A Practical Manual for Musculoskeletal Research

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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SINGAPORE

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HONG KONG

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TA I P E I

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CHENNAI

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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]

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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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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 <5% of the signals in the brightest spots are saturated and the ratio of the signals at control spots or control slides in two channels is close to 1. Save images acquired in red and green channels corresponding to test and reference samples, respectively.j Open the pair of reference and test images in image analysis and data extraction software. Place a grid to cover each spot according to the instructions of the software package. Extract and save the data.k

There are numerous other software programs that can be incorporated into statistical calculations and comparisons between replicate experiments. All of these can be included within the general category of bioinformatics, which has now become a vital discipline in itself for the purpose of interpreting masses of related biodata. The expressed genes can be analyzed for the establishment of interconnections between the different genes by using the program Ingenuity Pathway Analysis. We used the component plane presentation integrated selforganizing map (CPP-SOM) program from our institute microarray facility (http://www.arraylab.com/, http://www.ihs.ac.cn/cp4-12.htm); it provides a series of tools and approaches for the analysis and visualization of multidimensional data. j

Some scanners scan the red and green channels simultaneously (e.g. GenePix 4000 produced by Axon); in this case, a single composite image containing both red and green signals is saved. k Reproducibility of the results is also essential, although there has been no consensus on what constitutes a reproducible result. This is related to the issue of reproducible experimental conditions. But, if the whole experiment is repeated (as it should be, of course), can we be sure that the experimental conditions are precisely reproduced? This is doubtful, mainly because cell cultures and animals vary from one occasion to another and it is thus likely that some of the genes affected will tend to show somewhat different quantitative (and perhaps qualitative) changes on different occasions. To overcome this problem, some investigators average out the results between two or three experiments (with the help of software programs). This will probably lead to reinforcement of the results for a number of changes in gene expression, but may miss out on other significant changes that might not always be observed. Ideally, one would use a combination of technical replicates (in which three experiments are made with the same RNA) and biological replicates (in which the analyses are made with RNA extracted from three independent experiments).

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4. Data Analysis The raw data from microarray experiments consist of images from hybridized arrays. The exact nature of the images depends on the array platform or the type of array used. DNA arrays may contain thousands of features. Therefore, data acquisition and analysis must be automated. The software for initial image processing is normally provided with the scanner; this allows the boundaries of individual spots to be determined and the total signal intensity to be measured over the whole spot (signal volume). The signal intensity should be corrected for background, and control measures should be included to measure nonspecific hybridization and variable hybridization across arrays. The aim of data mining is to convert the hybridization signals into numbers which can be used to build a gene expression matrix. The interpretation of microarray experiments is carried out by grouping the data according to similar expression profiles. Clustering is a way of simplifying large data sets by partitioning similar data into specific groups. Many software applications are available for implementing microarray data analysis methods (Table 1). In the musculoskeletal research field, using cDNA microarray analysis (Research Genetics Pathways software), Hopwood et al. (2005) identified genes that were differentially expressed between osteoarthritic and normal trabecular bone from the intertrochanteric region of the proximal femur. Recently, GeneSpring software (Agilent Technologies) was used to analyze the gene expression profiling of bone marrow stromal cells from juvenile, adult, aged, and osteoporotic rats (Xiao et al. 2007).

5. Summary Microarray analysis is a powerful tool for simultaneously surveying the expression levels of many thousands of genes. It is increasingly being used to investigate the genetic basis of disease, leading to the generation of gene profiles for disease screening and gene targets for drug therapy. However, the technique is new and is still evolving, especially in connection with the methods used for data correlation and analysis

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DNA Microarray Table 1.

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Suppliers of microarray scanners and data analysis packages.

Suppliers Affymetrix Agilent Technologies Alpha Innotech Applied Precision Axon Instruments BioDiscovery BioGenex BioRad GeneFocus Genetic Microsystems Genomics Solutions Hitachi Genetic Systems Imaging Research Incyte Genomics Informax Iobion Informatics Lynx Therapeutics Media Cybernetics Molecular Dynamics MolecularWare NetGenics OmniViz Perkin Elmer Life Sciences Research Genetics Rosetta Biosoftware Silicon Genetics Spectral Genomics Spotfire Stanford University The Institute for Genomic Research Thermo Hybaid Vysis

URL www.affymetrix.com www.agilent.com www.alphainnotech.com www.appliedprecision.com www.axon.com www. biodiscovery.com www. biogenex.com www. bio-rad.com www. genefocus.com www. geneticmicro.com www. genomicsolutions.com www.miraibio.com www.imagingresearch.com www.incyte.com www. informaxinc.com www. iobion.com www. lynxgen.com www mediacy.com www. mdyn.com www. molecularware.com www. netgenics.com www.omniviz.com www. perkinelmer.com/lifesciences www.resgen.com www. rosettabio.com www. sigenetics.com www. spectralgenomics.com www. spotfire.com rana.lbl.gov www. tigr.org/tdb/microarray www. thermohybaid.com www.vysis.com

Note: This list of suppliers is far from complete, but should provide the reader with a few options enabling familiarization with some of the tools available.

(bioinformatics). We believe, on the basis of experience acquired by us and other investigators to date, that the technology of gene array analysis can make significant contributions to musculoskeletal research.

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References Balint E, Lapointe D, Drissi H et al. Phenotype discovery by gene expression profiling: mapping of biological processes linked to BMP-2–mediated osteoblast differentiation. J Cell Biochem 89:401–426, 2003. Beck GR, Zerler B, Moran E. Gene array analysis of osteoblast differentiation. Cell Growth Differ 12:61–83, 2001. Billiard J, Moran RA, Whitley MZ et al. Transcriptional profiling of human osteoblast differentiation. J Cell Biochem 89:389–400, 2003. Calvano SE, Xiao W, Richards DR et al. Inflammation and Host Response to Injury Large Scale Collaborative Research Program. A network-based analysis of systemic inflammation in humans. Nature 437:1032–1037, 2005. Clarke PA, te Poele R, Wooster R, Workman P. Gene expression microarray analysis in cancer biology, pharmacology, and drug development: progress and potential. Biochem Pharmacol 62:1311–1336, 2001. De Jong DS, Van Zoelen EJ, Bauerschmidt S et al. Microarray analysis of bone morphogenetic protein, transforming growth factor beta, and activin early response genes during osteoblast differentiation. J Bone Miner Res 17:2119–2129, 2002. Doi M, Nagano A, Nakamura Y. Genome-wide screening by cDNA microarray of genes associated with matrix mineralization by human mesenchymal stem cells in vitro. Biochem Biophys Res Commun 290:381–390, 2002. Du YZ, Wang KK, Fang H et al. Coordination of intrinsic, extrinsic and endoplasmic reticulum-mediated apoptosis by imatinib mesylate combined with arsenic trioxide in chronic myeloid leukemia. Blood 107: 1582–1590, 2006. Hopwood B, Gronthos S, Kuliwaba JS et al. Identification of differentially expressed genes between osteoarthritic and normal trabecular bone from the intertrochanteric region of the proximal femur using cDNA microarray analysis. Bone 36:635–644, 2005. Hudson J, Altamirano M. The application of DNA micro-arrays (gene arrays) to the study of herbal medicines. J Ethnopharmacol 108:2–15, 2006. Ignacimuthu S. Basic Bioinformatics. Alpha Science International, Harrow, UK, pp. 58–62, 2005. Locklin RM, Riggs BL, Hicok KC et al. Assessment of gene regulation by bone morphogenetic protein 2 in human marrow stromal cells using gene array technology. J Bone Miner Res 16:2192–2204, 2001.

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Qi H, Aguiar DJ, Williams SM et al. Identification of genes responsible for osteoblast differentiation from human mesodermal progenitor cells. Proc Natl Acad Sci USA 100:3305–3310, 2003. Raouf A, Seth A. Discovery of osteoblast-associated genes using cDNA microarrays. Bone 30:463–471, 2002. Sensen CW. Handbook of Genome Research: Genomics, Proteomics, Metabolomics, Bioinformatics, Ethical and Legal Issues, Vol. 1. WileyVCH, Weinheim, Germany, pp. 223–228, 2005. Seth A, Lee BK, Qi S, Vary CP. Coordinate expression of novel genes during osteoblast differentiation. J Bone Miner Res 15:1683–1696, 2000. Starkey MP. Genomics Protocols (Methods in Molecular Biology). Humana Press, Totowa, NJ, pp. 322–340, 2001. Vaes BL, Dechering KJ, Feijen A et al. Comprehensive microarray analysis of bone morphogenetic protein 2–induced osteoblast differentiation resulting in the identification of novel markers of bone development. J Bone Miner Res 17:2106–2118, 2002. Xiao Y, Fu H, Prasadam I et al. Gene expression profiling of bone marrow stromal cells from juvenile, adult, aged and osteoporotic rats: with an emphasis on osteoporosis. Bone 40:700–715, 2007. Zheng PZ, Wang KK, Zhang QY et al. Systems analysis of transcriptome and proteome in retinoic acid/arsenic trioxide combination-induced cell differentiation/apoptosis of acute promyelocytic leukemia. Proc Natl Acad Sci USA 102:7653–7658, 2005.

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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

The most exciting discoveries in the last two decades have been the mapping, sequencing, and understanding of genes in our body. In this chapter, we will discuss how to specifically map expression profiles of gene products — mRNA, protein, and reporter gene (X-gal) — using in situ hybridization, immunohistochemistry, and X-gal staining, respectively. Keywords:

Bone; cartilage; in situ hybridization; immunohistochemistry; X-gal staining.

1. Introduction Rapid progress in molecular biology has dramatically changed the way we study bone and cartilage. In particular, we need to know the tissue specificity of endogenous and foreign genes or proteins during normal and abnormal bone/cartilage development. The most powerful methods are in situ hybridization (with DIG-cRNA probe), used for detection of mRNA; immunohistochemistry, used to localize protein Corresponding author: Jian-Quan Feng. Tel: +1-214-3707235; E-mail: [email protected]

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expression; and staining for β-galactosidase, a foreign gene introduced into transgenic mice. All of these techniques are very sensitive, and offer a complementary way to analyze gene expression or gene products with a decalcified section. This chapter will provide a useful guide to each of these methods by detailing the sample preparation, solution preparation, procedures, and color reaction.

2. Techniques 2.1. In situ hybridization 2.1.1. Purpose and principle In situ hybridization is a method used to detect the mRNA expression of the gene of interest in a specific cell type in a histological section using a complementary RNA or DNA probe. In situ hybridization identifies cells expressing the gene of interest (Fig. 1). An in situ hybridization probe includes cRNA, oligonucleotide, and cDNA. The probe can be labeled with digoxigenin (DIG) or with radioisotope. In our laboratory, we routinely use a DIG-labeled cRNA probe for its sensitivity, stability, and safety. The cRNA probe is produced by T7, T3, or SP6 RNA polymerase using part of cDNA as a template and DIG-11-UTP as a substrate. The probe template normally contains only part of the full-length cDNA of the gene of interest. It can be obtained from its full-length cDNA by PCR or by digestion with proper restriction enzymes; sometimes, we also obtain the probe template from genomic DNA by PCR if the desired probe template is located in one exon for the gene of interest. The probe obtained by either PCR or enzyme digestion is cloned into the multiple cloning sites of a plasmid vector, flanked by T3, T7, or SP6 RNA polymerase promoter at both ends. The size of the probe is normally 300–600 nucleotides, and it can be as long as 1000 nucleotides. 2.1.2. Instruments •

Moisture chamber (e.g. Boekel Slide Moat, Model 240000; Boekel Scientific)

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Fig. 1. In situ hybridization, an assay for detection of mRNA in bone cells. The left panel shows very low levels of the FGF23 mRNA in the control osteoblast from a 3-week-old wild-type (WT) mandible. The right panel displays an ectopic expression of FGF23 in osteocytes from an agematched Dmp1-null knock-out (KO) mandible, suggesting that DMP1 may downregulate FGF23, a growth factor for inorganic phosphate (Pi) homeostasis. Note that a DIG-labeled RNA probe to the FGF23 gene is in red color and distributed in the cytoplasm, and the nuclei are in blue color. Adapted from Feng et al. (2006).

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• •

Plastic glass rack Centrifuge

2.1.3. Reagents • • • • • • • • • • • • • • • • • • • • • •

Acetic anhydride (reagent grade ≥98%, 200 mL) (Sigma A6404) Alkaline phosphate anti-DIG Fab fragment (Roche #1093274) BCIP/NBT stock solution (Roche #1681451) Blocking buffer (Roche #1096176) Formamide [≥99% (GC), 2.5 L] (Sigma F7503) Hybridization solution (6 mL) (Sigma H7782) Glycine (tissue culture grade, 500 g) (Fisher BP381-500) Glycogen (100 uL) (Invitrogen #10814-010) Diethyl pyrocarbonate (DEPC) [≥97% (NMR), 100 mL] (Sigma D5758) DIG-RNA labeling kit (SP6/T7) (Roche #1175025) DIG-RNA labeling mix 10× conc. (Roche #1277073) Lithium chloride [SigmaUltra, ≥99.0% (titration)] (Sigma L4408) Maleic acid (Sigma M0375) Paraformaldehyde (PFA) (Sigma P6148) Proteinase K (solution) (Invitrogen #25530-049) QIAquick gel extraction kit (Qiagen #28706) Sodium acetate buffer solution (Sigma S7899) Sodium citrate (poly bottle 1 kg) (Fisher BP327-1) T3 RNA polymerase (Roche #1031163) Triethanolamine (TEA) (Sigma T1377) VECTOR red substrate kit (Vector SK-5100) Wizard plus Minipreps DNA purification system (Promega A7510)

2.1.4. Solution preparation (1) 0.1% DEPC-treated water DEPC, 3 mL Distilled water, 3000 mL

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Seal the cap, mix well, and leave it for overnight. Autoclave, cool down, and then keep at room temperature.

(2) 8% PFA stock solution Paraformaldehyde, 80 g 1× PBSa (prepared with DEPC-treated water), 1000 mL •

Heat at 65°C and stir. Cool down, adjust pH to 7.4, and then keep at 4°C.

(3) 4% PFA working solution (freshly prepared) 8% PFA, 200 mL 1× PBS (made with DEPC-treated water), 200 mL (4) 15% EDTA solution for decalcification EDTA, 450 g 50% NaOH, 240 mL DEPC-treated water, 2760 mL Stir the solution until the solute is dissolved. Adjust pH to 7.2, and then autoclave. 2.1.5. Procedure (1) Dissection and fixation: quick dissection and immediate fixation of bone samples are very critical for the preservation of mRNAs • •

Do not remove all soft tissues attached to the bone, as this will remove the periosteum as well. Fix the dissected bone in freshly prepared 4% PFA for 1–3 days at 4°C.

(2) Decalcification •

a

Transfer the bone samples from the fixative solution into the decalcification bath, 15% EDTA solution (add 0.5% PFA prior to use) at 4°C.

Phosphate buffered saline.

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• •

Stir the decalcification bath with a stirrer bar by placing the container on a magnetic stirrer. Change the decalcification solution weekly.

(3) Dehydration and paraffin embedding • •

Wash the decalcified bone 3–4 times with DEPC-treated water. Place the samples in the tissue processor for dehydration, clearing, paraffin infiltration, and embedding.

(4) Sectioning and storage • •

Cut sections at a thickness of 4–6 µm and collect them on slides. Store the sections at 4°C after drying.

2.1.6. T3/T7 DIG-cRNA probe labeling (1) Preparation of template cDNA •

• • •

Isolate and purify plasmids containing cDNA of the gene of interest using the Wizard Plus Minipreps DNA Purification System. Linearize plasmid DNA at one end of the cDNA with the proper restriction enzyme. Purify the linearized plasmid DNA with the QIAquick Gel Extraction Kit. Concentrate the purified linearized DNA as follows: DNA (from step 3), 50 µL 3 M NaAc (4°C), 5 µL (1/10 volume) 100% ethanol (−20°C), 125 µL (2.5 volume) Glycogen (−20°C), 1.5 µL Mix and then incubate at −80°C for 30 minutes.

• • •

Centrifuge at 14 000 × g at 4°C for 10 minutes. Keep and wash the pellet by adding 500 µL of cooled 80% ethanol. Repeat the centrifugation as in step 5.

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Keep and air-dry the pellet. Suspend the pellet in 5 µL DEPC-treated water on ice. Ready the template cDNA for probe synthesis or store at −20°C.

(2) cRNA probe synthesis •

Prepare the riboprobe reaction on ice as follows: Linear template DNA (1–4 µg), 5 µL 10× NTP labeling mixture, 2 µL 10× transcription buffer, 2 µL RNase inhibitor (20 U), 0.5 µL RNase-free water, 8.5 µL RNA polymerase (T7, T3, or SP6), 2 µL (40 U) Total volume, 20 µL Mix and incubate at 37°C for 2 hours.

•

• • • • • • • •

•

DNase treatment: remove the DNA template by the addition of 2 µL DNase I (20 U, RNase-free) to the riboprobe reaction followed by incubation at 37°C for 15 minutes. Add 2 µL of 0.2 M EDTA (pH 8.0) to stop the activity of DNase I. Add 2.5 µL of 4 M LiCl and 75 µL of precold (−20°C) 100% ethanol. Mix and place the tube at −80°C for at least 30 minutes. Centrifuge at 14 000 × g at 4°C for 15 minutes. Keep and wash the pellet by adding of 50 µL of precold (−20°C) 70% ethanol. Centrifuge at 14 000 × g at 4°C for 5 minutes. Keep and air-dry the pellet. Add 50 µL of DEPC-treated water. Check the amount and size of the synthesized probe by agarose gel electrophoresis. A discrete band should be observed after electrophoresis. Aliquot and store the synthesized cRNA probe at −80°C.

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2.1.7. Procedure for in situ hybridization with DIG-cRNA probe Day 1 (RNase-free)b (1) Dewaxing and rehydration • •

Dewax sections in xylene with two changes, 10 minutes each. Rehydrate sections in series of graded ethanol as follows: 100% ethanol, 5 minutes 100% ethanol, 5 minutes 75% ethanol, 5 minutes 50% ethanol, 5 minutes 25% ethanol, 5 minutes

•

Wash with 1× PBST (0.1% Tween-20 in 1× PBS) for 5 minutes.

(2) Fixation • • • •

Fix sections in cold fresh 4% PFA for 20 minutes. Wash twice in 1× PBST for 5 minutes each. Incubate sections twice in 2% glycine in PBST for 5 minutes each. Wash twice in 1× PBST for 5 minutes each.

(3) Digestion • • •

Incubate sections in 20 µg/mL of proteinase K in PBST for 20 minutes at 37°C. Fix sections in 4% PFA for 20 minutes at room temperature to inactivate proteinase K. Wash twice with PBST for 3 minutes each.

(4) Acetylation •

b

Incubate sections in 0.1 M TEA (3.33 mL TEA + 250 mL DEPC-treated water) for 10 minutes at room temperature.

All reagents must be prepared with DEPC-treated water.

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• •

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Further incubate in freshly prepared 0.25% acetic anhydride in 0.1 M TEA (0.65 mL of acetic anhydride + 250 mL of 0.1 M TEA) for 10 minutes at room temperature. Wash twice with PBST for 3 minutes each. Air-dry slides for hybridization on the same day.

(5) Hybridization • • • • •

Dilute the cRNA probe to 1 µg/mL in hybridization solution. Incubate the diluted probe at 80°C for 15 minutes. Cool it on ice. Add 70–100 µL of hybridization solution on each slide. Cover the slide with the slide cover, and incubate the slides at 56°C–65°C overnight in a moisture chamber saturated with wash I buffer.

Day 2 (1) Solution preparation •

20× SSC 3 M NaCl 0.3 M sodium citrate Adjust pH to 7.0 with NaOH; autoclave.

•

Maleic acid buffer 100 mM maleic acid 150 mM NaCl Adjust pH to 7.5 with NaOH; autoclave.

•

Wash I buffer, 100 mL (50% formamide + 5× SSC + 1% SDS) Formamide, 50 mL 20 × SSC, 25 mL 20% SDS, 5 mL Distilled water, 20 mL

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•

Wash III buffer, 100 mL (50% formamide + 2× SSC) Formamide, 50 mL 20× SSC, 10 mL Distilled water, 40 mL

•

TBST, 500 mL (0.01 M NaCl + 0.025 M Tris-Cl + 0.5% Tween-20) 5 M NaCl, 1 mL 1M Tris-Cl (pH 7.5), 12.5 mL Distilled water, 484 mL Tween-20, 2.5 mL

(2) Procedure •

Washing: to remove excess probe









•

Blocking


•

Place slides with slide cover in preincubated 5× SSC at 70°C for 30 minutes. The slide cover will be dislodged while incubating. Wash slides twice in wash I buffer at 70°C for 30 minutes each. Wash twice in wash III buffer at 65°C for 30 minutes each. Wash three times in TBST at room temperature for 5 minutes each.

Block sections with 1% blocking reagent for 1 hour at room temperature (10% blocking reagent stored at 2°C–8°C; on the day of use, dilute it to 1% with maleic acid buffer).

Incubation with anti-DIG Fab fragment


Incubate sections in 1:5000 diluted anti-DIG Fab fragment (coupled with alkaline phosphatase) in TBST overnight at 4°C.

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Day 3 (1) Solution preparation •

Washing buffer, 500 mL (0.1 M maleic acid + 0.15 M NaCl + 0.3% Tween-20) Maleic acid, 5.8 g NaCl, 4.383 g Tween-20, 1.5 mL Distilled water, 450 mL Adjust pH to 7.5 with NaOH.

•

Detection buffer, 500 mL (0.1 M Tris-Cl + 0.1 M NaCl) 1 M Tris-Cl (pH 9.5), 50 mL 5 M NaCl, 10 mL Distilled water, 440 mL

(2) Procedure •

Washing



Wash slides in TBST for 5 minutes. Wash twice in washing buffer for 15 minutes each.

•

Color reactionc

•

Equilibrate slides in detection buffer twice for 5 minutes each.

Incubate slides in NBT/BCIP solution (1:50 NBT/ BCIP stock dilute with detection buffer to 1:1 or 44 µL NBT + 33 µL BCIP + 10 mL detection buffer) until the desired signal is obtained. Counterstaining






c

Wash slides in distilled water. Counterstain with methyl green.

VECTOR Red Substrate Kit is an excellent choice for reaction (staining the signal in red color), but it costs more.

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2.2. Immunohistochemistry 2.2.1. Purpose and principle Immunohistochemical staining is a technique to localize a target antigen by a specific antibody with a high specificity and a high affinity for the binding of antibody to antigen. In contrast to in situ hybridization, immunohistochemical staining localizes the protein, a gene product, but does not identify where it comes from, especially for extracellular proteins. This staining requires two types of antibodies: primary (with the specificity against the protein of interest) and secondary (without specificity, and coupled with an enzyme producing a color reaction). The avidin–biotin complex (ABC) method is the most widely used technique, whereby the secondary antibody is covalently coupled with biotin which is bound by either avidin or streptavidin. Because antigenic epitopes are masked by formalin fixation and paraffin embedding, antigen retrieval is required before application of the primary antibody. There are two approaches to antigen exposure: heat (95°C for 5 minutes) and enzymatic (trypsin) pretreatments. However, we only present the enzyme pretreatment, and the example given uses a polyclonal primary antibody raised against dentin matrix protein 1 (DMP1) in rabbits. 2.2.2. Instruments • • • • •

Cover slips (Fisher 12-544-12, 12-544-14) Humid box PAP pen (Vector H-4000) Plastic slide rack Staining jars (EMS Glass Coplin staining jar 70315, 70318-04)

2.2.3. Reagents • •

ABC kit (Vector PK-4000) DAB substrate (Sigma D563)

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Ethanol, 30%, 50%, 70%, 80%, and 100% Primary antibody (e.g. affinity purified, polyclonal rabbit antiserum) Secondary antibody (e.g. biotinylated goat anti-rabbit IgG)

2.2.4. Procedure (1) Dewaxing and rehydration •

Follow the same steps as in the in situ hybridization method.

(2) Quenching of endogenous peroxidase • •

Place slides in 3% H2O2 in PBS at room temperature for 10 minutes. Wash slides with 1× PBS 3 times.

(3) Antigen retrieval • •

Place slides in 0.1% trypsin in 0.1% CaCl2 (pH 7.8), and incubate them at 37°C for 30 minutes. Wash slides 3 times with 1× PBS for 3 minutes each.

(4) Blocking •

Cover sections with 3% BSA and 20% goat serum in 1× PBS (100 µL per slide) at room temperature for 1 hour or at 4°C overnight in a humid box.

(5) Primary antibody reaction • • •

No washing is necessary for the blocking solution. Remove excess blocking solution by tilting the slides using a paper towel. Add rabbit anti-mouse DMP1 IgG (affinity purified, code 784) (1:100 in 1% goat serum PBS for adult mouse; 1:150 for newborn) at room temperature for 2 hours or at 4°C overnight in the humid box.

(6) Secondary antibody reaction •

Wash slides 3 times in 1× PBS for 3 minutes each.

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•

•

Remove excess PBS using a paper towel; add secondary antibody, goat anti-rabbit (1:200 in 1% goat serum PBS), on the slides. Keep the slides in a humid box at room temperature for 1 hour.

(7) ABC kitd • • •

Mix 10 µL of each A and B in 1 mL of 1% goat serum in PBS 30 minutes before use. Wash slides with PBS for 3 minutes each. Cover sections with the ABC reagent and place the slides in a humid box at room temperature for 1 hour.

(8) Reaction with DABe • • •

Wash slides 3 times with PBS for 3 minutes each. Place slides in DAB solution and keep it in the dark for 2 minutes or longer to get a brown signal color. Wash the slides with ddH2O.

(9) Counterstaining (a) Using hematotoxylin: • • • • •

Wash the slides with dH2O. Place slides in hematoxylin for 1 minute. Rinse slides with tap water. Incubate slides in 0.5% NH4OH (v/v) for 20 seconds. Rinse slides with tap water.

d If secondary antibody is conjugated with peroxidase, then there is no need to use the ABC complex; just proceed to the next step. e The DAB solution should be freshly made before use, although ~25 mg of DAB per 50-mL tube can be prepared and stored at −20°C. An example of a working solution is listed below:

DAB, 25 mg 1 M Tris-HCl (pH 7.5), 2.5 mL Distilled H2O, 47.5 mL NaOH (2 N), ~100–150 µL to adjust pH to 7.4

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(b) Using methyl green: • • • • •

Wash slides with distilled water. Cover sections with methyl green for 1–5 minutes at room temperature. Rinse sections with dH2O until the water is clear. Dip slides 5–10 times in acetone containing 0.05% acetic acid (v/v). Proceed to dehydration.

(10) Dehydration 80% alcohol, 1 minute 90% alcohol, 1 minute 100% alcohol, 1 minute (11) Mounting •

Mount slides with hydrophobic mounting gel.

2.3. X-gal staining The bacterial β-galactosidase gene (lacZ ) and the genes encoding fluorescent proteins, e.g. green fluorescent protein (GFP) and red fluorescent protein (DsRed), have been widely used to generate transgenic mice (Fig. 2). These reporter genes have greatly enhanced and facilitated our understanding of molecular bone biology in various respects: (1) as a reflection of expression patterns of the endogenous gene of interest if the reporter gene is introduced into the locus of that gene by the gene targeting approach (called “knock-in”); (2) as a reflection of expression patterns of a specific promoter fragment if it is placed downstream of that promoter fragment; and (3) as a mark for a specific type of cells for cell lineage study. The methods to analyze the expression of reporter genes are fairly simple compared to in situ hybridization or immunohistochemistry. Here, the authors only describe a protocol for analyzing lacZ expression in frozen sections on slides.

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Fig. 2. X-gal stain and immunohistochemistry stain. (a) A whole-mount X-gal stain of an E15.5 Dmp1-lacZ knock-in embryo (left) and a mandible section restained with X-gal, showing high lacZ expression in osteoblasts (right). (b) A whole-mount X-gal stain of a skeleton from an 8-day-old Dmp1-lacZ knock-in pup, with lacZ expressed in osteocytes. (c) DMP1 immunostain of bone matrix surrounding osteocytes (signal in brown). Both assays suggest a transition expression of DMP1 from osteoblasts during embryo development to osteocytes during postnatal development. Adapted from Qin et al. (2007).

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2.3.1. Reagents • • • • • •

X-gal (Gold BioTechnology, Inc., X4281C) Potassium ferricyanide [K3Fe(CN)6] (Sigma P3667) Potassium ferrocyanide [K4Fe(CN)6 . 3H2O] (Sigma P3289) Deoxycholic acid, sodium salt (ACROS #21859-0250) IGEPAL CA-630 (NP-40) (Sigma I3021) N,N-dimethylformamide (DMF) (Sigma D4551)

2.3.2. Solution preparation •

500 mL X-gal buffer 1 M Mg2Cl, 1 mL NP-40, 100 µL Deoxycholic acid, sodium salt, 0.05 g 1× PBS, 499 mL

•

0.2 M K3 K3Fe(CN)6, 0.66 g Distilled water, 10 mL

•

0.2 M K4 K4Fe(CN)6, 0.85 g Distilled water, 10 mL

•

20 mL X-gal staining solution X-gal buffer, 19 mL K3, 0.5 mL K4, 0.5 mL 1 M Tris-HCl buffer (pH 8.0), 200 µL X-gal stock solution, 0.5 mL (see below) NaN3, 0.002%

•

X-gal stock solution (stored at −20°C)f X-gal, 10 mg DMF, 0.5 mL

f

The X-gal working solution has to be freshly prepared, and X-gal does not dissolve in water.

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2.3.3. Procedure • • • • • • • • •

Incubate the samples or slides in the staining solution at 30°C for 15–18 hours. Stop the staining by washing in 1× PBS until clear. Counterstain sections with hematoxylin for 10 seconds. Wash slides with tap water until clear. Dip slides in 0.5% NH3⋅H2O for 10 seconds. Wash slides with tap water. Stain slides with eosin Y for 15 seconds. Wash slides with tap water. Dehydrate slides, and cover slides with a cover slip.

References Feng JQ, Ward LM, Liu S et al. Loss of DMP1 causes rickets and osteomalacia and identifies a role for osteocytes in mineral metabolism. Nat Genet 38(11):1310–1315, 2006. Qin C, D’Souza R, Feng JQ. Dentin matrix protein 1 (DMP1): new and important roles for biomineralization and phosphate homeostasis. J Dent Res 86(12):1134–1141, 2007.

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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

In this chapter, bone marrow harvest and isolation as well as mesenchymal stem cell (MSC) culture are described, including sources from human, rabbit, and rat. The biological activity changes of MSCs in the pathogenesis of bone disorders are exemplified by the steroid-associated osteonecrosis rabbit model. As a cell model, the value of MSCs in screening candidate agents against bone disorders is exemplified by icariin, a single-molecule component purified from epimedium, a famous bone-tonifying herb in traditional Chinese medicine. Besides their role as a cell model for the underlying mechanisms, exploration, and corresponding molecular drug screening of bone disorders, MSCs are also a very important source of cell seeds for bone-related tissue engineering repair, which needs a large scale of stem cells. We therefore introduce the concept of MSC in vitro expansion in microcarrier by bioreactor, and the magnetic resonance imaging (MRI) dynamic tracking technique after superparamagnetic iron oxide (SPIO) particle labeling after in vivo transplantation. Keywords:

Mesenchymal stem cells; osteogenesis; adipogenesis; microcarrier; bioreactor; SPIO; icariin.

Corresponding author: Ling Qin. Tel: +852-26323071; fax: +852-26377889; E-mail: [email protected]

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1. Introduction Mesenchymal stem cells (MSCs) — a kind of multipotential differentiation of stem cells — are rich in bone marrow and can differentiate into osteoblasts, chondrocytes, adipocytes, and many other mesenchymederived tissue cells (Pittenger et al. 1999). MSCs in bone marrow are believed to serve as a reservoir for osteoblast differentiation during bone modeling and remodeling (Caplan 1991; Caplan 2005). The changes in MSC biological activities, including the decreased number of MSCs and/or their priority differentiation into adipocytes at the cost of decreased differentiation into osteoblasts, are implicated in the pathogenesis of steroid-associated and aging-associated osteoporosis and many other bone disorders (D’Ippolito et al. 1999; Sheng et al. 2007a; Walsh et al. 2001). For the evaluation of MSC activities in vitro, the number of MSCs from different donors can be evaluated by fibroblastic colonyforming unit (CFU-F) counting, which is believed to be derived from one single MSC (Castro-Malaspina et al. 1980). The change of differentiation potency into osteoblasts and adipocytes can be evaluated by measuring or quantifying the specific alkaline phosphate (ALP) activity, mineralized nodules, and lipid droplets (Sheng et al. 2003; Yang et al. 2003). Changes in MSC activities are therefore postulated to be one of the major contributors to many bone disorders including osteoporosis, osteonecrosis, and delayed fracture union or nonunion; and candidate agents against these disorders are also being screened using MSCs as an in vitro cell model (Gimble et al. 2006; Sheng et al. 2007c). Additionally, bone-related tissue engineering repair is a promising and challenging project. Tissue engineering applications for MSCs require the reproducible production of large numbers of well-characterized cells under well-controlled conditions. The routine expansion in twodimensional (2D) culture flasks suffers the disadvantage of frequent passages and large incubator room occupation. Expanding cells in mirocarrier by bioreactor in a three-dimensional (3D) environment is one alternative for large-scale MSC harvest in the future (King and Miller 2007; Li et al. 2006). The superparamagnetic iron oxide (SPIO)-labeling technique is also described here step by step for the dynamic monitoring of transplanted MSCs locally and systemically by

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magnetic resonance imaging (MRI) (Daldrup-Link et al. 2005; Frank et al. 2003; Sheng et al. 2007b).

2. Materials 2.1. Bone marrow harvest, mononuclear cell isolation, and MSC culture •

•

•

Animal anesthesia: equal volumes of xylazine (0.2 mL/kg) and ketamine (0.2 mL/kg) for New Zealand white rabbits and Sprague–Dawlay rats Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL of penicillin, 100 µg/mL of streptomycin, and 0.25% µg/mL of amphotericin (Gibco, UK) for MSC culture and expansion Phosphate buffered saline (PBS)






•

Add 8 g of NaCl, 0.2 g of KCl, 0.2 g of KH2PO4, 1.15 g of Na2HPO4, and 1000 mL of distilled water. Adjust the pH to 7.4. Sterilize by autoclaving at 121°C, 15 psi for 30 minutes.

Heparin (1000 IU/mL, DBL), transfixion pin, 75% ethanol, 10-mL sterile syringe, Ficoll–Histopague (density = 1.077; Sigma, USA), disposable plastic pipette, culture flasks, centrifuge tube

2.2. CFU-F staining and quantification •

0.2 M phosphate buffer





•

Add 2.6 g of NaH2PO4 . H2O, 29 g of Na2HPO4 . 12H2O, and 500 mL of distilled water. Adjust pH to 7.4.

2.5% glutaraldehyde fixation solution





Add 10 mL of 25% glutaraldehyde, 40 mL of distilled water, and 50 mL of 0.2 M phosphate buffer. Adjust pH to 7.3–7.4.

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•

Giemsa stock solution











•

Add 0.5 g of Giemsa powder, 33 mL of glycerol, and 33 mL of methanol. Mix a little glycerol with Giemsa powder and mill until the powder is dissolved, and then add the additional glycerol. Put the mixture in a 56°C water bath for 2 hours, and then add the methanol. Preserve the Giemsa stock solution in a dark-colored bottle.

Giemsa working solution


Dilute Giemsa stock solution with PBS at a volumetric proportion of 1/10. (Working solutions are stable for only a few hours and should be freshly prepared in small volumes. Deterioration of dye solutions is evidenced by the loss of red staining due to eosin precipitation.)

2.3. Osteogenic differentiation potency evaluation •

3 mM 4-nitrophenyl phosphate, hexahydrate (PNPP)



•

50 mM diethanolamine (DEA) buffer





•

Add 113.3 mg of PNPP and 100 mL of deionized water. Store in a −4°C freezer. Add 0.5 mL of 4.0 M HCl, 203.3 mg of MgCl2 . 6H2O, 0.5 mL of DEA, and 99 mL of deionized water. Adjust pH to 10.5.

10−9 M dexamethasone, 50 µg/mL ascorbic acid, 10 mM β-glycerophosphate, and 1 µM dexamethasone, all of which were prepared with plain DMEM

2.4. Adipogenic differentiation potency evaluation •

10% buffered formalin


Add 10 mL of formalin in 90 mL of PBS.

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•

60% propylene glycol solution


•

Add 60 mL of 100% propylene glycol and 40 mL of distilled water.

0.5% Oil Red O solution








•

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Add 0.5 g of Oil Red O powder in 100 mL of 100% propylene glycol. Add a small amount of propylene glycol to the Oil Red O with stirring, and then gradually add the remainder of the propylene glycol. Heat until the solution reaches 95°C.

1 µg/mL insulin, 0.5 mM isobutylmethylxanthine (IBMX), 10−9 M dexamethasone

2.5. MSC expansion in microcarrier by bioreactor •

2% methylsilicone oil


•

3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)






•

Weigh 100 mg of MTT powder. Dissolve with 20 mL of PBS. Sterilize with a 0.22-µm filter. Store in a −4°C freezer with a dark-colored bottle. Dilute with plain DMEM to 0.5 mg/mL before use.

0.4% (w/v) trypan blue staining solution



•

Add 2 mL of methylsilicone oil and 98 mL of acetoacetate.

Add 0.4 g of trypan blue powder in 100 mL of PBS. Sterilize with a filter and then store in a −4°C freezer.

Bioreactor (Techne, USA), biological stirrer machine (Techne, USA), glycan-collagen microcarrier CT-3 (patent product of State Key Laboratory of Bioreactor Engineering, East China University

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of Science and Technology, Shanghai, China), DMEM, FBS, 0.25% trypsin

2.6. MSC labeling with SPIO particles in vitro •

2% hydrochloric acid


•

2% potassium ferrocynide


•

Add 2 g of K4Fe(CN)6 . 3H2O and 100 mL of distilled water.

1% nuclear fast red (Kernechtrot) solution


•

Add 2 mL of concentrated hydrochloric acid and 98 mL of distilled water.

Add 1 g of nuclear fast red, 5 g of aluminum sulphate, and 100 mL of distilled water.

SPIO particles (Department of Diagnostic Radiology & Organ Imaging, The Chinese University of Hong Kong, Hong Kong, China), 400 ng/mL Poly-L-lysine (Sigma, USA), 2.5% glutaraldehyde

3. Methods 3.1. Bone marrow harvest, mononuclear cell isolation, and MSC culture 3.1.1. Bone marrow harvest from human, rabbit, and rat (Ouyang et al. 2004) (1) Bone marrow harvest from posterior superior iliac spine of human patients • • • •

Lie the patient on a bed in a lateral decubitus position. Check and mark the posterior superior iliac spine. Sterilize routinely, and perform local anesthesia with 1%–2% procaine. Puncture the bone marrow with a transfixion pin, and then use a 10-mL heparinized syringe to draw 5–10 mL of bone marrow.

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(2) Bone marrow harvest from iliac crest or digital femur of rabbits • • • •

Anesthetize rabbit with xylazine (0.2 mL/kg) and ketamine (0.2 mL/kg) via intramuscular injection. Sterilize the target area with 75% ethanol after cutting the hair. Puncture the bone marrow cavity at the iliac crest or distal femur with a transfixion pin. Use the heparinized syringe to draw bone marrow slowly and continuously (about 5–10 mL).

(3) Bone marrow harvest from rat femur and tibiae • • • •

Sacrifice the rat via overdose of pentobarbita. Dissect bilateral femurs and tibiae in sterile situation, and put in cold PBS. Remove the attached muscle. Cut one endplate off, and blow the marrow into plain DMEM.

3.1.2. Mononuclear cell isolation from one marrow • •

• •

Wash bone marrow with PBS twice to discard fat cells and blood clots (1500 rpm/5 min). Put Ficoll in the bottom of the centrifuge tube first, and then slowly add the bone marrow suspension with a pipette at a volumetric ratio of 2:3. Centrifuge for 30 minutes (700 g/min) at 20°C. Harvest the mononuclear cells in the middle layer between Ficoll and serum layer using a capillary pipette.

3.1.3. MSC culture • •

Wash mononuclear cells with PBS twice (1500 rpm/min for 5 minutes). Count and inoculate with DMEM + 10% FBS + 100 units/mL penicillin and 100 µg/mL streptomycin at a density of 1 × 105/cm2 in a 5% CO2, 37°C incubator.

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•

If not separated by Ficoll, seed the washed blood cells directly in a culture flask at a density of 5 × 107/cm2 in a 5% CO2, 37°C incubator. Refresh the medium 7 days after inoculation, and then every 2–3 days.

•

3.2. CFU-F staining and quantification • • • • • • •

CFU-F appears in the culture flask about 1 week after inoculation. Discard the culture media and wash with PBS once. Fix cells with 2.5% glutaraldehyde for 10 minutes at room temperature. Prepare Giemsa working solution: dilute the Giemsa stock staining solution with PBS at a volumetric ratio of 1:10. Filter the Giemsa working solution with filtration equipment to remove the undissolved particles. Stain with Giemsa working solution for 30 minutes at room temperature. Wash with distilled water completely. CFU-F will show purple color after staining. Take a picture and quantify the number of CFU-Fs in every well under microscopy with an imaging analysis system, e.g. Image Pro Plus 5.1 (Media Cybernetics, Inc., USA).

3.3. Osteogenic differentiation potency evaluation 3.3.1. Osteogenic differentiation induction culture (Chaudhary et al. 2004) • •

The passaged MSCs can be used for multipotential differentiation evaluation and candidate osteogenesis drug screening. For osteogenic differentiation potency evaluation:


Seed the MSCs at a density of 5000/cm2 in 96-well or 6-well plates in DMEM + 10% FBS.

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•

Three days later after confluence, change to osteogenic differentiation medium containing DMEM + 10% FBS + 10−9 M dexamethasone + 50 µg/mL ascorbic acid + 10 mM β-glycerophosphate.

For candidate agent screening as a cell model:





•

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Seed the MSCs at a density of 5000/cm2 in 96-well or 6-well plates in DMEM + 10% FBS. Three days later after confluence, change to DMEM + 10% FBS with or without icariin.

After 2 weeks’ induction, the fibroblastic-like MSCs will change to cubic osteoblastic cells, with high expression of alkaline phosphatase (ALP); after 3 weeks, they will form mineralized nodules. Quantify the osteogenic differentiation ability by measuring the ALP activity and mineralized nodules.

3.3.2. ALP activity analysis for osteogenic differentiation/ induction abilities • • • • •

Discard the medium in a 96-well plate and wash with PBS twice. Add DEA buffer (100 µL/well) to lysate the cells. Add PNPP (50 µL/well). Incubate at 37°C for 15 minutes. Measure the optical density (OD) value at a 405-nm wavelength by microplate reader for 45 minutes with an interval of 1 minute.

3.4. Adipogenic differentiation potency evaluation 3.4.1. Adipogenic differentiation induction culture (Pittenger et al. 1999) •

For adipogenic differentiation abilities:


Seed the MSCs at a density of 5000/cm2 in 48-well plates in DMEM with 10% FBS.

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Three days later after confluence, change to adipogenic differentiation medium containing DMEM + 10% FBS + 1 µg/mL insulin + 1 µM dexamethasone and 0.5 mM isobutylmethylxanthine for 3 weeks.

For adipogenic induction/inhibition ability evaluation:





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Seed the MSCs at a density of 5000/cm2 in 48-well plates in DMEM with 10% FBS. Three days later after confluence, change to adipogenic differentiation medium containing DMEM + 10% FBS + 1 µg/mL insulin + 1 µM dexamethasone and 0.5 mM isobutylmethylxanthine with or without icariin for 3 weeks.

After 3 weeks’ induction, the MSCs will differentiate into adipogenic cells characterized by lipid droplet formation in the cytoplasm. Stain with Oil Red and quantify spectrophotometrically at 510 nm.

3.4.2. Oil Red O staining for adipocyte quantification • • •

• • • • •

Discard the medium in a 48-well plate and wash with PBS twice. Add 10% buffered formalin to fix for 30–60 minutes (0.5 mL/well). Prepare Oil Red O working solution (mix 3 parts of Oil Red O stock solution and 2 parts of deionized water, allow to sit at room temperature for 10 minutes, and then filter by medium filter). Discard formalin solution and rinse with distilled water. Discard distilled water and add 0.5 mL/well of 60% propylene glycol; let sit for 5 minutes. Discard 60% propylene glycol and add 0.5 mL/well of Oil Red O working solution for 1 hour. Discard Oil Red O and rinse with distilled water (1 mL/well) three times. Add 0.45 mL/well of isopropanol to extract Oil Red O in vortex machine for 1 hour.

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Add 200 µL/well of extraction solution to a 96-well plate and measure spectrophotometrically at 510 nm.

3.5. MSC in vitro expansion by bioreactor 3.5.1. Bioreactor preparation • • •

Silicify the internal surface of the bioreactor by 2% methylsilicone oil. Let the bioreactor dry naturally at room temperature, and rinse with distilled water repeatedly. Put the bioreactor in a 120°C baker for 2 hours.

3.5.2. Microcarrier preparation • • • •

Weigh a 120-mg microcarrier into a 40-mL bioreactor (the density of the microcarrier is 3 mg/mL). Add 40 mL of PBS into the bioreactor to swell the microcarrier overnight. Sterilize with a high-pressure cooker (115°C, 15 lbs) for 15 minutes. Pipette out the PBS and add DMEM in the bioreactor.

