturbing the interphase, into a fresh tube. Measure the volume of the aqueous phase (the volume of the aqueous phase is about 60% of the volume of TRIZOL Reagent used for homogenization). Precipitate the RNA from the aqueous phase by mixing with isopropyl alcohol. Use 0.5 mL of isopropyl alcohol per 1 mL of TRIZOL Reagent used for the initial homogenization. Incubate samples at 15°C to 30°C for 10 minutes, and centrifuge at not more than 12 000 × g for 10 minutes at 2°C to 4°C. The RNA precipitate, often invisible before centrifugation, forms a gel-like pellet on the side and bottom of the tube. Remove the supernatant completely. Wash the RNA pellet once with 75% ethanol, adding at least 1 mL of 75% ethanol per 1 mL of TRIZOL Reagent used for the initial homogenization. Mix the samples by vortexing, and centrifuge at no more than 7500 × g for 5 minutes at 2°C to 8°C. Repeat the above washing procedure once. Remove all leftover ethanol. Air-dry the RNA pellet for 5–10 minutes. Dissolve RNA in DEPC-treated water by passing the solution a few times through a pipette tip.
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•
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3.2. Preparation of fluorescent probes (e.g. from total human mRNA)c In the alternative system, each mRNA in the population is enzymatically reverse-transcribed into its corresponding cDNA, which is labeled with a fluorescent nucleotide, usually one of the cyanine dyes abbreviated as Cy3 and Cy5 (green and red, respectively). Often, one dye is used for the control cDNA and the other dye for the treated cDNA; or both preparations can be simultaneously labeled with the same dye and independently hybridized with the reference probe labeled with the other dye.d •
• •
c
In a microcentrifuge tube, mix 5.0 µL of total polyA+ mRNA (1.0 µg/µL) (the polyA+ mRNA has to be purified from total RNA using a Qiagen Oligotex mRNA Kit, according to the manufacturer’s instructions), 1.0 µL of control mRNA cocktail (0.5 ng/µL) (control mRNAs from in vitro transcription are doped in at molar ratios of 1:10 000 and 1:100 000 for an average length of 1.0 kb for mRNAs), 4.0 µL of oligo-dT 21mer (1.0 µg/µL), and 27.0 µL of H2O (diethyl pyrocarbonate-treated). Denature the mRNA for 3 minutes at 65°C. Add 10.0 µL 5× first strand buffer (250 mM Tris-HCl, pH 8.3, 375 mM KCl, 15 mM KCl), 5.0 µL 10× DTT (0.1 M), 1.5 µL RNase Block (20 units/µL), 1 µL dATP/dGTP/dTTP cocktail (25 mM each), 2 µL dCTP (1 mM) (for labeling the mRNA with another fluorophore, substitute Fl 12-dCTP or Cy5-dCTP to the reaction), and 2 µL Cy3-dCTP (1 mM); and mix by gently tapping the microcentrifuge tube.
This step relies heavily on commercial kits, since it depends on the use of reliable and consistent enzymes to convert our RNAs into a form that can be labeled and measured. There are two basically distinct systems for doing this. The commercial systems such as Affymetrix make use of an enzyme-mediated transcription system, which converts all of the mRNA molecules into cRNAs that are labeled with a biotinylated nucleotide or radioactive nucleotide, although for various reasons the use of radioactive materials has recently fallen out of favor. d One of the major pitfalls of the entire methodology occurs at this stage, if it is assumed that the rate of incorporation of labeled nucleotides into the probes is equivalent for the two dyes (or radioactive labels) and equivalent among all of the probes. This is, however, not necessarily the case; and more recent analyses have attempted to correct this problem through various means.