3.5.3. MSC seeding on microcarrier and expansion in bioreactor •

• •

Seed MSCs at a density of 3000/cm2 on the surface of the microcarrier (the surface area of the CT-3 microcarrier is 5cm2/mg, so the 120-mg microcarrier in the 40-mL bioreactor has a surface area of 600 cm2). Move the bioreactor to the bioreactor machine in a humified incubator with 5% CO2 at 37°C. Adjust the bioreactor machine at a rotation speed of 25 r/min; 6 hours later after the MSCs adhere to the microcarrier, speed up to 50 r/mins. Change the medium every 3 days.

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•

After 1 week’s expansion, the MSCs would come to confluence in the microcarrier. Harvest the MSCs by trypsin for further expansion in advanced bioreactor or for biological evaluation.

3.6. MSC labeling with SPIO in vitro and dynamic detection in vivo by MRI 3.6.1. MSC labeling with SPIO in vitro • •

• • • • • • • •

Inoculate MSCs in a 6-well plate at a density of 1 × 104/cm2 in a 37°C, 5% CO2 incubator for 24 hours. Add 100 µL sterile SPIO solution to DMEM with 400 ng/mL Poly-L-lysine; the concentration of SPIO will be diluted to 100 µg/mL according to the stock solution concentration. Continue to culture MSCs in a 37°C, 5% CO2 incubator. Test the viability and proliferation of SPIO-labeled MSCs by 0.1% trypan blue staining and MTT method once a day for 2 weeks. Evaluate the change in differentiation potential of MSCs after SPIO labeling. Test SPIO-positive MSCs by Prussian blue staining after 18 hours. Collect expanded MSCs labeled with SPIO with 0.25% trypsin (1500 r, 5 minutes). Wash with 10 mL PBS twice (1500 r, 5 minutes). Adjust MSC density to 1 × 107/cm2 in 5 mL or 0.5 mL DMEM for local or systemic injection in rabbit model. Test the dynamic change of SPIO-labeled MSCs locally/systemically by MRI.

3.6.2. Perls’ Prussian blue staining for SPIO (Frank et al. 2003) • • • •

Discard culture medium in a 6-well plate and rinse with PBS twice. Fix with 2.5% glutaraldehyde for 10 minutes. Prepare Prussian blue solution (add equal volumes of 2% potassium ferrocynide and 2% HCl, and mix freshly before use). Rinse with PBS twice.

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Add Prussian blue solution and stain for 30 minutes. Rinse with PBS twice. Add 1% neutral red and let stand for 1 minute. Rinse with distilled water. Dehydrate with series of ethanol and mount routinely. SPIO particles show blue color in cytoplasm. Quantify the SPIOlabeled cells under microscopy using commercially available imaging analysis systems.

4. Results 4.1. Bone marrow harvest, mononuclear cell isolation, and MSC culture The MSCs from human, rabbit, or rat first appeared in culture flask within 1 week and reached confluence in 2 weeks. They showed a long spindle shape. Flow cytometry analysis showed that human MSCs were SH3-positive and CD34-negative (Fig. 1).

4.2. CFU-F staining and quantification CFU-Fs showed up in the culture disc within 1 week. They showed purple color after Giemsa staining. A colony containing more than 50 cells was counted as one CFU-F. The results showed that there were significantly less CFU-Fs in rabbits from the osteonecrosis group as compared with the normal control (Fig. 2).

4.3. Osteogenic differentiation potency evaluation Icariin stimulated MSC differentiation into osteoblasts. As shown in terms of shape, MSCs changed from long spindle stem cells to cubic osteoblast-like cells after icariin treatment. In ALP activity, icariin promoted ALP expression in a dose-dependent manner at a lower dose range (1–50 µM) and significantly inhibited ALP expression at a higher dose (as high as 100 µM). In mineralized nodules, icariin greatly enhanced the mineralization abilities of MSCs (Fig. 3).

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(a)

(b)

Fig. 1. Primary marrow MSCs appear in 1 week and show a long spindle shape. (a) MSCs from human; (b) MSCs from rabbit; (c) MSCs from rat (100×).

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(c)

Fig. 1.

(a)

(d)

(Continued )

(b)

(e)

(c)

(f )

Fig. 2. CFU-Fs from bone marrow of rabbit proximal femur. After Giemsa staining, there were much less CFU-Fs in the steroid-associated osteonecrosis group (a–c) than in the normal control group (d–f ).

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(a)

(b)

Fig. 3. Icariin promoted MSC differentiation into osteoblast-like cells. (a) MSCs changed from a spindle shape to a cubic osteoblast-like cell shape (200×). (b) Quantification data showed that icariin stimulated ALP expression in a dose-dependent manner. (c) Icariin also enhanced mineralization ability, as shown by the mineralized nodules formed by MSCs in week 4 and stained positive with alizarin red (100×).

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(c)

Fig. 3.

(Continued )

4.4. Adipogenic differentiation potency evaluation Icariin inhibited MSC differentiation into adipocytes. After 3 weeks’ induction in adipogenic medium with or without icariin treatment, there were much less adipocyte-like cells in the icariin treatment group as compared with the adipogenic induction control group. Quantification data by Oil Red O staining showed that icariin inhibited the adipogenesis of MSCs in a dose-dependent manner at a lower dose range (0.01–10 µM) and stimulated adipogenesis at a higher dose (as high as 100 µM) (Fig. 4).

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(a)

(b)

Fig. 4. Icariin inhibited MSC differentiation into adipocyte-like cells. (a) Oil Red O staining for MSC-differentiated adipocyte-like cells (150×). (b) Adipocyte quantification data showed that icariin inhibited MSC differentiation into adipocytes in a dose-dependent manner at lower doses, but stimulated adipogenesis at higher doses.

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(a)

(b)

Fig. 5. Human MSC expansion in microcarrier by bioreactor. (a) Bioreactor and MSC-104L biological stirrer. (b) Human MSC expansion in bioreactor (25×). (c) Human MSC expansion in routine culture flask (100×). (d) Higher expansion efficiency in bioreactor as compared with routine culture flask.

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(c)

(d)

Fig. 5.

(Continued )

4.5. MSC in vitro expansion by bioreactor MSCs adhered to the CT-3 microcarrier in bioreactor after 24 hours. They showed a much higher expansion efficacy as compared with routine 2D expansion. For biological activity evaluation, the expanded MSCs still showed a spindle shape. No difference was found in their differentiation potencies after 3D expansion (Fig. 5).

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4.6. MSC labeling with SPIO in vitro and dynamic detection in vivo by MRI The data showed that 100% of MSCs could be successfully labeled with the newly developed nano-sized SPIO particles. Viability analysis (a)

(b)

Fig. 6. SPIO-NP (nanoparticle) labeling. (a) Electron microscope images showed SPIO-NPs with a diameter of 7–10 nm. (b) Prussian blue staining showed many blue SPIO particles in the cytoplasm of rabbit MSCs (400×). (c) MSC pellet labeled with SPIO-NPs showed dark signal by MRI.

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(c)

Fig. 6.

(Continued )

showed that more than 97% of SPIO-labeled MSCs were still alive. Multipotential differentiation evaluation showed that there was no significant difference in osteogenic and adipogenic differentiation after SPIO labeling (Fig. 6).

5. Discussion and Summary MSCs, as multipotential stem cells, have shown great value as cell models in the pathogenetic exploration of all kinds of bone disorders and corresponding candidate molecule drug screening. More importantly, with the rapid development of tissue engineering, more and more tissues/organs are expected to be manually repaired or reconstructed. The 3D expansion of MSCs in microcarrier by bioreactor would satisfy the demand for large-scale and well-defined cell seeds in the future. Nano-sized SPIO particles could enhance the labeling efficacy of stem cells and lengthen the labeling time in vivo, while at the same time presenting little disadvantages against their biological characteristics. This would greatly facilitate the tracking of stem cells by MRI after transplantation and promote their clinical applications in the near future.

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Acknowledgments These studies were supported by grants from the RGC (CUHK4503/06M), ITF (ITS/012/06), and ITF (ITS/016/07) in Hong Kong; and by the Young Investigator Grant of the Shanghai Municipal Health Bureau (054y27) and the Young Teacher Grant of Fudan University (H. Sheng) in Shanghai, P. R. China.

References Caplan AI. Mesenchymal stem cells. J Orthop Res 9(5):641–650, 1991. Caplan AI. Review: mesenchymal stem cells: cell-based reconstructive therapy in orthopedics. Tissue Eng 11(7–8):1198–1211, 2005. Castro-Malaspina H, Gay RE, Resnick G et al. Characterization of human bone marrow fibroblast colony-forming cells (CFU-F) and their progeny. Blood 56:289–301, 1980. Chaudhary LR, Hofmeister AM, Hruska KA. Differential growth factor control of bone formation through osteoprogenitor differentiation. Bone 34(3):402–411, 2004. D’Ippolito G, Schiller PC, Ricordi C et al. Age-related osteogenic potential of mesenchymal stromal stem cells from human vertebral bone marrow. J Bone Miner Res 14(7):1115–1122, 1999. Daldrup-Link HE, Rudelius M, Piontek G et al. Migration of iron oxide–labeled human hematopoietic progenitor cells in a mouse model: in vivo monitoring with 1.5-T MR imaging equipment. Radiology 234(1):197–205, 2005. Frank JA, Miller BR, Arbab AS et al. Clinically applicable labeling of mammalian and stem cells by combining superparamagnetic iron oxides and transfection agents. Radiology 228(2):480–487, 2003. Gimble JM, Zvonic S, Floyd ZE et al. Playing with bone and fat. J Cell Biochem 98(2):251–266, 2006. King JA, Miller WM. Bioreactor development for stem cell expansion and controlled differentiation. Curr Opin Chem Biol 11(4):394–398, 2007. Li Q, Liu Q, Cai H, Tan WS. A comparative gene-expression analysis of CD34+ hematopoietic stem and progenitor cells grown in static and stirred culture systems. Cell Mol Biol Lett 11(4):475–487, 2006. Ouyang HW, Goh JC, Lee EH. Bone marrow stromal cells for tendon graftto-bone healing. Am J Sports Med 32:321–327, 2004.

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Pittenger MF, Mackay AM, Beck SC et al. Multilineage potential of adult human mesenchymal stem cells. Science 284(5411): 143–147, 1999. Sheng H, Qin L, Zhang G et al. Alternations in the differentiation potential of marrow mesenchymal stem cells in early steroid-associated osteonecrosis. J Orthop Surg Res 4:2–15, 2007a. Sheng H, Wang HF, Gao JJ et al. The effects of different doses of dexamethasome on the differentiation of rat mesenchymal stem cells into osteoblasts. J Fudan Univ 30(2):164–166, 2003. Sheng H, Wang YX, Zhang G et al. Silica-coated super-paramagnetic iron oxide nano-particles for in vitro mesenchymal stem cells labeling and in vivo MR dynamic monitoring. The 5th International Conference on Bone and Mineral Research & The 7th International Osteoporosis Symposium, Urumqi, China, pp. 66–67, 2007b. Sheng H, Zhang G, Qin L et al. Biphasic effects of icariin on adipogenesis of mesenchymal stem cells. Shanghai International Orthopaedics Conference, 2007c. Walsh S, Jordan GR, Jefferiss C et al. High concentrations of dexamethasone suppress the proliferation but not the differentiation or further maturation of human osteoblast precursors in vitro: relevance to glucocorticoidinduced osteoporosis. Rheumatology (Oxford) 40(1):74–83, 2001. Yang X, Tare RS, Partridge KA et al. Induction of human osteoprogenitor chemotaxis, proliferation, differentiation, and bone formation by osteoblast stimulating factor-1/pleiotropin. J Bone Miner Res 18(1):47–57, 2003.

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Chapter 4

Stem Cells and Their Role in Bone Formation and Regeneration Yi-Zhi Meng and Yi-Xian Qin

In recent years, mesenchymal stem cells (MSCs) have become more widely used in the treatment of bone fractures. Typically, MSCs are isolated from the bone marrow, expanded in cell culture, and implanted back into the subject using a polymeric material as the seeding scaffold. In the literature, there are a myriad of papers on the topic of MSC extraction, proliferation ability, and multipotency. As such, the characteristics of MSCs are not completely understood and their pluripotency is still being explored. The purpose of this chapter is to discuss the role of MSCs in bone remodeling and to highlight several methods for extracting, proliferating, and implanting MSCs into a defect. Keywords:

Mesenchymal stem cells (MSCs); MSC extraction; MSC proliferation; ECM; bone remodeling; bone adaptation; bone repair; surface interaction.

1. Introduction 1.1. Osteoporosis In the USA, more than 1.8 million people over the age of 65 years were treated in emergency departments for fall-related injuries in 2003, and more Corresponding author: Yi-Xian Qin. Tel: +1-631-6321481; fax: +1-631-6328577; E-mail: [email protected]

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than 421000 were hospitalized (CDCP 2005). Between 20% and 30% of fall victims suffer hip fractures or head traumas, are vastly debilitated due to reduced mobility and independence, and are at high risk for premature death (CDCP 2005). Hip fracture is also the leading cause of death among fall-related injuries and greatly reduces the quality of life for its victims (CDCP 2005). It is estimated that by the year 2040, the number of hip fractures will exceed 500 000 (Marcus et al. 1996). Every year, more than 300 000 hip fractures result from osteoporosis; and 50% of women and 25% of men over 50 years old will have an osteoporosisrelated fracture in their lifetime (Marcus et al. 1996). The cost of treating and caring for patients with osteoporosis was US$18 billion in 2002, and is expected to rise. Although some symptoms of osteoporosis may manifest themselves in the form of back pain, loss of height, and spinal deformities, many people are not aware of their bone loss until a sudden fracture occurs (Marcus et al. 1996). These statistics paint a very dire scenario for the elderly American population, and it is imperative that health practitioners and researchers work together to curb this epidemic. In the meantime, bone tissue engineering has emerged as a promising field to address fractures that have already occurred.

1.2. Link between osteogenesis and hemopoiesis The intramedullary cavity of bone is filled with soft bone marrow and blood vessels. Hematopoietic stem cells (HSCs) develop within this cavity and are released into the vascular system from the bone marrow upon maturity (Fliedner 1998). Also in the bone marrow cavity are mesenchymal stem cells (MSCs) that give rise to cells along the marrow stromal lineage — chondrocytes, osteoblasts, fibroblasts, adipocytes, endothelial cells, and myocytes (Conget and Minguell 1999; Muguruma et al. 2006; Reyes et al. 2001; Short et al. 2003). Transplanted whole bone marrow has tremendous bone-forming capabilities and contains cells that can differentiate along the osteoblastic lineage, forming mature osteocytes and bone-lining cells (Nilsson et al. 1999). Therefore, much interest has been generated over the expansion of the use of cultured bone marrow because of its potential relevance to therapeutic applications and its ease of being isolated and

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cultured. With the widening interest in this field, however, much still remains unknown regarding the basics of stromal cell biology. Much is to be learned about the location and lineage commitment of MSCs in vivo as opposed to those of HSCs, which have generally been characterized to a greater extent (Yin and Li 2006). Recently, osteoblasts, the bone-building cells responsible for osteogenesis, have been shown to regulate hemopoiesis in adult bone marrow (Calvi et al. 2003). This is a significant finding, for up until now the relationship between bone and blood has not been well established. The exact mechanism by which osteoblasts regulate hemopoiesis has not been established. It is known that they promote the in vitro adhesion of HSCs to osteoblasts. The osteoblastic niche is also thought to regulate the production of osteopontin, a bone protein expressed during late osteoblast differentiation that provides anchorage to integrins on HSCs (Kaplan et al. 2007). MSCs interact with HSCs to make up an important component of the bone marrow graft and mediate hemopoiesis by secreting cytokines (El-Badri 2006). The endosteal surface of bone is a rich source of HSCs (Gong 1978). Using murine bone marrow, Muguruma et al. (2006) recently showed that human HSCs and progenitor cells are generally localized near the endosteal areas of murine bone, whereas lineage-committed cells are more frequently found in the central marrow area. Zhang et al. (2003) also found that the majority of HSCs are found in the marrow cavity, whereas only one fourth are attached to the bone surface. These localizations of osteoblasts strongly suggest a close interaction between HSCs and osteoblasts. To further test the role of osteoblasts in the bone marrow niche, Calvi et al. (2003), Kaplan et al. (2007), and El-Badri (2006) used an in vivo model and found that the activation of a parathyroid hormone (PTH)-related receptor led to increased numbers of trabecular osteoblastic cells and hematopoietic cells in intertrabecular crevices.

1.3. Bone mechanosensory system Bone is a dynamic, living tissue that constantly remodels itself as the body adapts to various environmental stimuli. Mechanotransduction

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has been increasingly proposed to mediate cellular response through its induced interstitial fluid flow, by which bone cells perceive mechanical stimuli in the tissue and initiate remodeling (Piekarski and Munro 1977; Weinbaum et al. 2001; You et al. 2000). A recent finding by Qin and colleagues (Qin et al. 2003) showed that bone is extremely capable of adapting to mechanical loads and develops new bone more rapidly in the presence of mechanical stimulation. In addition, cultured osteocytes (Kleinnulend et al. 1995a; Kleinnulend et al. 1995b) and osteoblasts (Bancroft et al. 2002; Batra et al. 2005; Genetos et al. 2005; McAllister and Frangos 1999; Ogata 2000; Tanaka et al. 2005; Yu et al. 2004) have demonstrated osteogenic responses to fluid shear. More recently, bone marrow cells have also exhibited osteogenic behavior in response to mechanical stimulation (Yoshikawa et al. 1997). Knippenberg et al. (2005) found that differentiating rat MSCs responded to a pulsatile fluid flow by producing increased amounts of nitric oxide (NO) after 1 hour; the nondifferentiating cells produced amounts similar to those cultured under static conditions. Kreke et al. (2005) found that osteocalcin and osteopontin production is stimulated in bone marrow stromal cells which have been exposed to up to 2 hours of shearing flow. These recent developments are evidence that there is a strong link between bone development and its mechanosensory system. The objective of this chapter is to outline the extraction, proliferation, and implantation of bone-related stem cells, and their potential application in musculoskeletal tissue regeneration.

2. Materials •

• • • •

Tissue culture medium: minimum essential medium, alpha modification (α-MEM); fetal bovine serum (FBS); penicillin (100 U/mL); and streptomycin (100 µg/mL) Heparin (50 U/mL) Phosphate buffered saline (PBS) Trypsin (0.025% containing 0.02% EDTA) 20-gauge needle

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100-mm plastic tissue culture plates Antibodies: anti-CD34–fluorescein isothiocyanate (FITC), CD45-FITC, Sca-1–phycoerythrin (PE), and CD44–Cy-Chrome

3. Methods 3.1. Marrow cell extraction Marrow cells are harvested by inserting a 20-gauge needle into one end of the bone and flushing with α-MEM containing 15% FBS and heparin (50 U/mL). The released cells are collected in a 100-mm plastic dish containing 9 mL α-MEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin. The cells are cultured at 37°C in 5% CO2 and humidified air. The medium is changed after the first 3 days to remove nonadherent cells, and is subsequently changed every 3 days.

3.2. Cell culture and proliferation When the primary culture becomes nearly confluent (approximately 2 weeks after initial plating), the cells are detached using 0.025% trypsin containing 0.02% EDTA for 1 minute at room temperature. The action of the trypsin is stopped by adding one half of the volume of FBS. After two washes in PBS, the cells are suspended in complete medium supplemented with 10% bone-marrow–conditioned medium and then subcultured into 100-mm plastic tissue culture plates. Cells from this replating are designated as first-passage cells. At each passage, cells are typically diluted at a ratio of 1 : 2.

3.3. Fluorescence-activated cell sorting (FACS) Adherent cells are incubated with anti-CD34–fluorescein isothiocyanate (FITC), CD45-FITC, Sca-1–phycoerythrin (PE), and CD44–Cy-Chrome. Flow cytometry analysis is performed, and the CD34/44/45−Sca-1+ cells are identified as MSCs and are sorted aseptically. They are subsequently used to seed the scaffolds.

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3.4. Scaffold seeding The scaffolds are soaked in culture medium for 1 day prior to cell seeding. Excess medium is removed from the material and a 5 × 106 mL suspension of MSCs is slowly dripped into the material to prevent overflow. The seeded scaffold is incubated for 3 hours at 37°C with 5% CO2 and humidified air. Prior to in vivo implantation, the seeded scaffold is washed extensively and further cultured in serum- and antibiotic-free medium for 1 day.

3.5. Implantation Each scaffold seeded with a total of 1 × 106 MSCs is placed into the defect of the target animal. Defects receive either a seeded scaffold or an unseeded scaffold as control.

3.6. Micro-computed tomography (micro-CT) Quantitative three-dimensional (3D) analysis of bone ingrowth in the defects is performed at 4 and 16 weeks postoperation using an in vivo micro-CT system at a 38.5-µm voxel resolution (Fig. 1).

3.7. Histology Samples are fixed in 10% buffered formalin and stained with hematoxylin and eosin, Masson’s trichrome, and von Kossa (Fig. 2). 3.7.1. Hematoxylin and eosin staining Samples are washed twice with PBS and fixed in 2% fresh paraformaldehyde for 10 minutes at 4°C. After washing with deionized (DI) water, the samples are dehydrated in an ethanol series and stained with Mayer’s hematoxylin (filtered) for 5 minutes, followed immediately by several changes of water. The samples are immersed in acid alcohol (1% v/v concentrated hydrochloric acid in 70% alcohol)

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Fig. 1. Postmortem micro-CT images of 8-mm defects in the femurs of female Sasco Sprague–Dawley rats at 16 weeks postoperation. The defect contained either no scaffold (empty treatment group), a poly(L-lactide-coD,L-lactide 70:30) (PLDL) scaffold, or a PLDL scaffold enhanced with the growth factors (GFs) BMP-2 and TGF-β3. A constant volume of interest (VOI) centered over the defect site was selected for the analysis of all samples. This VOI was 118 slices thick (approximately 4.6 mm thick). A Gaussian filter (sigma = 1.2, support = 1) was used to suppress noise prior to the application of a consistent global threshold corresponding to 272 mg hydroxyapatite/cm3. The threshold was selected manually using evaluations of 10 single tomographic slices to isolate the bone tissue and preserve its morphology, while excluding soft tissues and polymer scaffold. Reproduced by courtesy of Oest et al. (2007).

for a few seconds and washed in water. Then, the samples are placed in eosin (1% w/v in DI water) for 5 minutes followed by washing in water. The nuclei will appear blue-black, and the cytoplasm will appear as varying shades of pink. 3.7.2. Masson’s trichrome Samples are washed twice with PBS and fixed with 2% fresh paraformaldehyde for 10 minutes at 4°C. After rinsing in DI water, samples are stained in Weigert’s iron hematoxylin working solution for 10 minutes and rinsed in running warm tap water for 10 minutes.

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Fig. 2. Histological sections taken from the PLDL-only treatment group at 16 weeks postoperation. (a) Hematoxylin and eosin stain; (b) Masson’s trichrome stain; (c) von Kossa stain. Reproduced by courtesy of Oest et al. (2007).

After washing in DI water, the samples are stained in Biebrich scarlet– acid fuchsin solution for 15 minutes (the solution can be saved for future use). Samples are washed in DI water and differentiated in phosphomolybdic-phosphotungstic acid solution for 15 minutes or until collagen is not red. The samples are transferred directly (without rinse) to aniline blue solution and stained for 5–10 minutes. After a brief rinse in DI water, the samples are differentiated in 1% acetic acid solution for 2–5 minutes. This is followed by washing in DI water, a quick dehydration series with 95% ethanol and 100% ethanol (this step will wipe off the Biebrich scarlet–acid fuchsin stain), and clearing

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in xylene. The collagen will appear blue, the nuclei will appear black, and the cytoplasm will appear red. 3.7.3. Von Kossa staining Samples are washed twice with PBS and fixed with 2% fresh paraformaldehyde for 10 minutes at 4°C. After washing with DI water, the samples are covered with a filtered silver nitrate solution (2 g/100 mL H2O) and exposed to ultraviolet light for 20 minutes. The samples are rinsed in DI water, and then 5% sodium thiosulfate is added for 3 minutes followed by a final rinse in DI water. Black areas represent regions where mineralized nodules have formed.

4. Discussion and Summary The vast majority of current stem cell research is being conducted using murine sources, and so the question is always asked as to its applicability and relevance to humans. A rigorous approach to a fundamental understanding of the molecular phenomena needs to be constantly pursued in order to delineate the distinction between the two species and to justify the validity of studies involving these two sources. In particular, the function of some antigens still remains a mystery (Gokhale and Andrews 2006). In the case of embryonic stem cells, human cells can be promoted toward differentiation by bone morphogenetic proteins (BMPs) (Xu et al. 2002); on the contrary, mouse embryonic stem cells can be maintained in an undifferentiated state in the presence of some BMPs (Ying et al. 2003). These are just a few examples of the differences between the two species that must be kept aware, and we must always keep an open mind when it comes to the clues that we glean from cell culture studies. Furthermore, because of the limited number of times each primary culture can be passaged before the phenotypical characteristics are lost, there is also room in the field of bone tissue engineering for a more materials-based approach to expand stem cells in vitro (Chai and Leong 2007).

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Acknowledgments This work was kindly supported by the NIH (R01 AR49286 and R01 AR52379) and U.S. Army Medical Research.

References Bancroft GN, Sikavitsast VI, van den Dolder J et al. Fluid flow increases mineralized matrix deposition in 3D perfusion culture of marrow stromal osteoblasts in a dose-dependent manner. Proc Natl Acad Sci USA 99(20):12600–12605, 2002. Batra NN, Li YJ, Yellowley CE et al. Effects of short-term recovery periods on fluid-induced signaling in osteoblastic cells. J Biomech 8(9):1909–1917, 2005. Calvi LM, Adams GB, Weibrecht KW et al. Osteoblastic cells regulate the haematopoietic stem cell niche. Nature 425(6960):841–846, 2003. CDCP (Centers for Disease Control and Prevention). Web-based Injury Statistics Query and Reporting System (WISQARS), 2005. Available at www.cdc.gov/ncipc/wisqars/ Chai C, Leong KW. Biomaterials approach to expand and direct differentiation of stem cells. Mol Ther 15(3):467–480, 2007. Conget PA, Minguell JJ. Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells. J Cell Physiol 181(1): 67–73, 1999. El-Badri NS. The mesenchymal stem cell advantage. Stem Cells Dev 15(4):473–474, 2006. Fliedner TM. The role of blood stem cells in hematopoietic cell renewal. Stem Cells 16(6):361–374, 1998. Genetos DC, Geist DJ, Liu DW et al. Fluid shear-induced ATP secretion mediates prostaglandin release in MC3T3-E1 osteoblasts. J Bone Miner Res 20(1):41–49, 2005. Gokhale PJ, Andrews PW. A prospective on stem cell research. Semin Reprod Med 24(5):289–297, 2006. Gong JK. Endosteal marrow: a rich source of hematopoietic stem cells. Science 199(4336):1443–1445, 1978. Kaplan RN, Psaila B, Lyden D. Niche-to-niche migration of bonemarrow-derived cells. Trends Mol Med 13(2):72–81, 2007.

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Klein-Nulend J, Semeins CM, Ajubi NE et al. Pulsating fluid flow increases nitric oxide (NO) synthesis by osteocytes but not periosteal fibroblasts — correlation with prostaglandin upregulation. Biochem Biophys Res Commun 217(2):640–648, 1995a. Klein-Nulend J, Vanderplas A, Semeins CM et al. Sensitivity of osteocytes to biomechanical stress in vitro. FASEB J 9(5):441–445, 1995b. Knippenberg M, Helder MN, Doulabi BZ et al. Adipose tissue-derived mesenchymal stem cells acquire bone cell-like responsiveness to fluid shear stress on osteogenic stimulation. Tissue Eng 11(11–12):1780–1788, 2005. Kreke MR, Huckle WR, Goldstein AS. Fluid flow stimulates expression of osteopontin and bone sialoprotein by bone marrow stromal cells in a temporally dependent manner. Bone 36(6):1047–1055, 2005. Marcus R, Feldman D, Kelsey J. Osteoporosis. Academic Press, San Diego, CA, 1996. McAllister TN, Frangos JA. Steady and transient fluid shear stress stimulate NO release in osteoblasts through distinct biochemical pathways. J Bone Miner Res 14(6):930–936, 1999. Muguruma Y, Yahata T, Miyatake H et al. Reconstitution of the functional human hematopoietic microenvironment derived from human mesenchymal stem cells in the murine bone marrow compartment. Blood 107(5):1878–1887, 2006. Nilsson SK, Dooner MS, Weier HU et al. Cells capable of bone production engraft from whole bone marrow transplants in nonablated mice. J Exp Med 189(4):729–734, 1999. Oest ME, Dupont KM, Kong HJ et al. Quantitative assessment of scaffold and growth factor–mediated repair of critically sized bone defects. J Orthop Res 25(7):941–950, 2007. Ogata T. Fluid flow–induced tyrosine phosphorylation and participation of growth factor signaling pathway in osteoblast-like cells. J Cell Biochem 76(4):529–538, 2000. Piekarski K, Munro M. Transport mechanism operating between blood supply and osteocytes in long bones. Nature 269(5623):80–82, 1977. Qin YX, Kaplan T, Saldanha A, Rubin C. Fluid pressure gradients, arising from oscillations in intramedullary pressure, is correlated with the formation of bone and inhibition of intracortical porosity. J Biomech 36(10):1427–1437, 2003.

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Reyes M, Lund T, Lenvik T et al. Purification and ex vivo expansion of postnatal human marrow mesodermal progenitor cells. Blood 98(9): 2615–2625, 2001. Short B, Brouard N, Occhiodoro-Scott T et al. Mesenchymal stem cells. Arch Med Res 34(6):565–571, 2003. Tanaka SM, Sun HB, Roeder RK et al. Osteoblast responses one hour after load-induced fluid flow in a three-dimensional porous matrix. Calcif Tissue Int 76(4):261–271, 2005. Weinbaum S, Guo P, You LD. A new view of mechanotransduction and strain amplification in cells with microvilli and cell processes. Biorheology 38(2–3):119–142, 2001. Xu RH, Chen X, Li DS et al. BMP4 initiates human embryonic stem cell differentiation to trophoblast. Nat Biotechnol 20(12):1261–1264, 2002. Yin T, Li LH. The stem cell niches in bone. J Clin Invest 116(5): 1195–1201, 2006. Ying QL, Nichols J, Chambers I, Smith A. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 115(3):281–292, 2003. Yoshikawa T, Peel SAF, Gladstone JR, Davies JE. Biochemical analysis of the response in rat bone marrow cell cultures to mechanical stimulation. Biomed Mater Eng 7(6):369–377, 1997. You J, Yellowley CE, Donahue HJ et al. Substrate deformation levels associated with routine physical activity are less stimulatory to bone cells relative to loading-induced oscillatory fluid flow. J Biomech Eng 122(4):387–393, 2000. Yu XJ, Botchwey EA, Levine EM et al. Bioreactor-based bone tissue engineering: the influence of dynamic flow on osteoblast phenotypic expression and matrix mineralization. Proc Natl Acad Sci USA 101(31):11203–11208, 2004. Zhang JW, Niu C, Ye L et al. Identification of the haematopoietic stem cell niche and control of the niche size. Nature 425(6960):836–841, 2003.

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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

Many musculoskeletal diseases and trauma can potentially be alleviated or cured by stem cell and tissue engineering approaches. Most musculoskeletal tissues are derived from mesenchymal stem cells (MSCs), which natively differentiate into chondrocytes, osteoblasts, adipocytes, fibroblasts, myocytes, etc. Although MSCs are assisted by other cell populations such as the hematopoietic lineages, there is no doubt that MSCs are the progenitors of the building blocks of the musculoskeletal system during development. The roles of MSCs in musculoskeletal regeneration have been demonstrated numerous times in various musculoskeletal tissues, and yet are not completely understood. Nonetheless, musculoskeletal regeneration often, but not always, requires biomaterial scaffolds that need to accommodate various cellular functions such as adherence, proliferation, and differentiation. Musculoskeletal scaffolds must also provide the structural similarity, mechanical strength, diffusion, and gas exchange needs of the tissues or organs to be regenerated. These conflicting needs of musculoskeletal scaffolds for cellular function and physical attributes of the regenerating tissue remain a challenge. Although a great deal can and should be learned from in vitro systems, follow-up in vivo studies are needed as a testing bed for cell-scaffold constructs. Corresponding author: Jeremy Jian Mao. Tel: +1-212-3054475; fax: +1-212-3420199; E-mail: [email protected]

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A Practical Manual for Musculoskeletal Research Additionally, there is a need to test the efficacy of musculoskeletal constructs in frequently inflammatory and diseased models. Furthermore, musculoskeletal regeneration often requires the engineering of two or more cell types, such as osteochondral, fibro-osseous, fibromyogenic, and myo-osseous tissues. This review provides a glimpse of selected approaches for the engineering of complex musculoskeletal tissue. Keywords:

Stem cells; generation; regeneration; differentiation; osteoblasts; osteocytes.

1. Introduction This chapter first describes several stem cells that are capable of differentiating into progenitor cells and tissue-forming cells which generate or regenerate the musculoskeletal system. The second portion of this chapter presents several experimental protocols for the isolation and differentiation of stem cells into osteoblasts and chondrocytes, two cell lineages that are of critical importance to the tissue engineering of musculoskeletal tissues, and several protocols for the fabrication of anatomically shaped musculoskeletal scaffolds that are of potential value in musculoskeletal regeneration.

2. Stem Cells Relevant to Musculoskeletal Tissue Engineering Stem cells can be defined in a number of ways. Many agree that stem cells must be able to self-replicate for some generations without losing their ability to further proliferate or differentiate. Also, stem cells must be able to differentiate into at least two distinctive cell types. In the realm of regenerative medicine, stem cells are believed — and have been demonstrated many times — to reconstitute the tissues and/or organs that they natively develop into. For the musculoskeletal system, stem cells have been coerced to generate elements of bone, cartilage, tendons, ligaments, muscles, and many dental and craniofacial structures.

2.1. Mesenchymal stem cells (MSCs) MSCs often refer to bone-marrow-derived mononuclear cells that (1) adhere to cell culture polystyrene, (2) are capable of self-replication,

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and (3) can differentiate into multiple cell lineages. In recent years, several stem- or progenitor-like cells have been isolated from adipose tissue, skeletal muscle, periosteum, tooth, and related tissues. Many of these stem- or progenitor-like cells share commonalities with bonemarrow-derived MSCs, although a number of differences have been noted. Bone marrow MSCs natively form connective tissues including bone, cartilage, adipose tissue, tendon, muscle, and many craniofacial structures (Anand and Maze 2001; Anon 2003; Bruder et al. 1994; Kogler et al. 2004; Melton et al. 2004; Osawa et al. 1996; Otero et al. 2004; Reyes et al. 2001; Sherley 2004; Mao et al. 2006; Taniguchi et al. 1996; Thorgeirsson and Grisham 2003; Wang et al. 2004; Westgren et al. 2002). MSCs have remarkable, but probably not unlimited, capacity for self-replication (Caplan 1991; Charbord et al. 1996; Kennea and Mehmet 2002; Lennon and Caplan 2006a; Lennon and Caplan 2006b; Purpura et al. 2004; Reyes et al. 2001). It is for certain that MSCs are capable of differentiation into multiple cell lineages including, but not limited to, chondrocytes, osteoblasts, and adipocytes (Aubin 1998; Alhadlaq et al. 2004; Barrilleaux et al. 2006; Mao 2005; Marion and Mao 2006; Prockop 2007). MSCs have been treated as the yardstick for the regeneration of musculoskeletal tissues, and have been utilized in the regeneration of other tissues such as cardiac and neural tissues. MSCs can be isolated from the patient who needs the treatment, and can therefore be used autologously without the fear of immunorejection. MSCs have also been used allogeneically and have been shown to heal large defects (Alhadlaq and Mao 2004; Barrilleaux et al. 2006; Marion and Mao 2006; Prockop 2007). MSCs are adult or somatic stem cells, and are therefore not associated with ethical concerns in comparison to embryonic stem cells (Long et al. 1995).

2.2. Adipose-derived stem cells (ADSCs) ADSCs are isolated from lipectomy or liposuction tissue aspirates. ADSCs have been differentiated into adipocyte, chondrocyte, myocyte, neuronal, and osteoblast lineages, and may provide hematopoietic support (De Ugarte et al. 2003; Estes et al. 2006; Gimble and Guilak 2003; Rodriguez et al. 2005; Zuk et al. 2002). ADSCs express some, but certainly not all, of the cell markers that

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bone marrow MSCs express (De Ugarte et al. 2003; Gimble et al. 2007; Schaffler and Buchler 2007; Zuk et al. 2002). For instance, CD105, STRO-1, and CD166 (ALCAM) are three common markers used to identify cells with multilineage differentiation potential and are consistently expressed by both human ADSCs and MSCs (Dennis et al. 2002; Gronthos et al. 2001). ADSCs can be readily expanded for many passages without losing their ability to further differentiate (De Ugarte et al. 2003; Gimble et al. 2007; Nakagami et al. 2006; Zuk et al. 2002). Many believe that ADSCs have advantages over other adult stem cell populations, for adipose tissue is abundant in certain individuals, readily accessible, and replenishable. However, the ability to reconstitute tissues and organs by ADSCs versus other adult stem cells has yet to be comprehensively compared and documented.

2.3. Umbilical cord blood stem cells (UCBSCs) UCBSCs are derived from the blood of the umbilical cord (Laughlin et al. 2001). There is a growing interest in their capacity for selfreplication and multilineage differentiation. UCBSCs have been differentiated into several cell types that resemble liver cells (Hurlbut and Doerflinger 2004), skeletal muscle cells (Bianchi et al. 1996), neural tissue (Gang et al. 2004), pancreatic cells (Theise et al. 2000), immune cells (Warnke et al. 2004), and mesenchymal stem cells (Young et al. 2004). Their high capacity for multilineage differentiation is likely related to the possibility that UCBSCs are not-too-distant derivatives of embryonic stem cells (ESCs); indeed, UCBSCs display some ESC markers (Parker et al. 2004). Several studies have shown the potential of human UCBSCs (hUCBSCs) in treating cardiac (Lee et al. 2005) and diabetic (Rebel et al. 1996; Tocci et al. 2003) diseases in mice. While it is probable that UCBSCs can be used for the tissue engineering of musculoskeletal tissues, their use will be limited to patients who receive allogeneic cells. Unless a patient’s UCBSCs have been banked, it is not possible to have autologous UCBSCs for the potential cure of musculoskeletal disorders in adulthood.

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2.4. Embryonic stem cells (ESCs) ESCs refer to the cells of the inner cell mass of the blastocyst during embryonic development. The key advantages of ESCs are twofold: their capacity to differentiate into any cell type of the organism, and their ability to self-replicate for numerous generations. One potential disadvantage of ESCs, besides ethical issues, is precisely their totipotency, i.e. being able to differentiate into any cell lineage and to proliferate endlessly unless controlled (Ryu et al. 2004). Furthermore, a nonhuman sialic acid molecule (a family of acidic sugars displayed on the surfaces of all cell types and on many secreted proteins) — N-glycolylneuraminic acid (Neu5Gc) — is expressed in at least one human ESC (hESC) line (Bartsch et al. 2005). This nonhuman sialic acid molecule may likely trigger immunorejection if the hESCs are implanted in humans. Recently developed new approaches have shown that hESCs can be cultured in an undifferentiated state without the use of animal feeder layers (van den Dolder et al. 2003).

3. Anatomically Designed Scaffolds for Musculoskeletal Tissue Engineering Musculoskeletal tissue engineering has an acute demand for scaffolds. Because musculoskeletal tissues are structural materials in addition to possessing physiological and biological functions, regeneration of the musculoskeletal tissue is likely difficult to accomplish without scaffolding materials. There are, however, exceptions; for example, existing musculoskeletal tissue may serve as its own scaffold, such as in the injection of stem cells or myogenic cells in atrophic skeletal muscles. Even in these exceptional cases, though, the argument that cell survival improves when delivered in a material which facilitates their viability and behavior prevails. A myriad of studies have demonstrated that three-dimensional (3D) scaffolds promote the regeneration of cells in the musculoskeletal system (Barrilleaux et al. 2006; Marion and Mao 2006). Various biodegradable and biocompatible scaffolds have been designed to support cell growth,

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cell adhesion, tissue formation, and transport of nutrients and metabolites, as well as to provide adequate mechanical strength required for the implantation and replacement of musculoskeletal tissues (Altman et al. 2003; Alhadlaq et al. 2004; Freeman et al. 2007; Funakoshi et al. 2005; Giordano et al. 1996; Landers et al. 2002; Lin et al. 1999; Liu et al. 2007; Vunjak-Novakovic et al. 2005; Williams et al. 2005; Troken et al. 2007). Continuous supply of nutrients and removal of metabolites are limited by perfusion or diffusion in cell culture and/or materials; it is for this reason that scaffolds for musculoskeletal tissue engineering require a porous internal structure to host most cell types (Landers et al. 2002). The diameter for cell growth into the scaffold has to be larger than 10–30 µm (cell size), desirably 200–400 µm (Lu and Mikos 1996). Tissue engineering without such a 3D pore structure is limited to nonvascularized cell types or thin sheets of cells (Landers et al. 2002). Especially for bone and cartilage, a 3D structure and interconnected inner pores appear to be essential (Altman et al. 2003; Alhadlaq et al. 2004; Freeman et al. 2007; Funakoshi et al. 2005; Giordano et al. 1996; Landers et al. 2002; Lin et al. 1999; Liu et al. 2007; Troken et al. 2007; Vunjak-Novakovic et al. 2005; Williams et al. 2005). The external shape and dimensions of the scaffold for musculoskeletal tissue engineering are at least equally important as the designed internal structures, again owing to the structural nature of musculoskeletal tissues. The production of a musculoskeletal scaffold with precisely defined outer shape and dimensions, in addition to internal structures such as interconnected pores, has been applied (Feinberg et al. 2001; Hutmacher 2000; Hutmacher 2001; Troken et al. 2007; Woodfield et al. 2002; Yang et al. 2002). A number of approaches have been applied towards the fabrication of scaffolds with both external shape and dimensions as well as internal structures. A key feature of rapid prototyping is the solid freeform fabrication (SFF) process: 3D computer models are cut into sequences of layers which are used to construct complex objects layer by layer. The layers are produced via solidification of melts, layer photopolymerization, or bonding of particles using either laser beam–induced sintering (selective laser sintering) or special binders (Giordano et al. 1996; Holman et al. 2002; Landers et al. 2002). Recently, a specialized

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rapid prototyping (RP) system (Bioplotter™; EnvisionTec, Germany) has been introduced, enabling the design and fabrication of anatomically shaped scaffolds with varying internal architectures, thereby allowing precise control over pore size, porosity, permeability, and stiffness (Landers et al. 2002; Landers and Pfister 2005). The Bioplotter™ technique, including 3D dispensing in a liquid medium, was successfully applied to the RP of scaffolds using a remarkably large variety of synthetic and natural materials including melts, solutions, pastes, thermosets, filled polymers, and reactive oligomers (Landers and Pfister 2005; Woodfield et al. 2004; Woodfield et al. 2005). Thus, bioplotted 3D scaffolds may have potential as clinically applicable tissue constructs by providing anatomical shape as well as an optimized internal microstructure for nutrient transportation and potential vascularization. The Bioplotter™ process for tissue-engineering scaffolds requires 3D architectural information of a target tissue or tissue defect that can be obtained by computed tomography (CT) or magnetic resonance imaging (MRI). Then, the 3D information about the tissue (defect) is used to design a functional scaffold via computer-aided design (CAD), and is transferred to the Bioplotter™ system with computerguided 3D dispensing of a large variety of biomaterials. The selected biomaterials can be melted by heat or dissolved in solvents and then dispensed layer by layer on a collecting plate or in a liquid medium, depending on the material properties. We describe the Bioplotter™ process for the fabrication of 3D polycaprolactone (PCL)a scaffolds,

a

Polycaprolactone (PCL) is an FDA-approved bioresorbable polymer that has potential for the tissue engineering of bone and cartilage (Williams et al. 2005). It has high solubility, a low melting point (50°C–64°C), and exceptional ability to form blends, thus stimulating research on its application as a biomaterial (Ang et al. 2007; Marra et al. 1999; Serrano et al. 2004; Widmer et al. 1998; Zhu et al. 2002). PCL degrades at a slow pace, remaining active for over 1 year (Ang et al. 2007). Scaffolds have previously been fabricated in PCL with a variety of SFF techniques including fused deposition modeling, shape deposition manufacturing, precision extruding deposition, 3D printing, low-temperature deposition, and multinozzle freeform deposition (Kweon et al. 2003; Marra et al. 1999; Zein et al. 2002). PCL scaffolds had compressive strength and modulus values within the range of trabecular bone, and supported the in-growth of bone in an in vivo model (Kweon et al. 2003; Marra et al. 1999; Williams et al. 2005; Zein et al. 2002).

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which can be applicable for engineering a variety of musculoskeletal tissues (or defects) such as ligaments, long bone defects, temporomandibular joint (TMJ), knee, and hip joint.