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•
Add 1.5 µL SuperScript II reverse transcriptase (200 units/µL) for a total reaction volume of 50 µL. Mix again by gently tapping the microcentrifuge tube. Anneal Oligo-dT to mRNA for 10 minutes at room temperature. Reverse-transcribe the polyadenylated RNA for 2 hours at 37°C. Add 5.0 µL of 2.5 M sodium acetate and 110 µL of 100% ethanol at room temperature. Centrifuge for 15 minutes at room temperature in a microfuge in order to pellet cDNA/mRNA hybrids. Remove and discard the supernatant, and wash the pellet carefully with 500 µL of 80% ethanol.e Dry the pellet in a SpeedVac and resuspend in 10 µL of 1× TE. Heat the sample for 3 minutes at 80°C to denature the cDNA/ mRNA hybrids. Put the sample on ice immediately thereafter. Add 2.5 µL of 1 N NaOH and incubate for 10 minutes at 37°C to degrade the mRNA. Neutralize the cDNA mixture by adding 2.5 µL of 1 M Tris-HCl (pH 6.8) and 2 µL of 1 M HCl. Add 1.7 µL of 2.5 M sodium acetate and 37 µL of 100% ethanol. Centrifuge for 15 minutes at full speed in a microfuge (to pellet the cDNA). Discard the supernatant and wash the pellet with 500 µL of 80% ethanol. Dry the pellet in a SpeedVac and resuspend it thoroughly in 13 µL of H2O. Add 5 µL of 20× SSC (3 M NaCl, 0.3 M sodium citrate, pH 7.0) and 2 µL of 2% SDS. Heat at 65°C for 30 seconds. Centrifuge for 2 minutes in a microfuge at high speed. Transfer the supernatant to a clean tube.f
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e
To avoid any loss of pellet, centrifuge 1 minute before the removal of 80% ethanol. The final cDNA concentration should be ~250 ng/µL per flour in 20 µL of 5× SSC and 0.2% SDS. f
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3.3. Microarray hybridization and washing The hybridization of complementary single-stranded RNA and DNA molecules, in which one of the molecules is labeled and therefore measurable, is carried out by means of standard buffered solutions (usually based on standard saline citrate, or SSC) containing hybrid-forming and hybrid-dissociating chemicals (e.g. formamide) at specific temperatures. These solutions can be purchased from reliable suppliers who guarantee that they will work in specific conditions. • • •
• •
g
Place the slide in a hybridization cassette. Add 5.0 µL of ddH2O to the slot in the cassette to prevent drying of the sample. Mix the Cy3-labeled reference probe with the Cy5-labeled test probe in a 1:1 ratio. Boil the mixed probes for 2 minutes and spin briefly at 13 000 g. Immediately add 1.7 µL/cm2 of the mixed probe onto the microarray, place a cover slip onto the slide using forceps,g close the hybridization cassette containing the microarray, and submerge the hybridization cassette in a water bath set at 62°C. Hybridize at 62°C for 6–12 hours. Wash the slide for 5 minutes at room temperature in 1× SSC and 0.1% (v/v) SDS with stirring.h The cover slip should slide off the microarray immediately during the wash step. If the cover slip does not slide off within 30 seconds, use forceps to gently remove it from the slide surface. Failure to remove the cover slip immediately may lead to elevated background fluorescence.
Cover slips must be free of oils, dust, and other contaminants. Lower the cover slip onto the microarray from left to right. Once it touches the liquid on the array, release it quickly so that the sample pushes out air bubbles as it forms a monolayer against the microarray surface. Small air bubbles trapped under the cover slip exit after several minutes at 62°C. h The microarray should be transferred quickly from the cassette to the washing buffer. Leaving the microarray at room temperature will lead to elevated background fluorescence. Either a microarray wash station (TeleChem) or staining dishes (Wheaton) can be used for this washing step. Permanent markers should not be used for labeling because the ink debris can deposit onto the array and cause elevated background fluorescence.
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Transfer the slides to 300 mL of a second wash solution containing 0.1× SSC and 0.1% (v/v) SDS. Wash the microarray for 5 minutes. Rinse the slide briefly in a third solution containing 0.1× SSC to remove the SDS. Dry the slide by spinning in a centrifuge at 500 g for 5 minutes.
• •
The other component in the hybridization procedure is the array itself, which contains DNA sequences representing the genes of the studied organism. The arrays may be specially prepared siliconized glass slides or nylon membranes that are spotted with as many DNA spots as possible, usually in duplicate. This process is usually done with the aid of a robot. The principles and methods, as well as some pitfalls, have been described in detail in several reviews (Clarke et al. 2001).
3.4. Image scanning and data acquisition The arrays, following the hybridization and washing protocols, need to be scanned or digitized into an optical scanner in order to quantify the intensities of each DNA spot using specific image analysis software such as the ImaGene version 6.0. This program can quantify the intensities of all the spots, subtract appropriate backgrounds, establish mean intensities for duplicate spots, allow for extraneous errors such as those caused by minute pieces of dust or particles on the slides, and make corrections for different labeling intensities in different regions of a slide caused by nonuniform hybridization. •
i
Scan the microarray for fluorescence emission in both 632-nm red and 543-nm green channels using a default setting such as 90% of laser power and 60% of photomultiplier voltage for ScanArray 3000.i
A number of microarray scanners are available, including instruments from General Scanning, Molecular Dynamics (Sunnyvale, CA, USA), Genetic MicroSystems (Woburn, MA, USA), Virtek Vision (Woburn, MA, USA), and Axon (Foster City, CA, USA). Because the power of the lasers and the photomultiplier voltage used in different scanners are different, the laser and photomultiplier settings should be adjusted according to the manufacturer’s recommendations.
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Adjust the laser power and photomultiplier settings for both channels such that