4. Materials 4.1. Isolation and differentiation of MSCs •

Human bone marrow sample (e.g. AllCells, LLC; Berkeley, CA, USA)


• • • •

•

10 mL marrow + 5 mL DPBS + 125 units/mL heparin (total volume, 15 mL)

Ficoll–Paque (e.g. StemCells, Inc.; Vancouver, BC, Canada) RosetteSep MSC enrichment cocktail (e.g. StemCells, Inc.) Basal culture media: 89% DMEM Low Glucose, 10% fetal bovine serum (FBS), 1% antibiotics Chondrogenic supplemented medium: 95% DMEM High Glucose, 1% 1× ITS+1 solution, 1% antibiotics, 100 µg/mL sodium pyruvate, 50 µg/mL L-ascorbic acid 2-phosphate (AsAP), 40 µg/mL L-proline, 0.1 µM dexamethasone, 10 ng/mL recombinant human TGF-β3 Osteogenic supplemented medium: 89% DMEM Low Glucose, 10% fetal bovine serum, 1% antibiotics, 50 µg/mL AsAP, 0.1 µM dexamethasone, 100 mM β-glycerophosphate

4.2. Bioplotting of PCL scaffolds •

3D Bioplotter™ system (EnvisionTec, Germany) (Fig. 1)


3D axis dispensing system   







Working area (XYZ ): 300 mm × 300 mm × 120 mm Resolution in XYZ: 50 µm Speed: 1–100 mm/s

Base plate (XY ): 400 mm × 400 mm, with controlled basement heating up to 100°C Controlled cartridge can be heated up to 300°C

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Fig. 1. The 3D Bioplotter™ system consists of (a) a control panel, (b) a 3-axis positioning system, (c) a dispensing-head mount, (d) HTV-DispenseHead, and (e) a cartridge. It is designed as an open frame structure so that maximum air flow from top to bottom is allowed if the system is placed into a laminar flow system for processing under a sterile environment. PCL melting at 120°C in the cartridge is dispensed through HTV-Dispense-Head by applying air pressure and an auger screw/stepping motor dispenser. The 3-axis positioning system guides the dispensing paths based on the predesigned internal microstructure and outer shape of scaffolds.





Automatic pressure control up to 10 bars Two types of dispensing heads  




•

High-temperature and high-viscous dispensing head (HTV-Dispense-Head) Low-temperature and low-viscous dispensing head (LTVDispense-Head)

Import of dispensing path per layer by CAD/CAM Bioplotter-SW

Polycaprolactone (Mn ~ 80 000; Sigma, St. Louis, MO, USA)

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•

25 G steel encapsulation needle (DL Technology, LLC; Haverhill, MA, USA)


Inner/outer diameter: 0.25 mm/0.508 mm

5. Methods 5.1. Isolation of MSCs from human bone marrow •

• • • • • • • • •

Transfer the bone marrow sample to a 50-mL conical tube. Add 750 µL RosetteSep (50 µL per 1 mL of bone marrow; 50 µL × 15 mL = 750 µL). Incubate for 20 minutes at room temperature. Add 15 mL of PBS–2% FBS–1 mM EDTA solution to bone marrow. The total volume is 30 mL. Add 15 mL Ficoll–Paque to two new 50-mL conical tubes. Layer the bone marrow solution gently on top of the Ficoll–Paque in each tube. Do not allow the marrow to mix with the Ficoll–Paque. Centrifuge for 25 minutes at 300 g with the brake off at room temperature. Remove enriched cells from the Ficoll–Paque interface. Wash enriched cells with the PBS-FBS-EDTA solution in a 50-mL tube, and centrifuge at 1000 rpm for 10 minutes with the brake off. Plate cells (0.5–1 million per Petri dish) with basal culture media. These are now referred to as primary cultures or passage 0 (P0). Change medium every 2 days. Remove nonadherent cells during medium changes. Some of the adherent colonies are of mesenchymal lineage.

5.2. Chondrogenic differentiation of human MSCs •

MSCs can be differentiated into chondrocytes in 3D biocompatible scaffolds, partially to circumvent the possibility of dedifferentiation and/or transdifferentiation in an extended two-dimensional (2D) culture system. Polymeric scaffolds such as alginate, agarose, chitosan, and poly(ethylene glycol) diacrylate (PEGDA) hydrogels have been used to provide a 3D environment for the chondrogenic

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differentiation of MSCs (Alhadlaq et al. 2004; Anseth et al. 2002; Hung et al. 2003; Kim et al. 2003; Williams et al. 2003). Centrifuge approximately 2.5 × 105 human MSCs (hMSCs) in a 15-mL conical tube at 500 g for 5 minutes at 4°C. Culture with human chondrogenic medium for at least 14 days and change medium biweekly. Remove pellets from the tube by inverting and gently tapping for quantitative and histological analyses.





1% Triton X100 may be used to disrupt cell pellets for quantitative biochemical assays such as DNA, collagen, and proteoglycans. Samples may be dehydrated and embedded in paraffin prior to sectioning and staining for histological analysis.

5.3. Osteogenic differentiation of human MSCs • • • •

Plate cells approximately 10 000 cells/cm2 in monolayer. Culture for 14–28 days with osteogenic supplemented medium and change medium biweekly. Fix the monolayered cultures for histological analysis or quantitative biochemical assays after 1% Triton X100 is used to disrupt the cells. Alkaline phosphatase activity may be detected within 2 weeks, whereas other bone markers may be detected later (Alhadlaq et al. 2004; Alhadlaq and Mao 2003; Alhadlaq and Mao 2005; Aubin 1998; Frank et al. 2002; Marion and Mao 2006; Rodan and Noda 1991).

5.4. Bioplotter™ PCL scaffolds for musculoskeletal tissue engineering (1) Prepare 3D volumetric data which define the outer shape of the scaffold. •

The embedded software imports a .DXF (AutoCAD drawing exchange format) or .STL (stereolithography) file.

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•

• • •

•

The 3D data file can be prepared by processing CT or MRI images using commercialized medical CAD programs (e.g. MIMICS; Materialise US, Ann Arbor, MI, USA). The 3D CAD model of the scaffold should be an integral solid, of which the body is surrounded by surface objects. The size of the 3D CAD model should not exceed the provided working area (300 mm × 300 mm × 120 mm). The 3D CAD model may need to be aligned by considering the layer-by-layer deposition of material as increasing Z. Basically, layers in lower Z must be larger than layers in higher Z to maintain the stability during the dispensing process. The 3D CAD model may be simplified by considering the dispensing resolution (50 µm).

(2) Set the dispensing parameters.b • • • • •

b

Applied air pressure: 3.5 bars Temperature: 120°C for cartilage and 50°C for basement plate Feeding rate (Fxy): 120 mm/min Strand distance (XY ): 1.0 mm Layer thickness: 0.2 mm (80% of needle inner diameter)

The dispensing head is to be heated by the preoptimized temperature to maintain appropriate viscosity of the polymer melt during the dispensing process. The basement is also to be heated up to 50°C in order to provide an optimized solidification time for the adhesion between layers. The structure of internal interconnected pores is controlled by (1) the size of each dispensed strand, (2) the space between strands, (3) the directional angle between neighboring layers, and (4) the pattern of the dispensing path. The strand size is determined by the viscosity of polymer melt, needle inner diameter, applied pressure in cartilage, and dispensing speed (feeding rate). For precise control of porosity, pore size, and structure, these parameters need to be determined prior to fabricating a whole 3D structure. The provided parameters are optimized to form a scaffold with 400-µm pores and 400-µm strands. The layer thickness indicates the Z-space between deposit layers, which is suggested to be set at 80% of the needle inner diameter in most materials.

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Other parameters can be adjusted by specific needs (e.g. pattern or dispensing path of layers: predefined at 0°–135° or arbitrary)

(3) Place the selected polymer material (PCL) in the cartilage of HTV-Dispense-Head, and wait for 5–10 minutes until all materials are melted. (4) Import the 3D model data file and construct a dispensing path set for each layer by applying a predetermined parameter set. (5) PCL melt will be dispensed following the layer paths constructed by 3D data. Once polymer strands come out from the heated

Fig. 2. (a) Chondrocytes derived from human MSCs showing positive staining to Alcian blue. Additional molecular and genetic markers (e.g. collagen type II and proteoglycan) can be used to further characterize MSCderived chondrocytes (Alhadlaq and Mao 2004; Lee et al. 2004; Pittenger et al. 1999; Marion and Mao 2006). (b) Osteoblasts derived from human MSCs showing positive von Kossa staining for calcium deposition (black) and active alkaline phosphatase enzyme (red). Additional molecular and genetic markers (e.g. osteocalcin, osteopontin, collagen type I) can be used to further characterize MSC-derived osteoblasts (Alhadlaq et al. 2004; Alhadlaq and Mao 2005; Marion and Mao 2006).

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cartilage, it will be cooled down and solidified. The shape of the 3D scaffold will be formed by depositing layers. (6) The process time varies from several minutes to hours, depending on the complexity of the internal structure, area of layers, and thickness of scaffold.

Fig. 3. (a) A 3D CAD model of human tibia was reconstructed from a patient’s computed tomography (CT) images. (b, c) The real size of the proximal tibial condyle was scaled down to 25% to save on material cost and to meet the demand for cell seeding in subsequent experiments, but can be made in real size. The prepared 3D CAD model was then converted into an AutoCAD.DXF file and used for bioplotting (via Bioplotter™) a human-shaped tibial condyle with PCL. (d) Fabricated scaffolds have 400 µm of interconnected channels shown in the cross-section pore size of 400 µm, and an interpore distance of 400 µm. (e, f ) PEGDA hydrogel was added to the porous PCL, providing an opportunity to create an osteochondral scaffold for accommodating the seeding of chondrocytes and osteoblasts as we reported before (Alhadlaq et al. 2004; Alhadlaq and Mao 2005; Marion and Mao 2006). In a subsequent experiment, MSC-derived chondrocytes were encapsulated in PEGDA hydrogel, whereas MSC-derived osteoblasts were seeded in the porous PCL.

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6. Results Examples of chondrogenic and osteogenic human MSCs are provided in Fig. 2. Each supplemented medium successfully induced differentiation into the selected musculoskeletal lineages, showing the selected markers for both chondrogenic and osteogenic differentiation. A human-shaped tibial condyle was bioplotted with PCL. The 3D structure of human tibia was obtained from CT scans and processed to a volumetric model (Fig. 3). The tibia was then scaled down to 25% of human tibia. The bioplotted PCL scaffold for human tibial condyle has 400-µm interconnected pores (Fig. 3) that may be suited for nutrient transportation and vascularization. In the same manner, a scaffold for human-shaped femoral head was fabricated (Fig. 4). For the cartilage portion of tibial condyle, poly(ethylene glycol) diacrylate (PEGDA) hydrogels can be added on the bioplotted PCL scaffolds as per our previous protocol (Alhadlaq et al. 2004; Alhadlaq

Fig. 4. (a) A 3D CAD model of human proximal femoral condyle was reconstructed from a patient’s CT images. (b, c) The real size of the proximal tibial condyle was scaled down to 25% to save on material cost and to meet the demand for cell seeding in subsequent experiments, but can be made in real size. The prepared 3D CAD model was then converted into an AutoCAD.DXF file and used for bioplotting (via Bioplotter™) a humanshaped femoral head with PCL.

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and Mao 2003; Alhadlaq and Mao 2005). Osteoblasts and chondrocytes derived from human MSCs can be seeded in each region, and the constructs can be implanted in animal models to engineer osteochondral tissues.

Acknowledgments We thank our colleagues whose works have been cited for their highly meritorious work that has energized the process of composing this review. We are especially grateful to the members of the Tissue Engineering and Regenerative Medicine Laboratory at Columbia University for their dedication and hard work. We thank Janina Acloque, Maryann Wanner, and Richard Abbott for administrative support. Generous support from the National Institutes of Health is gratefully acknowledged, through NIH grants DE015391, EB002332, and EB006261 to J. J. Mao.

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Wang HS, Hung SC, Peng ST et al. Mesenchymal stem cells in the Wharton’s jelly of the human umbilical cord. Stem Cells 22(7): 1330–1337, 2004. Warnke PH, Springer IN, Wiltfang J et al. Growth and transplantation of a custom vascularised bone graft in a man. Lancet 364(9436):766–770, 2004. Westgren M, Ringden O, Bartmann P et al. Prenatal T-cell reconstitution after in utero transplantation with fetal liver cells in a patient with Xlinked severe combined immunodeficiency. Am J Obstet Gynecol 187(2):475–482, 2002. Widmer MS, Gupta PK, Lu L et al. Manufacture of porous biodegradable polymer conduits by an extrusion process for guided tissue regeneration. Biomaterials 19(21):1945–1955, 1998. Williams CG, Kim TK, Taboas A et al. In vitro chondrogenesis of bone marrow-derived mesenchymal stem cells in a photopolymerizing hydrogel. Tissue Eng 9(4):679–688, 2003. Williams JM, Adewunmi A, Schek RM et al. Bone tissue engineering using polycaprolactone scaffolds fabricated via selective laser sintering. Biomaterials 26(23):4817–4827, 2005. Woodfield TB, Bezemer JM, Pieper JS et al. Scaffolds for tissue engineering of cartilage. Crit Rev Eukaryot Gene Expr 12(3):209–236, 2002. Woodfield TB, Malda J, de Wijn J et al. Design of porous scaffolds for cartilage tissue engineering using a three-dimensional fiber-deposition technique. Biomaterials 25(18):4149–4161, 2004. Woodfield TB, Van Blitterswijk CA, De Wijn J et al. Polymer scaffolds fabricated with pore-size gradients as a model for studying the zonal organization within tissue-engineered cartilage constructs. Tissue Eng 11(9–10):1297–1311, 2005. Yang S, Leong KF, Du Z, Chua CK. The design of scaffolds for use in tissue engineering. Part II. Rapid prototyping techniques. Tissue Eng 8(1): 1–11, 2002. Young HE, Duplaa C, Romero-Ramos M et al. Adult reserve stem cells and their potential for tissue engineering. Cell Biochem Biophys 40(1):1–80, 2004. Zein I, Hutmacher DW, Tan KC, Teoh SH. Fused deposition modeling of novel scaffold architectures for tissue engineering applications. Biomaterials 23(4):1169–1185, 2002.

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Zhu Y, Gao C, Shen J. Surface modification of polycaprolactone with poly(methacrylic acid) and gelatin covalent immobilization for promoting its cytocompatibility. Biomaterials 23(24):4889–4895, 2002. Zuk PA, Zhu M, Ashjian P et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 13(12):4279–4495, 2002.

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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

Osteoblast isolation from neonatal rat calvariae and human trabecular bone, as well as osteoblast coculture with proximal tubular epithelial cells, is described in this chapter. These cells also serve as cell models for pharmaceutical evaluation in anabolic drug screening, thus providing a technical platform for the regulation of bone turnover and potentially aiding the development of new agents for prevention and treatment of metabolic bone disorders. Keywords:

Osteoblasts; renal tubular epithelial cells; alkaline phosphatase; mineralized nodules; coculture.

1. Introduction Osteoblasts, the bone-forming cells, play an important role in the regulation of bone modeling and remodeling. The primary osteoblasts cultured in vitro have many similar characteristics with the osteoblasts in vivo, and can act as cell models for bone biology studies and candidate agent screening (Wang et al. 2007). The methods of osteoblast isolation, either from bone extracellular matrix by collagenase digestion Corresponding author: Hong-Fu Wang. Tel: +86-21-64048126; fax: +86-21-64436239; E-mail: [email protected]

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(Birnbaum and Wiren 1994; Wang et al. 1991) or outgrowth from bone fragments (Siggelkow et al. 1999), could easily harvest the welldefined and more homogeneous osteoblasts. The primary osteoblasts — derived from rat, mice, rabbit, chick, and human — are widely used to study the physiology of bone formation and the mechanisms of bone disorder, and for candidate therapeutic agent screening or cell therapy in tissue engineering (Harada and Rodan 2003). Additionally, proximal tubular epithelial cells (PTECs) — characterized by high expression of alkaline phosphatase (ALP) and 1α-hydroxylase in in vitro culture — are usually used in renal physiology and toxicological studies, and their use has been extended to investigate the mechanisms of renal osteodystrophy and other bone mineral disorders. For example, primary cultures of renal proximal tubule cells were initiated from a suspension of proximal tubule fragments following either the enzymatic release procedure or outgrowth procedure (Datta et al. 2007). The outgrowth procedure after mesh screening and trypsin digestion described below is a rapid and effective way to obtain PTECs for coculture with osteoblasts. This chapter describes the techniques of osteoblast and PTEC isolation and their coculture for pharmaceutical evaluation screening based on our established protocols (Wang et al. 2001).

2. Materials 2.1. Osteoblast isolation from neonatal rat calvariae • •

• •

Animals: newborn rats, 3–4 days of age Culture medium: minimum essential medium (MEM), supplemented with 10% NCS, 100 U/mL of penicillin, and 100 µg/mL of streptomycin sulfate Collagenase solution: 1 mg of collagenase II per milliliter of PBS (dissolve and sterilize with filter) 0.25% trypsin, phosphate buffered saline (PBS), trypan blue stain, ethylenediaminetetraacetic acid (EDTA; Gibco)

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Forceps, scissors, scalpels, Petri dishes, flasks, Falcon tissue culture plastics, Eppendorf tube, 50-mL centrifuge tubes with caps, syringes and pipettes, 70% alcohol, vortex mixer, tissue incubators, glass coverslips, sterile hood, inverted phase-contrast microscope, refrigerated centrifuge

2.2. Osteoblast isolation from adult human cancellous bone • •

•

Human samples from femoral heads Culture medium: DMEM/HamF12 (1:1) with 100 U/mL of penicillin, 100 µg/mL of streptomycin sulfate, and 15% fetal borine serum (FBS) Collagenase type IV (Gibco), 0.25% trypsin, PBS

2.3. Renal tubular epithelial cell and osteoblast coculture •

•

•

Culture medium: RPMI 1640, supplemented with 5 µg/mL of insulin, 5 µg/mL of transferrin, 5 ng/mL of sodium selenite, 36 ng/mL of hydrocortisone, 4 pg/mL of 3′,3′,5-triiodo-Lthyronine, 10 ng/mL of epidermal growth factor, 800 IU/mL of penicillin, and 1 mg/mL of streptomycin Pharmacological medium: culture medium plus 10−8 M rhPTH1–34 and 10−8 M 25(OH)D3; culture medium with 10−8 M 25(OH)D3 as control Stainless steel sieve (80-mesh, 100-mesh), Transwell dish (Corning Costar Corp.), FBS, 0.25% trypsin

2.4. Proliferation rate test (MTT method) •

5 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) stock solution
Add 100 mg of MTT powder into 100 mL of PBS.
Dissolve and sterilize with filter.
Keep in black-colored bottle in a −4°C freezer.

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96-well plates, dimethyl sulfoxide (DMSO), PBS, universal microplate reader

2.5. ALP activity test (PNPP method) •

3 mM 4-nitrophenylphosphatehexahydrate (PNPP) Add 113.3 mg of PNPP and 100 mL of deionized water.
Store in a −4°C freezer. 50 mM diethanolamine (DEA) buffer
Add 0.5 mL of 4.0 M HCl, 203.3 mg of MgCl2 . 6H2O, 0.5 mL of DEA, and 99 mL of deionized water.
Adjust pH to 10.5.


•

2.6. Mineralized nodule staining and quantification •

5% sodium thiosulfate Add 5 g of sodium thiosulfate, 0.2 mL of 0.1 mol/L(N)NaOH, and 100 mL of distilled water. 50 µg/mL of ascorbic acid, 10 µM β-glycerophosphate, 0.1% Alizarin red staining solution, tetracycline, 2% silver nitrate




•

3. Methods 3.1. Osteoblast isolation from neonatal rat calvariae • • • • •

a

Sacrifice rats by decapitation and sterilize with 75% alcohol. Strip away the skull skin, and then cut the calvaria and place it in a Petri dish with PBS. Remove the soft connective tissue in calvaria with curved forcepsa and wash in dishes with PBS. Cut the calvaria into small pieces (1 mm × 1 mm), and then transfer them to a 10-mL centrifuge tube. Incubate the bone pieces in 0.25% trypsin solution (5 mL) in a 37°C shaking water bath for 20 minutes.

Remove all soft tissues thoroughly before collagenase digestion so as to minimize the fibroblast contamination.

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•

• •

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Transfer the slices to a 0.1% collagenase solution (5 mL)b and incubate in a 37°C shaking water bath for 60 minutes, collect the cellcontaining supernatant by centrifugation at 1000 rpm for 10 minutes at room temperature, and then resuspend the cell pellets with MEM. Repeat step 6 with new collagenase solution, and then combine the cell suspension with that in step 6 and adjust to the desired cell density with MEM. Seed into dishes or flasks, and culture in a humidified incubator with 5% CO2 at 37°C. Change the medium 1 day after culture, and then refresh the medium each 2–3 days.

3.2. Osteoblast isolation from adult human cancellous bone c • • • • •

•

Place cancellous bone, obtained from surgery or biopsy, in a sterile container with PBS or MEM. Remove soft connective tissue by scraping with a sterile scalpel blade, and cut off the cortex if necessary. Wash the bone fragments in PBS three times for 10 seconds using a vortex mixer. Transfer the cancellous bone fragments to a new Petri dish containing 2–3 mL of PBS, and cut into pieces (1 mm3) with scissors. Rinse the bone pieces in PBS and transfer to a 10-mL bottle with 0.25% trypsin for 20 minutes in a 37°C water bath, and then discard the supernatant. Resuspend the bone pieces with 10 mL of culture medium, and spread on the bottom of Petri dishes or flasks at a density of 50 pieces per 100-mm-diameter Petri dish or 75-cm2 flask.

b Make sure to prepare the collagenase solution freshly each time and warm up to 37°C before use. c An excellent source of human samples for osteoblast harvest is the upper femur of patients.

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Culture the bone explants at 37°C in a humidified atmosphere with 5% CO2.d Refresh the medium after 4 days of incubation; be careful not to shake the explants during this period. Check for outgrowth of cells each 3 or 4 days. Discard the bone chips with pipettes or forceps after 14 days and refresh the medium. Refresh the medium twice weekly thereafter until confluent.

3.3. Renal tubular epithelial cell and osteoblast coculture • • • • •

• •

• •

Isolate kidneys from rats under anesthesia with 22% urethane (0.5 mL/100 g). Immediately transport on ice to culture room for cell isolation. Strip the outer member and split the kidney in half with blade. Separate the cortex from the medulla by scraping it with a scalpel, cut into small pieces (1 mm3), and wash with ice-cold PBS three times. Squash these pieces through an 80-mesh stainless steel sieve mechanically and collect the renal tubule segments with a 100-mesh sieve, followed by centrifugation at 700 rpm for 5 minutes at 4°C and washing with PBS. Add 0.25% trypsin solution to tubule segments (3 mL/kidney) and incubate in a 37°C shaking water bath for 20 minutes. Centrifuge the digested mixture at 1000 rpm for 5 minutes at 4°C, discard the supernatant, and resuspend the pellets with culture medium supplement with 3% FBS. Plate into Transwell dishes and incubate at 37°C in a humidified atmosphere of 5% CO2.e Change the medium after 3 days’ incubation and refresh the medium every 2–3 days.

d Be careful not to shake dishes when checking each time, and not to move dishes during the first 3 to 4 days when harvesting human osteoblasts from explants. e An alternative method is to obtain the tubular epithelial cells from tubular fragments by the enzymatic digestion procedure (from step 7) and wash the tubule segments with RPMI 1640 medium; digest in 0.1% collagenase IV (Gibco) and 0.05% hyaluronidase (Sigma) in RPMI 1640 medium without supplements at 37°C for 20 minutes, and collect the supernatant and resuspend with culture medium; and then combine the cell suspension by both digestions.

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Change with pharmacological medium and negative control for 24 hours of incubation. Culture the rat calvaria-derived osteoblasts in a six-well plate (about 80% confluence) as described above. Coculture tubular epithelial cells (in the upper chamber) with osteoblasts (in the lower chamber).

3.4. Pharmacological evaluation on osteoblasts in vitro 3.4.1. Proliferation rate test (MTT method) • • • • • •

Seed calvaria-derived osteoblasts in 96-well plates at a density of 2000/well with drugs for 7 days. Discard the culture medium and wash with PBS twice. Dilute MTT stock solution with plain culture medium to 0.5 mg/mL, and add to the 96-well plate. Incubate in a 37°C incubator with 5% CO2. Discard MTT solution and dissolve with DMSO for 20 minutes. Measure the absorbance value (OD570 nm) by auto-microplate reader (EL×800; BioTek, USA).

3.4.2. ALP activity test (PNPP method) • • • • • • •

Seed the calvaria-derived osteoblasts at a density of 5000/well in 96-well plates. Refresh the medium with drugs 2 days after the osteoblasts reach confluence. Change the medium every 2–3 days for 2 weeks. Discard the culture medium and wash with PBS twice. Add DEA buffer to lysate the cells. Add PNPP as a substrate to react with ALP. Measure the absorbance value (OD405 nm) dynamically by automicroplate reader (EL×800; BioTek, USA), and then normalize the ALP activity to total protein.

3.4.3. Mineralized nodule staining and quantification Seed the second passage of calvaria-derived osteoblasts in 6-well or 24-well plates with drugs in the presence of 10 mM β-sodium

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glycerophosphate and 50 µg/mL ascorbate. The mineralized nodules — which usually appear in 2–3 weeks — can be detected by the Alizarin red staining, von Kossa staining, or tetracycline labeling method. The mineral nodule number and area can be quantified by computer-assisted image analysis. (1) Alizarin red staining (ARS) for mineralized nodules • • •

Wash cells with PBS twice, fix in 95% ethanol for 10 minutes at room temperature, and then wash with distilled water. Add 0.1% Alizarin red–Tris-HCl (pH 8.3) and incubate at 37°C for 30 minutes. Wash with distilled water and dry in air.

(2) Modified von Kossa staining for mineralized nodules • • • • •

Wash cells with PBS twice, fix in 95% ethanol for 10 minutes at room temperature, and then wash with distilled water. Stain in 2% silver nitrate for 1 hour at room temperature in the dark. Wash in running water. Stain in 5% sodium thiosulfate for 1 hour. Wash with distilled water and dry in air.

(3) Tetracycline labeling for mineralized nodules • • • •

Add tetracycline (50 µg/mL) to cells and incubate for 30 minutes. Refresh the medium for another 30 minutes. Wash with PBS and fix in 95% ethanol. Wash with PBS and observe under a fluorescent microscope.

3.4.4. Bone matrix protein • •

Seed the second passage of calvaria-derived osteoblasts in 6-well or 24-well plates with drugs. Measure the BGP, PICP, and IGF-1 protein levels in the medium using commercialized enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA) kits.

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Assess the gene expressions of BGP, Col-1, and IGF-1 in osteoblasts by reverse transcription–polymerase chain reaction (RT-PCR).

4. Results 4.1. Osteoblast isolation from neonatal rat calvariae About 1–3 × 105 cells/rat enriched with osteoblasts and osteoblast progenitors were harvested following this protocol and used directly for experiments. Primary osteoblasts from neonatal rat showed cuboidal morphology and reached confluence in about 7 days (Fig. 1).

4.2. Osteoblast isolation from adult human cancellous bone Osteoblasts grew out from explanted cancellous bone in 3–10 days and reached confluence in 2–4 weeks, depending mainly on the donor age and culture conditions (Fig. 2).

Fig. 1. Phase-contrast micrograph of primary neonatal rat calvaria-derived osteoblasts (200×).

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Fig. 2. Phase-contrast micrograph of human osteoblasts from cancellous bone (200×).

4.3. Mineralized nodule staining and quantification Mineral nodes showed red color after Alizarin red staining [Fig. 3(a)], black color after modified von Kossa staining [Fig. 3(b)], and yellow color after tetracycline labeling [Fig. 3(c)].

4.4. Bone matrix protein Tubular fragments attached to the dishes in 12 hours, and cells grew out of the fragments in 24–36 hours. After refreshing the medium in 72 hours’ incubation, the attached flat cells with typical epithelial morphology were seen around fragments growing in a cobblestonelike monolayer under phase-contract microscopy (Fig. 4), and had high ALP activity and positive staining for cytokeratin. The cultured

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PTECs with osteoblasts after 24 hours of 10−8 M rhPTH1–34 treatment showed stimulated effects on osteoblast differentiation. The ALP, BGP, and Col-I mRNA levels in osteoblasts increased by 60%, 26%, and 36%, respectively. (a)

(b)

Fig. 3. Mineralized nodes formed in rat calvaria-derived osteoblast cultures, shown as (a) red nodes by Alizarin red staining, (b) black nodes by modified von Kossa staining, and (c) yellow nodes by tetracycline labeling procedure (×40).

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(c)

Fig. 3.

Fig. 4.

(Continued )

Micrograph of primary rat PTECs (200×).

5. Summary Osteoblasts belong to the fibroblastic lineage and grow adherently in vitro. Functional active osteoblasts show a cuboidal morphology and feature several biochemical markers such as alkaline phosphatase (ALP), parathyroid hormone (PTH) receptor, osteocalcin (or bone Gla protein

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[BGP]), 1,25(OH)2D responsiveness, and the ability to form mineralized nodules in vitro (Langub et al. 2000; Wang et al. 2007). Although many markers are not unique to osteoblasts, they express at different stages during osteoblast differentiation and development. ALP, a marker of early osteogenic differentiation, can be measured by enzyme assay or by cytochemical staining with NBT/BCIP as the substrate. Osteocalcin, a marker specific to mature osteoblasts, can be measured by commercialized kits or by RT-PCR. The formation of mineralized nodules, in the presence of ascorbate and β-sodium glycerophosphate, can be analyzed by histochemical staining. The value of cultured primary osteoblasts in assessing the pharmacological effects of antiosteoporosis agents has been validated, though some limitations must be acknowledged. The parameters used for assessing the anabolic effects on osteoblasts include the proliferation rate; ALP activity; mineral-nodule–forming ability; and measurement of cytokines such as osteocalcin (BGP), procollagen type I carboxyterminal propeptide (PICP), and IGF-1. The promising candidate agents are expected to stimulate osteoblast proliferation, osteoblast differentiation, and bone matrix formation (Wang et al. 2001; Wang et al. 2007).

Acknowledgments These studies were supported by grants from the National Nature Science Foundation of China (NSFC) to H. Wang (39170790), W. Jin (39570774, 39970809), and J. Gao (30170439).

References Birnbaum RS, Wiren KM. Changes in insulin-like growth factor-binding protein expression and secretion during the proliferation, differentiation, and mineralization of primary cultures of rat osteoblasts. Endocrinology 135(1):223–230, 1994. Datta D, Dormond O, Basu A et al. Heme oxygenase-1 (HO-1) modulates the expression of the anti-angiogenic chemokine CXCL10 in renal tubular epithelial cells. Am J Physiol Renal Physiol 293:F1222–F1230, 2007.

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Harada S, Rodan GA. Control of osteoblast function and regulation of bone mass. Nature 423(6937):349–355, 2003. Langub MC, Reinhardt TA, Horst RL et al. Characterization of vitamin D receptor immunoreactivity in human bone cells. Bone 27(3):383–387, 2000. Siggelkow H, Rebenstorff K, Kurre W et al. Development of the osteoblast phenotype in primary human osteoblasts in culture: comparison with rat calvarial cells in osteoblast differentiation. J Cell Biochem 75(1):22–35, 1999. Wang HF, Jin WF, Gao JJ (eds.). An Atlas of Bone Cells and Cell Culture Techniques. Shanghai Scientific & Technical Publishers, Shanghai, China, 2001. Wang HF, Jin WF, Gao JJ, Sheng H. Changes of biological functions of bone cells and effects of anti-osteoporosis agents on bone cells. In: Qin L, Genant HK, Griffith JF, Leung KS (eds.), Advanced Bioimaging Technologies in Assessment of the Quality of Bone and Scaffold Materials, Springer, Berlin, pp. 205–222, 2007. Wang HF, Sekimoto H, Lin GG. In vitro study of cultured bone cells from calvaria of rat embryos. J Shanghai Med Univ 18(6):475–477, 1991.

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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

Osteoblast harvest from rat bone and bone marrow by mechanical isolation and induction culture methods, respectively, is described in this chapter. Osteoclasts act as a cell model for pharmaceutical evaluation on anti–bone resorption drug screening by providing a technical platform to study the regulation of bone resorption and to evaluate the agents developed for prevention and treatment of metabolic bone disorders. Keywords:

Osteoclast; resorption pit; TRAP; apoptosis.

1. Introduction Osteoclasts, the bone resorption cells, are usually fewer in number than other cell types in bone and are very fragile in isolation for their large size and high content of proteolytic enzymes. Many strategies have been developed to improve the quantity and purity of isolated osteoclasts in the past 20 years. The mechanical isolation of mature osteoclasts directly from bone, which was first developed by Chambers and Magnus (1982) and then used in resorption assay by Corresponding author: Wei-Fang Jin. Tel: +86-21-64048126; fax: +86-21-64436239; E-mail: [email protected]

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Chambers et al. (1984), is still the most basic and best way to obtain authentic osteoclasts. Additionally, various osteoclastogenetic culture systems have been developed, such as bone marrow cells, spleen cells, and other hemopoietic stem cells in vitro; these are usually cocultured with bone marrow stromal cells (MSCs) or cell lines under the stimulation of 1,25(OH)2D or dexamethasone (Takahashi et al. 2003), or induced by M-CSF and RANKL only (Fuller et al. 2006). The protocol described here is a routine and validated procedure used in our laboratory (Wang et al. 2001) for the evaluation of some potential antiresorbing agents on osteoclast formation.

2. Materials 2.1. Osteoclast isolation from neonatal rat long bones • •

•

Animals: newborn rats, 2–3 days of age Culture medium: minimum essential medium (MEM) supplemented with 10% fetal borine serum (FBS), 100 U/mL of penicillin, 100 µg/mL of streptomycin sulphate, and 2 mM Lglutamine; pH 7.2 Fixative: 2.5% glutaraldehyde in phosphate buffered saline (PBS)


•

Prepare fresh before use.

Sharp scissors, scalpel, fine forceps, glass dishes, PBS, diamond saw microtome (Leitz 1600; Leitz, Germany), microscope, Nikon digital camera, image analysis software (Image-Pro Plus version 4.1)

2.2. Osteoclast formation in bone marrow culture • •

•

Animals: 6–9-week-old male mice Tissue culture medium: MEM supplemented with Earle’s salts, 100 IU/mL of penicillin, 100 µg/mL of streptomycin, 2 mM L-glutamine, and 10% FBS; pH 7.2 Pharmacological medium: MEM (100 U/mL of penicillin, 100 µg/mL of streptomycin sulfate, and 2 mM L-glutamine; pH 7.2) containing 20% serum of 10-month-old rats from Sham, Ovx, and Ovx+Estrodial groups, respectively

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Elcatonin: 10−7, 10−9, and 10−11 mol/L of elcatonin in TC199 medium with 10% FBS Sterile instruments: sharp scissors, fine forceps, syringes, and needles

2.3. TRAP + multinucleated cell (MNC) counting • •

10−2 mol/L of ibandronate prepared in normal saline and 10−4–10−12 mol/L of working solution diluted by MEM Tartrate-resistant acidic phosphatase (TRAP) staining solutiona


Acetate solution (solution A)   




100 mM tartrate (solution B) 




Dissolve 2.3 g of sodium tartrate in 100 mL of acetate buffer.

Hexazonium pararosanilin solution (solution C) 

 

a

Solution (a): Dissolve 1.64 g of anhydrous sodium acetate in 100 mL of distilled water. Solution (b): Take 1.2 mL of glacial acetic acid and make up to 100 mL with distilled water. Mix 75 mL of solution (a) with 25 mL of solution (b), and adjust to pH 5.0 with solution (b).

Add 1 g of pararosanilin to 20 mL of distilled water and add 5 mL of concentrated hydrochloric acid, heat carefully for 15 minutes in a 90°C water bath while stirring, and then filter once the solution has cooled down. Store at 4°C and protect from light. Mix with the same volume of 4% sodium nitrite just before use.

Cytochemical staining for TRAP is widely used to identify osteoclasts in vivo and in vitro. As an alternative to the protocol described here, a staining kit from Sigma (Acid Phosphatase Leukocyte TRAP Kit; Sigma 386A) is also commercially available. Fast garnet GBC is used as the dye in this kit, and this leads to a dark purple stain.

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Naphthol AS-BI phosphate stock (solution D) 




Dissolve 20 mg/mL of naphthol AS-BI phosphate in dimethyl formamide.

Mix 8 mL of solution B, 1 mL of solution C, and 1 mL of solution D.

2.4. Apoptosis detection •

•

Binding buffer: 0.2383 g of HEPES, 0.8766 g of NaCl, 0.02646 g of CaCl2 . 2H2O, 0.0203 g of MgCl2, and 0.03728 g of KCl dissolved in 100 mL of H2O Annexin-PI (povidone idoine) solution: 5 uL of annexin V-FITC (A9210; Sigma) and the same volume of PI (50 µg/mL in PBS) (P4170; Sigma) mixed in 500 µL of binding buffer

2.5. Pit-forming assay •

Bone slices: dentine slices (diameter, 12 mm) of ivory blocks





•

1% toluidine blue: 1 g of toluidine blue and 1 g of sodium borate


•

Clean by ultrasonication in distilled water and sterilize under ultraviolet light for 4 hours on both sides. Sterilize further in PBS with 1000 IU/mL of penicillin and 1 mg/mL of streptomycin overnight. Add distilled water to 100 mL and filter before use.

0.25 M ammonium hydroxide

3. Methods 3.1. Osteoclast isolation from neonatal rat long bones • •

•

Sacrifice rats by decapitation and sterilize the skin with 75% alcohol. Cut off all limbs and put in iced PBS dish. Dissect out the femora, tibiae, and humeri using fine forceps and a scalpel. Remove most soft tissue, and keep the dissected bones in 0.5 mL of iced MEM per rat. Quickly curette the bones with a scalpel in the iced glass dish, gently pipetting several times with a blunt-ended pipette. If necessary,

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•

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transfer to a 10-mL centrifuge tube (hold in ice) and gently vortex twice for 10 seconds each. Settle for 15–30 seconds. Collect the supernatant to a new tube. Repeat pipetting the bone chips with fresh MEM and combine the supernatant. Seed the cell suspension immediately or after dilution to the desired volume onto glass coverslips, bone slices, or directly into tissue culture plate, as required. After 30–60 minutes’ incubation, gently wash off the nonadherent cells with warmed culture medium, add fresh medium, and maintain at 37°C in a humidified atmosphere of 5% CO2.

3.2. Osteoclast formation in bone marrow culture • •

•

• • •

Sacrifice rats by cervical dislocation and wipe the skin with alcohol. Remove the femora, tibiae, humeri, ulnae, and radii, and cut off the bone ends with scissors. Flush out the marrow cells with 1 mL of MEM by injecting at one end of the bone using a sterile 27-gauge needle. Squeeze the cell suspension through a 22-gauge needle to get single-cell suspension. Dilute to 5 × 106 cells/mL, and seed 2 mL/well in 6-well plates or 1 mL/well on bone slices in 24-well plates as required. Add 50 ng/mL of M-CSF and 50 ng/mL of RANKL in culture medium, and incubate in a humidified atmosphere of 5% CO2. Refresh the medium every 2–3 days. At the end of the experiment, fix the cells in 2.5% glutaraldehyde for 7–10 minutes at 4°C, and stain for TRAP as described above or perform resorption pit assay on bone slices by a toluidine blue staining procedure after cleaning by ultrasonication.

3.3. Evaluation of pharmacological effects on osteoclasts in vitro 3.3.1. TRAP + multinucleated cell (MNC) counting •

Isolate osteoclasts from long bone of 10-day-old rabbits as described above.

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Plate on bone slices (5 mm × 5 mm) in a tissue plate and culture for 3 hours. Refresh the medium and separate the bone slices into 96-well plates randomly, one piece in each well. Add three doses of ibandronate to the medium (working concentration of 10−8, 10−10, and 10−12 mol/L) and the same volume of MEM as negative control (NC) for 3 days’ incubation. At the end of the experiment, wash bone slices with PBS and fix in 2.5% glutaraldehyde for 7–10 minutes at 4°C. Stain in TRAP staining solution for 40–50 minutes at 37°C in the dark, and then wash with distilled water. Count TRAP-positive multinucleated cells with three or more nuclei and red or purple color under a light microscope.

3.3.2. Apoptosis detection • • • • • •

• •

Isolate osteoclasts from long bone of neonatal rats as described above. Plate on 5-mm-diameter glass slices and culture for 30 minutes. Wash with warmed medium, and then change to pharmacological medium for 6 hours’ incubation. At the end of the experiment, wash cells with binding buffer. Cover the cells with annexin-PI solution and incubate for 5–10 minutes at room temperature in the dark. Quickly count the apoptotic osteoclasts (green-stained multinucleated large cells) and necrotic osteoclasts (red-stained large cells) under a fluorescent microscope. Count the total osteoclasts, followed by TRAP staining (redstained multinucleated large cells) on the same slice. Calculate the percentage of apoptotic cells to total osteoclasts.

3.3.3. Pit-forming assay • •

Place dentine slices in 24-well plates containing 1 mL of TC199 medium with 10% FBS (1 slice/well). Transfer about a 1-mL aliquot of the crude osteoclast preparation onto the surface of slices.

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Incubate for 3 hours and refresh the medium. Add elcatonin to the concentration of 10−8, 10−10, and 10−12 mol/L, and the same volume of blank medium as a negative control (NC). Culture for 3 days and refresh 50% of the medium each day. At the end of the experiment, remove the medium. Strip cells by ultrasonication in 0.25 mol/L of NH4OH solution three times, 1 minute for each time, and then dehydrate routinely in series of ethanol. Stain in 1% toluidien blue (TB) for 3–4 minutes at room temperature and wash with distilled water. Count the number of resorption pits on dentine slices under a light microscope, and then photograph the resorbed area using a Nikon digital camera and analyze by Image-Pro Plus analysis software.

4. Results 4.1. Osteoclast isolation from neonatal rat long bones Small numbers of osteoclasts were obtained by this protocol (several hundred per rat). After 1–2 hours’ incubation, the osteoclasts spread out well and were easily recognizable — based on their characteristic morphology of large size and multinuclearity (more than three nuclei) — from other adherent cell types under a phasecontrast microscope (Fig. 1).

4.2. Osteoclast formation in bone marrow culture Large numbers of TRAP-positive multinucleated cells (osteoclasts) were formed uniformly all over the culture dishes in 1 week according this protocol (Fig. 2).b

b

In the absence of bone-resorbing factors, TRAP-positive multinucleated cells will rarely appear until the formation of alkaline phosphatase (ALP)-positive colonies. Cocultures of primary calveria osteoblasts with bone marrow cells produce more osteoclasts than bone marrow cultures alone.

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Fig. 1. (×200).

Phase-contrast micrograph of rat osteoclasts with several nuclei

Fig. 2. Rat osteoclasts stained for tartrate-resistant acid phosphatase (TRAP) by histochemical procedure (×200).

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4.3. Evaluation of pharmacological effects on osteoclasts in vitro 4.3.1. TRAP + multinucleated cell (MNC) counting About 130 TRAP+ multinucleated cells per slice were available, and the number of MNCs was lower in treated groups. The inhibition rates of ibandronate, calculated as (NC − treatment group)/NC, were 13.5%, 33.6%, and 71.3% at doses of 10−12, 10−10, and 10−8 mol/L, respectively (Table 1).

Table 1.

Number of TRAP-positive multinucleated cells (MNCs).

Groups

Number of MNCs

Inhibitory rate (%)

± ± ± ±

13.5 33.6 71.3

Control 10−12 10−10 10−8

26.75 23.00 17.75 7.00

7.8 13.6 7.4 6.9*

* p < 0.01 vs. control.

4.3.2. Apoptosis detection (Table 2) Table 2.

Percentage of apoptotic cells to total osteoclasts.

Groups Sham Ovx Estrodial

Apoptotic cells (%) 13.62 ± 6.16 11.09 ± 3.18 23.61 ± 5.70*

* p < 0.01 vs. control.

4.3.3. Pit-forming assay Resorption lacunae showed violet color in bone slices after toluidine blue staining. The resorption pit number was found decreased in elcatonin groups in a dosage-dependent manner (Fig. 3).

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Fig. 3.

Inhibitory rate of elcatonin on osteoclast bone resorption.

5. Summary Osteoclasts are sensitive to changes in pH, and low pH is an essential requirement for osteoclast resorption pit formation in vitro. Arnett and Spowage (1996) suggested that culture medium should be acidified by directly adding small amounts of concentrated HCl or by increasing the concentration of CO2 to produce a working pH close to 6.95–7.0, which is optimal for resorption pit formation. The improved purity of osteoclasts can be obtained by shortening of the isolation time, which favors osteoclasts over other cell types, or by enzymatic treatment such as 0.001% pronase in PBS containing 0.02% EDTA (Coxon et al. 2003); but absolute yield is reduced. Density gradient and immunomagnetic isolation (Coxon et al. 2003) will be used when high purity of osteoclasts is needed; this usually applies to large-number osteoclast sources such as long bone of 10-day-old rabbits or osteoclast-like cells in suspension of osteoclastomas. Osteoclasts can be identified by tartrate-resistant acid phosphatase (TRAP) staining or calcitonin receptor analysis (Wang et al. 2007).

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(a)

(b)

Fig. 4. Lacunae on the dentine slice, observed by (a) light microscopy stained with toluidine blue (×200) and (b) SEM (×500).

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More than 1 day of incubation on bone slices and osteoclasts will form resorption pits, which can be visualized either by toluidine blue staining [Fig. 4(a)] or by scanning electron microscopy (SEM) [Fig. 4(b)] (Wang et al. 2001; Wang et al. 2007). In some species, osteoclasts can also be identified by vitronectin receptor staining or by F-actin ring detection with phalloidin coupled to rhodamine staining (Lakkakorpi and Vaananen 1991; Wang et al. 2001).

Acknowledgments These studies were supported by grants from the National Nature Science Foundation of China (NSFC) to H. Wang (39170790), W. Jin (39570774, 39970809), and J. Gao (30170439).

References Arnett TR, Spowage M. Modulation of resorptive activity of rat osteoclasts by small changes in extracellular pH near the physiological range. Bone 18(3):277–279, 1996. Chambers TJ, Magnus CJ. Calcitonin alters behaviour of isolated osteoclasts. J Pathol 136(1):27–39, 1982. Chambers TJ, Revell PA, Fuller K, Athanasou N. Resorption of bone by isolated rabbit osteoclasts. J Cell Sci 66:383–399, 1984. Coxon FP, Frith JC, Benford HL, Rogers MJ. Isolation and purification of rabbit osteoclasts. In: Helfrich MH, Ralston SH (eds.), Bone Research Protocols, Methods in Molecular Medicine, Vol. 80, Humana Press, Totowa, NJ, pp. 89–100, 2003. Fuller K, Kirstein B, Chambers TJ. Murine osteoclast formation and function: differential regulation by humoral agents. Endocrinology 147: 1979–1985, 2006. Lakkakorpi PT, Vaananen HK. Kinetics of the osteoclast cytoskeleton during the resorption cycle in vitro. J Bone Miner Res 6(8):817–826, 1991. Takahashi N, Udagawa N, Tanaka S, Suda T. Generating murine osteoclasts from bone marrow. In: Helfrich MH, Ralston SH (eds.), Bone Research Protocols, Methods in Molecular Medicine, Vol. 80, Humana Press, Totowa, NJ, pp. 129–144, 2003.

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Wang HF, Jin WF, Gao JJ (eds.). An Atlas of Bone Cells and Cell Culture Techniques. Shanghai Scientific & Technical Publishers, Shanghai, China, 2001. Wang HF, Jin WF, Gao JJ, Sheng H. Changes of biological functions of bone cells and effects of anti-osteoporosis agents on bone cells. In: Qin L, Genant HK, Griffith JF, Leung KS (eds.), Advanced Bioimaging Technologies in Assessment of the Quality of Bone and Scaffold Materials, Springer, Berlin, pp. 205–222, 2007.

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Chapter 8

Primary Cultures of Human Periosteal Cells Wing-Hoi Cheung, Wing-Sze Lee, C. Zhang and Kwok-Sui Leung

Enhancing fracture healing and other related conditions (like delayed union or nonunion) can help to shorten hospitalization, reduce complications, and improve rehabilitation. Understanding the underlying mechanism of fracture healing will help to explore new treatment approaches in this aspect. Periosteal cells, as a cell type with differentiation ability to become bone-forming cells, are crucial in the fracture healing process. Research on the biology of periosteal cells or understanding the response of the cells to external stimulation may be useful to open up a new area to treat fracture healing. This chapter describes the isolation of human periosteal cells for research on biophysical interventions or other stimulations developed to enhance fracture healing. Keywords:

Periosteal cell; explant culture; alkaline phosphatase (ALP); cytostaining; osteocalcin; fluorescence-activated cell sorter.

1. Introduction Fracture is a common clinical problem, especially in the elderly. With the increase in the aging population, osteoporotic fracture healing has Corresponding author: Wing-Hoi Cheung. Tel: +852-26323312; fax: +852-26324618; E-mail: [email protected]

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become a major medical concern all over the world. The worldwide cost of hip fracture treatment alone is projected to be US$131.5 billion in the year 2050 (Cooper et al. 1992). The situation becomes more complicated when normal fractures develop into delayed union or nonunion. Therefore, research on the enhancement of fracture healing has been a major trend in orthopedics in recent years. The periosteum is the outermost layer of bone, in which periosteal cells are a critical cell type with differentiative potential to become bone-forming osteoblasts in the fracture healing process (Hanada et al. 2001; Ozaki et al. 2000; Hutmacher and Sittinger 2003). Investigations into the biology of periosteal cells will thus help us understand the underlying mechanisms of fracture healing, from which we may explore potential approaches to accelerate fracture healing. For example, Mierisch et al. (2002) reported the induction of chondrogenesis in periosteum using insulin-like growth factor 1; positive effects of various biophysical interventions on periosteal cells have also been demonstrated (Leung et al. 2004; Tam et al. 2005). Moreover, delayed union has been defined as the cessation of periosteal healing response before the fracture is successfully bridged, and nonunion as the cessation of both the periosteal and endosteal healing responses without bridging (Marsh 1998). In addition, periosteal cells may play an important role in distraction osteogenesis, as reported by the upregulation of Runx2 and osteogenic factor of periosteal cells under tensile mechanical strain (Kanno et al. 2005). Therefore, research on periosteal cells will help in developing knowledge on fracture healing, delayed union, nonunion, and distraction osteogenesis. This chapter will provide a detailed protocol of explant culture for periosteal cell isolation, from the collection of periosteum in the operating room to the characterization of periosteal cells.

2. Materials •

Dulbecco’s modified Eagle’s medium (DMEM; Sigma) — for providing nutrients to periosteum tissues during treatment and rinsing

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Phosphate buffered saline (PBS) — for washing Penicillin-streptomycin-neomycin (PSN) antibiotics (PSN; Gibco Laboratories) — for sterilizing the periosteum obtained from the operation theater before aseptic culturing procedures Tissue culture medium: DMEM supplemented with 10% FBS (Gibco Laboratories), 0.8% PSN complex, 10 mmol/L of betaglycerophosphate (Sigma), and 50 µg/mL of ascorbate — for routine monolayer culturing of human periosteal cells 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) (Sigma) — for reacting with alkaline phosphatase (ALP) and forming insoluble black-purple precipitates 2-amino-2-methyl-1-propanol buffer (ALP buffer; Biosystems): reagent A — 0.35 mol/L of 2-amino-2-methyl-1-propanol, 1 mmol/L of zinc sulfate, 2 mmol/L of N-hydroxyethylethylenediaminetriacetic acid, 2 mmol/L of magnesium acetate, pH 10.4; reagent B — 12 mmol/L of 4-nitrophenylphosphate once dissolved Eosin yellow (1%) — for counterstaining

3. Methods (Leung et al. 2004; Tam et al. 2005; Zhang et al. 2006a; Zhang et al. 2006b) 3.1. Obtaining periosteum from operating theater •

•

With prior consent obtained from the patient, obtain a 5 mm × 5 mm periosteum from the anteromedial surface in the middiaphysis of the normal tibia in patients having joint replacement surgery and implant removal. Store the biopsy sample in sterile normal saline solution and transfer to cell culture laboratory immediately.

3.2. Explant culture of human periosteal cells • • •

Under aseptic conditions, rinse the periosteum biopsy five times with DMEM containing 3% PSN antibiotics. Remove the flesh on the periosteum biopsy using a pair of scissors. Mince the periosteum into small pieces.

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Fig. 1.

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Morphology of isolated human periosteal cells (200×).

Explant the minced periosteum onto a 25-cm2 culture flask by a spreader in tissue culture medium at 37°C, 5% CO2 and humidified incubator (Fig. 1). When the explanted cells become confluent, disperse by trypsin and transfer to a new culture flask. Change the tissue culture medium every 3 days.

3.3. Characterization of human periosteal cells Characterize part of the isolated periosteal cells by standard cytostaining of ALP. • • • •

Coat the cells on a cover slip overnight. Wash the cells with PBS twice and fix with 70% ethanol. Add 1 mL of ALP buffer (to facilitate the reaction of BCIP/NBT in the following step) and then discard the solution. Add BCIP/NBT at 37°C for 30 minutes and protect the cells from light with aluminum foil.

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Purple color will develop if ALP is present (BCIP/NBT reacts with ALP to form intense, insoluble black-purple precipitate). If no purple color develops after 30 minutes, then prolong the incubation time. Stop the reaction with deionized water if no further purple color develops. Counterstain the slides which have no purple color development with eosin.

4. Discussion The present protocol uses ALP cytostaining to characterize human periosteal cells, as ALP is a well-known biochemical marker of periosteal cells (Shedden et al. 1976). However, ALP is not specific to periosteal cells, as it can be expressed by many cell types like osteoblasts (Yamada et al. 2007) and chondrocytes (Ho et al. 2006; Lee et al. 2003). Therefore, ALP cytostaining cannot absolutely confirm the identity of periosteal cells. The quality of obtaining human periosteal cells can be further enhanced if (1) surgeons obtain a piece of periosteum while trying to get rid of other surrounding nonperiosteal tissues, and (2) the standardized isolation protocol is performed by a well-trained technician. To further secure the nature of periosteal cells, in situ hybridization of osteocalcin expression may be conducted as well (Calvi et al. 2001). Fluorescence-activated cell sorter (FACS) — a technique to identify and distinguish cell types from each other on the basis of their differential expression of cell surface proteins using specific antibodies — may be useful for the identification of periosteal cells, as observed by previous studies on other cell types (Kim et al. 2007; Ozdogu et al. 2007). However, since periosteal cells do not currently have a confirmed specific biochemical marker other than ALP expression, ALP characterization is still the most reliable and most convenient method to identify periosteal cells.

5. Remarks •

During flesh removal, it is important to clear the periosteum from the flesh before periosteal cell explant culture; otherwise,

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the released periosteal cells may be contaminated and overwhelmed by fibroblasts, which have a higher proliferation rate than periosteal cells. When mincing the periosteum into small pieces, the size of the periosteum should be as small as possible to facilitate the release of periosteal cells from the biopsy and to shorten the time for releasing cells. After spreading the minced periosteum onto a 25-cm2 flask, it should be placed in a 37°C incubator for 30–45 minutes before adding the culture medium. This can partially dry the periosteum, leading to a better attachment to the bottom of the culture flask, which is critical for successful cell release of explant culture. No cells can be obtained once the periosteum detaches from the flask. Minced periosteum should be evenly distributed to let the cells explant evenly onto the culture flask. Uneven distribution may lead to regional overcrowding of released cells, which may induce the differentiation of cells into other lineages. The morphology of released periosteal cells should be monitored frequently. Since the cell is a highly differentiated cell type, it may potentially differentiate after several passages or in an overcrowded situation. Discard the cells if a change in cell morphology is identified. This technique should be applicable to release cells from animal periosteum, provided that the periosteum is enough to perform such procedures.

Acknowledgments This work was supported by the RGC Earmarked Grant (CUHK4257/99M, CUHK4153/02M), Research Grants Council, Hong Kong, China.

References Calvi LM, Sims NA, Hunzelman JL et al. Activated parathyroid hormone/ parathyroid hormone-related protein receptor in osteoblastic cells

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differentially affects cortical and trabecular bone. J Clin Invest 107(3): 277–286, 2001. Cooper C, Campion G, Melton LJ. Hip fractures in the elderly: a world-wide projection. Osteoporos Int 2:285–289, 1992. Hanada K, Solchaga LA, Caplan AI et al. BMP-2 induction and TGF-beta 1 modulation of rat periosteal cell chondrogenesis. J Cell Biochem 81:284–294, 2001. Ho ML, Chang JK, Wu SC et al. A novel terminal differentiation model of human articular chondrocytes in three-dimensional cultures mimicking chondrocytic changes in osteoarthritis. Cell Biol Int 30(3):288–294, 2006. Hutmacher DW, Sittinger M. Periosteal cells in bone tissue engineering. Tissue Eng 9(Suppl 1):S45–S64, 2003. Kanno T, Takahashi T, Ariyoshi W et al. Tensile mechanical strain up-regulates Runx2 and osteogenic factor expression in human periosteal cells: implications for distraction osteogenesis. J Oral Maxillofac Surg 63(4):499–504, 2005. Kim J, Moon SH, Lee SH et al. Effective isolation and culture of endothelial cells in embryoid body differentiated from human embryonic stem cells. Stem Cells Dev 16(2):269–280, 2007. Lee KM, Cheng ASL, Cheung WH et al. Bioengineering and characterization of physeal transplant with physeal reconstruction potential. Tissue Eng 9(4):703–711, 2003. Leung KS, Cheung WH, Zhang C et al. Low intensity pulsed ultrasound stimulates osteogenic activity of human periosteal cells. Clin Orthop Relat Res 418:253–259, 2004. Marsh D. Concepts of fracture union, delayed union, and nonunion. Clin Orthop Relat Res 355(Suppl):S22–S30, 1998. Mierisch CM, Anderson PC, Balian G, Diduch DR. Treatment with insulinlike growth factor-1 increases chondrogenesis by periosteum in vitro. Connect Tissue Res 43(4):559–568, 2002. Ozaki A, Tsunoda M, Kinoshita S, Saura R. Role of fracture hematoma and periosteum during fracture healing in rats: interaction of fracture hematoma and the periosteum in the initial step of the healing process. J Orthop Sci 5:64–70, 2000. Ozdogu H, Sozer O, Boga C et al. Flow cytometric evaluation of circulating endothelial cells: a new protocol for identifying endothelial cells at several stages of differentiation. Am J Hematol 82(8):706–711, 2007.

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Shedden R, Dunham J, Bitensky L et al. Changes in alkaline phosphatase activity in periosteal cells in healing fractures. Calcif Tissue Res 22:19–25, 1976. Tam KF, Cheung WH, Lee KM et al. Delayed stimulatory effect of lowintensity shockwave on human periosteal cells. Clin Orthop Relat Res 438:260–265, 2005. Yamada S, Ganno T, Ohara N, Hayashi Y. Chitosan monomer accelerates alkaline phosphatase activity on human osteoblastic cells under hypofunctional conditions. J Biomed Mater Res A 83(2):290–295, 2007. Zhang C, Leung KS, Cheung WH. Biological effect of low intensity pulsed ultrasound on human periosteal cells. J Chin Clin Rehabil 10(5):78, 2006a. Zhang C, Liang G, Zhang Y, Hu Y. Response to dynamic strain in human periosteal cells grown in vitro. Sheng Wu Yi Xue Gong Cheng Xue Za Zhi 23(3):546–550, 2006b.

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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

Although osteocytes account for over 90% of bone cells, little progress has been made in their study partly due to the limitations of methodology. In this chapter, we will describe a few much improved techniques to visualize their morphology using both light microscope and electron microscope approaches. These include (1) fluorochrome labeling in conjunction with 4′-6-diamidino-2-phenylindole (DAPI) nuclear counterstaining, (2) imaging of the osteocyte canalicular system with Procion red, and (3) resincasted scanning electron microscopy (SEM). Keywords:

Osteocyte; canalicular system; confocal microscope; double labeling.

1. Introduction The osteocyte, making up over 90% of all bone cells, is derived from the osteoblast, which synthesizes the bone matrix containing collagen and noncollagen proteins. The newly formed osteocyte with few dendrites is incorporated within the osteoid, nonmineralized matrix. The mature osteocyte, with volume reduction of cell body but volume Corresponding author: Jian-Quan Feng. Tel: +1-214-3707235; E-mail: [email protected]

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increase of cell process, is embedded in fully mineralized bone matrix. Its cell body is within spaces called lacunae, and its cell processes named dendrites are within spaces called canaliculi. Like neural cells, these dendrites connect with each other through gap junctions. For many years, osteocytes have been considered to be dormant, metabolically inactive cells. Therefore, little attention has been given to osteocyte studies in comparison to studies of osteoblast (the cellforming bone) and osteoclast (the cell-resorbing bone) cells. However, there is now evidence that these cells are mechanosensors which transmit mechanical signals to the osteoblast and osteoclast for modeling and remodeling of the skeleton. Furthermore, these cells synthesize collagen, respond to systemic and local growth factors, control systemic phosphate homeostasis and mineralization, and are possibly involved in bone resorption. Interestingly, new evidence provided by Dallas (2006) shows that osteocyte cell body movement occurs within lacunae, and that the dendrites of these cells retract within canaliculi. However, osteocyte function is still largely unknown, partly due to a lack of knowledge of genes restricted in osteocytes as well as limited technologies in the exploration of high-mineral environments where osteocytes are embedded. In this chapter, we will describe useful tools for still views of osteocyte canalicular systems that have been modified and improved in our laboratory. These techniques are (1) fluorochrome labeling combined with 4′-6-diamidino2-phenylindole (DAPI) nuclear staining, a new assay developed for visualizing the position of osteocytes within the mineralizing matrix during bone formation; (2) imaging of the osteocyte canalicular system with Procion red, a small molecular dye; and (3) an acid-etched resin casting method which gives a threedimensional (3D) view of the osteocyte canalicular system. For a better understanding of the importance of osteocytes in bone biology, we have also used dentin matrix protein 1 (DMP1)-null mice as an example to show readers how to apply these methods step by step for analyzing changes in the osteocyte canalicular system when a gene is deleted in osteocytes. Note that our recent data showed that hypophosphatemia, osteomalacia, and rickets in Dmp1-null

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mice and DMP1 human mutations are mainly due to defects in osteocytes (Feng et al. 2006).

2. Materials and Methods 2.1. Fluorochrome labeling in conjunction with DAPI staining 2.1.1. Purpose and principle Fluorochromes such as calcein or Alizarin red are widely used for the quantitative measurement of bone formation and bone remodeling in animal research. Tetracycline, an antibiotic, is mainly used in human studies. These dyes, after administration through intraperitoneal (i.p.) injection, quickly enter the bloodstream and preferentially bind to bone minerals at the surface of newly formed apatite crystals through chelation of calcium ions. This binding is irreversible and can last for a long time in the body. In general, for measurement of the mineral apposition rate, bone formation rate, or bone remodeling dynamics, two-time labeling is sufficient. The time interval between these two time injections varies depending on species and age. For mice younger than 2 months of age, we recommend an interval of 5–7 days; and for mice older than 2 months of age, 7–10 days. For rats, an interval of 15–25 days is ideal. The optimal labeling time for each dye injection is 2–3 days, and animals are sacrificed 2 days after the second dye injection. For visualization of the mineralization front and its relationship with osteocytes, DAPI is added to the slide for staining the nuclei of osteocytes.a 2.1.2. Reagents • • • a

DAPI (Sigma, Catalog No. D8417) Alizarin red (Fisher, Catalog No. 155830050) Calcein (MP Biomedical, Catalog No. 190167)

DAPI will form fluorescent complexes with natural double-stranded DNA.

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2.1.3. Stock solution (10×) •

Prepare the resolving solution: 2% NaHCO3 in 0.9% NaCl solution. NaCl, 0.9 g NaHCO3, 2.0 g Distilled H2O, 100 mL

•

•

Use the resolving solution to make 20 mg/mL of Alizarin red (50 mL of resolving solution + 1.0 g of Alizarin red) and 10 mg/mL of calcein (50 mL of resolving solution + 500 mg of calcein). Adjust the pH to 7.4 and store in dark vials at 4°C.

2.1.4. Working solution (1×) • •

Dilute the stock solution 10× with the resolving solution (see above), filter with Millipore filters, and store in dark vials at 4°C. The solution can be kept at 4°C for several weeks.

2.1.5. Injection procedure •

•

The dosage for i.p. injection of calcein is 5 mg/kg; and for Alizarin red, 20–25 mg/kg (note that there are no apparent side effects in cases of overdosage). Carry out two time injections 5–7 days apart, and sacrifice animals 2 days after the second injection.

2.1.6. Sample collection, plastic embedding, and sectioning •

•

Dissect out the bone samples; wash with PBS solution; and fix in either 70% EtOH, 10% formalin, or 4% paraformaldehyde for 36–48 hours at 4°C. Embed the samples in plastic and section at a thickness of 6–20 µm.

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2.1.7. DAPI staining of nuclei of osteocytes (Fig. 1) • • •

Dissolve 1 mg of DAPI in 1 mL of dH2O. Aliquot into a 50-µL vial and store the stock at −20°C. Dilute 50 µL of stock solution into 12.5 mL of dH2O (4 µg/mL of working stock). Add a small volume of this working solution to the double-stained slides (enough to cover tissues) and leave on for 5–10 minutes.

Fig. 1. Confocal microscopy images of fluorochrome labeling, counterstained with DAPI for visualization of osteocyte nuclei. Calcein (yellowgreen) was i.p. injected into 3-week-old animals (1st), followed by the injection of Alizarin red (red, 2nd) and then calcein again (3rd) with 5 days apart. DAPI was stained to the cell nuclei (blue). Normal bone labeling lines are sharp, and osteocytes are separated from the labeling line (upper panel). In contrast, Dmp1-null osteocytes are buried in diffuse fluorochrome labels (arrows, lower panel), suggesting that osteocytes play an important role in the process of mineralization. Adapted from Feng et al. (2006).

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Aspirate and wash the slides three times with phosphate buffered saline (PBS). Mount and view under confocal microscope using a DAPI filter.b

2.2. Imaging of the osteocyte canalicular system with Procion red 2.2.1. Purpose and principle Procion red is a small molecule. This small molecular dye, when injected into the tail vein, fills in the lacunae and canaliculi of osteocytes but does not enter the mineralized matrix. Thus, the dye can be used to give a visual representation of the organization of the lacunocanalicular system within the skeleton (Knothe-Tate et al. 1998). 2.2.2. Reagent •

Procion red (Sigma, #404365)

2.2.3. Solution •

Prepare the solution: 0.8% Procion red solution. Procion red, 400 mg Distilled H20, 50 mL

•

Filter the solution with Millipore filters.

2.2.4. Procedure • • • • •

b

Inject 0.01 mL of solution/g of body weight (for example, a 30-g mouse needs 0.3 mL of solution). Inject slowly and finish in 1 minute through the tail vein. Sacrifice the mouse 20 minutes after injection. Fix fresh bone in 40% ethanol for 2 hours. Section samples at a thickness of 100–200 µm using a diamond wafer saw (Buehler IsoMet) or a microtome (Leica).

Wavelength for red: EX, 530–550 nm; BA, 590–650 nm. Wavelength for green: EX, 460–500 nm; BA, 510–560 nm. Wavelength for blue: EX, 330–380 nm; BA, 435–485 nm. A better image can be obtained if the photographs are taken separately first and then combined into one.

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Grind the sections to 50–100 µm in thickness. Place a coverslip using a mounting medium that does not cause fluorescence at 550–630 nm. Photograph the samples using a confocal microscope with green light excitation (565 nm) and observe the emission maximum at 610 nm (an example is presented in Fig. 2).

Fig. 2. Visualization of the osteocyte canalicular system via the Procion red injection method. A confocal image (at 565/610 nm, 40×) from crosssections of 3-month-old mouse tibia showed that the wild-type (WT) osteocyte lacunae (filled with Procion red) are highly organized and spaced apart regularly, generally in linear arrays (left panel). In contrast, the osteocyte lacunae in Dmp1 knock-out (KO) mice appear much larger, the distribution of the osteocytes appears less organized, and the canaliculi are less straight and more randomly oriented (right panel). In addition, the nonmineralized matrix (osteoid) in Dmp1 KO mice is filled with Procion red, while the normal mineralized matrix is not stained with Procion red. Adapted from Feng et al. (2006).

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2.3. Resin-casted scanning electron microscopy (SEM) 2.3.1. Purpose and principle SEM uses electrons instead of white light to view a specimen with a magnification of up to 100 000 times. Rather than “seeing through” a sample with a light microscope, SEM only views the surface details in black and white. Because this work is typically carried out by specially trained personnel in EM laboratories, this chapter will only provide a basic discussion of SEM without detailed procedures. However, this fundamental knowledge will guide the reader in sample preparations for SEM. The resin-casted SEM method, which has been used for imaging dentin (Martin et al. 1978), gives a 3D view of the osteocyte lacunocanalicular system. In this technique, a polished surface from a plasticembedded bone is etched with acid to remove minerals, leaving a relief cast of the nonmineralized areas that have been infiltrated by resin, including the osteocyte lacunocanalicular system, osteoid (nonmineral matrix), and bone marrow. 2.3.2. Reagents and equipment • • • • •

37% phosphoric acid (Sigma, Catalog No. 438081) 5.25% sodium hypochlorite (bleach solution) Sputter coater (Denton Vacuum) FEI/Philips XL30 field-emission environmental SEM (Hillsboro, OR, USA) Water-cooled low-speed diamond saw (e.g. Buehler IsoMet™ 1000 Precision Saw; Buehler Ltd, Lake Bluff, IL, USA)

2.3.3. Sample preparation •

•

Dissect out bone samples; wash with PBS solution; and fix in either 70% EtOH, 10% formalin, or 4% paraformaldehyde for 36–48 hours at 4°C. Embed the samples in plastic and trim the tissue blocks using a water-cooled diamond saw.

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Grind the surface of the specimen with a series of silicon carbide papers (600-, 800-, and 1200-grit), and then polish with Metadi Supreme Polycrystalline Diamond Suspensions from 1 micro to 0.25 micro to 0.5 micro (Buehler 40-6630, 40-6629, 40-6627) to get a fine and smooth surface. Immerse in ultrasonic wash for 1 minute.

2.3.4. Acid etching and coating • • • • • •

Etch the samples with 37% phosphoric acid for 5–15 seconds (depending on age). Wash in distilled water for 2 minutes five times. Immerse in 5.25% sodium hypochlorite solution for 5–15 minutes. Wash in distilled water for 2 minutes five times. Air-dry overnight. Coat the surface of the specimen with a very thin layer of goldpalladium using a sputter coater. The reason for the coating is that SEM illuminates sample surfaces with electrons, so they have to be made to conduct electricity.

2.3.5. Scanning process [Fig. 3(a)] • •

•

• •

Place the sample inside the microscope’s vacuum column through an air-tight door. Pump air out of the column. An electron gun (at the top) then emits a beam of high-energy electrons; this beam travels downward through a series of magnetic lenses, condenser lens, to focus the electrons to a very fine spot. Near the bottom, use the set of scanning coils to move the focused beam back and forth across the specimen, row by row. Adjust the objective lens to further focus the electron beam. As the electron beam hits each spot on the sample, secondary electrons are knocked loose from its surface. A detector counts these electrons and sends the signals to an amplifier.

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Fig. 3. SEM scanning process of a resin-casted osteocyte canalicular system. (a) A plastic embedded femur, after acid etching and coating, is placed into a vacuum column and then air is pumped out. A beam of high-energy electrons, emitted from an electron gun, goes through a series of magnetic lenses named condenser lens (upper) and objective lens (lower). These lenses

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The final image, recorded on a computer, is built up from the number of electrons emitted from each spot on the sample. The high-energy electron signal gives a backscattered SEM image, mainly reflecting mineral content in bone samples [white color, Fig. 3(b)]. The low-energy electron signal, detected by a different detector/amplifier set, gives a regular SEM image that provides a detailed 3D image of the osteocyte canalicular system in cortical bone surface [Fig. 3(c)] and alveolar bone surface [Fig. 3(d), with a much higher magnification].

References Dallas SL. Dynamics of bone extracellular matrix assembly and mineralization. J Musculoskelet Neuronal Interact 6:370–371, 2006. Feng JQ, Ward LM, Liu S et al. Loss of DMP1 causes rickets and osteomalacia and identifies a role for osteocytes in mineral metabolism. Nat Genet 38:1310–1315, 2006. Knothe-Tate ML, Niederer P, Knothe U. In vivo tracer transport through the lacunocanalicular system of rat bone in an environment devoid of mechanical loading. Bone 22:107–117, 1998. Martin DM, Hallsworth AS, Buckley T. A method for the study of internal spaces in hard tissue matrices by SEM, with special reference to dentine. J Microsc 112:345–352, 1978.

focus the electrons to a very fine spot. A set of scanning coils moves the beam back and forth across the specimen. Secondary electrons are produced when the beam hits the specimen. The high-energy electrons, called backscattered electrons, will provide information particularly on mineral status [(b), white color] through a detector/amplifier set. The low-energy electrons, called secondary electrons, will provide a 3D image of the osteocyte canalicular system through a different detector/amplifier set [(c) cortical bone; (d) alveolar bone]. Partly adapted from Feng et al. (2006).

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Chapter 10

Tissue Culture of Giant Cell Tumor of Bone Lin Huang and Ming-Hao Zheng

The interplay between neoplastic cells and multinucleate osteoclast-like giant cells found in giant cell tumor has been considered as a model of the cellular interactions that occur during bone resorption in both primary and metastatic neoplasms. This chapter describes the tissue culture techniques of giant cell tumor of bone. The main proliferating cells maintained in culture are the spindle-shaped stromal-like mononuclear cells, which represent the neoplastic component of this tumor. Keywords:

Giant cell tumor; tissue culture; multinucleate giant cells; multinuclear giant cells; stromal-like mononuclear cells; osteoclast; proliferating cell; macrophage.

1. Introduction Giant cell tumor of bone (GCT) is a primary, benign skeleton neoplasm characterized by richly vascularized tissue consisting of spindle-shaped and ovoid mononuclear cells and a great number of large, multinucleate giant cells. The giant cells resemble osteoclasts, as they express calcitonin and vitronectin receptors as well as osteoclastic bone-resorbing enzymes (tartrate-resistant acid phosphatase Corresponding author: Lin Huang. Tel: +852-26322003; fax: +852-26324675; E-mail: [email protected]

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or TRAP) (Athanasou et al. 1985; Goldring et al. 1987). The ovoid mononuclear cells are considered to be macrophages; they do not persist in long-term culture, and express monocyte-macrophage markers such as CD68 antigen (Goldring et al. 1987; Zheng et al. 1998). The spindle-shaped mononuclear cells are the only proliferating cell population in GCT and represent the neoplastic component of the tumor; they phenotypically resemble a connective tissue stromal cell, expressing no macrophage surface antigens, producing collagen types I and III, and possessing parathyroid hormone receptors (Goldring et al. 1987). Thus, GCT is now regarded as a neoplasm of connective tissue stromal cells, which have the ability to recruit and interact with macrophages and multinucleate osteoclastlike giant cells. GCT is the model of a lesion in which the neoplastic nature of the stromal component drives the hematopoietic precursors to undergo fusion, producing aggressive bone resorption and resulting in extensive skeletal destruction (Zheng et al. 2001).

2. Materials • • • • •

Dulbecco’s modified Eagle’s medium (DMEM) L-glutamine Fetal bovine serum (FBS) Penicillin-streptomycin Phosphate buffered saline (PBS)

3. Methods •

• •

Freshly collect tumor specimens and store at 4°C in DMEM containing 100 U/mL of penicillin and 100 µg/mL of streptomycin for no longer than 24 hours. Wash the tumor tissue several times with PBS containing 100 U/mL of penicillin and 100 µg/mL of streptomycin. Chop the tumor tissue finely with a scalpel blade in a sterile dish.

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Collect the tissue fragments and the resultant cell suspension in DMEM containing 100 U/mL of penicillin and 100 µg/mL of streptomycin in a 50-mL Falcon™ tube. Spin down the cells together with the small tissue fragments and resuspend in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL of penicillin, and 100 µg/mL of streptomycin. Transfer the cell suspension together with small pieces of tissues to 25-cm2 flasks for culture at 37°C in 5% CO2 and 95% air.a Change half of the culture media every 3 days with fresh DMEM containing 10% FBS, 2 mM L-glutamine, 100 U/mL of penicillin, and 100 µg/mL of streptomycin. Upon reaching confluence, primary cultures can be subcultured and cultured cells at various passages can be prepared for experimental manipulation.

4. Results Adherent cells in primary culture of GCT include mononuclear cells and multinuclear giant cells. The majority of mononuclear cells are spindleshaped, but a small portion display a rounded epithelioid appearance and are considered to be macrophages [Fig. 1(a)]. Most multinuclear giant cells display a round “fried-egg” appearance with centrally located nuclei and a wide, thin peripheral rim of cytoplasm [Fig. 1(b)]. After 3 to 5 days’ culture, multi-nuclear giant cells are swollen, whereas their nuclei aggregate into small groups. Subsequently, multinuclear giant cells cannot be found, but macrophage-like cells still remain. After the third passage, only spindle-shaped mononuclear cells remain and the cultures continue to proliferate. Figure 2 shows the morphological appearance of these spindle-shaped cells at the eighth passage. a

The small tissue fragments contain many multinuclear giant cells and mononuclear cells that will migrate across the culture flask surface and contribute to the cell population in culture, and therefore should not be removed.

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(a)

(b)

Fig. 1. Morphology of cultured GCT. The primary GCT culture is composed of (a) spindle-shaped stromal cells and round-shaped macrophage-like cells and (b) multinuclear giant cells.

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Fig. 2. GCT culture at the eighth passage. Only spindle-shaped stromal cells remain.

5. Summary Since there are no commercially available representative cell lines for CT up to now, the primary culture of GCT is the only way to extract the cells for in vitro studies. This chapter described the tissue culture techniques of GCT of bone and typical in vitro findings.

Acknowledgments Financial support received for the related work from the Research Grants Council, Hong Kong SAR, China, is herewith acknowledged.

References Athanasou NA, Bliss E, Gatter KC et al. An immunohistological study of giant-cell tumour of bone: evidence for an osteoclast origin of the giant cells. J Pathol 147(3):153–158, 1985.

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Goldring SR, Roelke MS, Petrison KK, Bhan AK. Human giant cell tumors of bone — identification and characterization of cell types. J Clin Invest 79(2):483–491, 1987. Zheng MH, Fan Y, Smith A et al. Gene expression of monocyte chemoattractant protein-1 in giant cell tumors of bone osteoclastoma: possible involvement in CD68+ macrophage-like cell migration. J Cell Biochem 70(1):121–129, 1998. Zheng MH, Robbins P, Xu J et al. The histogenesis of giant cell tumour of bone: a model of interaction between neoplastic cells and osteoclasts. Histol Histopathol 16(1):297–307, 2001.

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Chapter 11

Chondrocyte Mechanotransduction in Three-Dimensional Cell Culture Xu Yang, Riaz Gillani and Qian Chen

Chondrocytes in the growth plate undergo a relatively linear differentiation process. The progression of a chondrocyte from the proliferative stage to the hypertrophic stage is governed by complex interactions with the extracellular matrix within which it resides. A network of peptides, ion channels, and second messengers affects the transcription of certain genes that are ultimately translated into peptides which control cellular activity. Much effort has been invested into replicating this environment under in vitro conditions. It has been found that the three-dimensional (3D) cell culture is a more accurate representation of the in vivo environment in comparison to the traditional monolayer culture. It has also been found that a variety of stimuli may be used to induce the proliferation and differentiation of chondrocytes; one such stimulus is the mechanical stimulation of chondrocytes embedded in a 3D Gelfoam sponge. Chondrocytes are obtained from the chicken sternum. After the cells are cultured and cyclically loaded, mRNA levels of various mechanosensitive genes are quantified by real-time reverse transcription–polymerase chain reaction (RT-PCR). Mechanical stimulation has been shown to upregulate the expression of type X collagen mRNA in early hypertrophic chondrocytes. The entire process, beginning with the obtainment of chondrocytes and ending with the quantification

Corresponding author: Qian Chen. Tel: +1-401-4445676; fax: +1-401-4445872; E-mail: [email protected]

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A Practical Manual for Musculoskeletal Research and interpretation of gene expression, is detailed in the following chapter. Keywords:

Chondrocytes; growth plate; culture; proliferation; hypertrophy; extracellular matrix.

1. Introduction During endochondral bone formation, chondrocytes in the cartilaginous anlagen of long bones progress through a spatially and temporally regulated differentiation process before being replaced by bone (van der Weyden et al. 2006; Kobayashi et al. 2005). This differentiation is the end result of several second messenger systems that act by enhancing or repressing the transcription of genes linked to the hypertrophy of chondrocytes. Interactions with the extracellular matrix (ECM) regulate gene expression, which in turn regulates cell development and function. Second messenger cascades are triggered by a variety of stimuli in vivo, including secreted extracellular ligands that bind to cell surface receptors, and mechanical forces that — through the opening of channels which allow for the flux of ions and proteins — regulate transcriptional activity by the same type of ligand–receptor activity (Curtis 1994). When changes in gene expression are induced by the latter stimulus, mechanotransduction is said to occur. Mechanically induced ECM deformation plays a fundamental role in regulating cellular development and activity (De Croos et al. 2006; Grodzinsky et al. 2000). This in vivo phenomenon lends itself to replication in an in vitro experimental setting; chondrocytes may be isolated and external mechanical stimulation may be applied to simulate the deformation of the ECM. In vitro experiments that make use of such a setup allow researchers to obtain results which have direct implications for gene expression induced or repressed by matrix deformation in vivo. In setting up an in vitro chondrocyte cell culture, a conscious effort must be made to accurately replicate the environment of chondrocytes in the growth plate. Chondrocytes in a monolayer cell culture often dedifferentiate and regress to a fibroblastic phenotype; therefore, the monolayer cell culture does not ideally simulate in vivo growth plate conditions. The three-dimensional (3D) cell culture is a

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much more accurate representation of the in vivo environment. Recent reports have observed distinct cellular behaviors in the 3D culture that are not present in the standard monolayer culture (Cukierman et al. 2001; Schmeichel and Bissell 2003). Agarose gels, collagen sponges, alginate beads, and high-density cultures are some examples of this more accurate 3D cell culture (Freeman et al. 1994; Wu et al. 2001; Guo et al. 1989; Handley and Lowther 1976). Our laboratory has adapted a 3D cell culture system in order to better simulate the chondrocytes’ matrix environment (Wu and Chen 2000). We have developed a protocol in which chondrocytes are cultured in a sponge of 3D collagen networks; these sponges may be loaded mechanically with a computer-controlled Bio-Stretch device. Chondrocytes maintain their phenotypes and form organotypic structures in this 3D culture (Wu and Chen 2000). Primary chondrocytes isolated from the proliferative zone of the epiphyseal growth plate in the culture can proceed through the same differentiation process as they do in the cartilaginous rudiments (Chen et al. 1995). An alternative source of primary growth chondrocytes is the embryonic chick sternum, which is a more convenient and easily available tissue from which to isolate chondrocytes in various stages of differentiation. This chapter describes the isolation of primary hypertrophic chondrocytes from embryonic chick sterna and the setup of the 3D chondrocyte culture in the Bio-Stretch loading system. The quantification of mRNA, which is used as a measure of cell differentiation, using the real-time reverse transcription–polymerase chain reaction (RT-PCR) technique is also described. In addition, some basic techniques used in the study of mechanotransduction are provided in this chapter.

2. Materials • • • •

Eggs: specific pathogen free (SPF) eggs (Charles River Laboratories) Sterilized scissors, tweezers, and scalpel Phosphate buffered saline (PBS) — for storing and washing tissues Digestion solution: 0.1% trypsin (Gibco), 0.3% type II collagenase (Worthington), 0.1% hyaluronidase (Sigma)

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Sponge digestion solution: 0.03% type II collagenase (Worthington) Cell culture medium: Ham’s F-12 medium (Gibco) with 10% fetal bovine serum (FBS) 3D culture scaffold: Gelfoam sponge (Pharmacia & Upjohn Co.) Mechanical loading system: Bio-Stretch (ICCT Technologies) RNeasy Mini Kit (Qiagen) — for total RNA isolation iScript cDNA Synthesis Kit (Bio-Rad) — for cDNA synthesis QuantiTect SYBR Green PCR Kit (Qiagen) — for real-time PCR

3. Methods 3.1. Isolation of embryonic chick sterna 3.1.1. Procedure Chondrocytes isolated from the caudal region of embryonic chick sterna differ markedly from those isolated from the cephalic region. The cephalic region of chick sterna at 17 to 18 days of development contains a largely uniform population of hypertrophic chondrocytes within the ECM, distinguished by the presence of type X collagen (Castagnola et al. 1987). Because the chondrocytes isolated from this region at this developmental stage continue to express even higher levels of type X collagen (Fig. 1) and other differentiation marker genes under in vitro culture conditions, they are classified as early hypertrophic chondrocytes. In our laboratory, early hypertrophic chondrocytes are routinely isolated from the sterna of 17-day-old chick embryos. Resorption of the sterna, with associated vascular invasion, chondrocyte apoptosis, and bone formation, begins at days 19 and 20 of development (Gibson et al. 1997). Therefore, this is not a good time to isolate pure hypertrophic chondrocytes from the cephalic regions of sterna. 3.1.2. Protocol • •

Wipe eggs (17 days of age) with 70% ethanol swabs. Use sterilized scissors to cut a circle in the top hemisphere of the egg to expose the embryo chick; and free the body from the egg

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Fig. 1. The cephalic region of a 17-day-old chicken sternum is outlined. This is the region from which early hypertrophic chondrocytes are extracted.

•

•

by cutting the umbilical cord and the neck, completely dislodging the head and vessel cords. Place the body on its back in a Petri dish on the operation table. Using two sterile tweezers, pull feathers away from the chest area and separate the superficial tissue over the sternum. Isolate the sternum and place it in cool sterile PBS. Repeat the procedure, placing all sterna in PBS.

3.2. Isolation of chondrocytes 3.2.1. Procedure When subcultured in a monolayer, chondrocytes may gradually assume a more fibroblast-like morphology (Grundmann et al. 1980). Morphological changes are followed by a decrease in the expression of chondrocyte matrix components, including type II collagen. Concomitantly, low levels of type I collagen — characteristic of the ECM of fibroblasts in skin, bone, and tendon — are produced. To prevent chondrocytes from losing the differentiated phenotype

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in vitro, many special serum-free media have been developed to support cell proliferation and preserve the differentiation potential (Baudysova and Michl 1983). However, serum-free medium is not necessary in our 3D cell culture. Advantageously, chondrocytes normally maintain their phenotypes in the 3D cell culture.

3.2.2. Protocol •

•

•

•

•

Wash sterna with PBS, and remove the cephalic portion of each sternum to obtain early hypertrophic chondrocytes. The cephalic portion is the lower third portion of the sternum, and is characterized by a wide base. Collect all cephalic regions and mince them into tiny pieces using a scalpel. Mincing should take ~10 minutes, with the end result being pieces <1 mm in diameter. Transfer the minced tissue pieces to a 15-mL conical tube, and add digestion solution for predigestion for 20 minutes in a 37°C water bath. Carefully remove the supernatant, add fresh digestion solution, swirl the tube, and place in a 37°C water bath until fully digested (1–1.5 hours). Pass cell suspension through a 100-micron filter. Centrifuge the solution and obtain cell pellet.

3.3. 3D cell culture and mechanical loading 3.3.1. Procedure We have found that chondrocytes evenly distribute across a sponge after a drop of cell suspension is added. This requires some time, however. We therefore allow the seeded sponge to remain undisturbed overnight so that chondrocytes may attach to it. Mechanical loading is applied after this period. Previous studies using this system have demonstrated that 5% elongation of the sponge provides

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Fig. 2. The Bio-stretch system. (a) The cell chamber rests in front of a solenoid panel that induces cyclic strain using magnetism. (b) A schematic of the cell chamber. The collagen Gelfoam sponge rests in a Petri dish clamped at one end, and is attached to a coated metal bar at the other end. The coated metal bar is parallel to the solenoid panel.

the most ideal mechanical strain for seeded chondrocytes (Yang et al. 2006). 3.3.2. Protocol •

• • • • •

Set up 3D cell culture chambers, following the instructions in the manufacturer’s manual (Fig. 2). Soak sponges with PBS for 1 hour in an incubator until ready for seeding with chondrocytes. Make the sponges relatively dry by using a suction pipette to remove extra fluid. The sponges should still be saturated. Adjust the concentration of cell suspension to 1 million/100 µL, and add 100 µL of cell suspension to the sponge. After seeding the sponges, place them in an incubator at 37°C for 1 hour to let them attach to the sponge. Add 4 mL of 10% FBS in F-12 plating medium to each sponge, and place the cell culture chambers in the incubator overnight. Aspirate the medium and add new medium on the next day. Begin stretching using the Bio-Stretch system.

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3.4. Isolation of total RNA from cells in sponge and real-time RT-PCR 3.4.1. Procedure Mechanically stress-induced matrix deformation plays a fundamental role in regulating cellular activities. The Bio-Stretch system applies mechanical stimulation to the chondrocytes through cyclic deformation of the sponge. We quantify the mRNA level of type X collagen, a frequently measured mechanoresponsive gene, to determine the impact of mechanical loading (Figs. 3 and 4). We were curious to see if the digestion of the sponge for 20 minutes at 37°C prior to the mRNA extraction would have an impact on gene expression. We therefore compared this method, known as the collagenase method, to the collagenase-free tissue isolation method. The collagenase method is described in the protocol above. The collagenase-free tissue isolation method comprises taking the sponge and attached cells as a small piece of tissue, and extracting total mRNA using the RNeasy Fibrous Tissue Mini Kit. Both RNA isolation methods are followed by the same procedure of real-time quantitative

Fig. 3. Upregulation of type X collagen mRNA level in early hypertrophic chondrocytes in response to cyclic loading.

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Fig. 4. Continuing upregulation of type X collagen mRNA in early hypertrophic chondrocytes cultured in the 3D Gelfoam system under nonload conditions.

RT-PCR. We did not find any significant difference in mRNA levels between these two methods. However, the collagenase-free tissue isolation method yielded a relatively low concentration of total RNA. Real-time RT-PCR is used to quantify the impact of mechanical stimulation on gene expression. Real-time RT-PCR is preferred over RT-PCR because it may be used to detect more minute changes in mRNA levels. One of several techniques may be used to quantify protein expression after obtaining the cell pellets, including flow cytometry, Western blot, and immunocytochemistry. 3.4.2. Protocol •

• •

Cut the sponges into small pieces so that digestion may proceed more quickly. Place the sponges into separate 15-mL tubes containing sponge digestion solution. Allow the tubes to remain in the water bath for up to 20 minutes so that the collagen sponges may be completely digested. Centrifuge the tubes at 1800 rpm for 5 minutes to create a cell pellet. Aspirate the fluid from the tubes without removing the pellet.

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Use 1 µg of total RNA for each RT reaction in a reaction buffer containing 1 µL of oligo(dT) and 1 µL of 10 mM dNTP Mix. Perform real-time quantitative PCR amplification using the QuantiTect SYBR Green PCR Kit with DNA Engine Opticon 2 Continuous Fluorescence Detection System. Retrieve gene-specific primers from GeneBank. Normalize all of the target genes’ mRNA levels to the housekeeping gene 18S RNA level, and calculate mRNA values as previously described (Wu and Chen 2000). Amplify the 18S RNA at the same time and use it as an internal control. Calculate the cycle threshold (Ct) values for 18S RNA and for samples using computer software. Calculate relative transcript levels as x = 2−∆∆Ct, in which ∆∆Ct = ∆E − ∆C, ∆E = Ctexp − Ct18s, and ∆C = Ctctl − Ct18s. Present the data as mean ± standard error of mean, and analyze using two-way analysis of variance (ANOVA); p < 0.05 when compared with wild-type (WT) controls.

Acknowledgments This study was supported by grants AG14399 and AG17021 from NIH as well as a grant from the Arthritis Foundation to Q. Chen.

References Baudysova M, Michl J. Proliferation and differentiation of chondrocytes in cell culture. Physiol Bohemoslov 32:346–351, 1983. Castagnola P, Torella G, Cancedda R. Type X collagen synthesis by cultured chondrocytes derived from the permanent cartilaginous region of chick embryo sternum. Dev Biol 123:332–337, 1987. Chen Q, Johnson DM, Haudenschild DR, Goetinck PF. Progression and recapitulation of the chondrocyte differentiation program: cartilage matrix protein is a marker for cartilage maturation. Dev Biol 172: 293–306, 1995. Cukierman E, Pankov R, Stevens DR, Yamada KM. Taking cell–matrix adhesions to the third dimension. Science 294:1708–1712, 2001. Curtis ASG. Biomechanics and Cells. Cambridge University Press, Cambridge, UK, 1994.

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De Croos JNA, Dhaliwal SS, Grynpas MD et al. Cyclic compressive mechanical stimulation induces sequential catabolic and anabolic gene changes in chondrocytes resulting in increased extracellular matrix accumulation. Matrix Biol 25:323–331, 2006. Freeman PM, Natarajan RN, Kimura JH, Andriacchi TP. Chondrocyte cells respond mechanically to compressive loads. J Orthop Res 12:311–320, 1994. Gibson G, Lin DL, Roque M. Apoptosis of terminally differentiated chondrocytes in culture. Exp Cell Res 233:372–382, 1997. Grodzinsky AJ, Levenston ME, Jin M, Frank EH. Cartilage tissue remodeling in response to mechanical forces. Annu Rev Biomed Eng 2:691–713, 2000. Grundmann K, Zimmermann B, Barrach HJ, Merker HJ. Behaviour of epiphyseal mouse chondrocyte populations in monolayer culture. Morphological and immunohistochemical studies. Virchows Arch A Pathol Anat Histol 389:167–187, 1980. Guo JF, Jourdian GW, MacCallum DK. Culture and growth characteristics of chondrocytes encapsulated in alginate beads. Connect Tissue Res 19:277–297, 1989. Handley CJ, Lowther DA. Inhibition of proteoglycan biosynthesis by hyaluronic acid in chondrocytes in cell culture. Biochim Biophys Acta 444:69–74, 1976. Kobayashi T, Lyons KM, McMahon AP, Kronenberg HM. BMP signaling stimulates cellular differentiation at multiple steps during cartilage development. Proc Natl Acad Sci USA 102:18023–18027, 2005. Schmeichel KL, Bissell MJ. Modeling tissue-specific signaling and organ function in three dimensions. J Cell Sci 116:2377–2388, 2003. van der Weyden L, Wei L, Luo J et al. Functional knockout of the matrilin3 gene causes premature chondrocyte maturation to hypertrophy and increases bone mineral density and osteoarthritis. Am J Pathol 169: 515–527, 2006. Wu Q, Chen Q. Mechanoregulation of chondrocyte proliferation, maturation and hypertrophy: ion-channel dependent transduction of matrix deformation signals. Exp Cell Res 256:383–391, 2000. Wu Q, Zhang Y, Chen Q. Indian hedgehog is an essential component of mechanotransduction complex to stimulate chondrocyte proliferation. J Biol Chem 276:35290–35296, 2001. Yang X, Vezeridis PS, Nicholas B et al. Differential expression of type X collagen in a mechanically active 3-D chondrocyte culture system: a quantitative study. J Orthop Surg Res 1:15, 2006.

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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

Cartilage damage is irreversible due to its properties of avascularity and lack of undifferentiated cells. Studying the biology of chondrocytes and cartilage is therefore critical to understand the underlying mechanism and to explore the potential repair approaches. It has been reported that three-dimensional (3D) chondrocyte culture behaves very differently from two-dimensional (2D) monolayer culture. Therefore, a native 3D culture of chondrocytes is valuable for such a research purpose, and may also be an option for repairing cartilage defects. This chapter describes a detailed protocol for high-yield chondrocyte isolation and the modified pellet culture technique (using a 3D chondrocyte culture), which can synthesize a bioengineered tissue up to 8 mm in diameter. The authors aim at providing a platform for researchers to study chondrocyte behavior in a 3D environment and to explore the application of scaffold-free tissue for transplantation into a large cartilage defect model. Keywords:

Chondrocytes; cartilage; pellet culture; 3D culture; centrifugation.

Corresponding author: Wing-Hoi Cheung. Tel: +852-26323312; fax: +852-26324618; E-mail: [email protected]

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1. Introduction Cartilage is a durable musculoskeletal tissue to sustain loading in joints. Due to its avascularity and shortage of undifferentiated cells, cartilage is difficult to heal if injured (Buckwalter et al. 2000). Therefore, isolating chondrocytes to study their biology or bioengineering three-dimensional (3D) cartilaginous tissues is important in cartilage research. One of the components of cartilage is chondrocyte, which is critical for cartilage research. Since chondrocytes occupy only less than 10% of tissue volume while over 90% are constituted by the extracellular matrix, it is technically difficult to obtain a high yield of viable chondrocytes from the matrix. Good control of the enzymatic digestion of the matrix is crucial for a high yield of released chondrocytes. The chondrocytes can be kept in monolayer for biochemical investigation or in 3D culture as cartilaginous tissues, which mimic the in vivo behavior better than the chondrocytes in monolayer culture (Kato et al. 1988; Niethard et al. 2007). Pellet culture is a 3D culture technique using a defined cell number, whereby the cells are centrifuged into a pellet and cultured in a floating environment. The cells are at ultrahigh density. The advantages of pellet culture are that they (1) resemble normal in vivo morphogenesis, (2) allow cell–cell interactions, (3) detect the synthesis and degradation of the matrix temporally and concurrently, and (4) enable strict control of culture conditions. Therefore, the pellet culture technique is a better model with the 3D matrix structure than the monolayer culture as a simulated cartilaginous tissue. This technique has been reported to apply to chondrocytes (Ballock and Reddi 1994; Kato et al. 1988; Stewart et al. 2000), retinal neuroepithelial cells (Watanabe et al. 1997), bone-marrow–derived mesenchymal progenitor cells (Huang et al. 2004), and melanoma cells (Fletcher et al. 1999), among others. The first trial can be traced back to Kato et al. (1988), who used this technique to investigate terminal differentiation and calcification in rabbit chondrocyte culture.

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The original protocol used a 15-mL centrifuge tube to produce a chondrocyte pellet of 3 mm in diameter. The present study has made some modifications to the technique to bioengineer a scaffold-free tissue of 8 mm in diameter, which provides a good platform for investigating the behavior of chondrocytes in a 3D environment and for animal research on transplantation in a large cartilage defect model.

2. Materials •

• •

•

•

•

•

Dulbecco’s modified Eagle’s medium (DMEM) (Sigma, St. Louis, MO, USA) — for providing nutrients to the tissues during serial enzymatic digestions Phosphate buffered saline (PBS) — for washing purpose throughout the entire procedure Trypsin: trypsin powder (Sigma) dissolved in DMEM at a final concentration of 0.1% (w/v) and filtered through a 0.22-µm membrane (Millipore, Billerica, MA, USA) — for breaking down the core and link proteins of cartilage matrix into smaller peptides or amino acids Hyaluronidase: hyaluronidase crystal (Sigma) dissolved in DMEM at a final concentration of 0.1% (w/v) and filtered through a 0.22-µm membrane (Millipore) — for digesting the proteoglycan aggrecan of cartilage matrix Collagenase: collagenase crystals (Sigma) dissolved in DMEM at a final concentration of 0.075% (w/v) and filtered through a 0.22-µm membrane (Millipore) — for digesting collagen fibers of cartilage matrix to release chondrocytes Tissue culture medium: DMEM supplemented with 10% fetal bovine serum (FBS; Gibco Laboratories, Grand Island, NY, USA) and 0.8% penicillin-streptomycin-neomycin antibiotics (PSN; Gibco Laboratories) — for routine monolayer culturing of released chondrocytes Culture containers: 50-mL conical polypropylene tube (Falcon, Lincoln Park, NJ, USA) — for modified pellet culture

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3. Methods (Cheung 1999; Cheung et al. 2001a; Cheung et al. 2001b; Lee et al. 1996; Lee et al. 2001) Day 1

3.1. Isolation of cartilage from rabbit rib cage • • •

Anesthetize a 6-week-old New Zealand white rabbit with ketamine and xylazine. Expose the rib cage and dissect aseptically. Euthanize the rabbit by an overdose of pentobarbital and dispose the carcass properly.

3.2. Isolation of chondrocytes from cartilage •

• •

a

In culture room, remove the soft tissues of the rib cage to isolate the rib cartilage. Take out the resting zone of the cartilage near the osteochondral junctions of all the ribs and temporarily store in plain DMEM. After weighing, cut the cartilage into 0.1-mm3 small pieces. Digest the minced cartilage tissues with serial enzymes with 0.1% trypsin for 30 minutes,a 0.1% hyaluronidase for 1 hour, and 0.075% collagenase overnightb on a shaking machine. All enzymatic treatments are performed in a 37°C incubator. Ten milliliters of each enzyme solution are used for every gram of cartilage tissues.

Trypsin digestion is very critical in the isolation of chondrocytes from the cartilage matrix. After trypsin digestion, stop the trypsin reaction immediately using FBS-containing DMEM. Inadequate trypsin treatment will lead to a low yield of chondrocytes, while overtime trypsin digestion will severely affect the viability of released chondrocytes. Careful tuning of the trypsin concentration for enzymatic digestion is required for the first few trials, particularly if using newly purchased trypsin powder. The recommended dosage in this protocol is 0.1% trypsin. b Collagenase digestion is also important in the isolation of chondrocytes. The recommended dosage is 0.075%, but this depends on the enzyme activity of the purchased collagenase. Pay attention to the international unit (IU) on the enzyme bottle label. Carefully tune the collagenase concentration during the first few trials. Due to the overnight treatment, collagenase concentration is not recommended to be very high.

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Day 2 •

•

After centrifugation (300 × g for 5 minutes) of the digested mixture, filter the released chondrocytes through a glass wool filter and wash twice with plain DMEM to remove the digested matrix debris.c Resuspend the released cells in tissue culture medium (with 10% FBS and 0.8% PSN) as listed above (Fig. 1).d

Fig. 1. Morphology of the released chondrocytes in monolayer culture under a light microscope (100×).

c

If the released chondrocytes are used for monolayer culture, the cells should be transferred to a new flask once the cells reach 80% confluence; otherwise, the chondrocyte culture may become multilayered and difficult to trypsinize. In some cases, a sheet of chondrocyte culture may form when the chondrocytes secrete the matrix to wrap themselves, which leads to very difficult handling. Also, in the late cell passages (over four passages), confluent chondrocytes may clump together and become calcified (look shiny under microscope) on some occasions. d Unlike the monolayer culture, the chondrocyte pellet culture requires ascorbate in tissue culture medium to maintain the 3D structure of the chondrocyte pellet. The recommended dose is 50 µg/mL. Without adequate ascorbate supplement, the cell pellet structure cannot be maintained and will break down in a couple of days.

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3.3. Modified pellet culture e • • • •

• •

Count the number of viable released chondrocytes by the trypan blue exclusion method using a hematocytometer.f Suspend 2 × 106 viable released chondrocytes in 5 mL of tissue culture medium in a 50-mL conical polypropylene tube. Centrifuge the cell suspension at 350 × g at room temperature for 5 minutes to form a pellet (Fig. 2). Culture the cell pellet in a 37°C, 5% CO2 and 100% humidity incubator within the same tube, with the cap loosely closed and standing vertically. Change the tissue culture medium every 2 days and centrifuge the pellets after every medium change.g The chondrocyte pellet formed using the modified technique is 8 mm in diameter after 14 days’ culture (Fig. 3). The histology of the chondrocyte pellet demonstrated a typical physeal zonal pattern with the resting zone at the bottom, the proliferative zone in the middle, and the hypertrophic zone at the top (Fig. 4).

e 2 × 106 viable chondrocytes are used in the modified chondrocyte pellet culture to bioengineer enlarged tissues; this is fourfold of the cell number for the conventional pellet culture (Lee et al. 2003; Wong et al. 2003). The cell density has been optimized, since too high or too low cell density may cause unsuccessful formation of pellet. Meanwhile, 5 mL of tissue culture medium is used to keep the pellet in order to provide enough nutrients to the high-density chondrocytes. The tissue culture medium should be monitored and renewed when the medium color becomes yellowish. f The number of viable released chondrocytes is not very critical for monolayer culture; the viable ones will attach to the bottom of cultureware within a few hours while the dead cells will float in the medium, and they can be distinguished after the medium change. However, the viable cell number is very critical if chondrocytes are used for pellet culture, in which the percentage of viable chondrocytes is strictly required to be very high (>90% recommended); otherwise, the 3D structure of cell pellet cannot be formed and chondrocytes will disperse in the medium on the second day. Even if the number of viable chondrocytes reaches 2 × 106, the formation of cell pellet will still be unsuccessful if the percentage of dead cells is high. g One of the modifications in the modified pellet culture technique is repeated centrifugations at every medium change. This procedure can provide mechanical stimulation to the chondrocytes in the cell pellet to proliferate and synthesize the matrix in order to strengthen the structure of the bioengineered tissue. This method has high potential to further enhance the size of the chondrocyte pellet in the future. Without the repeated centrifugations, the enlarged chondrocyte pellet may not form well, with some holes appearing in the center of pellets in some cases.

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Fig. 2. Chondrocyte pellet formed in a 50-mL conical polypropylene tube (Falcon, Lincoln Park, NJ, USA) using the modified pellet culture technique.

Fig. 3.

The culture pellet grew to a diameter of 8 mm on day 14.

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Fig. 4. Cross-sectional histology of the day 14 chondrocyte pellet showed a typical physeal zonal pattern, with high proteoglycan content as reflected by high red intensity (Safranin O staining; 200×).

4. Discussion The modified pellet culture technique is different from the conventional protocol. The modifications include using suitable polypropylene cultureware, higher chondrocyte density, and multiple centrifugations during medium changes. Compared with the conventional pellet culture (Ballock and Reddi 1994; Kato et al. 1988; Stewart et al. 2000), this modified protocol can synthesize a larger chondrocyte pellet of 8 mm in diameter (an approximately sevenfold increase in area) with all of the physeal-like characteristics (physeal zonal pattern, high proliferative potential, high proteoglycan content, and active alkaline phosphatase activity) similar to our previous study (Lee et al. 2003; Wong et al. 2003). Furthermore, the tissue is scaffoldfree, which may be advantageous for transplantation experiments that

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do not need to consider the in vivo degradation of scaffolds, as compared with other studies on the tissue engineering of articular cartilage using polyglycolic acid scaffolds and bioreactor (Vunjak-Novakovic et al. 1999). In this protocol, multiple centrifugations are critical for maintaining the high proliferation rate of the bioengineered growth plates. Centrifugation, a kind of mechanical stimulation, is crucial for the early development of bioengineered tissue. This is consistent with other studies which showed that the cyclic mechanical force is a potent anabolic stimulus for chondral growth in the growth plate (Wang and Mao 2002a; Wang and Mao 2002b), and that the rate of chondrocytic proliferation is also modulated by mechanical loading (Stokes et al. 2002). In addition, Montufar-Solis et al. (1996) demonstrated that increased loading enhances the differentiation of chondrocytes. A larger cultureware was used in this study to culture bioengineered tissue instead of the 15-mL polypropylene tube used in the conventional protocol (Lee et al. 2003; Wong et al. 2003) — another important factor for large tissue cultures, as more space is available for the development of tissues. These findings imply that, with suitable culturewares and multiple centrifugations, more breakthroughs in the size of bioengineered tissue may be possible in the future.

5. Summary This is the first report to show the successful synthesis of an enlarged chondrocyte pellet with an 8-mm diameter using the modified pellet culture technique. The culture conditions have been optimized. The technique and the chondrocyte pellet are expected to have wide applications in cartilage research in the future.

Acknowledgments This study was supported by the AO Research Fund, Switzerland (reference no. : 2001-L16); and the RGC Earmarked Grant, Research Grant Council of Hong Kong (CUHK 4510/05).

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References Ballock RT, Reddi AH. Thyroxine is the serum factor that regulates morphogenesis of columnar cartilage from isolated chondrocytes in chemically defined medium. J Cell Biol 126(5):1311–1318, 1994. Buckwalter JA, Einhorn TA, Simon SR (eds.). Orthopaedic Basic Science: Biology and Biomechanics of the Musculoskeletal System, 2nd ed. American Academy of Orthopaedic Surgeons, Rosemont, IL, 2000. Cheung WH. A study on the mechanism of retardation to osteosarcoma growth and spread by cartilaginous tissues. PhD thesis, The Chinese University of Hong Kong, Hong Kong, 1999. Cheung WH, Lee KM, Fung KP, Leung KS. Growth plate chondrocytes inhibit neo-angiogenesis — a possible mechanism for tumor control. Cancer Lett 163(1):25–32, 2001a. Cheung WH, Lee KM, Fung KP, Leung KS. TGF-β1 is the factor secreted by proliferative chondrocytes to inhibit neo-angiogenesis. J Cell Biochem S36:79–88, 2001b. Fletcher JN, Mew D, DesCoteaux JG. Dissemination of melanoma cells within electrocautery plume. Am J Surg 178(1):57–59, 1999. Huang CY, Reuben PM, D’Ippolito G et al. Chondrogenesis of human bone marrow-derived mesenchymal stem cells in agarose culture. Anat Rec A Discov Mol Cell Evol Biol 278(1):428–436, 2004. Kato Y, Iwamoto M, Koike T et al. Terminal differentiation and calcification in rabbit chondrocyte cultures grown in centrifuge tubes: regulation by transforming growth factor beta and serum factors. Proc Natl Acad Sci USA 85(24):9552–9556, 1988. Lee KM, Cheng ASL, Cheung WH et al. Bioengineering and characterization of physeal transplant with physeal reconstruction potential. Tissue Eng 9(4):703–711, 2003. Lee KM, Fung KP, Leung PC, Leung KS. Identification and characterization of various differentiative growth plate chondrocytes from porcine by countercurrent centrifugal elutriation. J Cell Biochem 60(4):508–520, 1996. Lee KM, Ye GL, Yung WH et al. In situ model for studying potassium currents in various growth plate chondrocyte subpopulations. Life Sci 69(6):721–728, 2001. Montufar-Solis D, Duke PJ, D’Aunno D. In vivo and in vitro studies of cartilage differentiation in altered gravities. Adv Space Res 17:193–199, 1996.

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Niethard M, Schneider U, Wallich R. Differential behaviour of human adult arthrotic chondrocytes under 2D- and 3D-cultivation set-ups in a collagen I gel. Z Orthop Ihre Grenzgeb 145(1):102–107, 2007. Stewart MC, Saunders KM, Burton-Wurster N, Macleod JN. Phenotypic stability of articular chondrocytes in vitro: the effects of culture models, bone morphogenetic protein 2, and serum supplementation. J Bone Miner Res 15(1):166–174, 2000. Stokes IA, Mente PL, Iatridis JC et al. Enlargement of growth plate chondrocytes modulated by sustained mechanical loading. J Bone Joint Surg 84A:1842–1848, 2002. Vunjak-Novakovic G, Martin I, Obradovic B et al. Bioreactor cultivation conditions modulate the composition and mechanical properties of tissue-engineered cartilage. J Orthop Res 17:130–138, 1999. Wang X, Mao JJ. Accelerated chondrogenesis of the rabbit cranial base growth plate by oscillatory mechanical stimuli. J Bone Miner Res 17:1843–1850, 2002a. Wang X, Mao JJ. Chondrocyte proliferation of the cranial base cartilage upon in vivo mechanical stresses. J Dent Res 81:701–705, 2002b. Watanabe T, Voyvodic JT, Chan-Ling T et al. Differentiation and morphogenesis in pellet cultures of developing rat retinal cells. J Comp Neurol 377(3):341–350, 1997. Wong MW, Qin L, Lee KM et al. Healing of bone–tendon junction in a bone trough: a goat partial patellectomy model. Clin Orthop Relat Res 413:291–302, 2003.

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Part II Histology and Histomorphometry

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Chapter 13

Tissue Preparations Yong-Bo Lu, Yi-Xia Xie and Jian-Quan Feng

Perhaps the most difficult task in bone histology work is the preparation of a good specimen. It is impossible to obtain a good image of bone or cartilage without careful preparation and processing of specimens. This chapter will describe very basic skills in the preparation of specimens. These techniques include the paraffin method, plastic method, and frozen method. Keywords:

Bone; cartilage; fixatives; decalcification.

1. Introduction Bone and cartilage are unique in their extracellular matrices. For example, the bony matrix is calcified, forming mainly calcium phosphate, whereas the cartilage matrix contains mostly glycosaminoglycans and proteoglycans. There are two classic methods for the visualization of cell morphologies: paraffin and plastic. The former method requires decalcification, but has broad applications such as in varieties of dye stainings (for matrices, nuclei, and cytoplasm), in situ hybridization (for mRNA detection), and immunohistochemistry (for protein detection). The latter method preserves tissue structure much better, but applications are limited, mainly for electron microscopy and mineral labeling. In addition, the frozen method is a useful tool Corresponding author: Jian-Quan Feng. Tel: +1-214-3707235; E-mail: [email protected]

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for visualization of reporter gene expression such as β-galactosidase (lacZ ). Good preparations of tissue samples, such as fixation and decalcification, are critical for the preservation of a real cellular morphology; otherwise, it would be very difficult to define whether variations or differences observed are caused by real pathological changes in tissues or artifacts from poorly prepared tissues. This chapter will provide a useful guide to these approaches.

2. Paraffin Method 2.1. Fixation 2.1.1. Purpose and principle Fixation is a process in which the fixative penetrates and fixes the tissue in order to preserve the constituents of the tissue as they were in the living state. Fixatives serve four functions in this process: (1) hardening tissues; (2) blocking enzymatic autolysis (degradation of proteins into amino acids) of the tissue; (3) increasing affinity to staining; and (4) inactivating endogenous RNases to preserve mRNAs, a critical step in the detection of mRNA level by in situ hybridization. There are two forms of fixatives: aldehydes, including formaldehyde and glutaraldehyde, and alcohols (e.g. ethanol). Aldehydes fix tissues by cross-linkages formed between protein molecules, particularly between the basic lysyl residues. Alcohol (ethanol) is a protein denaturant, acting by coagulation and precipitation of the proteins in the tissue. Different fixatives have different properties and serve different functions, depending on the needs. All of these are summarized in Table 1. In addition, ethanol and formalin are routinely used as preservatives that protect the specimen from decay or deterioration and that give it a normal appearance and protection from mechanical disturbance when handled. 2.1.2. Formaldehydes Commercially available forms of formaldehyde include paraformaldehyde, 37% formaldehyde solution, and 10% formalin (further diluted

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Property and application of fixatives. Formaldehyde

Fixatives

Formalin

Glutaraldehyde

Paraformaldehyde

Ethanol

Application For general histology

For TEM when For general For noncombined with histology decalcified paraformaldehyde and TEM tissues Pros Fast fixation Fast penetration Fast fixation Fast fixation Cons Slow Slow fixation Slow penetration; Small sample penetration fresh solution volume required required Preservative Yes Yes TEM: Transmission electron microscopy. Note: (a) Ethanol fixation prevents the removal of mineral from bone and preserves the fluorochrome labels; therefore, it is preferable for the fixation of nondecalcified bone for mineral analysis. (b) All aldehyde solutions are toxic and carcinogenic.

formaldehyde). Paraformaldehyde cannot be directly used as a fixative, as it is insoluble. When heated to ~60°C, paraformaldehyde depolymerizes into an active form and dissolves in water solution; on the other hand, when exposed to light, paraformaldehyde polymerizes spontaneously in water solution and gradually loses its efficiency of fixation. Therefore, paraformaldehyde work solution should be freshly prepared. Note that formaldehyde solution (37%) contains 10%–15% methanol as a preservative to slow down the polymerization process. 2.1.3. 4% paraformaldehyde (PFA) (1) Equipment •

Stirring hot plate or 60°C water bath

(2) Reagents • •

Paraformaldehyde (PFA; Sigma P6148) Phosphate buffered saline (PBS) (1× powder concentrate white granular powder, 50 L; Fisher BP661-50)

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(3) Procedure •

• • •

Dissolve paraformaldehyde in Ca++/Mg++-free PBS (pH 7.4) with stirring at ~60°C by placing the container on a stirring hot plate. Alternatively, PFA can be dissolved by placing the container in a 60°C water bath with frequent swirling. Cool to 4°C. Store it at 4°C in the dark, and use it up in 1 week. Place the bone samples in 4% paraformaldehyde in a volume at least 10 times of the sample volume. Fix bones at 4°C for 24–48 hours, depending on the size of the specimen, followed by either decalcification (see Sec. 2.2) or long-term storage, either in 0.5% paraformaldehyde in PBS at 4°C or dehydrated and stored in 100% ethanol at −20°C.a

2.1.4. 10% formalin (1) Reagents •

10% formalin (Fisher 23-245-685; ready for direct usage): 10% (v/v) of 36.5% formaldehyde in a buffer containing 0.4% sodium phosphate monobasic and 0.65% sodium phosphate dibasic in deionized water; or 37% formaldehyde solution (Fisher BP531) diluted into 10% formalin in PBS

(2) Procedure • •

a

Place the bone samples in 10% formalin in a volume at least 10 times of the sample volume. Fixation takes place for 1–2 days at −4°C, and can be facilitated by placing the container on an orbital shaker.

For in situ hybridization, PBS should be RNase-free and stored in baked glass or disposable plastic containers.

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2.1.5. Glutaraldehyde (for nondecalcified transmission electron microscopy) (1) Reagents • •

25% glutaraldehyde (Ted Pella, Inc., Cat. No. 18426) Paraformaldehyde (PFA; Sigma P6148)

(2) Fixative solution • • • •

2% freshly prepared paraformaldehyde 2% glutaraldehyde 0.1 M sodium cacodylate, pH 7.4 1% osmium tetroxide

(3) Procedure • •

• • •

Dissect the bone of interest. The sample should be ~1–3 mm3 and as clean as possible. After dissection, the sample undergoes immediate primary fixation (solution of 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M sodium cacodylate at pH 7.4) for 6–8 hours. Rinse three times (20 minutes each time) in 0.1 M cacodylate at pH 7.4. The sample undergoes secondary fixation (solution of 1% osmium tetroxide in 0.1 M cacodylate at pH 7.4) for 1 hour. Store samples in 0.1 M sodium cacodylate buffer at pH 7.4, 4°C until processing.

2.1.6. Alcohol (ethanol) (1) Reagent •

b

70% ethanolb

All aldehyde fixatives should be prepared in buffers, e.g. PBS or cacodylate, as samples will become decalcified when too acidic or will clear when too basic.

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(2) Procedure • • •

•

Place the bone samples (tissue) in 70% ethanol (fixative) in a volume at least 10 times of the sample volume. Facilitate the fixation by placing the container on an orbital shaker. Fix the sample in ethanol for hours to 2 days, depending on the size of the sample (for complete penetration and fixation). It can be kept in ethanol indefinitely. After fixation, wash the fixed bone samples in PBS or tap water to remove the fixative solution in the tissue.c

2.2. Decalcification There are two ways to decalcify bone: chelating agent (e.g. EDTA) decalcification and acid decalcification.

2.2.1. EDTA decalcification (1) Purpose and principle •

Ethylenediaminetetraacetic acid (EDTA) is a chelating agent that reacts with calcium ions and forms a stable complex. This method is slower, but shows a minimum of artifacts and less risk of overdecalcification.

(2) Instrument •

Stirrer (e.g. Corning stirrer/hot plate)

(3) Reagents • • c

Ammonium hydroxide, concentrated (JT Baker 9721-2) EDTA (Fisher AC11843-0010)

Shrinkage of proteins near the nucleus and between cell boundaries is unavoidable even with right fixation.

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(4) Procedure •

Prepare 10% EDTA, pH 7.2:











•

Add 280 mL of concentrated ammonium hydroxide into 3 L of distilled water. Add 400 g of EDTA and stir until it is completely dissolved.d Adjust pH to 7.2 by adding more concentrated ammonium hydroxide. Add more distilled water to make the final volume 4 L. If too much ammonium hydroxide is added, add enough EDTA to get the pH down to 7.2.

Place the fixed bone in EDTA bath (at least 20 times the volume of the bone samples). The decalcification times are as follows: Calvaria, 7 days Limbs, 14 days

• •

Change the EDTA bath weekly until decalcification is completed. Wash the decalcified bone samples briefly in water to remove any residual EDTA solution that could interfere with subsequent processing and staining.e

2.2.2. Acid decalcification (1) Purpose and principle The lower pH solution will dissolve the hydroxyapatite and move calcium from the bone to the solution. Stronger

d

EDTA in free acid form is insoluble in water and must be dissolved in a diluted basic solution, whereas sodium EDTA can be dissolved in water but is more expensive. e Adequate removal of the EDTA occurs during the dehydration/clearing process.

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acids (e.g. hydrochloric acid, taking less time) or weaker acids (e.g. formic acid and picric acid) are effective in removing calcium from the bone at a much faster speed than EDTA; however, acid decalcification leads to a greater risk of overdecalcification than EDTA decalcification, removing proteins from the remaining cells and organic matrix and resulting in a poorly stained section. Therefore, we recommend a combination of chelator and acid solution using products such as Cal-Ex* Decalcifier from Fisher (described below). This solution with chelating agent in dilute hydrochloric acid completely removes and binds all calcium in the specimen with minimal distortion of tissue or interference with subsequent staining. (2) Reagent •

Fisher Cal-Ex* Decalcifier (Fisher, Cat. No. CS510)

(3) Procedure • • • •

f

Use Cal-Ex solution at a ratio of 50 parts to 1 part bone sample. Place fixed bone samples in Cal-Ex solution overnight.f Wash free of Cal-Ex in running tap water for 3–4 hours. Check the decalcification.g For larger bone samples, it may be required to cut the bone into the desired sizes and place in fresh Cal-Ex solution for another 12–15 hours.

Agitation will promote the diffusion of ions in solution, but it will not increase the diffusion of the calcium ions through the bone to the surrounding decalcifant. Therefore, it is not necessary to have the decalcifant stirred. Agitation of the decalcifant two to three times daily is optimal. We normally place the decalcification container on an orbital shaker. g Complete decalcification can be checked simply by bending the bone samples to determine whether they are soft enough to section. A more accurate approach is to take a radiograph to determine whether the calcium has been removed from a bone sample; it is very convenient when a digital, high-resolution X-ray is available.

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2.3. Dehydration/Clearing/Infiltration 2.3.1. Purpose and principle Because fixatives and decalcifying agents are aqueous solutions, water must be removed before tissues are infiltrated with paraffin wax or plastic embedding agents. This is accomplished by passing the bone block through a series of progressively higher concentrations of ethanol, which is hydrophilic and is miscible with water and with many organic solvents, until dehydration is complete. To facilitate the infiltration of wax, the dehydrated tissue needs to be “cleared”, that is, made translucent by xylene which is miscible in both paraffin and ethanol. Lastly, the cleared sample is now ready for infiltration, a procedure whereby the xylene is replaced by paraffin. 2.3.2. Equipment • • • •

Automated tissue processor (e.g. Shandon Hypercenter XP) Plastic tissue cassettes (Fisher, Cat. No. 15182701K, 15182706) Plastic or glass beaker Small glass bottles

2.3.3. Reagents • • •

Ethanol Xylene (Fisher, Cat. No. X5-4) Tissue embedding medium (Fisherbrand paraplastTM; Fisher, Cat. No. 23-021-399)

2.3.4. Procedure • • •

Transfer the bone samples through a graduated series of increasing ethanol concentrations as indicated below. Use ethanol in a volume at least 10 times of the tissue volume. Allow the samples to remain in each concentration for the indicated time as in Table 2.

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Dehydration, clearing, and infiltration procedure.

Steps 1 2 3 4 5 6 7 8 9 10 11 12

Procedure

Time (h)

50% alcohol 70% alcohol 95% alcohol 95% alcohol 100% alcohol 100% alcohol 100% alcohol Xylene Xylene Xylene Wax (58°C) Wax (58°C)

1 1½ 1½ 1½ 2 2 2 2 2 2 2 2

2.4. Paraffin embedding 2.4.1. Purpose and principle After complete infiltration with wax, the bone samples need to be embedded with extra wax to form a square or rectangular block for easy sectioning. 2.4.2. Equipment •

• •

Automatic embedding center (Leica, EG1160), consisting of a warming oven for preheating molds and forceps, a wax bath and reservoir, and a heated work surface Cold surface for quickly cooling the wax-filled molds once the specimen has been positioned Molds (Shandon stainless steel base molds 6401015, 6401016, 6401017, and 6401018)

2.4.3. Reagent •

Tissue embedding medium, melting point 56°C (Fisherbrand paraplastTM; Fisher, Cat. No. 23-021-399)

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2.4.4. Procedure • •

•

•

Transfer the bone samples to a wax bath in the embedding center. Place the bone sample(s) in a mold on a heated work surface with forceps, and position the sample(s) in an orientation suitable for cutting good sections. To reposition the sample(s) in the wax, put the mold back on the heated work surface and reposition the sample(s) with the forceps once the wax has melted. Store blocks at 4°C in a plastic wrap or bag.

2.5. Sectioning 2.5.1. Purpose and principle Light microscopy can only provide an image of a three-dimensional structure of bone in two dimensions. Ironically, the goal of this section is to obtain very thin bone slices with very little depth. Thus, it is important to plan and orientate bone blocks before sectioning. Eventually, one must attempt to mentally reconstruct a more complete image from bone sections for the interpretation of changes observed in a three-dimensional view.

2.5.2. Equipment • • • • • • • • •

Compressed air spray (Fisher 23-022523) Drying oven or hot plate (Fisher slide warmer 12-594) Fine paintbrush or forceps Flotation bath (TBS) Glass slides (Thermo Electron Corp. 6776214) Kimwipes Microtome (Microm GmbH HM315) Pencil Disposable blades (Sakura 4689)

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2.5.3. Reagent •

Diethyl pyrocarbonate (DEPC; Sigma D5758)-treated autoclaved water (for in situ hybridization only)

2.5.4. Procedure •

•

•

•

•

•

•

Trim the wax block to remove extra wax, and leave a perfect square or rectangle with parallel sides. The bottom edge of the wax block should be parallel to the microtome blade when it is mounted. Section the wax blocks 5–7 µm thick so that they are appropriate for general staining, in situ hybridization, or immunohistochemistry. The wax blocks should be as cold as possible when sectioning. The best sectioning is to obtain a single, continuous ribbon (straight and long). The ribbon is held with a fine paintbrush or forceps; the side of the ribbon facing the blade is shiny, and the other side is matte. Clean the ribbons stacked on the microtome blade with a compressed air spray, or wipe them with Kimwipes. The blade can be wiped with xylene and air-dried if sections curl. Transfer the ribbons of sections on a flotation bath filled with distilled water (DEPC-treated water for in situ hybridization) at 42°C–45°C, below the melting point of the wax. The shiny side of the ribbon must be placed downward on the flotation bath with a fine paintbrush or forceps, as this side will attach to the slides. The slight drag produced when placing the ribbon on the water is sufficient to remove most, if not all, of the folds on the ribbons of sections. Hold a glass slide at a 45° angle and immerse halfway into the water to collect the ribbons of sections, and slowly lift the slide out of the water. The sections will adhere to the slide. Label the slides with a pencil on the frosted surface, and dry the slides on a hot plate or an oven at 44°C overnight or longer. Put the slides into the box and store in a refrigerator until the tissue stains.

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2.6. Dewaxing and rehydration of sections 2.6.1. Purpose and principle Because all tissue stainings are processed in solution, the wax infiltrated into tissue sections has to be removed by xylene followed by rehydration. 2.6.2. Equipment • •

Slide rack (Miles Tissue-Tek 4465A) Staining jars

2.6.3. Reagents • • • • •

Xylene 100%, 90%, 70%, 50%, and 30% ethanol Water 1× PBS 1× PBST: 0.1% Tween-20 (Fisher BP337) in 1× PBS

2.6.4. Procedure •

Place the slides in a rack and transfer them sequentially as follows. Incubate them for a proper time in each solution. Table 3.

Dewaxing and rehydration procedure.

Xylene Xylene 100% ethanol 90% ethanol 75% ethanol 50% ethanol 30% ethanol PBS (or 1× PBST for in situ hybridization)

10 10 5 5 5 5 5 5

min min min min min min min min

3. Plastic Method 3.1. Purpose and principle The plastic method serves two purposes: studies of nondecalcified bone and tissue preparations for electron microscopy. Methyl methacrylate

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(MMA) is one of the commonly used resins in the laboratory (Feng et al. 2006). It produces a hard plastic that allows a distinction between mineralized bone and unmineralized osteoid, with an excellent preservation of the cellular structures. The resin infiltrated into the bone can be removed with solvents to achieve better staining (see below).

3.2. Instruments • • •

Incubator, 37°C Plastic or glass beaker 20-mL glass vials (Wheaton Science 986540)

3.3. Reagents • • • • • • • •

Benzoyl peroxide (BPO) reagent grade 97% (Sigma 179981) Dibutyl phthalate ReagentPlus ≥99% (Sigma D2270) 70% ethanol Formalin (1:10 dilution) (Fisher 23-245-685) Methylmethacrylate purum ≥99.0% (GC) (Sigma 64200) Paraformaldehyde (PFA; Sigma P6148) Polyethylene glycol 400 (PEG-400; EM Science PX1286B-2) N,N-dimethyl-p-toluidine (DMT; Aldrich D18-900-6)

3.4. Procedure 3.4.1. Fixation Although 10% neutral buffered formalin or 4% paraformaldehyde (PFA) can be used for fixation, 70% ethanol is highly recommended, as formalin or PFA may dissolve mineral from the bone at low pH and remove the fluorescent dye labeling. In addition, dehydration begins with fixation when ethanol is used as a fixative. Bone samples can be preserved in 70% ethanol indefinitely in a refrigerator.

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3.4.2. Dehydration The resin is immiscible with water. The dehydration process removes all water within the bone samples to allow complete infiltration of undecalcified bone by resin. The water is removed by immersing the samples in a series of increasing concentrations of ethanol (see below) in a plastic or glass beaker. 95% ethanol, 2 hours 95% ethanol, 2 hours 95% ethanol, 2 hours 100% ethanol, 2 hours 100% ethanol, 2 hours 100% ethanol, 2 hours 100% ethanol, 2 hours Xylene, 30 min Xylene, 30 min 3.4.3. Infiltration • •

Place each bone sample in a small glass bottle. Add 10 mL of freshly prepared embedding solution for each sample, and seal the cap tightly. The embedding solution needs to be freshly prepared under a fume hood as follows: Methylmethacrylate, 8.4 mL Dibutyl phthalate, 1.4 mL Polyethylene glycol 400 (PEG-400), 100 µL Benzoyl peroxide (BPO), 70 mg

•

Allow the infiltration to proceed at room temperature for 3–4 days.

3.4.4. Embedding and polymerization Polymerization can be accomplished either by heating or by the addition of an accelerator. Resin present either in solution or within tissues becomes solidified during polymerization, and a hard block containing the bone sample is eventually obtained.

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Directly place the bottles with the embedding solution at 37°C in an incubator, and heat for 2–3 days to allow it to polymerize; or replace the embedding solution with 10 mL of freshly prepared embedding solution containing an accelerator as follows: Methylmethacrylate, 8.4 mL Dibutyl phthalate, 1.4 mL Polyethylene glycol 400, 100 µL Benzoyl peroxide (BPO), 70 mg N,N-dimethyl-p-toluidine, 33 µL

•

Purge the air out of bottles with nitrogen and cap tightly.h Polymerize at 4°C.

3.4.5. Labeling of blocks The glass bottle has to be broken to obtain the block. • • • • • •

Write down the sample ID on a piece of paper. Wrap the glass bottle with a piece of paper towel to avoid injury from the broken glass. Break the bottle with a hammer. Remove the glass pieces and discard them in a proper container for broken glass.i Write down the sample ID on the block with a marker pen. Place the samples with resin in darkness.

3.4.6. Sectioning (1) Instruments •

Water-cooled low-speed diamond saw (e.g. Buehler IsometTM 1000 Precision Saw; Buehler Ltd, Lake Bluff, IL, USA)

h The glass bottles must be sealed tightly after the inside air is replaced by nitrogen or the bottles are opened under vacuum, as the oxygen in the air inhibits the polymerization process. i The container used must be pretested to ensure that there is no reaction between its material and the resin.

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Leica SM 2500S microtome (Leica, Deerfield, IL, USA) Leitz 1600 saw microtome (Ernst Leitz Wetzlar GmbH, Wetzlar, Germany) Polyethylene film Slides

(2) Reagents • • • •

30% ethanol Gelatin (ICN Biomedicals 960102) Glycerol (Fisher BP229-1) Permount (Fisher SP15)

(3) Procedure (a) Preparing the slides — coating: • •

Place the new slides on a slide rack. Immerse the slides in slide coating solution (as follows) for 5 minutes at 45°C–50°C. Gelatin, 1 g Glycerol, 15 mL Distilled water, 85 mL

• •

Take the slides out and place them at 37°C–45°C in an incubator overnight. Store the coated slides at 4°C.

(b) Trimming the block The block has to be trimmed to be placed on the microtome holder before cutting. Trimming can be performed using a band saw or a precision slow-speed saw (e.g. Buehler Isomet 2000 precision saw). • •

Determine the orientation of the bone sample in the block to the microtome blade. Make four cuts for a cylindrical block to get a cubic block. For intact ulna bone, this should make the ulna parallel to the length of the block in order to get crosssections of ulna.

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•

Make one more cut at the end of the block on which the initial cut for sectioning began. This cut removes the excess plastic as well as the end of the ulna bone. It will reduce the amount of trimming when cutting sections, thereby increasing the longevity of the diamond blade.

(c) Cutting sections — thin sections Although plastic sections can be 1–2 µm thick, a 4–7-µm thickness is good for Goldner’s stain and von Kossa stain, which allows the differentiation of osteoid from mature mineralized bone matrix. •

•

• • •

•

• •

Mount the trimmed block in the microtome chunk tightly. Instability can induce chatter which produces wrinkled sections, and can also damage the knife edge. Adjust the clearance angle that the knife edge makes with the block face in order to obtain a flat and even thickness of sections. Cut the block at a slow speed. Apply 30% ethanol to the block surface with a fine paintbrush to keep it damp and soft for cutting each time. Collect the sections with a soft paintbrush moistened with 30% ethanol onto the gelatin-coated slides,j and cover with a piece of polyethylene film. Use a clamp to stack the slides together to make the sections flat and to make the sections adhere to the slides evenly. Place the slides with sections at 42°C–45°C in an incubator for 3 days. The sections should adhere to the slides. Remove the polyethylene film prior to staining.

(d) Cutting sections — thick sections Sections of 100–200 µm in thickness can be obtained using a horizontal rotation sawing system (e.g. a Leitz 1600 saw microtome). j Caution must be taken to avoid damaging the knife edge when collecting sections onto the slides, especially with forceps. A fine paintbrush is highly recommended for collecting sections.

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•

•

197

Mount the trimmed block firmly to the arm-type holder that moves toward the diamond saw. Turn on the tap water to cool the block while cutting. Set the speed of the block at 5–10 when cutting the block. Adjust the thickness of sectioning. Assuming the thickness of the saw is 300 µm, screw up the arm-type holder to 400 µm if a section 100 µm thick is desired. Collect the sections with a pair of forceps, place on a slide, and cover with a coverslip. The coverslip is attached to the slide at the four corners with permount. View the unstained sections under epifluorescent illumination using a Nikon E800 microscope.

3.4.7. Deplasticization The plastic on thin sections has to be removed prior to staining. The entire procedure should be performed in a ventilated fume hood. (1) Instrument •

Plastic slide rack

(2) Reagents • •

2-methoxyethylacetate (Sigma 308269) Acetone (Fisher A929-4)

(3) Procedure • • • •

Place the slides on a plastic slide rack. Immerse the rack with slides in three changes of 2-methoxyethylacetate for 20 minutes each. Immerse slides in two changes of acetone for 5 minutes each. Wash slides in two changes of deionized water for 5 minutes each.

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4. Frozen Method 4.1. Purpose and principle This method is mainly used to detect the enzyme activity of transgenes such as LacZ (Lobe et al. 1999). Occasionally, it is also used for immunohistochemistry staining when the paraffin method does not work. Because crystal formed at temperatures below zero disrupts the cell structure, decalcified bone samples or embryos have to be infiltrated with sucrose, which prevents crystal formation in cells.

4.2. Instrument •

Cryostat (e.g. Leica CM 3050S; Leica Microsystems GmbH, Germany)

4.3. Reagents • •

PBS powder concentrate (Fisher Scientific BP661-50) Sucrose (Sigma S0389)

4.4. Cryoprotection solution • •

15% sucrose: add 75 g of sucrose in 500 mL of 1× PBS, and shake until dissolved. 30% sucrose: add 150 g of sucrose in 500 mL of 1× PBS, and shake until dissolved.

4.5. Procedure 4.5.1. Tissue preparation • • k

Dissect out the bone or the whole-mount embryos.k Wash the bone samples with 1× PBS.

Embryo skin must be removed at this step, as skin makes a very good barrier that affects the penetration of both fixative and X-gal substrates.

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4.5.2. Fixation •

•

Fix the dissected tissues in 4% paraformaldehyde for 30 minutes to 2 hours at 4°C or on ice with shaking. The fixation time is dependent on the age of the animal and the sample size: for embryos, 30 minutes; for newborns to 10-day-old mouse tissue, 1 hour; and for old mouse tissue, 2 hours.l Wash the tissues in 1× PBS three times at 4°C for 15–30 minutes each with shaking.

4.5.3. Decalcification •

Decalcify tissues in 10% EDTA solution (pH 7.2–7.4), and adjust the decalcification time as follows: Newborn and embryonic tissue, 1 day 4-day-old tissue, 3 days 10-day-old tissue, 7 days 4-month-old tissue, 10 days

•

The time listed above will not completely decalcify the tissues, especially for the adult tissues. However, this has no apparent effect on tissue sectioning. Wash the decalcified tissues three times in 1× PBS at 4°C for 15–30 minutes each with shaking.

4.5.4. Sucrose infiltration • •

Incubate the tissues in 15% sucrose until they sink to the bottom at 4°C. Change 15% sucrose to 30% sucrose until the tissues sink to the bottom at 4°C.

4.5.5. Optimum cutting temperature (OCT) embedding medium and cryosectioning • l

Set the cabinet temperature of a cryostat at −15°C to −24°C.

Overfixation will inactivate the activity of X-gal, so do not overfix the bone samples.

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Place the bone sample in OCT embedding medium on a sample holder. Place the holder with sample in OCT embedding medium on the sample bar to freeze the sample. Mount the sample holder. Cut the sections at a thickness of 10–12 µm. Rest the cut section on the surface of the blade holder after cutting. Collect the sections on the slides. Air-dry the sections 2–4 hours at room temperature. Store the slides at 4°C until use.

References Feng JQ, Ward LM, Liu S et al. Loss of DMP1 causes rickets and osteomalacia and identifies a role for osteocytes in mineral metabolism. Nat Genet 38(11):1310–1315, 2006. Lobe CG, Koop KE, Kreppner W et al. Z/AP, a double reporter for cremediated recombination. Dev Biol 208(2):281–292, 1999.

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Chapter 14

Acceleration of Bone Decalcification by Ultrasound Xia Guo and Wai-Ling Lam

Decalcification is a technique used to process bone specimens for histopathology or to produce surgical graft material. However, it usually takes a long period of time for a bone specimen, especially large bone samples, to be completely decalcified. The long decalcification process could cause delay in obtaining experimental results and loss of signals as well as reduce staining qualities. This chapter describes the use of a new invention that incorporates ultrasonic waves into the decalcification process so as to shorten the period required for decalcification. The effectiveness of ultrasonic decalcification is also demonstrated. The application of such a technique would help both basic and clinical research scientists and technicians to accelerate their routine work in dealing with bone decalcifications. Keywords:

Decalcification method; ultrasound; bone; bone bank.

1. Introduction Bone is composed of bone cells, collagen networks, and crystals of hydroxyapatite (HA) [Ca10(PO4)6(OH)2] on or within the collagen fibers. Decalcification is a process in which HA is removed from the Corresponding author: Xia Guo. Tel: +852-27666720; fax: +852-23308656; E-mail: [email protected]

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Ca10 (PO4)6(OH)2 + 8H+




calcifying tissue to soften the tissues so as to enable paraffin sectioning for histomorphological and histopathological studies. HA can be removed from the tissues either by ionization in acid solution or by chelation with a chelating agent (Gruber and Stasky 1999; Page 1996), as is represented by Eq. (1). However, it usually takes a long period of time, e.g. several weeks to several months, for a bone specimen to be completely decalcified. The long period of time for decalcification not only causes delay in obtaining experimental results, but also results in loss of signals in in situ hybridization (Arber et al. 1997; Kaneko et al. 1999) and reduces staining qualities (Verdenius and Alma 1958). 10Ca+2 + 6HPO4−2 + 2H2O

(1)

Many attempts have been made to accelerate bone decalcification, such as through elevation of temperature, a higher concentration of the decalcifying agent, agitation, a vacuum, electric current, and ultrasonic waves for decalcification (Madden and Henson 1997). However, not all of the interventions produced promising results in accelerating decalcification; for example, some researchers (Kiviranta et al. 1980; Verdenius and Alma 1958) found that heat could accelerate the rate of decalcification, but some others disagreed (Lillie et al. 1951; Lillie and Fullmer 1976). Until now, the issue of the optimal temperature for decalcification remains a contentious one. It has been shown that a higher concentration of the decalcifying agent could accelerate the decalcifying process, but at the same time it was detrimental to the morphology of the specimens (Clark 1954; Hoole 1971; Lillie et al. 1951; Lillie and Fullmer 1976; Verdenius and Alma 1958). Among those mentioned interventions, ultrasonic waves have shown a satisfactory result in shortening the period of time for decalcification without introducing morphological damages to the natural structure of bone (Milan and Trachtenberg 1981; Muller et al. 1990; Poston 1967; Thorpe et al. 1963). The use of ultrasonic waves to accelerate the decalcification of bone was first introduced by Thorpe et al. (1963), but little information and data were presented. It is believed that ultrasonic waves

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accelerate the decalcification of bone by cavitations. Even though several studies have been conducted to prove their accelerating effects in decalcification (Milan and Trachtenberg 1981; Muller et al. 1990; Poston 1967; Thorpe et al. 1963), it is still uncommon to see ultrasonic decalcification being employed in both laboratory and clinical practice. This chapter describes the use of a new invention that incorporates ultrasonic waves into the decalcification process so as to shorten the period required for bone decalcification. The effectiveness of ultrasonic decalcification is demonstrated by comparing the bone mineral density (BMD) obtained from peripheral quantitative computed tomography (pQCT) during decalcification.

2. Materials •

• • •

Specimens: any bony part of the body can be used for decalcification. The soft tissues surrounding the bone are removed before chemical fixation. Bone from various parts of the body (e.g. humerus, tibia, femur) and from different species (e.g. human, rabbit, bovine, rat) have been tested by the authors for fast decalcification. Sodium hydroxide: 1 M NaOH (e.g. RDH, Germany) — for adjusting pH Hydrochloric acid: 1 M HCl (e.g. RDH, Germany) — for adjusting pH Phosphate buffered saline (PBS)





•

Dissolve 8 g of sodium chloride (NaCl), 0.2 g of potassium chloride (KCl), 0.2 g of potassium dihydrogen phosphate (KH2PO4), and 1.15 g of disodium hydrogen phosphate (Na2HPO4) (all from RDH, Germany) in 1 L of distilled water. Adjust the final pH to 7.4 by adding drops of HCl or NaOH.

Fixative: 4% phosphate buffered paraformaldehyde


Dissolve 4% (w/v) paraformaldehyde powder (e.g. Fluka, Switzerland) in a mixture of 1:1 distilled water and PBS in a fume hood.

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Adjust the final pH to 7.4 for fixing the specimens before decalcification. Depending on the size of the specimens, the fixation period ranges from a few hours to a few days.a

Decalcifying agent: 10% ethylenediaminetetraacetic acid (EDTA)


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Dissolve 10% (w/v) EDTA powder (e.g. RDH, Germany) in distilled water. NaOH pellets could be added to assist powder dissolution.b

There are different types of chemical fixatives available for specimen fixation. These include ethanol, acetic acid, picric acid, glutaraldehyde, osmium tetroxide, etc. Different fixatives are used for different purposes with regard to subsequent embedding and microscopic observation. Formaldehyde, available as a solution (formalin) and as a solid polymer (paraformaldehyde), is the most widely used fixing agent for pathologic histology probably because of its ability in preserving most of the elements in the tissue structure (Kiernan 1981; Lillie and Fullmer 1976); therefore, formaldehyde was chosen for the present study. A 4% solution at pH 7.2–7.4 is commonly used. This substance dissolves very slowly in water but more quickly in near-neutral buffer solutions, so PBS can be used to dissolve the powder. Heating the mixture to around 55°C–60°C can further accelerate the dissolution of the formaldehyde. Specimens may be fixed by immersion in at least 20 times their own volumes of the appropriate fixatives. Tissue may remain in the fixative for several months, but it may become harder after a long fixation period, say 1 or 2 weeks (Kiernan 1981). The rate of fixation is influenced by the concentration of the reagent and the temperature. The specimen could be completely fixed in a shorter period of time if a higher concentration of fixative and higher temperature are used. However, for chemical fixation using formaldehyde, autolysis may occur if the specimen is fixed at a higher temperature (e.g. 55°C). Some researchers thus recommend fixation at lower temperatures (e.g. 4°C) (Kiernan 1981; Lillie and Fullmer 1976). b Specimens for decalcification must be properly fixed. The fixative must be completely washed out of the tissue prior to decalcification in order to avoid undesirable chemical reactions. There are two main types of decalcifying agents, i.e. acids and chelating agents. Hydrochloric, nitric, and formic acids are the acids most often used for decalcification. Decalcification with strong acids may result in hydrolysis of nucleic acids and tissue distortion; using strong acids for decalcification may also interfere with the interpretation of results obtained through histochemical techniques. EDTA, at a concentration of 5%–10% (w/v), is the most common chelating agent used for decalcification. Unlike strong acids, EDTA is used at a neutral or near-neutral pH (7.2–7.4); therefore, it does not cause any deleterious effects on labile substances such as nucleic acids and enzymes during decalcification (Page 1996). The main disadvantage of EDTA is that it acts much more slowly than the acids (Milan and Trachtenberg 1981). The solution should be freshly prepared. NaOH pellets could be added to bring up the pH and at the same time assist the dissolution of EDTA. Since the chelating agent is insoluble in alcohol, tissue treated with EDTA should be washed in water before dehydration (Gruber and Stasky 1999; Kiernan 1981). Similar to fixatives, the volume of decalcifying fluid should be at least 20 times that of the specimen. The decalcifying agent is changed frequently in terms of the type and concentration used, ranging from 1 to 5 days, to prevent the decalcifying agent from being saturated (Kiernan 1981). The acceptable temperature range for decalcification processing is 15°C to 60°C from a previous research (Lillie and Fullmer 1976).

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• • •

•

•

• • • • •

Custom-made ultrasound machine — for ultrasonic decalcification (US patent US-2007-0092863-A1) Plastic tank with a similar size as the tank in custom-made ultrasound machine — for traditional decalcification Orbital shaker (e.g. Model OR50, Analog Orbital Shaker; Daigger, USA) — for providing continuous agitation for traditional decalcification Peripheral quantitative computed tomography (pQCT) (e.g. Stratec XCT 2000; Stratec, Germany) — for monitoring the changes of bone mineral density (BMD) in decalcifying bone every 24 hours after decalcification X-ray machine (e.g. Faxitron X-ray machine, Model 43855C; Wheeling, IL, USA) — for estimating the endpoint of decalcification Tissue processor (e.g. Shadon, England) — for tissue processing Embedding center (e.g. Leica, Germany) — for tissue embedding Microtome (e.g. RM2145; Leica, Germany) and blade (e.g. DB80L; Leica, Germany) — for sectioning tissue blocks Hematoxylin and eosin (H&E) — for staining tissue sections Scott’s tap water


•

Add 1% (v/v) concentrated HCl to 70% alcohol (RDH, Germany) for H&E staining.

Eosin


•

Mix 0.2% (w/v) potassium bicarbonate (KHCO3) or sodium bicarbonate (NaHCO3) with 2% (w/v) magnesium sulphate (MgSO4) (all from RDH, Germany).

1% acid alcohol


•

205

Add 1% (w/v) eosin yellowish (e.g. Sigma-Aldrich, St Louis, MO, USA) in distilled water for H&E staining.

DPX (e.g. Fluka, Switzerland)


Dilute DPX by adding xylene (e.g. RDH, Germany) for mounting slides.

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Microscope: transmitted light microscope (e.g. Eclipse 80i; Nikon, Japan) — for studying the morphology of the traditionally and ultrasonically decalcified specimens

3. Methods 3.1. Ultrasonic decalcification • • • • •

• • •

c

Remove all of the soft tissues from the specimen. Fix the specimen in 4% paraformaldehyde overnight. Fill the decalcification tank with 400 mL of 10% EDTA to the custom-made ultrasound machine. Place the specimen in the tank and turn on the ultrasound machine for decalcification. Measure the BMD of the midshaft and the distal region of the specimen (three slides each, 1 mm apart) by using the pQCT machine to monitor the amount of calcium loss at different regions every 24 hours after decalcification. Change the EDTA in the tank every 24 hours during decalcification. Take an X-ray for endpoint detection.c When the endpoint is reached, process the specimen in the tissue processor, embed it with paraffin by using the embedding center, and section it to 5 µm thick with the use of a microtome and a blade.

Several methods can be used to confirm the completion of decalcification. Physical tests such as bending and needling are relatively cheap and simple, but they may introduce damages to the tissue structure. Chemical test (which relies on the detection of calcium in the decalcifying agent) using ammonia is only applicable to decalcification using certain types of acid, and is not sensitive in situations decalcified with an acid of high concentration (over 10%) (Page 1996). Loss of bone weight is another index which has been used to estimate the level of bone decalcification (Sanderson et al. 1995). During decalcification, there is a rapid or progressive, uninterrupted loss of specimen weight until decalcification is completed. However, the weight loss method is inaccurate, since weight could be regained during the decalcifying procedure because of hydration of the collagen fibers exposed by the removal of calcium (Sanderson et al. 1995). Among all of the methods available, X-ray is the most reliable and accurate method. However, the main disadvantage of X-ray evaluation is that it cannot be used to determine the level of specimen decalcification if specimens are fixed with radio-opaque material such as mercury chloride (Kiernan 1981). pQCT can quantitatively measure both bone mineral content (BMC) and BMD. We find this to be the most accurate and safe method for monitoring the decalcification level.

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Stain the section with H&E. Study the tissue morphology under a light microscope.

3.2. Traditional decalcification Repeat all of the steps as in Sec. 3.1. However, instead of placing the bone in a custom-made ultrasound machine, place the bone in a plastic tank for decalcification. Continuous agitation is introduced throughout the decalcification period by using an orbital shaker.

3.3. H&E staining • • • • • • • • •

Take the sections to water (xylene, 100% alcohol, 95% alcohol, 70% alcohol, and then water). Stain the sections by placing them in hematoxylin for 10–30 minutes, depending on the type of hematoxylin used.d Wash in tap water. Wash and blue in Scott’s tap water for about 1 minute. Differentiate the sections by placing the sections in 1% acid alcohol for a few seconds. Wash in tap water. Place sections in eosin for 4–5 minutes. Wash in tap water. Dehydrate, clear (70% alcohol, 95% alcohol, 100% alcohol, and then xylene), and finally mount in DPX. The nuclei are stained blue, while the other tissue components are stained shades of red and pink.

4. Statistics For this study, we compared ultrasonic decalcification (n = 4) with the traditional method as control (n = 4) for the entire rat femur (average length, 4.5 cm; average diameter, 0.55 cm). Paired t-test was used to compare the total volumetric BMD (amount of calcium present in bone) between day 0 (before decalcification) and day 1 (24 hours d

Hematoxylin is oxidized to hematein when it stays in oxygen for a long period of time, after which it is no longer useful. It is therefore recommended that hematoxylin be filtered before it is used to ensure good staining quality (Page 1996).

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after decalcification) at different regions of interest. Independent t-test was used to analyze the percentage loss of calcium at different regions between the ultrasonic decalcification group and the control group on day 0 and day 1.

5. Results 5.1. Dynamic change of BMD of bone specimens Paired t-test showed significant differences (p < 0.05) in total BMD at the midshaft, distal region, and entire bone (measurements of midshaft and distal region were taken together into consideration) between day 0 and day 1 for the ultrasonic decalcification group; while significant differences (p < 0.05) in total BMD were found only at the midshaft and entire bone between day 0 and day 1 for the control group (Table 1). It was found that the percentage of bone mineral loss in 24 hours as interpreted by changes of BMD was greater in the group with ultrasonic decalcification (24%–38% of BMD loss) when compared to the control group (6%–24% of BMD loss), and the reduction of BMD between these two groups was significantly different (p < 0.05). The results from pQCT measurement also indicated that the decrease in bone mineral content (BMC; an index on total calcium) was faster in the midshaft than in the distal region in both groups, either treated with ultrasonic waves or in controls. This may be due to the fact that the diameter of the midshaft region is much thinner than that of the distal region; therefore, it is much easier for the decalcifying agent to penetrate (Table 2). Figure 1 shows the results of pQCT measurements on a specimen which has been decalcified for 24 hours. Bone specimens with a white or brighter background indicate regions with higher BMD (calcium content); regions with lower BMD are shown in red, yellow, or gray. By comparing the results from day 0 to day 1 between the two groups, a greater loss of BMC was seen in the ultrasonic decalcification group than in the control group (p < 0.05). The results of pQCT measurements on day 4 (specimens being decalcified for 96 hours) showed that no mineral component could be detected from those femora treated with ultrasonic waves, while

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Ultrasound-treated

Control

Day 1

p-value

Day 0

Day 1

p-value

Midshaft Distal

644.85 ± 47.91 511.78 ± 59.24

395.83 ± 43.95 388.70 ± 70.73

*0.000 *0.000

636.98 ± 46.38 488.88 ± 16.11

477.26 ± 47.37 454.98 ± 24.24

*0.000 0.174

Total

578.32 ± 86.00

392.26 ± 57.70

*0.000

562.93 ± 90.78

466.12 ± 13.81

*0.000

*p < 0.05.

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Dynamic change in bone mineral density (BMD) (mg/cm3) in rat femora.

Acceleration of Bone Decalcification by Ultrasound

Table 1.

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Percentage of bone mineral loss in 24 hours (%) in rat femora. Ultrasound-treated

Control

p-value

Midshaft Distal

38.41 ± 7.23 24.38 ± 7.29

24.89 ± 7.68 6.59 ± 16.48

*0.000 *0.002

Total

31.40 ± 10.09

15.73 ± 15.67

*0.000

*p < 0.05.

Fig. 1. pQCT results on day 1 (24 hours after decalcification) for ultrasonictreated femurs.

a relatively large amount of calcium could still be detected by the pQCT machine on those femora in the control group. This indicated that decalcification was completed on day 4 (decalcified for 96 hours) by using ultrasonic waves, while the decalcification was still incomplete in those femora in the control group.

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Fig. 2.

Fig. 3.

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X-ray for determining the level of decalcification.

Growth plate of femur treated with ultrasonic decalcification (100×).

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Fig. 4.

Fig. 5. (100×).

Growth plate of control femur (100×).

Cortical region of femur treated with ultrasonic decalcification

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Fig. 6.

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Cortical region of control femur (100×).

Fig. 7. Trabecular region of femur treated with ultrasonic decalcification (100×).

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Fig. 8.

Fig. 9.

Trabecular region of control femur (100×).

Chondrocytes of femur treated with ultrasonic decalcification (400×).

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Fig. 10.

Fig. 11.

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Chondrocytes of control femur (400×).

Osteoblasts of femur treated with ultrasonic decalcification (250×).

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Fig. 12.

Osteoblasts of control femur (250×).

The level of decalcification was semiquantitatively revealed by radiography. Figure 2 shows the results of radiographs taken on day 4, after specimens were decalcified for 96 hours, for all eight femora used in this study. F3, F4, F5, and F6 were decalcified by ultrasonic waves; while F7, F8, F9, and F10 were controls. F11 was a femur that did not undergo decalcification. Figure 2 indicates that those femora treated with ultrasonic waves were completely decalcified at day 4, while those femurs in the control group were incompletely decalcified at the distal regions.

5.2. H&E staining Sections from both the ultrasonic decalcification and control groups were stained with Harris hematoxylin. The growth plate, cortical bone, trabecular bone, chondrocytes, and osteoblasts were assessed under a light microscope for both groups to test whether any artifacts or detrimental effects were introduced to the tissues on decalcification by using ultrasonic waves. The results of H&E staining suggested that

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decalcification of bone did not result in any architectural change or distortion to any of the mentioned regions or cells in morphology as compared with tissues processed in the conventional fashion. The cellular details of the tissues could be easily demonstrated and observed after being treated with ultrasonic waves (Figs. 3–12).

6. Summary This chapter described the use of a new invention that incorporates ultrasonic waves into the decalcification process, accelerating the decalcifying process and thus shortening the period required for decalcification. The application of such a technique would help both basic and clinical research scientists and technicians to accelerate their routine work in dealing with bone decalcifications.

Acknowledgment The authors would like to thank Dr Zheng Yong Ping, Associate Professor of the Department of Health Technology and Informatics at The Hong Kong Polytechnic University, for his professional advice on constructing the ultrasound machine. The ultrasound machine described in this chapter has been patented in the USA (US-20070092863-A1) and China.

References Arber JM, Weiss LM, Chang KL et al. The effect of decalcification on in situ hybridization. Mod Pathol 10:1009–1014, 1997. Clark PG. A comparison of decalcifying methods. Am J Clin Pathol 24:1113–1116, 1954. Gruber HE, Stasky AA. Histological study in orthopaedic animal research. In: An YH, Friedman RJ (eds.), Animal Models in Orthopaedic Research, CRC Press, Boca Raton, FL, pp. 115–131, 1999. Hoole PF. Rapid decalcification of bone for diagnostic histology. Med Lab Technol 28:201–204, 1971. Kaneko M, Tomita T, Nakase T et al. Rapid decalcification using microwaves for in situ hybridization in skeletal tissues. Biotech Histochem 74:49–54, 1999.

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Kiernan JA (ed.). Histological and Histochemical Methods: Theory and Practice. Pergamon Press, Oxford, UK, 1981. Kiviranta I, Tammi M, Lappalainen R et al. The rate of calcium extraction during EDTA decalcification from thin bone slices as assessed with atomic absorption spectrophotometry. Histochemistry 68:119–127, 1980. Lillie RD, Fullmer HM (eds.). Histopathologic Technic and Practical Histochemistry. McGraw-Hill, New York, 1976. Lillie RD, Laskey A, Greco J et al. Decalcification of bone in relation to staining and phosphatase technics. Am J Clin Pathol 21:711–722, 1951. Madden VJ, Henson MM. Rapid decalcification of temporal bones with preservation of ultrastructure. Hear Res 111:76–84, 1997. Milan L, Trachtenberg MC. Ultrasonic decalcification of bone. Am J Surg Pathol 5:573–579, 1981. Muller S, Pleul J, Gotz M et al. A method to determine the end point of decalcification of hard tissue and bone. Stain Technol 65:77–83, 1990. Page KM. Bone. In: Bancroft JD, Stevens A (eds.), Theory and Practice of Histological Techniques, Churchill Livingstone, Edinburgh, Scotland, pp. 309–339, 1996. Poston F. Bone decalcification expedited by ultrasonic sound. Am J Med Technol 33:263–268, 1967. Sanderson C, Radley K, Mayton L. Ethylenediaminetetraacetic acid in ammonium hydroxide for reducing decalcification time. Biotech Histochem 70:12–18, 1995. Thorpe EJ, Bellomy BB, Sellers RF, Tennessee K. Ultrasonic decalcification of bone. J Bone Joint Surg 45A:1257–1259, 1963. Verdenius HHW, Alma L. A quantitative study of decalcification methods in histology. J Clin Pathol 11:229–236, 1958.

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Chapter 15

Stains of Bone and Cartilage Yong-Bo Lu, Yi-Xia Xie and Jian-Quan Feng

Bone or cartilage cells are essentially colorless, and it is difficult to distinguish their morphologies under a light microscope. To better visualize these structures, various staining techniques have been developed; due to page constraints, we will only describe the most common methods for readers. These staining assays include hematoxylin and eosin (H&E) stain, used for general histology of the cell; Safranin O stain, used for the cartilage; and von Kossa and van Gieson stains as well as modified Goldner’s trichrome stain, used for nondecalcified tissues. Keywords:

Bone; cartilage; mineralized bone; osteoid.

1. Introduction Tissues such as bone or cartilage cells are colorless, and it is very difficult to distinguish them under a light microscope unless they are stained. Thus, the greatest aid in imaging cells or the extracellular matrix (ECM) is the use of various staining techniques, depending on the nature of cell structure and the content (especially pH value). For example, nuclei contain high levels of nucleic acids and have an affinity for basic dyes such as hematoxylin or basic fuschin; in contrast, the cytoplasm contains more alkaline substance (high pH value) and attracts acid dyes such as eosin, orange G, or fast green. In addition, Corresponding author: Jian-Quan Feng. Tel: +1-214-3707235; E-mail: [email protected]

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dyes can be used to stain ECMs, such as van Gieson for collagen fibers and silver nitrate for interaction with calcium carbonate in nondecalcified bone matrices. This chapter provides a useful guide to the most common staining techniques, namely, hematoxylin and eosin (H&E) stain; safranin O stain, von Kossa and van Gieson stains, and modified Goldner’s trichrome stain.

2. Materials and Methods 2.1. Hematoxylin and eosin (H&E) stain 2.1.1. Purpose and principle Hematoxylin with mercuric oxide is used to stain nuclei, and eosin is used to stain cytoplasm. The staining results are as follows: the nuclei show dark blue and are distinct; the muscle cytoplasm shows bright red; while the osteoclast cytoplasm and osteoblast cyctoplasm show pink (Fig. 1). 2.1.2. Reagents • • • • • • • • • •

Aluminum ammonium sulfate (Sigma A2140) Ammonium water (0.5% NH4OH) Eosin Y (Sigma E4382) Ethanol Hematoxylin (Sigma H3136) Mercuric oxide (mercury II oxide, red) (Aldrich) Permount (Fisher SP15) Phloxine B (Sigma P4030) Orange G, sodium salt (Sigma 01625) Xylene (Fisher X5-4)

2.1.3. Solution preparation (1) Harris’s acidified hematoxylin •

Add aluminum ammonium sulfate to water in a 500-mL Erlenmeyer flask (Pyrex), and heat with stirring until dissolved. Remove from heat.

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Fig. 1. Hematoxylin and eosin (H&E) stain, a useful assay for the initial screening of bone cell and matrix status. The left panel shows a well-formed lamellar bone with smooth bone matrix from a 2-month-old wild-type (WT) mouse tail. The right panel displays a poorly formed cortical bone from an age-matched BMP receptor 1a conditional knock-out (CKO) mouse tail. Note that fibrosis-like tissues (arrow) are filled in the cortical bone.

Aluminum ammonium sulfate, 25 g Distilled water, 235 mL •

Add the two powders below to a 50-mL Erlenmeyer flask, and stir to mix. Pour this mixture into the aluminum ammonium sulfate solution, rinsing with distilled water to make a complete transfer. Hematoxylin, 1.25 g Mercuric oxide, 0.6 g Distilled water, 15 mL

•

•

Return the flask to heat and bring the entire solution to a boil with high heat for 2–3 minutes, then plunge into an ice bath to cool. Store covered in darkness for at least 24 hours. Just before use, filter and add 8 mL of glacial acetic acid. Filter the solution before each use. It would be best if used within 1 week. Note that mercuric oxide is toxic.

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(2) Eosin •

0.6% eosin Y Eosin Y, 6 g Distilled water, 100 mL Ethanol, 900 mL Stir to dissolve eosin. Add 50 mL of glacial acetic acid until the pH is between 4.6 and 5.0. The color will change from opaque green to clear red.

• • •

1% phloxine B: 1 g in 100 mL dH2O 2% orange G (sodium salt): 2 g in 100 mL dH2O Working solution 0.6% eosin Y, 238 mL 1% phloxine, 6 mL 2% orange G, 6 mL The concentration of orange G may have to be adjusted because of variations in dye content.

2.1.4. Procedure Table 1.

Hematoxylin and eosin (H&E) staining procedure.

Xylene 100% ethanol 90% ethanol 80% ethanol Hematoxylin Water (clean) Ammonium water (0.5% NH4OH) Water 80% ethanol 95% ethanol Eosin 95% ethanol 100% ethanol Xylene Coverslip with permount

3× 2 min 3× 1 min 1× 1 min 1× 1 min 1× 30 s Change until rinse water is clear 1× 10 s 1× 3 min 1× 1 min 1× 1 min 1× 2 min 3× 1 min 3× 1 min 3× 1 min

Source: Bone Metabolism Lab, Pathology Dept., The University of Texas Health Science Center at San Antonio, San Antonio, TX, USA.

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2.2. Safranin O stain 2.2.1. Purpose and principle The ECM of cartilage consists mainly of glycosaminoglycans, proteoglycans, collagen, and hyaluronic acid. Safranin O, a cationic dye, binds to glycosaminoglycans and polyanionic proteoglycans, but not to collagen. Therefore, it stains cartilage in red color and the background in green color. This staining method works best on undecalcified, acid-decalcified, or very short EDTA-decalcified (within 6d) skeletal sections (Fig. 2). 2.2.2. Reagents • •

Acetic acid (Fisher A490-212) 70% and 80% alcohol

Fig. 2. Safranin O stain, an assay for the detection of proteoglycan in cartilage. The left panel shows nicely formed, vertical columns from a 3-week-old wild-type (WT) mouse growth plate. The right panel displays an expanded growth plate from an age-matched Dmp1-null knock-out (KO) mouse.

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Fast green FCF (Sigma F7258) Ferric chloride (EMS 15510) Hematoxylin C. I. (EMS 16620) Hydrochloric acid, concentrated (Fisher A144-212) Safranin (Sigma S8884)

2.2.3. Solution preparation (1) Modified Weigert’s iron hematoxylin (a) Stock solution A (good for 4 months) Hematoxylin C. I., 4.0 g 80% alcohol, 200 mL (100% alcohol, 160 mL; distilled water, 40 mL) (b) Stock solution B (good for 4 months) Ferric chloride, 8.0 g Distilled water, 190 g Hydrochloric acid, concentrated, 2.0 mL (2) Working modified Weigert’s iron hematoxylin (good for 7 days) •

Mix equal parts of solutions A and B.

(3) 1% acid alcohol (good for 6 months) 70% alcohol, 1000 mL Hydrochloric acid, concentrated, 10 mL (4) 0.02% fast green (good for 1 month) Fast green, 0.02 g Distilled water, 100 mL (5) 1% acetic acid (good for 4 months) Distilled water, 100 mL Acetic acid, 1 mL

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(6) 0.1% Safranin O (good for 1 month) Safranin O, 0.1 g Distilled water, 100 mL 2.2.4. Procedure • • • • • • • • • •

Deparaffinize the slides and hydrate to water. Dip in Weigert’s iron hematoxylin for 5 minutes. Rinse in distilled water. Dip in 1% acid alcohol to destain. Rinse in distilled water. Dip in 0.02% fast green for 5 minutes (no rinse). Dip in 1% acetic acid for 30 seconds (no rinse). Dip in 0.1% Safranin O for 20 minutes (no rinse). Rinse in 95% alcohol. Dehydrate and coverslip with permount.

(Cristan’s procedure, Department of Orthopaedics/Pathology, University of Rochester, Rochester, NY, USA)

2.3. von Kossa and van Gieson stains 2.3.1. Purpose and principle von Kossa stain (silver nitrate) alone is used to stain mineralized bone matrices, resulting in a brownish-black color due to chemical reactions between silver nitrate and calcium carbonate. van Gieson stain is used to stain collagen fibers and osteoids (nonmineralized matrices), leading to a red color. The combination of von Kossa and van Gieson stains is used to differentiate nonmineralized bone (osteoid) from mature mineralized bone. This method is performed on methylmethacrylate (MMA)embedded undecalcified bone sections 4–7 µm thick [Fig. 3(a)]. 2.3.2. Reagents • •

95% alcohol Absolute alcohol

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Fig. 3. von Kossa/van Gieson stain and Goldner’s stain, assays for the distinction of good and poor mineralization. (a) In von Kossa/van Gieson stain, the mineral is black in color (arrowheads) and the osteoid (nonmineralized matrix) is pink/pale in color (arrows). (b) In Goldner’s stain, the mineral is blue/green in color (arrowheads) and the osteoid (nonmineralized matrix) is red in color (arrows). The left panel shows a well-mineralized tibia from a 3-month-old wild-type (WT) mouse, while the right panel displays a poorly formed tibia from an age-matched Dmp1-null knock-out (KO) mouse.

• • • • • •

Acid fuchsin (Sigma A3908) Permount (Fisher SP15-500) Picric acid, saturated (Sigma 925-40) Silver nitrate (Sigma S7276) Sodium thiosulfate (Sigma S7026) Xylene (Fisher X5-4)

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2.3.3. Solution preparation (1) 5% silver nitrate solution Silver nitrate, 2.5 g Distilled water, 50 mL •

Store at 4°C in a dark bottle, and discard it if the solution precipitates or is not clear.

(2) 5% sodium thiosulfate Sodium thiosulfate, 5 g Distilled water, 100 mL (3) 1% acid fuchsin Acid fuchsin, 1g Distilled water, 100 mL (4) van Gieson’s solution 1% acid fuchsin, 2.5 mL Picric acid, saturated, 97.5 mL

2.3.4. Procedure • • •

• • • •

Deplasticize MMA-embedded sections. Rinse the slides well with distilled water. Apply 5% silver nitrate solution on the slides to completely cover the sections, and then expose to sunlight or strong light until the mineralized bone turns black. This usually takes 45 minutes to 1 hour. Rinse the slides with distilled water. Cover the sections with 5% sodium thiosulfate solution for 5 minutes. Rinse the slides with distilled water. Counterstain in van Gieson’s solution for 1–3 minutes.

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Dehydrate in 95% alcohol and absolute alcohol; and then clear in xylene, two changes each, for 2 minutes each. Mount with permount.

(John Tarpley, Histology Lab)

2.4. Goldner’s trichrome stain 2.4.1. Purpose and principle Similar to the combined von Kossa and van Gieson stains, Goldner’s trichrome stain is used to differentiate nonmineralized bone (osteoid) from mineralized bone: bone with distinctive lamella in light blue, osteoid in red, and nuclei in purple to blue-black. This method is performed on MMA-embedded undecalcified bone sections 1–7 µm thick [Fig. 3(b)]. 2.4.2. Reagents • • • • • • • • • • •

Acid fuchsin (Sigma A3908) Ethanol Light green SF yellowish (ICN Biomedicals 152641) Glacial acetic acid Ferric chloride (FeCl3 6H2O) (EMS 15510) Hematoxylin crystals (Sigma H9627) Hydrochloric acid (HCl) Orange G (Sigma O1625) Phosphomolybdic acid (Sigma P7390) Ponceau Xylidine (Fluka 81465) Phosphotungstic acid (Sigma-Aldrich P4006)

2.4.3. Solution preparation (1) Weigert’s solution A Hematoxylin crystals, 1.0 g 95% ethanol, 100 mL

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(2) Weigert’s solution B HCl, 1.0 mL Distilled water, 100 mL Ferric chloride (FeCl3 6H2O), 1.16 g •

Mix equal parts of solutions A and B prior to use.

(3) Ponceau-acid fuchsin Glacial acetic acid, 0.6 mL Ponceau Xylidine, 0.4 g Acid fuchsin, 0.1 g Distilled water, 300 mL (4) Phospho-orange G Phosphotungstic acid, 6 g Phosphomolybdic acid, 6 g Distilled water, 250 mL Orange G, 5 g (5) Light green SF yellowish Light green SF yellowish, 1.5 g Distilled water, 500 mL Glacial acetic acid, 1 mL 2.4.4. Procedure • • • • • • • • •

Deplasticize MMA-embedded sections using 2-methoxyethyl acetate, four changes, for 15 minutes each. Dip in Weigert’s hematoxylin for 15 minutes. Wash in running water (tap or distilled) for 5 minutes. Dip in Ponceau-acid fuchsin for 20 minutes. Rinse in 1% acetic acid water, two changes, each for 30 seconds. Dip in phospho-orange G for 5–10 minutes. Rinse in 1% acetic acid water, two changes, each for 30 seconds. Dip in light green SF yellowish for 15 minutes. Rinse in 1% acetic acid water, two changes, each for 30 seconds.

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Dip quickly in 70% ethanol. Dip quickly in 95% ethanol. Dehydrate in absolute ethanol, two changes, for 1–2 minutes each. Clear in xylene, two to three changes, for 3 minutes each. Mount in permount.

References Bone Metabolism Lab, Pathology Dept., The University of Texas Health Science Center at San Antonio, San Antonio, TX, USA. Cristan’s procedure, Department of Orthopaedics/Pathology, University of Rochester, Rochester, NY, USA. John Tarpley, Histology Lab.

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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

This chapter introduces a Scion Image based on contact microradiography for evaluating the degree of secondary mineralization in basic structure units (BSUs) of cortical bone. The effects of long-term bisphosphonate (incadronate disodium) administration on the degree of secondary mineralization in osteons in Beagle dogs are evaluated as an example of the application of this technique. The relevant evaluation parameters used and validated for comparison include the mean degree of secondary mineralization in osteons and the distribution curves of mineralization frequency. Scion Image based on contact microradiography is a simple and precise method that can accurately evaluate the mean degree of secondary mineralization in BSUs of bone. Experimental findings suggest that long-term incadronate administration significantly increases the degree and uniformity of secondary mineralization of osteons in a dose-dependent manner, but does not cause hypermineralization of bone tissue. Keywords:

Scion Image; contact microradiography; degree of mineralization; bone remodeling; bisphosphonate.

Corresponding author: Yong-Ping Cao. Tel: +86-10-66551122 ext. 2268; E-mail: [email protected]

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1. Introduction Bone strength is determined not only by the bone mass and bone structure, such as geometrical shape, microstructure, trabecular thickness, and connectivity, but also by the degree of mineralization of the matrix (Meunier and Boivin 1997; Adolphson et al. 2000; Boivin and Meunier 2001; Turner 2002). In bone disorders with high bone resorption, the mean degree of mineralization of bone is reduced by shortening the duration of the secondary mineralization of basic structure units (BSUs) due to an increase in the activation frequency of new remodeling units. Currently, bisphosphonates — a kind of bone antiresorptive agent — are available in the treatment of bone diseases with high bone resorption, such as postmenopausal osteoporosis, Paget’s disease, metastatic bone disease, and osteogenesis imperfecta (Russell and Rogers 1999; Rodan and Martin 2000). They can effectively prevent or restore the loss of bone mass by decreasing bone turnover through rapidly suppressing osteoclastic resorption (Rodan and Fleisch 1996; Fleisch 1997). With the widespread use of bisphosphonates, it is only recently that investigations have addressed the effects of changes in the bone remodeling rate by bisphosphonates on the degree of mineralization at the tissue level (Roschger et al. 1997; Boivin and Meunier 2001; Roschger et al. 2001), although some have reported that bisphosphonate treatment increases the mean degree of mineralization in the whole matrix (Boivin and Meunier 2001; Boivin and Meunier 2002). To date, it is not clear what the long-term effects of bisphosphonate administration are on the degree of secondary mineralization in BSUs, and whether or not this change can cause hypermineralization in bone. This chapter introduces a precise Scion Image for accurate evaluation of the degree of secondary mineralization in individual BSUs, which are particularly interesting due to their high bone remodeling rate. Scion Image is an image processing system for evaluating bone mineralization based on contact microradiography, which directly evaluates the degree of secondary mineralization in individual BSUs (Martin et al. 1992; Xu et al. 2000). We introduce this system to investigate the effects of long-term bisphosphonate administration on the mean degree of secondary mineralization in osteons.

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2. Materials and Methods 2.1. Animals and experimental design 2.1.1. Animals and grouping Twenty-nine adult Beagles (7.3–13.0 kg; 1 year old; 15 males and 14 females) were housed at 26°C ± 4°C and 75°C ± 25% humidity in a 12-hour day/night light condition with free access to water and a standard dry diet (400 g/day; Special Diet Services Ltd, Withman, Essex, UK). Animals were allocated randomly into three groups based on their body weight: control group (CON; 5 males and 5 females), low-dose group (LD; 5 males and 5 females), and high-dose group (HD; 5 males and 4 females). 2.1.2. Drug delivery Animals in the CON group were orally given lactose at a dose of 12 mg/kg/day, while those in the LD and HD groups were orally given incadronate disodium (YM-175; Yamanouchi Pharmaceutical Co., Tokyo, Japan) at a dose of 0.3 mg/kg/day and 0.6 mg/kg/day, respectively. These drugs were enwrapped in gelatin capsules (ParkeDavis, Morris Plains, NJ, USA) and given daily 4 hours before morning feeding. The administration procedure lasted for 3 years. 2.1.3. Animal sacrifice and sample collection Animals were sacrificed by an overdose of a sodium pentobarbital derivative (Abbott Laboratories, North Chicago, IL, USA). After necropsy, the left ninth ribs were excised for evaluation.

2.2. Specimen preparation 2.2.1. Specimen preparation for histology After dissection of soft tissues, the excised ribs were fixed in 70% ethanol, dehydrated in graded ethanol, defatted in acetone, and embedded in methyl methacrylate (MMA).a a During this process, attention is paid to avoid either acidic solutions provoking complete or partial demineralization of the samples, or mixtures reacting with the mineral deposits.

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2.2.2. Specimen sectioning About 150-µm-thick undecalcified sections were cut perpendicularly to the orientation of Haversian canals by a diamond microtome saw (SP1600; Leica Institute, Nussloch, Germany), and then were progressively ground to about 100 µm in thickness between two glass plates which were roughened with black silicon carbide powder (grain = 320; ESCIL, Chassieu, France).b The mean thickness of each section was obtained from the measurement at anterior, posterior, medial, and lateral aspects of the section via a high-precision vernier (Mitutoyo, Japan) with an accuracy of less than 1 µm. Finally, the samples were cleaned in a distilled water bath for 5 minutes to remove any abrasive grains. The clean sections for contact microradiography were preserved between two clean glass slides.

2.3. High-resolution contact microradiography Contact microradiography (CMR) was performed with a Sofron X-ray diffraction unit (SRO-M50; Tokyo, Japan). A high-resolution film (SO-343; Kodak, USA) that contacted the section tightly by a thin polyester sheet in a specimen holder was explored for 20 minutes under the condition of 10 kVp and 2 mA, and then was developed in Kodak D19 solution for 5 minutes at 20°C before finally fixing it in fix solution (Hi-RENFIX, Japan) for 5 minutes at 20°C. After being washed and dried in a dust-free room, the film was mounted between a slide and a coverslip.

2.4. Digital images captured from contact microradiography (CMR) The digital CMR images were captured sequentially at 40× magnification using a digital camera system (DXM1200; Nikon, Japan) equipped on a microscope (ECLIPSE E800; Nikon, Japan) that connected a computer with the Image-Pro Plus system (version 4.0011; b

It is important to keep the thickness of each section uniform and consistent.

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Media Cybernetics, Inc.). The computer system was supported by great power manostat so as to keep consistent voltage during the process described below.

2.5. Imaging acquisition After starting up the Image-Pro Plus image system, the Acquire button was opened and the condition under Time Interval was set up at a 10-ms resolution with 1281 × 1025 and 8-bit Gray Scale Acquire. The targeted field of microradiography was previewed and Snap was activated to capture the given field, and then the image was saved as a .TIFF file with an 8-bit Gray Scale Acquire [Fig. 1(a)]. 2.5.1. Selection of region of interest (ROI) Under Adobe Photoshop (version 6.0; Adobe, USA), every matured osteon on the digital images captured by Image-Pro Plus was selected manually by the lasso tool, excluding those osteons that were undergoing bone formation and resorption; and then other fields were deleted. The digital images were saved as .TIFF files for mineralization measurement [Fig. 1(b)]. 2.5.2. Measurement for the degree of mineralization in osteons The degree of mineralization for individual osteons was measured directly by Scion Image (Beta 4.0.2; NIH, USA). 2.5.3. Starting up of Scion Image The Open command in the File menu was used, and a digital image from the image library that was previously saved by Adobe Photoshop was selected. To activate Density Slice under the Options menu, the gray images of osteons were now colored in red because all pixels between lower and upper thresholds were highlighted in red in the Density Slicing mode, while background pixels were left unchanged. The look-up table (LUT) toolbar was used to adjust the red band

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(a)

(b)

Fig. 1. (a) 8-bit grayscale digital image from contact microradiography (25×). (b) Selected osteons, excluding those that were being absorbed or were forming, through Adobe Photoshop. (c) The whole field of each osteon, except the Haversian canal, is chosen by adjusting the look-up table (LUT) under Scion Image. (d) Each osteon is automatically coded when activating Analyze Particles in the Analyze menu of Scion Image.

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(c)

(d)

Fig. 1.

(Continued )

within the grayscale range by dragging the mouse to get the whole field of each osteon, except the Haversian canal [Fig. 1(c)].c c

Importantly, when dragging the red band in the LUT window to adjust brightness and contrast, the measured density values would not change since the LUT (other than the image’s pixel values) is manipulated.

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2.5.4. Thresholding of ROI The Threshold command in the Options menu was clicked to discriminate objects of interest from the surrounding background based on their gray values. The threshold was set to 1, allowing all gray shades above 1 to be visible and all those below 1 to be ignored. Once the threshold was set, the Analyze menu was opened and the Set scale was clicked. The units were changed to pixels, and then OK was clicked. In the Analyze menu, Options was clicked to choose the Area box, Mean Density box, and X-Y Center box as well as to change the Max. The measurements were valued to 8000, the Label Particles box was activated, and the Measurement box was reset; and then OK was clicked. 2.5.5. Activation of Analyze Particles This was achieved in the Analyze menu, where each osteon was automatically coded. Then, Measure and Show Results in the Analyze menu was activated, and the mean area and mean density for every osteon were shown in a text file [Fig. 1(d)]. When activating the Histogram in the Analyze menu, the plot of density could also be displayed dynamically.

2.6. Data normalization In order to normalize the data from the variation of the X-ray exposure for each sample, a calibration aluminum step-wedge was used beside the sample for every X-ray exposure. The aluminum stepwedge was composed of five stepped foils, with 8.9 µm for each foil [Fig. 2(a)]. Aluminum foil was chosen because its atomic number is not far from the effective atomic number of apatite (Boivin and Meunier 2002). The particle density of each step in the aluminum step-wedge beside the sample was measured using the same processes as those for the mean degree of secondary mineralization in osteons [Fig. 2(b)].d Each ROI was measured in the order of d

The step-wedge must be measured in order, starting from the lowest gray value (darkest) to the highest gray value (lightest) standard.

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(a)

(b)

Fig. 2. (a) Calibration aluminum step-wedge and (b) regression curve. The five-stepped aluminum step-wedge beside the sample for every X-ray exposure was used to normalize the data from the variation of the X-ray exposure for each sample.

increasing density until all of the steps were measured. The calibration curve and regression equation of each sample were established on the particle density of the step-wedge. The mean degree of mineralization for each osteon was normalized correspondingly by the mean thickness of the section measured before taking the microradiograph and by the regression equation from the stepwedge data beside the sample.

2.7. Statistical analysis Differences among groups were tested by two-way analysis of variance (ANOVA) using Statview (SAS Institute Inc., Cary, NC, USA). If the overall ANOVA was significant, comparisons between pairs of groups were tested by Fisher’s protected least significant difference test (PLSD), where p < 0.05 was considered statistically significant.

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3. Results 3.1. Mean degree of secondary mineralization The mean degree of secondary mineralization in both LD and HD groups was significantly higher than that in the CON group (p < 0.05 and p < 0.001, respectively); while the difference between LD and HD groups did not reach statistical significance, although the value of mineralization in the HD group was higher than that in the LD group (Fig. 3).

3.2. Distribution of secondary mineralization The data of secondary mineralization for each group was in symmetrical distribution. The degree of mineralization of osteons in the LD and HD groups shifted ordinally towards higher degrees of mineralization concomitantly with a decrease of the lowest degree, and the higher values in both LD and HD groups were more numerous but of a similar magnitude to the highest ones measured in the CON

Fig. 3. Comparison of mean degree of mineralization among the control (CON) group, low-dose (LD) group, and high-dose (HD) group.

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% distribution

30 25 20

CON LD HD

15 10 5 0

1

2

3

4

5

Osteonal degree of mineralization Fig. 4. Distribution of osteons based on degree-of-mineralization comparison among the control (CON) group, low-dose (LD) group, and highdose (HD) group.

group; this trend was more obvious in the HD group (Fig. 4). In addition, the peak values in the mineralization frequency distribution curve did not show any significant difference among the three groups.

3.3. Mineralization frequency distribution The plot of mineralization frequency distribution showed a broader range of the curve in the CON group than in the LD and HD groups (Fig. 4).

4. Discussion In the present study, Scion Image based on contact microradiography was introduced to evaluate the mean degree of secondary mineralization in BSUs of cortical bone. Its validation was testified by evaluating the long-term effects of bisphosphonate (incadronate) on the mean degree of mineralization in osteons.

4.1. Strength of Scion Image analysis compared with clinical bone densitometry Clinically, bone mineral density (BMD) and bone mineral content (BMC) are commonly measured by dual-energy X-ray absorptiometry

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(DXA) for the diagnosis and management of osteoporosis. However, DXA cannot differentiate between changes in bone volume at the organ level or in the degree of mineralization of bone matrix at the tissue level. The same amount of bone tissue having either a high or a low degree of mineralization will correspond to a higher or a lower BMD, respectively. Although peripheral quantitative computed tomography (pQCT) can measure the true volume density in three dimensions, it is still determined by the amount of bone and its degree of mineralization.

4.2. Bone matrix mineralization The mineralization process of bone matrix consists of a rapid primary mineralization on the calcification front that starts 5–10 days after osteoid formation, followed by secondary mineralization, which is a slow and gradual maturation of the mineral component to complete the outer shape of BSUs — osteons in cortical bone and trabecular packets in cancellous bone (Frost 1985; Wu et al. 1998). Primary mineralization can be detected directly in vivo from a histomorphometry parameter — mineral apposition rate (MAR) — using double tetracycline or calcein labeling (Cochran et al. 1994). The degree of secondary mineralization at the tissue level is very difficult to be evaluated. With the rapid development of the electronic industry, the degree of mineralization in local bone can now be measured by backscattered electron imaging (Bloebaum et al. 1997; Roschger et al. 1995; Roschger et al. 1998; Roschger et al. 2001), by small-angle X-ray scattering (Fratzl et al. 1992; Fratzl et al. 1996; Rinnerthaler et al. 1999), and more recently by the microradiographic microdensitometric technique (Boivin and Meunier 2002). Even though the marrow cavity, vascular spaces, and resorption cavities in bone can be excluded by all of the above methods, however, the measured field is still the whole matrix rather than the BSUs, which have a high remodeling rate and are considerably easy to be interfered by therapeutic agents.

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4.3. Scion Image for measurement of bone matrix mineralization Scion Image, as a public domain image processing and analysis program, can be freely downloaded from Scion Corporation. It is a simple and efficient tool for the measurement of particle density. Its validation has recently been testified by its increasing usage in biological science (Xu et al. 2000; Cress 2000), especially in molecular biology where extremely high sensitivity, precision, and accuracy are needed to analyze the density of PCR or RT-PCT products in electrophoresis gel (Masters et al. 1992; Goldfarb 1999; Goldfarb 2001). We introduced Scion Image to evaluate the mean degree of mineralization in osteons at the tissue level by measuring the particle density based on contact microradiography, and its validation was testified by evaluating the long-term effects of bisphosphonate on the degree of mineralization in osteons. Attention should be paid to calibrating data during densitometric analysis. Otherwise, the data collected from different images cannot be used directly for comparison with each other because of the possibility of minute variations in X-ray exposure, film development and fixation, and section thickness and illumination settings when capturing images. In the present study, the measured data on the degree of mineralization for each sample via Scion Image were calibrated correspondingly to the mean thickness of the section, and also to the stepwedge system beside the sample exposed simultaneously to the same X-ray beam. During this processing, other key points should also be adequately noted. First, the electric voltage should be kept consistent whenever capturing images with the Image-Pro Plus image system and doing analysis via Scion Image; therefore, a manostat with great power is an indispensable choice. Only then can the captured images have a consistent condition with the original. Secondly, the digital images captured from contact microradiography should be in good quality. To avoid high contrast and noisy images, the original digital images must be in the full range of 8-bit values (0–255). An appreciation for the grayscale content of the images captured through Image-Pro Plus is necessary. It is most common to examine a

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histogram of the image if the distribution of grayscale values on the x-axis has a range of 0–255. Ideally, the histogram should be spread over the full range of values.

4.4. Comparison of bone mineralization among bisphosphonate-treated bone matrices In the present study, the validity of Scion Image was testified by investigating the effects of long-term bisphosphonate (incadronate) administration on the mean degree of secondary mineralization of osteons in dog cortical bone. The 3-year administration of both low-dose and highdose incadronate significantly increased the mean degree of mineralization in osteons, indicating that incadronate administration effectively suppresses bone turnover, prolongs the duration of mineral apposition on osteons, and subsequently increases the mean degree of mineralization in a dose-dependent manner. In addition, the peak values of mineralization frequency distribution did not show any significant difference among the groups, indicating that long-term treatment with bisphosphonate (incadronate) either at low dosage or high dosage does not cause hypermineralization of matrix tissue even though the degree of mineralization in osteons substantially increases. The increased degree of mineralization by bisphosphonate has also been testified by backscattered electron imaging (Bloebaum et al. 1997; Roschger et al. 1995; Roschger et al. 1997; Roschger et al. 1998; Roschger et al. 2001), smallangle X-ray scattering (Fratzl et al. 1992; Fratzl et al. 1996; Rinnerthaler et al. 1999), and more recently the microradiographic microdensitometric technique (Boivin and Meunier 2002); but the measured field was the whole cortical or trabecular bone. In the adult cortical bone, the mean degree of mineralization depends mainly on the rate of remodeling, that is, the biological determinant of mineralization is the rate of bone turnover (Parfitt 1993; Chavassieux et al. 1997; Boivin and Meunier 2001). Under the condition of increased bone turnover, such as osteoporosis, the activation frequency and birthrate of BSUs are augmented while their lifespan is shortened; therefore, BSUs do not have enough time to reach the full degree of mineralization before being prematurely absorbed by

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osteoclasts and starting a new remodeling cycle. During bone remodeling, old bone is absorbed by osteoclasts and replaced by a new bone matrix which is subsequently mineralized. Therefore, the young bone initially has a lower degree of mineralization than the neighboring old bone. Antiresorptive therapies for osteoporosis, such as bisphosphonates (BPs), substantially reduce bone turnover concomitantly with a decreased activation frequency, a reduced birthrate of BSUs, and a prolonged lifespan of BSUs. Consequently, BP treatment leads to more mature bone in which most BSUs approach an approximately normal level in the degree of mineralization. This has been testified in the present study by long-term incadronate administration, which increased the degree and uniformity of secondary mineralization in dog cortical bone in a dose-dependent manner. The degree of mineralization of the collagenous matrix as well as the size, shape, and arrangement of the mineral particles are crucial parameters that influence the mechanical and functional properties of the whole structure (Wagner and Weiner 1992). Therefore, it is very important to make sure of the correction of mineralization and biomechanical characters of bone for either normal or disordered bone. Increasing the mineralization density can enhance the bending strength of bone; but if the ash content is over 60% by weight, the cortical bone will lose partial ability to absorb impact energy and will be increasingly liable to fracture (Currey 1969). Antiresorptive agent treatment, such as bisphosphonate, reduces bone turnover and gives BSUs enough time to be fully mineralized, thus increasing bone mineral density (BMD) and bone strength. On the other hand, decreased bone turnover also results in microdamage accumulation, which may increase bone fragility and impair the intrinsic properties of bone. With regard to the net effects of long-term administration of these antiresorptive agents on the mineralization, microdamage accumulation, and biomechanical properties of bone, further studies are needed.

5. Summary Scion Image based on contact microradiography is a simple, accurate, and efficient method for evaluating the mean degree of secondary

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mineralization in BSUs. Its application has been validated in an experimental study, where long-term bisphosphonate treatment was found to substantially increase the degree and uniformity of mineralization in osteons, but did not cause hypermineralization of bone tissue.

References Adolphson P, Abbaszadegan H, Boden H et al. Clodronate increases mineralization of callus after Colles’ fracture: a randomized, double-blind, placebo-controlled, prospective trial in 32 patients. Acta Orthop Scand 71:195–200, 2000. Bloebaum RD, Skedros JG, Vajda EG et al. Determining mineral content variations in bone using backscattered electron imaging. Bone 20:485–490, 1997. Boivin G, Meunier PJ. Changes in bone remodeling rate influence the degree of mineralization of bone which is a determinant of bone strength: therapeutic implications. Adv Exp Med Biol 496:123–127, 2001. Boivin G, Meunier PJ. The degree of mineralization of bone tissue measured by computerized quantitative contact microradiography. Calcif Tissue Int 70:503–511, 2002. Chavassieux PM, Arlot ME, Reda C et al. Histomorphometric assessment of the long-term effects of alendronate on bone quality and remodeling in patients with osteoporosis. J Clin Invest 100:1475–1480, 1997. Cochran M, Neville A, Marshall EA. Comparison of bone formation rates measured by radiocalcium kinetics and double-tetracycline labeling in maintenance dialysis patients. Calcif Tissue Int 54:392–398, 1994. Cress AE. Quantitation of phosphotyrosine signals in human prostate cell adhesion sites. Biotechniques 29:776, 778, 780–781, 2000. Currey JD. The relationship between the stiffness and the mineral content of bone. J Biomech 2:477–480, 1969. Fleisch H. Mechanisms of action of the bisphosphonates. Medicina (B Aires) 57:65–67, 1997. Fratzl P, Groschner M, Vogl G et al. Mineral crystals in calcified tissues: a comparative study by SAXS. J Bone Miner Res 7:329–334, 1992. Fratzl P, Schreiber S, Klaushofer K. Bone mineralization as studied by smallangle X-ray scattering. Connect Tissue Res 34:247–254, 1996. Frost HM. The pathomechanics of osteoporoses. Clin Orthop Relat Res 200:198–225, 1985.

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Goldfarb M. Two-dimensional electrophoresis and computer imaging: quantitation of human milk casein. Electrophoresis 20:870–874, 1999. Goldfarb MF. Analysis of casein using two-dimensional electrophoresis, Western blot, and computer imaging. Adv Exp Med Biol 501:535–539, 2001. Martin BR, Prescott WR, Zhu M. Quantitation of rodent catalepsy by a computer-imaging technique. Pharmacol Biochem Behav 43:381–386, 1992. Masters DB, Griggs CT, Berde CB. High sensitivity quantification of RNA from gels and autoradiograms with affordable optical scanning. Biotechniques 12:902–906, 908–911, 1992. Meunier PJ, Boivin G. Bone mineral density reflects bone mass but also the degree of mineralization of bone: therapeutic implications. Bone 21: 373–377, 1997. Parfitt AM. Bone age, mineral density, and fatigue damage. Calcif Tissue Int 53(Suppl 1):S82–S85; discussion S85–S86, 1993. Rinnerthaler S, Roschger P, Jakob HF et al. Scanning small angle X-ray scattering analysis of human bone sections. Calcif Tissue Int 64:422–429, 1999. Rodan GA, Fleisch HA. Bisphosphonates: mechanisms of action. J Clin Invest 97:2692–2696, 1996. Rodan GA, Martin TJ. Therapeutic approaches to bone diseases. Science 289:1508–1514, 2000. Roschger P, Fratzl P, Eschberger J, Klaushofer K. Validation of quantitative backscattered electron imaging for the measurement of mineral density distribution in human bone biopsies. Bone 23:319–326, 1998. Roschger P, Fratzl P, Klaushofer K, Rodan G. Mineralization of cancellous bone after alendronate and sodium fluoride treatment: a quantitative backscattered electron imaging study on minipig ribs. Bone 20:393–397, 1997. Roschger P, Plenk H, Klaushofer K, Eschberger J. A new scanning electron microscopy approach to the quantification of bone mineral distribution: backscattered electron image grey-levels correlated to calcium K alpha-line intensities. Scanning Microsc 9:75–86; discussion 86–88, 1995. Roschger P, Rinnerthaler S, Yates J et al. Alendronate increases degree and uniformity of mineralization in cancellous bone and decreases the porosity in cortical bone of osteoporotic women. Bone 29:185–191, 2001.

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Russell RG, Rogers MJ. Bisphosphonates: from the laboratory to the clinic and back again. Bone 25:97–106, 1999. Turner CH. Biomechanics of bone: determinants of skeletal fragility and bone quality. Osteoporos Int 13:97–104, 2002. Wagner HD, Weiner S. On the relationship between the microstructure of bone and its mechanical stiffness. J Biomech 25:1311–1320, 1992. Wu Y, Ackerman JL, Chesler DA et al. Evaluation of bone mineral density using three-dimensional solid state phosphorus-31 NMR projection imaging. Calcif Tissue Int 62:512–518, 1998. Xu YH, Sattler GL, Edwards H, Pitot HC. Nuclear-labeling index analysis (NLIA), a software package used to perform accurate automation of cell nuclear-labeling index analysis on immunohistochemically stained rat liver samples. Comput Methods Programs Biomed 63:55–70, 2000.

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Chapter 17

Undecalcified Histology in Studying Hard Tissue Implanted with Calcium Phosphate–based Ceramics Chun-Wai Chan and Ling Qin

For the analysis of calcified tissues, nondestructive bioimaging techniques such as X-ray, peripheral quantitative computed tomography (pQCT), and micro-CT are becoming popular to measure their structural architecture and degree of mineralization. However, these techniques do not provide details on the biological changes of tissues. Thus, histology is still an important technique to demonstrate the interaction of mineralized tissue with other soft tissues. A special histological technique known as undecalcified histology or hard tissue histology is used to preserve the calcification information of tissues. Moreover, calcium phosphate–based ceramics has recently been applied in orthopedic research and clinics. Undecalcified histology involving bioceramics is described and discussed, using an experimental rabbit spinal model as an example, to illustrate how to attain good histology. Keywords:

Undecalcified histology; sawing; sectioning; staining; calcium phosphate ceramics.

Corresponding author: Chun-Wai Chan. Tel: +852-26323309; fax: +852-26377889; E-mail: [email protected]

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1. Introduction Histology is a classical technique to study and evaluate tissues and cells microscopically. In hard tissues such as bone, tooth, and pathologically calcified tissue (e.g. calcified blood vessel), a special histological method called undecalcified histology can reserve the bone mineral in tissue matrix, especially under the presence of in vivo injected calcium-binding fluorochromes (e.g. calcein, xylenol orange deposit) into newly formed osseous tissue (Chan et al. 2007a; Chan et al. 2007b). By sequential injection of these fluorochromes at different time intervals, the mineral apposition rate of both remodeled and/or regenerated bone is calculated. Furthermore, research on composite biomaterials and cells-biomaterials is a currently emerging area in tissue engineering and orthopedics. Experimental spinal fusion is a frequently used model because it is clinically relevant to spinal fusion surgery in treating spinal deformity (e.g. scoliosis) (Deviren and Metz 2007; Zarzycki et al. 2005) and degenerative disease (e.g. intervertebral disc degeneration) (Mirza and Deyo 2007; Levin et al. 2007). It also can serve as a study model to compare the efficacy of biomaterials with the gold standard implant, i.e. autograft (Cheng et al. 2002; Guo et al. 2002). To investigate the outcome of biomaterials, both nondestructive bioimaging techniques such as X-ray, peripheral quantitative computed tomography (pQCT), and micro-CT as well as destructive histology are used. The bioimaging techniques utilize the principle of X-ray attenuation by bone mineral and biomaterials. Bone mineral content (BMC), bone mineral density (BMD), and bone microarchitecture can be measured. However, these techniques are not able to reveal the biological changes of tissues. On the contrary, undecalcified histology illustrates the interaction of mineralized tissue with other soft tissue as well as the interface biology of biomaterials and tissue. This chapter describes undecalcified histology, using spinal fusion implanted with calcium phosphate ceramics (CPC), to illustrate how to attain histology-containing calcium phosphate ceramics. The flowchart of the undecalcified histology procedure is summarized in Fig. 1.

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Animal model

Sequential fluorochrome labeling in living animal

Sample harvesting

Fixation

Embedding with resinous methyl methacrylate

Sectioning Thick section 200−500 µm

Thin section 10−15 µm

Sawing serial sections

Micro-X-ray

Heavy-duty microtome Select optimal calcified section with region of interest Goldner trichrome staining

Grinding and polishing 70−100 µm

Fluorochrome labeling analysis

Surface staining: toluidine blue

Fig. 1. Flowchart of undecalcified histology. The flowchart summarizes the procedure of undecalcified histology, starting from the animal model.

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2. Materials and Methods 2.1. Fluorochrome labeling 2.1.1. Materials Fluorochrome is able to bind to calcium and deposit into the bone matrix during bone formation. Several fluorochromes are used to label bone mineralization in the literature (Pautke et al. 2005; Pautke et al. 2007). We describe the commonly used fluorochromes, calcein and xylenol orange, as examples in this chapter. All of the chemicals were purchased from Sigma-Aldrich (St. Louis, USA), unless otherwise specified. (1) Calcein 0.5 g of calcein is dissolved in 80 mL of distilled water. The pH of the calcein solution is adjusted to 7.2–7.4 with sodium hydroxide. The total volume is made up to 100 mL. The final concentration is 5 mg/mL. The dosage is 5 mg/kg of body weight of the animal. (2) Xylenol orange 0.9 g of xylenol orange is dissolved in 80 mL of distilled water, and then the pH of the xylenol orange solution is adjusted to 7.2–7.4 with sodium hydroxide. The total volume is made up to 100 mL. The final concentration is 90 mg/mL. The dosage is 9 mg/kg of body weight of the animal. To sterilize fluorochrome solutions, the filtration set (pore size, 0.45 µm; Corning) is used as a cell culture. 2.1.2. Injection •

After the animal is sedated, the fluorochrome solution is injected subcutaneously on the backside.

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•

253

For large animals, e.g. goat, where the weight is about 30–40 kg, multiple sites of injection can be applied.

2.1.3. Results •

•

• •

After the samples are embedded by resin and sectioned, ground, and polished (these procedures will be described in later sections), the fluorochrome-labeled bone is observed under an ultraviolet fluorescent microscope. The mineral apposition rate is calculated by the distance between two fluorochome-labeled lines over a period of time. It can reflect the bone formation rate (Eriksen et al. 1994; Gabet et al. 2005) (Fig. 2). The fluorochrome-labeled bone area fraction (Lu et al. 2006) can be measured to imply bone mineralization at the injection time (Fig. 3). During bone formation such as spinal fusion, the volume of transverse processes increases gradually. The contour area of fluorochrome-labeled bone reflects the increasing rate of volume of newly formed bone (Fig. 4).

Fig. 2. The dynamic bone formation rate can be reflected by the mineral apposition rate, which is calculated as the distance d between two fluorochomelabeled lines over a known time interval of two injections.

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Fig. 3. The bone is labeled by fluorochromes during mineralization. The fluorochrome-labeled bone area can be measured to imply bone mineralization at the injection time.

Fig. 4. The contour area of fluorochrome-labeled bone reflects the increasing rate of volume of newly formed bone. Xylenol orange and calcein are injected at week 4 and week 6 after spinal fusion surgery of rabbit. (a) The contour area of the xylenol orange–labeled transverse process; and (b) the contour area of the calcein-labeled transverse process.

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2.1.4. Remarks •

•

•

•

•

It is easier to dissolve calcein powder when the pH is adjusted to 7.2. The fluorochrome-containing bottle should be kept in the dark or wrapped in aluminum foil. After xylenol orange injection, the rabbit eye will change to purple color and the urine will contain purple xylenol orange. These are normal phenomena and will fade out in the following days. The fluorochrome is retained in the animal body for 3–4 days. The animal is euthanized after at least 4 days postinjection; otherwise, the soft tissue may still maintain fluorochrome and lead to a blurred fluorescent background in images. There are two sequential labeling methods. The first one is double labeling, in which two dosages of the same fluorochrome are injected within a certain period of time. This method is usually used in rather slow bone formation conditions (e.g. osteoporosis). The second method is to inject several different fluorochromes sequentially in the animal. It is appropriate to use in fast bone formation conditions (e.g. spinal fusion). The fluorescent signal shows not only a clear line, but also a blurred fluorescent area. Double injection of the same fluorochrome is overlapped and cannot be easily distinguished in spinal fusion. After the sample is embedded with methyl methacrylate (MMA) and sawed with a thickness of 200–500 µm, the polychrome-labeled section is appropriate for further grinding to 100 µm and is then polished and observed under fluorescent microscopy.

2.2. Fixation of fusion complex 2.2.1. Materials • • •

4% phosphate buffered formaldehyde Ethanol Vacuum desiccator

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2.2.2. Method •

• • • • •

• •

The spinal segments with bioceramic implants are carefully harvested. Since the implants are usually placed between L5 and L6 transverse processes, a bone cutter is used to cut between L4 and L5. A hard stainless steel rod is inserted into the spinal canal to fix the L5 and L6 vertebrae in order to prevent any breakage of bone tissue. All of the soft tissue is removed. The samples are cut by a wire saw into left and right halves. The specimens are put in a container with fixative. For the cross-linking type of fixative, 4% phosphate buffered formaldehyde and 4% paraformaldehyde, the fixation time is about 1–2 days. For the dehydration type of fixative, 70% ethanol, the fixation time can be longer (e.g. 2–3 days). During the fixation period, the sample can be under a vacuum desiccator for about 1 hour. This can facilitate the infiltration of fixatives into the tissue.

2.2.3. Remarks •

•

•

Mild fixation is optimal for undecalcified histology; otherwise, the tissue will become harder to be sectioned. Thus, the fixation is quite short at about 1–2 days. Mild fixation is also optimal for immunohistochemistry after sectioning. The infiltration of the fixative, ethanol, and embedding materials is essential for the following step of sectioning. The sample under vacuum can extract any air bubble out of the tissue. After releasing the air pressure, the surrounding solution infiltrates into the space. However, the vacuum step should be done after initial fixation; otherwise, loose tissue (e.g. bone marrow, fatty tissue) may be extracted out from the bone. For an intact bone, the infiltration rate is very slow, about 1 mm/day. The unimportant region of the bone specimen can be removed to expose the interior tissue with the region of interest. The

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surrounding solution easily infiltrates into the deeper zone of the specimen.

2.3. Embedding 2.3.1. Materials (1) Preparation of water-free methyl methacrylate (MMA) This is for the removal of inhibitor in the stock MMA (mainly hydroquinone). Washing solution is used to extract it from MMA by partition separation. All of the procedures are performed in a fume hood. • • • •

• • • • • •

The washing solution contains 200 g of sodium chloride and 50 g of sodium hydroxide per 1 L of distilled water in a separating funnel. 550 mL of MMA is poured into the funnel [Fig. 5(a)], and then 300 mL of washing solution is measured into the funnel. The MMA with washing solution is mixed vigorously [Fig. 5(b)]. The solution is left to stand for about 5 minutes until the solution is separated into an upper MMA layer and a lower washing solution layer [Fig. 5(c)]. The lower layer is drained into the waste bottle for aqueous waste of MMA. The above steps are repeated two more times to add washing solution (three extractions in total). Distilled water is added instead of washing solution for three times. 60-g CaCl2 pellets or large granules are added to the MMA solution to dehydrate the remaining water. MMA is filtered with filter paper, and the water-free MMA is drained into the water-free MMA bottle. The water-free MMA bottle (about 500 mL) is sealed with parafilm before storing at 4°C.

(2) Preparation of dry benzoyl peroxide •

Benzoyl peroxide should be kept in moisture (about 25% H2O) for long-term storage in a refrigerator.

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(a)

(b)

(c)

Fig. 5. (a) Methyl methacrylate (MMA) is transferred to a pear-shaped separating funnel in a fume hood. (b) The whole system is mixed well to dissolve the MMA inhibitors into sodium hydroxide wash solution. (c) The whole system is left to stand for about 5 minutes. The solution is separated into an upper MMA layer and a lower washing solution layer.

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Before using it as a catalyst of polymerization, benzoyl peroxide should be dried in a dessicator with desiccate. The whole setup should be stored in a dark drawer. It usually takes at least 3 days for 12 g of benzoyl peroxide.

(3) Preparation of MMA solution •

• • •

MMA solution I: 60 mL of water-free MMA solution is added with 35 mL of butyl methacrylate (stored at 4°C), 5 mL of methyl benzoate, and 1.2 mL of polyethylene glycol 400. MMA solution II: 100 mL of MMA solution is added with 0.4 g of dry benzoyl peroxide. MMA solution III: 100 mL of MMA solution is added with 0.8 g of dry benzoyl peroxide. Polymerized MMA solution (PMMA, freshly prepared): 100 mL of MMA solution III is added with 400 µL of N,N-dimethyl-ptoluidine.

2.3.2. Method • •

All of the solutions should cover the specimen. After fixation, dehydration and embedding (for 3 cm × 3 cm samples) are listed as follows:

Table 1. Undecalcified histology tissue processing of fixed specimen in resinous methyl methacrylate. Reagent

Days per change

Conditions

70% ethanol

3 days

95% ethanol

3 days

100% ethanol

3 days

Xylene

3 days

MMA I MMA II MMA III

1 day 1 day 1 day

Room temp., vacuum intermittently twice a day, refresh every day Room temp., vacuum intermittently twice a day, refresh every day Room temp., vacuum intermittently twice a day, refresh every day Room temp., vacuum intermittently twice a day, refresh every day Store at 4°C, vacuum intermittently twice a day Store at 4°C, vacuum intermittently twice a day Store at 4°C, vacuum intermittently twice a day

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Freshly prepared 100 mL of MMA solution III and 400 µL of N,N-dimethyl-p-toluidine are transferred to specimens. Nitrogen gas is sprayed on the surface of the PMMA solution for more than 2 minutes to expel oxygen, and then the samples are mounted with parafilm. The samples can be put in an ice bath to prevent overheating of samples. The samples are put in an air-tight plastic container in a −20°C freezer. The solidification will be completed in about 2–4 weeks.

2.3.3. Remarks •

•

•

• •

•

•

Sometimes, the washing solution becomes a dark brown to light brown color after the first separation step. It is a normal phenomenon because the inhibitor dissolves in washing solution. Excess calcium chloride granules are required for the dehydration of MMA. Calcium chloride powder is not effective to absorb moisture in MMA. Using insufficient calcium chloride granules or a powder form will cause calcium chloride crystals to form. An MMA-resistant plastic container or glass container should be used. A plastic specimen cup composed of polypropylene or highdensity polycarbonate is suitable for MMA embedding. Do not let the sample solidify at room temperature, since the filtration of MMA into the samples is not yet completed. Water and oxygen will hinder the polymerization of MMA. Dehydration and spraying of nitrogen gas should be thoroughly done. To make sure of the completeness of solidification, PMMAembedded samples can be stored from a −20°C freezer to room temperature for 1 more week. Safety guide of MMA


MMA is a resin that releases an irritating and choking smell. It is toxic with high concentration in closed working areas. All MMA experiments should be done in a fume hood.

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In case the MMA smell is found in the lab, open the windows and let fresh air come into the lab. MMA is flammable. Dry benzoyl peroxide is extremely flammable; it can explode by naked fire or electric spark. Do not turn on any electrical appliances when using benzoyl peroxide. Put the benzoyl peroxide in a cold drawer with desiccate during dehydration. The polymerization of MMA is exothermic and an increase in temperature also accelerates the reaction, so polymerization is a chain reaction. Vigorous polymerization builds up a high pressure in the containing bottle. The pressure can break this glass bottle. Normally, MMA I, MMA II, and MMA III are relatively stable at 4°C; and the storage can be in an MMA-resistant plastic bottle or glass bottle. Do not leave the MMA bottle at room temperature overnight. MMA can be reused in the steps for MMA I and MMA II. The more impurities there are in reused MMA or MMA waste, the more easily polymerization may occur. The reused MMA and MMA waste should be stored in an MMA-resistant plastic bottle and put in an air-tight large container (double package) at 4°C immediately after use. MMA waste should be added with benzoyl peroxide for solidification in a −20°C freezer as part of the preparation of PMMA.

2.4. Sectioning 2.4.1. Materials • • • • • •

261

Saw microtome [Fig. 6(a)] Band cutting machine [Fig. 6(b)] Grinder/Polisher [Fig. 6(c)] Polycut [Fig. 6(d)] Different grades of grinding paper Gelatin-coated glass slide

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(a)

(b)

Fig. 6. The equipments and accessories for undecalcified histology. (a) The saw microtome machine is used for cutting thick sections (200–500 µm). (b) The band cutting machine is used for cutting large samples (more than 300 µm). (c) The grinder/polisher is used to grind thick sections and then polish the surface for observation under a microscope. The sections can be stained by surface staining such as toluidine. (d) The polycut machine is used to cut MMA samples 15 µm thick.

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(c)

(d)

Fig. 6.

(Continued )

2.4.2. Method After the bone samples are embedded, there are two ways of sectioning: sawing and cutting.

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(1) Sawing The embedded samples have to be trimmed around the excess surrounding solid medium to make the plane of the sample base parallel to the section plane. The section plane should include all of the tissue regions of interest (ROIs). (If the section is further cut using Polycut, then the surrounding MMA medium should leave about 5 mm outward from the samples.) • •

•

•

• • • •

Grinding can be used to flatten the base of the sample. The trimmed samples can be fixed either on stands by cyanoacrylatebased glue in a saw microtome or on a clamp in a contact point sawing machine. The samples can be cut by saw microtome using a circular saw (Leica SP1600; Leica, Germany) or a contact point sawing machine (Exakt, Denmark). For every sectioning step, the samples are adjusted according to the thickness of the section plus the thickness of the saw. For example, for a 500-µm-thick section, adjust 800 µm of the sample upwards in a saw microtome (500-µm-thick section plus 300-µm-thick saw). Sample contact on the operating saw should be slow and gentle to let the sample be sawed. Excess water or coolant should be used to minimize heating during sawing. A heating effect makes the section curve. Serial sections can be obtained for the same thickness of section and labeled with numbers. For some samples, newly formed bone cannot be easily identified. Microradiography of the serial sections is taken by using a highresolution X-ray film (Agfa) (40 MeV, 20 minutes). The calcified tissue can be examined in radiography. The optimal section can be subjected to surface staining or further sectioning by heavy-duty Polycut (SE 2600; Germany).

Remarks: •

During sawing, the thickness of samples is decreased; otherwise, in cases where the samples are relatively small, serial sections cannot be obtained.

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•

•

265

The section plane is very important for histological sectioning. The ROI should be shown in one section, preferably with positive or negative control tissue. For spinal fusion samples, two L5 and L6 transverse processes should be shown in one section. The sagittal section plane perpendicular to the transverse process is used. Excess water is used to reduce the heating effect; otherwise, the histological section will be curved. If the section is bent, it can be flattened by putting it between two flat metals and transferring it to an oven under 50°C–60°C for 4–6 hours, and then cooling down the whole setup to room temperature. The sawed section is too thick to be observed under a microscope and also too coarse on the surface. Therefore, grinding and polishing should be performed for histology.

(2) Cutting • • • •

• •

•

• •

The samples to be sectioned can be a whole sample or a thick section (300–500 µm). The sections or samples can be fixed by the machine holder or stuck on a custom-made holder using cyanoacrylate-based glue. About 5 mm of MMA from the tissue is reserved for Polycut. After fixing the sample in the Polycut, the leveling of the sample is adjusted well by bubble level. This can help to reduce trimming. The control panel is operated to move the sample for sectioning. DPX mountant is spread on the surface of calcium phosphate ceramic (CPC)-implanted samples. After the mountant is dried up, sectioning can begin. During sectioning, 70%–100% ethanol can be added onto the surface of the sample for lubrication to soften the surface of the sample. For hard MMA samples (no additive), 100% ethanol is used. For soft MMA samples (butyl methacrylate or glycol methacrylate is added), higher-grade ethanol will make the section too soft for handling, so 70%–90% ethanol can be used.

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A gelatin-coated glass slide is added with drops of water to dissolve the gelatin. The section is put on top of the water on the glass slide and left to stand for about 1 minute. Excess water is removed, and then excess 100% ethanol is added to soften the section. The section is gently spread by a brush, and then a polyethylene film is placed on top. Excess ethanol is removed by squeezing. The glass slide is put between two plastic glass plates (same size as the glass slide). The section is placed on a metal press and then in an oven at 60°C–70°C overnight. The section is taken out after the whole setup is cooled down on the following day.

Remarks: •

•

For Polycut sectioning, the time-consuming part is the setting of the sample and the trimming. The leveling of samples can help to reduce trimming time. The sections range in thickness from 10 µm to 15 µm for Polycut.

2.5. Surface staining for thick sections (200–500 µm) — toluidine blue staining 2.5.1. Materials •

Toluidine stain solution Toluidine blue (C. I. 52040), 0.3 g Sodium carbonate, 2.5 g dH2O, 100 mL

•

0.7% formic acid

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2.5.2. Method Table 2.

Toluidine blue staining.

Description 0.7% formic acid Toluidine blue stain 70% EtOH for differentiation 95% EtOH 100% EtOH Air dry

Time 10 min × 2 30 min Rinse ~15–30 s Rinse ~15–30 s Rinse ~15–30 s

2.5.3. Results Different shades of blue → Calcified bone cells and soft tissue Metachromatic red-purple → Cartilage and mast cell granules Dark blue → Calcified cartilage •

•

The toluidine blue signal is more intense in newly formed bone because the extent of calcification is relatively less in newly formed bone than in intact original bone [Fig. 7(a)]. After toluidine blue staining, the number of osteocytes can be counted over the blue background of the bone matrix. This reflects the cellular activity of newly formed bone [Fig. 7(b)].

2.5.4. Remarks • •

The stain is effective within 2 weeks. Toluidine blue can bind negatively charged tissue. The slight decalcification by formic acid can increase the blue color in the section. The articular cartilage also exhibits an intense blue-topurple color because it is composed of high, negatively charged proteoglycan.

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(a)

(b)

Fig. 7. Toluidine blue is used to stain the surface of MMA sections. (a) The bony tissue of the rabbit spinal fusion model is shown in blue. The black hydroxyapatite/tricalcium phosphate implant is located on top. (b) The number of osteocytes can be counted after toluidine blue staining. This reflects the bone formation activity.

2.6. Staining for thin sections (10–15 µm) — Goldner’s trichrome stain 2.6.1. Materials (1) Weigert’s hematoxylin Solution A Hematoxylin, 10 g Distilled water, 1000 mL (Ripened for at least 2 weeks before use)

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Solution B Ferric chloride (hydrated), 11.6 g Distilled water, 1000 mL 2% hydrochloric acid, 10 mL •

For use, mix equal proportions of solutions A and B immediately before required. Do not keep premade working solution.

(2) Ponceau 2R/acid fuchsin Ponceau 2R (Xylidine Ponceau 2R) (Sigma P2395, C. I. 16150), 1.5 g Acid fuchsin, 0.5 g Acetic acid (glacial), 2 mL Distilled water, 98 mL (3) Azophloxine (acid red 1, C. I. 18050) Azophloxine, 0.5 g Acetic acid (glacial), 0.6 mL Distilled water, 99.4 mL (4) Ponceau/acid fuchsin/azophloxine (working solution) Ponceau 2R/acid fuchsin, 12 mL Azophloxine, 8 mL 0.2% acetic acid, 80 mL (5) Phosphomolybdic acid/orange G Phosphomolybolic acid, 6 g Orange G, 4 g Distilled water, 1000 mL (6) Light green Light green, 2 g Acetic acid (glacial), 2 mL Distilled water, 1000 mL

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2.6.2. Method • • • • • • • • • • • • • • • •

The MMA section (10–15 µm thick) is put in acetone for 30 minutes two times. After removal of MMA, the section is transferred to a low concentration of ethanol to hydrate the tissue. The section is then transferred to water for 15 minutes two times. The section is placed in Weigert’s hematoxylin for 20 minutes. The section is washed in water. The section is differentiated with 0.5% acid alcohol. The section is washed in water for 20 minutes. The section is placed in Ponceau/acid fuchsin/azophloxine for 5 minutes. The section is rinsed in 1% acetic acid for 10 seconds. The section is placed in phosphomolybdic acid/orange G for 20 minutes. The section is washed in water for 20 minutes. The section is placed in light green for 5 minutes. The section is rinsed in water. The section is blotted dry. The section is transferred to a high concentration of ethanol. The section is mounted with DPX.

2.6.3. Results Tissues are stained in different colors as shown in Table 3. Table 3. Result of stained tissue by Goldner’s trichrome staining. Tissue Calcified bone Calcified cartilage Osteoid Nucleus Cytoplasm Erythrocyte

Color Bright green Pale green Red Blue-black Reddish brown Orange-red

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Fig. 8. Goldner’s trichrome stain is used to stain the unmineralized bone matrix in red and the mineralized bone in greenish blue. Bone formation is observed when the osteoid is accompanied by active bone-lining cells; while bone resorption is observed when the osteoid is without bone lining-cells, and sometimes with a resorption pit by osteoclastic activity.

2.6.4. Remarks •

•

•

Goldner’s trichrome stain distinguishes osteoids in red color (noncalcified bone matrix) from the bony tissue (Fig. 8). However, red osteoids alone cannot imply a noncalcified bone matrix under bone formation or resorption. To identify an osteoid in the bone formation stage, active bonelining cells (columnar shape with highly intense nucleus) are attached on the osteoid. To identify an osteoid in the bone resorption stage, less active or inactive bone-lining cells (spindle-shaped and less nuclear stain) are found. Sometime, a resorption pit by osteoclasts may be observed.

2.7. Contact microradiography 2.7.1. Materials •

High-resolution X-ray film (Struct D4 Pb VacuPac; Agfa-Gevaert Group, Mortsel, Belgium)

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X-ray machine Image analysis system (MetaMorph version 4.6; Molecular Devices Corp., Sunnyvale, CA, USA)

2.7.2. Method • • • •

After the MMA samples are cut by saw microtome into 200– 500 µm thick, the sections are put on a high-resolution X-ray film. The aluminum step-wedge (Fig. 9) is placed adjacent to the sections. A low energy of X-ray (40 kV, 20 minutes) is used to expose the sections. The X-ray is developed.

2.7.3. Results •

The mineralized tissue is shown in the X-ray film with aluminum step-wedge (Fig. 10).

Fig. 9. Contact microradiography. The aluminum step-wedge is used for the calibration of radio-opacity of different X-ray films and also for the analysis of standard length of dimensions such as bone volume. The design is shown in different views: (a) front view, (b), side view, and (c) back view.

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Fig. 10. Microradiograph of serial sections of the rabbit spinal fusion segment implanted with hydroxyapatite/tricalcium phosphate block. The aluminum step-wedge is shown in the middle.

• •

The bone volume density (i.e. bone volume over tissue volume) can be measured to demonstrate the bony formation. In the spinal fusion model, the intertransverse process gap distance can be measured to show the progress of spinal fusion (Cheng et al. 2002; Guo et al. 2002).

2.7.4. Remarks •

•

The section should be flat to put on the X-ray film. During sawing, the section may become curvy, especially with soft tissue. To flatten the section in such cases, the plastic section is gently put on a metal press and in an oven at 60°C for 2–3 hours, and then the metal press with the section is cooled down to room temperature. The MMA samples are usually cut into serial sections. After contact microradiography is taken, the desirable section (e.g. the middle region of samples or the section containing abundant bony tissue) can be selected for further thin-sectioning by Polycut.

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3. Summary Undecalcified histology is an essential histological technique to study hard tissue at the cell-to-tissue level. It can also be used to quantify cell and matrix content by histomorphometry analysis. By the method of sequential fluorochrome labeling, dynamic histomorphometrical analysis can be performed to assess the bone formation rate. Moreover, it is also used to study one-biomaterial interface biology.

Acknowledgments This study was supported by the AO Research Fund of the AO Foundation, Switzerland (Ref. No. 03-C40 and S-06-89C), and by the Li Ka Shing Institute of Health Sciences, Hong Kong.

References Chan CW, Qin L, Lee KM et al. Bio-engineered mesenchymal stem cell– tricalcium phosphate ceramics composite augmented bone regeneration in posterior spinal fusion. Key Eng Mater 334–335:1201–1203, 2007a. Chan CW, Wong KHK, Lee KM et al. Can basic fibroblast growth factor pre-treatment enhance mesenchymal stem cell therapy in undecorticated posterior spinal fusion? Key Eng Mater 330–332:1137–1140, 2007b. Cheng JCY, Guo X, Law LP et al. How does recombinant human bone morphogenetic protein-4 enhance posterior spinal fusion? Spine 27(5):467–474, 2002. Deviren V, Metz LN. Anterior instrumented arthrodesis for adult idiopathic scoliosis. Neurosurg Clin N Am 18(2):273–280, 2007. Eriksen EF, Axelrod DW, Melsen F (eds.). Bone Histomorphometry. Raven Press, New York, 1994. Gabet Y, Kohavi D, Muller R et al. Intermittently administered parathyroid hormone 1–34 reverses bone loss and structural impairment in orchiectomized adult rats. Osteoporos Int 16(11):1436–1443, 2005. Guo X, Lee KM, Law LP et al. Recombinant human bone morphogenetic protein-4 (RhBMP-4) enhanced posterior spinal fusion without decortication. J Orthop Res 20:740–746, 2002.

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Levin DA, Hale JJ, Bendo JA. Adjacent segment degeneration following spinal fusion for degenerative disc disease. Bull NYU Hosp Jt Dis 65(1):29–36, 2007. Lu HB, Qin L, Fok PK et al. Low intensity pulsed ultrasound accelerates bone-tendon-junction healing — a partial patellectomy model in rabbits. Am J Sports Med 34(8):1287–1296, 2006. Mirza SK, Deyo RA. Systematic review of randomized trials comparing lumbar fusion surgery to nonoperative care for treatment of chronic back pain. Spine 32(7):816–823, 2007. Pautke C, Tischer T, Vogt S et al. New advances in fluorochrome sequential labelling of teeth using seven different fluorochromes and spectral image analysis. J Anat 210(1):117–121, 2007. Pautke C, Vogt S, Tischer T et al. Polychrome labeling of bone with seven different fluorochromes: enhancing fluorochrome discrimination by spectral image analysis. Bone 37(4):441–445, 2005. Zarzycki D, Winiarski A, Makiela G et al. Early outcome in the surgical treatment of idiopathic scoliosis by “bone on bone” anterior interbody fusion. Ortop Traumatol Rehabil 7(2):137–142, 2005.

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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

The use of micro-computed tomography (micro-CT) in orthopedic research has flourished in recent years. It has been shown to be an objective measurement of the trabecular bone structure and a useful tool in the study of osteoporosis. With its application base in osteoporosis, the present chapter introduces a protocol for work on other orthopedic conditions such as fracture healing, spinal fusion, and anterior cruciate ligament tunnel healing. In addition to the quantification of structural parameters of mineralized tissue, protocols for the quantification of blood vessels and porous structure of biomaterials are also presented. Keywords:

Micro-CT; osteoporosis; bone mineral density; fracture healing; spinal fusion; vasculature.

1. Introduction The use of micro-computed tomography (micro-CT) in orthopedic research has flourished in recent years. In the field of osteoporosis, the Corresponding author: Hiu-Yan Yeung. Tel: +852-26323309; fax: +852-26324618; E-mail: [email protected]

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application of micro-CT has given a new dimension to researchers in terms of clinical observations of bone and measurement of bone mineral density (BMD). Nowadays, one of the most important parameters in osteoporosis research is the microstructure of the trabecular bone. It has long been recognized that osteoporosis is a disease associated with not only the loss of bone mineral content, but also the loss of the structural element of the trabecular bone. The microarchitecture of trabecular bone reflects its biomechanical strength. With an increase in scanning resolution up to the micron level, micro-CT provides an assessment of the three-dimensional (3D) images of the trabecular bone microstructure with objective quantification. This gives a more comprehensive evaluation to the bony network when compared with conventional histology, which is based on two-dimensional (2D) images with stereological estimation of the 3D structure. The trabecular bone structure is a dynamic scaffold within an anatomical site, as bone modeling and remodeling occurs throughout one’s life. Therefore, a better quantification of the bone structure is to evaluate the structure directly on the 3D image, which can be done through micro-CT and the 3D bone structure evaluation program. This chapter provides micro-CT evaluation protocols established for the analysis for animal studies using different species and scaffold biomaterials developed for orthopedic applications.

2. Materials and Methods 2.1. Basic key operation for trabecular bone structural analysis 2.1.1. Micro-CT scanner and workstation The micro-CT procedures described below are based on the µCT40 system from SCANCO Medical AG, Bassersdorf, Switzerland. 2.1.2. Phantom calibration for mineral density protocols Calibration with the phantom should be done weekly to keep track of the performance of the scanner and the high precision of the density measurement.

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Before scanning the phantom for mineral density calibration, a special scanning profile has to be created to tell the computer to analyze multiple regions of interest (ROIs) at the same time. For details of the profile setup, the scanner manufacturer will provide a guideline as the setup may be dependent on the phantom and the scanner configuration. Basically, it is the correlation of the series of standardized known mineral content of the phantom with the corresponding X-ray attenuation values. For regular phantom scanning, the procedure is no different from any regular scanning protocol of a sample. The number of slices for the phantom scan is dependent on the size of the detector. It is necessary to check with the manufacturer about the number of slices that a scanner can take in one turn. After the phantom image acquisition, evaluation of the X-ray attenuation values is done and is correlated with the known mineral content of the individual phantom by linear regression. The system will record the new calibration curve. 2.1.3. Sample preparation In general, the sample preparation step of hard and soft tissues for micro-CT is dependent on the subsequent experiment. The choices of medium for the sample to soak into are saline, neutral buffer, formalin solution, and ethanol. It is suggested that foam can be used to fix the sample inside the sample-holding tube so that, when the sample tube is inverted, the sample will stay in place without any movement.

2.2. Definition of micro-CT measurement parameters The following are the definitions of the parameters shown in a 3D evaluation report, which has been adopted from a previous study (Hildebrand et al. 1999). (1) Primary indices •

BS: bone surface. It is the total surface area of bone within the volume of interest (VOI).

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BV: bone volume. It is the volume of bone within the VOI. TV: total volume. It is the whole VOI volume, including the mineralized tissue and other tissues as well as the void volume. BV/TV: bone volume fraction. It is the percentage of mineralized tissue within the total volume of the VOI (TV). BS/TV: bone surface area fraction within the total volume of the VOI (TV)

(2) Traditionally derived indices •

•

•

Tb.Th*/Tb.Th: trabecular bone thickness. It is derived from two methods. One is based on the plate model with the application of the stereological technique, while the other is a direct method that determines the thickness by filling maximal spheres into the structure with the distance transformation. Tb.Sp*/Tb.Sp: thickness of the marrow cavities or the separation of trabecular bone. It can be derived either from the marrow surface-to-volume ratio, or from the calculation with the same procedure as that used for Tb.Th but with the voxels as the nonbone parts. Tb.N*/Tb.N: number of plates/trabecular bones per unit volume. It can be derived from the inverse sum of Tb.Th and Tb.Sp. From the direct method, it is taken as the inverse of the mean distance between the indices of the observed structure.

(3) Directly assessed nonmetric indices • •

•

SMI: structure model index. It is an estimation of the platerod characteristic of the structure. DA: geometrical degree of anisotropy. It is the ratio between the maximal and minimal radii of the mean intercept length (MIL) ellipsoid. Conn.D: connectivity density. It is calculated by the ConnEuler method of Odgaard and Gundersen (1993).

3. In Vitro Study In general, for in vitro studies, it is important to know the anatomical orientation of samples before the samples are put into the sample

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tube for scanning. If a particular ROI is well known before the scanning, it is recommended to place the sample such that the ROI is best aligned with the orthogonal axis; this will greatly help in selecting the right VOI during subsequent analyses.

3.1. Fracture healing The following example of fracture healing is based on the rat closed fracture model. A midshaft transverse fracture was observed in the right tibia of each rat after the insertion of an intramedullary Kirschner wire (Bonnarens and Einhorn 1984).The animals were sacrificed at different time points for micro-CT evaluation of the fracture callus.

3.1.1. Objective •

To visualize and evaluate the external callus formation and the degree of mineralization during the healing process

3.1.2. Sample preparation and scanning • • • •

Remove the internal fixation if necessary. Fix the harvested femur vertically along the longitudinal axis of the sample tube for micro-CT scanning. Make sure that the scan range covers the whole segment of the external callus with a resolution of 16 µm. Carry out the scan with isotropic voxels. The X-ray energy is 70 kV and 102 mA.

3.1.3. Analysis procedure • •

Contour the callus with ROI iteration to define the outer surface of the callus. Perform 3D reconstruction using a low-pass Gaussian filter (sigma = 1.2; support = 2).

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Define the highly mineralized osseous tissue and normal mineralized tissue by obtaining the threshold values of the proximal end of the cortical bone of the sample and the trabecular bone of the proximal tibia, respectively. Use the threshold values obtained in step 3 and an additional threshold value lower than that of the trabecular bone for the segmentation of three different degrees of mineralized callus (e.g. cortical bone threshold value, 325; trabecular bone threshold value, 155; and low degree of mineralized callus, 120).a Reconstruct the callus with the different threshold values for a simple qualitative analysis. With the 3D direct evaluation method, the callus extracted from the different threshold values is evaluated as the standard histomorphometry (Fig. 1). Obtain bone volume (BV) with different degrees of mineralization by subtracting different BVs from different threshold values.

Fig. 1. Rat fracture callus after 4 weeks of healing. The yellow color is the callus with low mineral density, while the orange color is the original cortical bone and the high mineral density tissue formed around the fracture site. a

Please note that these values are dependent on the individual micro-CT systems of different brands and require adjustment for the individual research centers.

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For the lowest mineralized tissue, BV is calculated by subtracting the volume of the lowest-threshold tissue from that of the medium-threshold tissue. For medium-mineralized tissue, it is simply the difference in volume between medium-threshold and high-threshold tissues. The BV from different threshold values is then normalized with the total volume (TV) shown in the histomorphometry report. Isolate the callus with different densities. Normal, direct 3D histomorphometry analysis is then provided by the micro-CT system.

3.1.4. Data interpretation The gross morphology of different densities of the fracture callus can be used for a descriptive qualitative analysis of fracture healing. High-threshold tissue represents the original cortical bone and callus with high mineral content. Medium-threshold tissue represents a better mineralized and mature callus as compared to the lowthreshold tissue. Low-threshold tissue represents a newly formed osseous tissue at the fracture site and a partially mineralized osseous tissue. A detailed report on the histomorphometry will give further quantitative parameters for comparison between different treatments (Shi et al. 2007). 3.1.5. Parameters •

• •

•

Normalized bone volume (BV) of different thresholds: the percentage of osseous tissue with different degrees of mineralization within the callus Tissue volume (TV): the volume of the callus formed during fracture healing Trabecular bone thickness (Tb.Th*): the thickness of the trabecular bone in the callus with different densities during the healing phase, as the callus is undergoing an active remodeling toward the formation of cortical bone at the fracture site Mineral density (material) (mg HA/ccm): the BMD of different threshold calluses

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3.1.6. Limitations •

•

•

During the healing of this closed fracture model, it is known that endochondral ossification is the major mode of bone formation within the callus. Conventional histomorphometry under microscope is complementary to micro-CT analysis for the evaluation of the cartilaginous tissue. Τhe BMD of the callus at different levels may reflect the degree of mineralization within different regions of the callus. However, this may be affected by the initial threshold value set for the segmentation. During the fracture healing, new bone formed through endochondral ossification at the fracture site consists mainly of trabecular bone (Einhorn 1995; Einhorn 1998; Yeung et al. 2002; Yeung et al. 2001). The callus undergoes rapid remodeling within a few months. Therefore, the change of the trabecular bone should be observed as a transitional phenomenon and cannot be interpreted as conventional histomorphometry. It reflects the potential for bone formation at the fracture site. The potential for remodeling to return to the normal cortical bone may be different, as the remodeling process involves bone formation and bone resorption.

3.2. Spinal fusion This protocol is based on the rabbit posterior spinal fusion model (Boden et al. 1995; Guo et al. 2002). If a different species is used for the spinal fusion model, modification of the sample preparation and the scanning parameter may require adjustment accordingly. 3.2.1. Objective •

To evaluate the volume of bone formed at the intertransverse process region between lumbar spine L5 and L6 as well as the fusion gap

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3.2.2. Sample preparation •

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After euthanasia, dissect the experimental animal from the dorsal side for sample harvesting. For the rabbit model, the fusion site is usually at the L5 and L6 levels due to the initial model establishment (Boden et al. 1995). Carefully remove the muscles and the three spinous processes at the dorsal side of the spine without disturbing the implant and the fusion site. At the midsagittal plane of the vertebral bodies, cut the sample into two halves and trim off the long transverse process with a saw without disturbing the implant. Place the sample in 4% buffered formaldehyde solution overnight. Change the fixative to 70% alcohol. The sample in 70% alcohol is ready to be scanned in micro-CT.

3.2.3. Scanning procedure • • • •

Use a sample tube 36 mm in diameter and a resolution of 36 µm. Fit two samples into the tube and make the transverse processes of the two samples aligned to the same plane. Carry out the scan with isotropic voxels. The X-ray energy is 70 kV and 102 mA. Preview the scan to double-check the positioning of the sample along the long axis of the sample tube. Scan the whole implant from top to bottom with an appropriate margin so that the new bone is formed around the ends.

3.2.4. Data analysis (1) Gap distance of the fusion mass between two transverse processes • •

Use the VOI (Volume of Interest Only) command to start the 3D reconstruction of the whole fusion mass. Contour the whole implant and the transverse process. Set the breakpoint at a regular interval for morphing the volume of interest.

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• • • • •

•

Morph between the breakpoints to create the volume of interest. Use the 2D Evaluation program to set the optimal segmentation parameters for all three parameters (sigma, support, and threshold). For example, in our samples, sigma is 1.2; support, 2.0; and threshold, 153. Correct the “IPL_VOI_ONLY.COM” file with the segmentation parameters. Click the 3D Evaluation program in the VOI window and press Start Evaluation. After completion of the 3D reconstruction, view the 3D image with the built-in 3D Image Viewer. Rotate the image to a suitable plane so that the viewer is looking at the image from the right ventral side. While pressing the Shift button, point to the fusion mass where the two points are closest together to obtain the x-, y-, and zcoordinates. The two sets of (x,y,z) are used for the calculation of the distance. Alternatively, export the best dorsal view of the fusion mass parallel to the axial plane to a .TIFF-format picture, and further analyze it with a simple image analysis program for the gap distance of the fusion mass developed from the transverse processes.

(2) Fusion mass volume at the fusion site • •

•

Use the VOI (Volume of Interest Only) command to start the 3D reconstruction of the whole fusion mass. Examine the fusion mass and use the cursor with the Shift button to point to the fusion mass in order to obtain the x-, y-, and z-coordinates for the most proximal and distal ends of the fusion mass. These coordinates provide an approximation of which slices of 2D images are the beginning and end of the fusion mass. Redraw the contour for the fusion mass with the Iteration function in the Contouring function to generate a new fusion mass .GOBJ file.

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Use the Evaluation program to recalculate the fusion mass with the normal, direct 3D histomorphometry analysis.

3.2.5. Data interpretation • • • • •

Fusion gap: the degree of fusion made Fusion mass volume (TV): the volume of bone formed at the fusion site Bone volume fraction (BV/TV): the bone volume density within the fusion mass Mineral density (apparent): similar to the BV/TV value, indicating the amount of bone mineral of the fusion mass Mineral density (material): the degree of mineralization of the fusion mass

3.3. Anterior cruciate ligament (ACL) tunnel healing This protocol uses a rabbit ACL reconstruction model (Anderson et al. 2001; Wang et al. 2005). 3.3.1. Objective •

To evaluate the bone ingrowth in the tunnel and the volumetric changes of calcium phosphate injection into the tunnel

3.3.2. Procedure •

•

•

Create a volume of interest (VOI) — including both high-threshold and low-threshold objects — with the “IPL_VOI_ONLY” evaluation protocol, which does not have 3D histomorphometry. Since calcium phosphate injection has a high attenuation value, a high threshold value is set for the biomaterial. For the host trabecular bone, the threshold value is lower. The values can be defined through the 2D Evaluation program of the micro-CT system. Use the “IPL_TITAN_ARTIF_BONE” function in the microCT system to separate the high-threshold-value material and the low-threshold-value bone. Before carrying out the function, modification of the function parameter is required to ensure proper segmentation of the two materials.

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“xxxxx_ARTIF.AIM”: high-threshold-value objects (hi-th-objs) “xxxxx_SEG.AIM”: low-threshold-value objects (lo-th-objs) “xxxxx_TRANSP.AIM”: combined image of high- and lowthreshold objects, with the latter object slightly transparent.

With the two newly generated .AIM files (“_ARTIF.AIM” and “_SEG.AIM”), evaluate the 3D histomorphometry by “IPL_EVAL_ ONLY” on the three .AIM files. To have the output, use “uct_threedee_batch” at the “$” of the DECterm window to print the evaluation report.

3.3.3. Limitations • • •

Proper selection of the ROI to represent the tunnel is required. It is suggested to use concentric ROI for the contouring of the tunnel through the tibia and the femur. The thresholding values are for the beta-TCP granules and the surrounding bony tissues. They are not accounted for the matrix injection.

3.3.4. Data interpretation Figure 2 shows the 3D reconstruction of ACL tunnel healing with the tricalcium phosphate cement injection. (1) High-threshold-value object histomorphometry • • •

Bone volume fraction (BV/TV): the bone growth in the tunnel region Bone surface (BS): the surface area of bone in the tunnel region Structure model index (SMI): the morphology of the bony ingrowth

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Fig. 2. The sagittal view of anterior cruciate ligament (ACL) tunnel healing with the injection of tricalcium phosphate beads. The transparent bony structure (with low threshold value) is the bone tissue around the tunnel, while the yellow beads (with high threshold value) at the center are the tricalcium phosphate beads in the tunnel. The dotted lines represent the tunnel wall.

• • • • •

Bone surface fraction (BS/BV): the approximation of the bone and tendon attachment surface Trabecular bone thickness (Tb.Th*): the size of trabecular bone in the tunnel Trabecular bone number (Tb.N*): the number of bone ingrowth to the tunnel Mineral density (apparent) (mg HA/ccm): the amount of bone in the tunnel Mineral density (material) (mg HA/ccm): the BMD of bony tissue in the tunnel, indicating the degree of mineralization of the bone

(2) Low-threshold-value object histomorphometry •

Bone volume fraction (BV/TV): the volume of tricalcium phosphate granules in the tunnel region

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Bone surface fraction (BS/BV): the surface area of tricalcium phosphate granules in the tunnel Trabecular bone thickness (Tb.Th*): the diameter of the tricalcium phosphate granules, assuming that the granules are close to a spherical shape Trabecular bone number (Tb.N*): the number of tricalcium phosphate granule clusters Mineral density (material) (mg HA/ccm): the mineral content of the tricalcium phosphate granules Structure model index (SMI): the shape of the granules

3.4. Blood vessels in steroid-associated osteonecrosis The following micro-CT protocol is for the analysis of vascularization at the proximal femur region of the steroid-associated osteonecrosis model (Qin et al. 2006; Zhang et al. 2007). 3.4.1. Objective •

To quantify both the intact and damaged blood, and to have a descriptive analysis of the blood vessel distribution of blood vessels in the proximal femur region

3.4.2. Procedure •

•

After the Microfil injection, histological fixation, and decalcification for 4–6 weeks in EDTA solution, check the completeness of the decalcification by plain X-ray imaging before the micro-CT scanning. When the decalcification is completed, the contour of the bone cannot be seen clearly while the blood vessels will be more obvious in the plain X-ray image. Place the fully decalcified sample in the sample tube with the proximal femur pointing to the bottom of the sample tube. The alignment is parallel to the long axis of the sample tube.

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In this model, the sample tube size is 36 mm in diameter. The X-ray energy setting is 70 kV and 112 mA. The scanning setting is isotropic and the resolution is standard. After a scout view of the proximal region of the femur, define the scanning region of 10 mm in height from the most proximal end of the femur. Define the ROI by contouring. The segmentation parameters of blood vessels are defined as sigma = 1.2, support = 2, and threshold = 85. After the 3D evaluation, a thickness distribution of the blood vessels is generated as a text file ending with “_TH.TXT”.

3.4.3. Data interpretation Figure 3 shows the 3D network of blood vessels at the proximal femur of the steroid-associated osteonecrosis model. •

Bone volume fraction (BV/TV): the volume of blood vessels within the volume of interest

Fig. 3. The 3D structure of blood vessels at the proximal femur of a steroidassociated osteonecrosis rabbit model. The micro-CT 3D reconstruction shows that different sizes of blood vessels can be coded for easy viewing.

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Trabecular bone thickness (Tb.Th*): the overall thickness of the blood vessels Trabecular bone number (Tb.N*): the number of vessels per unit volume Trabecular bone separation (Tb.Sp*): the separation of the blood vessels Connectivity density (Conn.D): the connectivity of the blood vessels, which is greatly dependent on the Microfil injection technique, which in turn may introduce small bubbles into the vessels

3.4.4. Limitations •

•

The minimal detection limit for blood vessels in this scanning protocol is 36 µm, if the noise of the scan is well controlled. For vessels smaller than 36 µm, it is necessary to trim the sample to suit the smaller sample tube for scanning. The visualization of the vessel network is greatly dependent on the Microfil injection technique, the scanner setting, and the segmentation parameters. Thus, it is necessary to control the injection pressure, the resolution of the scan, and the definition of the segmentation parameters by trial and error for batch analysis.

3.5. Biomaterials The following example is based on the analysis of two commercially available bioceramics in which the main constituent is calcium phosphate. The detailed data have been published previously (Yeung et al. 2005). 3.5.1. Objective •

To study the histomorphometry of the material and the porosity of the bioceramics

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3.5.2. Procedure •

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Trim the biomaterial to a desirable size that fits into the sample tube for scanning. In this example, the sample tube is 16 mm in diameter. The X-ray energy is 70 kV and 112 mA. The resolution is standard and isotropic. After the image acquisition, define the ROI to inscribe 90% of the material and carry out a standard 3D evaluation procedure for standard histomorphometry. After the evaluation, process the 3D reconstructed image of the material further to visualize the pores by changing the value of the material (default value: 127) to 0 and the value of the space between the material (default value: 0) to 127. Write out the converted image to a new file as “pore.aim”. Use the “pore.aim” file for 3D histomorphometry analysis by “IPL_EVAL_ONLY.COM”.

3.5.3. Data interpretation Figure 4 shows the 3D images of the biomaterial and its pores. (1) Analysis of the material • • • • • •

Bone volume fraction (BV/TV): the material volume fraction Bone surface fraction (BS/BV): the surface fraction of the material with respect to the material volume Trabecular bone thickness (Tb.Th*): the thickness of the materials Trabecular bone number (Tb.N*): the number of materials per unit volume Trabecular bone separation (Tb.Sp*): the overall average pore size of the material Mineral density (apparent) (mg HA/ccm): the overall mineral density of the material (only applicable to the calcium-containing biomaterial)

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(a)

(b)

Fig. 4. 3D reconstruction of the micro-CT–scanned scaffold biomaterial. (a) The 3D image of the reconstructed interconnected porous biomaterial. (b) The 3D image of interconnected pores within the material.

•

Mineral density (material) (mg HA/ccm): the material density of the bioceramic which contains micropores within the materials. Therefore, if the two similar materials with different manufacturing processes are compared, this value is also a representation of the micropore density within the material.

(2) Analysis of the pores • • • • • •

Bone volume fraction (BV/TV): the porosity of the materials Bone surface fraction (BS/BV): the surface fraction of the pores Trabecular bone thickness (Tb.Th*): the average pore size in diameter Trabecular bone number (Tb.N*): the number of pores per unit volume Connectivity density (Conn.D): a quantitative value of the interconnectivity of the pores Structure model index (SMI): the shape of the pores

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3.5.4. Limitations •

•

The scanning resolution limits the minimal size of the pores that can be analyzed. Since the voxel size of the micro-CT system can be as fine as 6 µm, it is possible to study micropores smaller than 16 µm in more detail. It is relatively easy to analyze the bioceramic, as calcium phosphate is the main inorganic component of bone. For other scaffolds, it is possible to study their histomorphometry using different X-ray energy levels for the scanning. It is also possible to introduce a contrast agent to coat the scaffolds before performing the micro-CT scan.

4. Summary Micro-CT is a powerful noninvasive technique that assesses material histomorphometry and soft tissue such as blood vessels. It provides an objective, quantitative analysis of the materials and biological samples. An important note to make for micro-CT analysis is that, in a previous study on the osteoporosis rat model, when structural parameters such as trabecular bone number, bone volume fraction, and connectivity density decreased during the course of osteoporosis development, trabecular bone thickness plateaued in the middle of osteoporosis development (Siu et al. 2004). This plateau is likely due to the evaluation algorithm in which, when the thinning of the trabecular bone is severe and the connectivity density is dramatically decreased, the algorithm no longer takes account of those disconnected trabecular bones; the remaining disconnected trabecular bones are counted as one entity, which will remain as a big trabecular bone during resorption. Hence, the trabecular bone thickness is plateaued (Lai et al. 2005). This shows that it is not absolutely applicable for all individual microstructural parameters in addressing biological events during fracture repair. During fracture repair, the bone is formed by endochondral ossification at the initial phase; then, the callus undergoes active remodeling to restore the original cortical bone. Therefore, the interpretation of the trabecular bone evaluation

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by micro-CT should be careful within the context of background biology. The recent development of in vivo micro-CT has made the scanning of live small animals possible with high resolution. The image acquisition and image analysis techniques are basically the same as those for in vitro micro-CT described above. Basically, all of the protocol analyses suggested should be able to be transferred to in vivo micro-CT; however, there will be major modifications for the blood vessel visualization protocol in the steroid-associated osteonecrosis model. Appropriate contrast agents can be employed so that the blood vessels can be imaged within the scanning time of the image acquisition of in vivo micro-CT.

References Anderson K, Seneviratne AM, Izawa K et al. Augmentation of tendon healing in an intraarticular bone tunnel with use of a bone growth factor. Am J Sports Med 29:689–698, 2001. Boden SD, Schimandle JH, Hutton WC. An experimental lumbar intertransverse process spinal fusion model. Radiographic, histologic, and biomechanical healing characteristics. Spine 20:412–420, 1995. Bonnarens F, Einhorn TA. Production of a standard closed fracture in laboratory animal bone. J Orthop Res 2:97–101, 1984. Einhorn TA. Enhancement of fracture-healing. J Bone Joint Surg Am 77:940–956, 1995. Einhorn TA. The cell and molecular biology of fracture healing. Clin Orthop Relat Res 355(Suppl):S7–S21, 1998. Guo X, Lee KM, Law LP et al. Recombinant human bone morphogenetic protein-4 (rhBMP-4) enhanced posterior spinal fusion without decortication. J Orthop Res 20:740–746, 2002. Hildebrand T, Laib A, Muller R et al. Direct three-dimensional morphometric analysis of human cancellous bone: microstructural data from spine, femur, iliac crest, and calcaneus. J Bone Miner Res 14:1167–1174, 1999. Lai YM, Qin L, Yeung HY et al. Regional differences in trabecular BMD and micro-architecture of weight-bearing bone under habitual gait loading — a pQCT and microCT study in human cadavers. Bone 37:274–282, 2005.

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Odgaard A, Gundersen HJ. Quantification of connectivity in cancellous bone, with special emphasis on 3-D reconstructions. Bone 14:173–182, 1993. Qin L, Zhang G, Sheng H et al. Multiple bioimaging modalities in evaluation of an experimental osteonecrosis induced by a combination of lipopolysaccharide and methylprednisolone. Bone 39:863–871, 2006. Shi HF, Cheung WH, Qin L et al. Effect of low-magnitude, high-frequency vibration therapy on fracture healing in rats: pilot study. 53rd Annual Meeting of Orthopaedic Research Society, Vol. 32, San Diego, CA, poster 0935, 2007. Siu WS, Qin L, Cheung WH, Leung KS. A study of trabecular bones in ovariectomized goats with micro-computed tomography and peripheral quantitative computed tomography. Bone 35:21–26, 2004. Wang CJ, Wang FS, Yang KD et al. The effect of shock wave treatment at the tendon–bone interface — an histomorphological and biomechanical study in rabbits. J Orthop Res 23:274–280, 2005. Yeung HY, Lee KM, Fung KP, Leung KS. Sustained expression of transforming growth factor-beta 1 by distraction during distraction osteogenesis. Life Sci 71:67–79, 2002. Yeung HY, Lee SKM, Fung KP, Leung KS. Expression of basic fibroblast growth factor during distraction osteogenesis. Clin Orthop Relat Res 385:219–229, 2001. Yeung HY, Qin L, Lee KM et al. Novel approach for quantification of porosity for biomaterial implants using microcomputed tomography (µCT). J Biomed Mater Res B Appl Biomater 75B:234–242, 2005. Zhang G, Qin L, Sheng H et al. Epimedium-derived phytoestrogen exert beneficial effect on preventing steroid-associated osteonecrosis in rabbits with inhibition of both thrombosis and lipid-deposition. Bone 40:685–692, 2007.

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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

In order to understand the mechanisms of fracture healing, especially the neovascularization of the callus, we have established a closed femoral fracture model in rats. This chapter describes a microangiography technique that has been adopted to investigate temporal changes in the three-dimensional (3D) vasculature of the healing callus. Quantitative evaluation protocols for vessel size distribution, total vessel volume, and volume fraction have also been established for comparative studies. Keywords:

Angiography; neovascularization; fracture; rat; micro-CT; callus; decalcification; osteogenesis; mineralization; scanning resolution; binarization threshold.

1. Introduction Fracture healing is a well-characterized cascade of events that includes hematoma formation, inflammation, soft cartilaginous callus formation, neovascularization, osteoblastic callus mineralization, and osteoclastic remodeling of the hard callus back to mature lamellar bone (Einhorn 1998). Angiogenesis precedes osteogenesis and also plays a critical role in Corresponding author: Xiao-Zhong Zhou. E-mail: [email protected]

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the process of endochondral ossification during bone formation (Gerber et al. 1999). Enhanced angiogenesis promotes fracture healing, whereas treatment with angiogenesis inhibitors blocks callus formation and produces atrophic nonunions (Eckardt et al. 2005; Hausman et al. 2001). In order to evaluate vascular structures in animal models, many techniques have been utilized. Histology is a common way to analyze capillary density, but it is two-dimensional (2D) and relatively subjective (Amano et al. 2003). Laser Doppler perfusion imaging has been used to analyze functional blood flow because it can offer semiquantitative data and a measure of functionality (Scholz et al. 2002); however, this technique cannot provide anatomic information of vasculature and is limited by the fact that only cutaneous blood flow can be measured. Another popular technique, X-ray microangiography, provides high-resolution 2D angiograms of the vascular anatomy, but lacks the ability to employ a quantitative, volumetric analysis (Duvall et al. 2004). Other imaging modalities such as magnetic resonance angiography and positron emission tomography (PET) serve as viable methods for analyzing vascular function, but the resolution of these methods is usually not enough for studies using small animals. To more thoroughly understand the nature of neovascularization during fracture healing, a quantitative, three-dimensional (3D), highresolution micro-computed tomography (micro-CT) imaging approach has been developed in mice and rabbits (Duvall et al. 2004; Qin et al. 2006). Such a technique may also be adopted to study revascularization in long bone fracture repair in a rat femoral fracture model. This chapter describes the essential steps in using such a technique, which serves as a platform for evaluating the potential interventions that promote fracture site revascularization in both a qualitative and quantitative manner.

2. Materials • • •

Animals: Sprague–Dawley rats (body weight, 260 g ± 20 g) 20-gauge needle, 1.2-mm-diameter K-wire, gauze, guillotine-like fracture apparatus X-ray machine for fracture site evaluation (Model 43855C; Faxitron X-ray Systems, Wheeling, IL, USA)

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Prewarmed (37°C) heparinized saline: 0.9% normal saline containing heparin sodium (50 IU/mL) (10 mL for each rat) Prewarmed 0.9% normal saline (60 mL for each rat) Prewarmed formalin: 10% neutral buffered formalin (10 mL for each rat) Liquid compound (Microfil; Flow Tech, Inc., Carver, MA, USA) for microvascular injection agent (9 mL for each rat with 350–400 g in body weight): prewarmed 4.75 mL of MV-Diluent, prewarmed 3.8 mL of MV-117 Orange, and 0.45 mL of MV Curing Agent (working time is 20 minutes, beginning with the addition of curing agent into MV-Diluent–MV-117 Orange) Operational apparatus: scissors, scalpel, forceps, microforceps, bulldog clamp, 3-0 silk 9% formic acid

3. Methods 3.1. Establishment of fracture model The midshaft femoral closed fracture is established using a standard protocol (Bonnarens and Einhorn 1984; Zhou et al. 2007). • • • • •

• •

Anesthetize the rats intraperitoneally with 36 mg/mL of chloral hydrate (1 mL/100 g of body weight). Shave and sterilize with alcohol and povidone-iodine solution on the limbs. Make a lateral parapatellar knee incision to expose the distal femoral condyle. Use a 20-gauge needle to bore a hole from the trochlear groove into the femoral canal for reaming. Insert a 1.2-mm-diameter K-wire retrogradely until the wire exits through the greater trochanter and the skin. Position the distal end of the wire deeply to the articular surface of the knee. Make a curve close to the greater trochanter and cut the rest of the wire. Close the wounds with 3-0 nylon suture.

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Fig. 1. A custom-made guillotine-like fracture apparatus for establishing a closed transverse fracture in the K-wire–fixed rat femora.

• •

Establish a closed transverse fracture in the K-wire–fixed femur by using a custom-made guillotine-like fracture apparatus (Fig. 1). Radiographically assess the fracture site to confirm proper placement of the pins and alignment of the fractures (Fig. 2). Fractures with an obvious oblique fracture line or comminuted ones are excluded.

3.2. Specimen harvesting and preservation • • • •

Sacrifice the rats at a time interval of postoperative weeks 2, 3, and 4. Carefully harvest femoral specimens without damaging the fracture callus. Wrap the specimens in saline-soaked gauze, and store at −20°C after removing the intramedullary wires from the insertion site. To study the neovascularization of the callus, perform microangiography just before euthanasia according to the following protocols.

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Fig. 2. Radiographic monitoring on the fracture site to confirm proper placement of the pins and alignment of the fractures (arrow: transverse fracture line).

3.3. Perfusion and decalcification • • • • • • • • •

Anesthetize the rats intraperitoneally with 36 mg/mL of chloral hydrate (1 mL/100 g of body weight). Remove hair using a hair clipper. Open the abdomen cavity, retract the bowels laterally, and carefully separate the abdominal aorta from the vessel sheath (Fig. 3). Ligate the abdominal aorta, and gently clamp the distal aorta with a bulldog clamp. Insert a scurf-needle (with its sharp needlepoint smoothly cut) into the aorta between the clamp and ligation points. Loosen the bulldog clamp to make sure that the needle is in the aorta, and then fix the needle with aorta using 3-0 silk. Cut the inferior vena cava to allow outflow of the perfusate. Link the pump apparatus (PHD 22/2000; Harvard Apparatus, USA) to the needle (Fig. 4). Inject 10 mL of prewarmed heparinized saline (50 IU/mL).

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Fig. 3. The abdomen cavity is opened, the bowels are retracted laterally, and the abdominal aorta is carefully separated from the vessel sheath (white arrow).

Fig. 4. The pump apparatus is linked to the needle. Prewarmed heparinized saline, normal saline, formalin, and the contrast agent are injected at a flow speed of 20 mm/min (syringe diameter, 25 mm).

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Continuously flush using prewarmed normal saline (about 60 mL) until the outflow from the inferior vena cava is limpid. Inject 10 mL of prewarmed formalin to fix the nourished skeletal specimen. Rapidly inject MV-Diluent–MV-117 Orange–MV Curing Agent (http://www.flowtech-inc.com) as soon as they are mixed (flow speed, 20 mm/min; syringe diameter, 25 mm). The animals are then euthanized with an overdose of chloral hydrate. Store the cadaver (not only the perfused lower extremities) at room temperature for 1 hour and then at 4°C overnight to ensure polymerization of the contrast agent. Dissect the fractured femora free from the surrounding musculature carefully. Fix the samples by using 10% neutral buffered formalin for 24 hours, and then decalcify them by using 9% formic acid (generally, 72 hours are needed). Take anteroposterior-view radiographs of the samples to confirm the success of decalcification by using a cabinet X-ray system (e.g. Specimen Radiography System, Faxitron 43855C; Faxitron X-ray Corp., Wheeling, IL, USA) under an exposure condition of 55 kV/3 s.

3.4. Microangiography A high-resolution (8–36-µm isotropic voxel size) micro-CT imaging system (µCT 40; Scanco Medical, Bassersdorf, Switzerland) is used to perform callus scanning and produce 3D vasculature images. • • •

•

Set the scanner to a voltage of 55 kVp and a current of 114 µA. Set the resolution to standard, which creates a 1024 × 1024 pixel image matrix. Fix the femoral shaft in a plastic tube (12.3 mm in diameter) with its long axis perpendicular to the bottom of the tube. Add 70% ethanol and then seal with paraffin film. Initiate the scan 3.15 mm (or at any fixed distance to include the whole callus area for the same batch of samples) above the fracture line, with an entire scan length of 6.3 mm (Fig. 5).

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Fig. 5. A scan 3.15 mm above the fracture line with an entire scan length of 6.3 mm is initiated. Left: 3D CT reconstruction of the whole fractured femur. Right: anteroposterior view of the decalcified femur with perfusate.

•

•

•

For segmentation of blood vessels from the background, remove the noise using a low-pass Gaussian filter (sigma = 1.2, support = 2) and then define the blood vessels at a threshold of 265. Use the semiautomatically built-in Contouring Program to draw contours at each 2D section for an automatic reconstruction of 3D vascular images in the decalcified sample. Subsequently, generate a histogram to display the distribution of the vessel size; and map a color-coded scale to the surface of the 3D images to produce a visual representation of the vessel size distribution, total vessel volume, and volume fraction (Fig. 6).

4. Discussion Fracture healing, a complex biological phenomenon, requires the exquisite coordination of programs for angiogenesis and osteogenesis

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Fig. 6. Comparison of 3D vascular images of rat femoral fracture healing callus at 2 weeks after fracture. More callus mineralization and better mechanical characteristics are found in group B (b) than in group A (a), which shows a higher total vessel volume and volume fraction, indicating that enhanced angiogenesis does increase osteogenesis (green, 10–50 µm; yellow, 50–100 µm; orange, 100–150 µm; red, 150–250 µm).

(O’Keefe et al. 1994; Vu et al. 1998). Angiogenesis precedes osteogenesis, and it is also a critical step in the process of endochondral ossification during bone formation (Gerber et al. 1999). Enhanced angiogenesis was reported to promote bone formation (Eckardt et al. 2005). In the present study, more callus mineralization and better mechanical characteristics were found in the group with a higher total vessel volume and volume fraction at different time points postoperation, indicating that enhanced angiogenesis does increase osteogenesis. Such a method (quantitative angiographic measurements) was also successfully employed in evaluating the relationship of callus angiogenesis and mineralization in osteopontin-deficient mice (Duvall et al. 2007). Micro-CT imaging, combined with the use of perfused contrast agents and bone decalcification, provides a robust methodology for the evaluation of vascular networks in the femoral callus. Specifically, micro-CT is advantageous because it provides high-resolution, quantitative, 3D, and objective data analysis.

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In a pilot study, the investigators also explored whether clinical angiography using the radiopaque substance barium sulfate would be a better approach to monitor neovascularization in the fracture site. However, due to its larger particle size and rather lower solubility in solution, only blood vessels larger than 250 µm were visualized perfused radiographically. Microfil (lead chromate) is a radiopaque solution with a smaller particle size that has recently been used for quantitative micro-CT analysis of collateral vessel development after ischemic injury (Duvall et al. 2004) and for a study on avascular necrosis (Qin et al. 2006); it has also been successfully adopted into the present fracture callus angiographic study. When using micro-CT imaging for analysis of vascular structures, two important methodological cautions must be considered: the binarization threshold and the scanning resolution. When defining the binarization threshold, a value that is too high will delete small vessels from the image, but a threshold that is too low can make major vessels appear artifactually large. We recommend that the user make a visual determination of the optimal threshold by assessing several specimens and then keep this value constant (e.g. 265) for all evaluations within a study. This value should be chosen based on the threshold that allows the capture of intricate details with minimal overestimation of broader structures. The voxel size of the scan should be chosen based on the proposed application. Scanning with smaller voxel sizes does provide more information by resolving smaller vessels that cannot be detected at larger voxel sizes; however, scanning at larger voxel resolutions can be more effective for some applications. For example, to gain a global perspective of collateral growth in the upper thigh (as is commonly done in two dimensions by investigators who utilize X-ray microangiography), a voxel size of 36 µm is optimal for focusing the analysis on larger, arteriole-sized vessels, which are the best indicators of ischemic limb recovery. In contrast, for local vascular anatomy in cleared small tissue sections such as rat callus, higherresolution scans are necessary to image smaller vascular structures. In the case of our neovascularization study of the rat femoral callus, a voxel size of 8 µm or smaller was required because our aim was to

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measure angiogenesis or small blood vessel structure within a confined area. However, when smaller voxel sizes are used, the major disadvantage is the significantly increased scan time along with the increased computational time and complexity required to analyze a much larger data set. One possible drawback of this technique is that it does not allow for longitudinal analyses at different time points within the same animal. Because this is a postmortem analysis, the number of animals required for completion of a time course study is higher compared with methods such as laser Doppler perfusion imaging, which offers the ability to acquire multiple scans on the same living animal at different time points after surgery. The recent commercial availability of high-speed in vivo micro-CT scanners that provide maintenance of animal anesthesia within the scanning system may remedy this limitation. Nevertheless, the development of nanotoxic and nontoxic contrast agents for in vivo application is essential.

5. Summary Microangiography is a robust methodology for evaluating vascular networks in the healing fracture callus, as it provides high-resolution, quantitative, 3D, and objective data analysis. As the micro-CT available for this study is an in vitro model and the lead chromate is a toxic substance not for in vivo application, high-resolution dynamic magnetic resonance imaging (MRI) and positron emission tomography (PET) may also be used to monitor the local perfusion disturbance of corresponding skeletal sides.

References Amano K, Matsubara H, Iba O et al. Enhancement of ischemia-induced angiogenesis by eNOS overexpression. Hypertension 41:156–162, 2003. Bonnarens F, Einhorn TA. Production of a standard closed fracture in laboratory animal bone. J Orthop Res 2:97–101, 1984. Duvall CL, Robert Taylor W, Weiss D, Guldberg RE. Quantitative microcomputed tomography analysis of collateral vessel development after

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ischemic injury. Am J Physiol Heart Circ Physiol 287:H302–H310, 2004. Duvall CL, Taylor WR, Weiss D et al. Impaired angiogenesis, early callus formation, and late stage remodeling in fracture healing of osteopontindeficient mice. J Bone Miner Res 22:286–297, 2007. Eckardt H, Ding M, Lind M et al. Recombinant human vascular endothelial growth factor enhances bone healing in an experimental nonunion model. J Bone Joint Surg Br 87:1434–1438, 2005. Einhorn TA. The cell and molecular biology of fracture healing. Clin Orthop Relat Res: S7–S21, 1998. Gerber HP, Vu TH, Ryan AM et al. VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation. Nat Med 5:623–628, 1999. Hausman MR, Schaffler MB, Majeska RJ. Prevention of fracture healing in rats by an inhibitor of angiogenesis. Bone 29:560–564, 2001. O’Keefe RJ, Crabb ID, Puzas JE, Rosier RN. Effects of transforming growth factor-beta 1 and fibroblast growth factor on DNA synthesis in growth plate chondrocytes are enhanced by insulin-like growth factor-I. J Orthop Res 12:299–310, 1994. Qin L, Zhang G, Sheng H et al. Multiple bioimaging modalities in evaluation of an experimental osteonecrosis induced by a combination of lipopolysaccharide and methylprednisolone. Bone 39:863–871, 2006. Scholz D, Ziegelhoeffer T, Helisch A et al. Contribution of arteriogenesis and angiogenesis to postocclusive hindlimb perfusion in mice. J Mol Cell Cardiol 34:775–787, 2002. Vu TH, Shipley JM, Bergers G et al. MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell 93:411–422, 1998. Zhou X, Dong Q, Zhang J. Establishment and evaluation of a standard closed fracture model on rat femur. J Southeast Univ Med Sci Ed 26:60–62, 2007.

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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

Noninvasive three-dimensional imaging of live animals is a powerful research tool that has become prevalent in many biomedical fields including cancer, aging, cardiovascular disease, and cognitive behavior. Micro-computed tomography (micro-CT) distinguishes itself from other imaging techniques in its ability to acquire high-resolution images based on the physical density of the material, facilitating precise assessments of tissue density and morphology of physiological systems such as bone, muscle, vasculature, and fat. To this end, in vivo micro-CT can measure temporal changes in tissue morphometry, under the influence of genetic and epigenetic factors during development, homeostasis, or repair, for testing the efficacy of pharmacological and nonpharmacological treatments or for evaluating the mechanical behavior of the tissue. Here, an in vivo micro-CT protocol is described with specific examples for bone, muscle, and fat. Keywords:

Micro-computed tomography; in vivo; bone; muscle; fat; small animal models.

Corresponding author: Stefan Judex. Tel: +1-631-6321549; fax: +1-631-6328577; E-mail: [email protected]

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1. Introduction Advances in biomedical imaging technology have been critical for the advanced detection and more effective treatment of diseases (Goldstein et al. 2005; Judex et al. 2003b; McCormack et al. 2007). For bone, the gold standard is X-ray–based technology that can image and contrast tissues according to their densities. In the clinic, the most popular method to assess the health of the skeleton is dualenergy X-ray absorptiometry (DXA), which provides information on the quantity of bone present in a subject (Rubin et al. 2004; Watts 2004). However, the risk of skeletal fracture cannot simply be determined by the amount of bone that is present, as the quality of the bone may be just as important (Judex et al. 2003b; Mashiba et al. 2000). Bone quality is influenced, among other factors, by morphometric features such as the bone’s cross-sectional geometry, its trabecular connectivity, the architecture of the trabecular elements themselves, and the physical density of the material. In preclinical studies, these parameters can be quantified by histological and ashing techniques. Unfortunately, histology is extremely labor-intensive, typically two-dimensional (2D), and can suffer from large variability (Birkenhager-Frenkel et al. 1988). In contrast, high-resolution micro-computed tomography (micro-CT) allows for a direct and nondestructive description of three-dimensional (3D) structures without tedious preparations (Feldkamp et al. 1989; Muller et al. 1994; Muller and Ruegsegger 1997; Ruegsegger et al. 1996). Not surprisingly, over the past 5 to 10 years, micro-CT has become the standard technique for the ex vivo evaluation of bone quantity, architecture, and density in animal models (Muller and Ruegsegger 1997; Ruegsegger et al. 1996). More recently, this technique has been adapted to allow for in vivo scanning. Clearly, assessing longitudinal changes in tissue morphometry holds great advantages by decreasing data variability, increasing statistical power, and reducing the required number of animals (Boyd et al. 2006a; Cowan et al. 2007). Furthermore, changes in any given animal can be determined without having to rely on group averages.

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Fig. 1. In vivo micro-computed tomography of mouse, scanned between the neck and knees, delineating the contrast between calcified tissue and soft tissue (fat, muscle, and internal organs). In vivo micro-CT presents a robust method for quantifying differences in tissue quantity and quality in a host of tissues.

Because micro-CT images are 3D density maps of the volume of interest (VOI), any biological structure that provides an adequate density gradient (contrast) can be imaged (Fig. 1). If the contrast is insufficient, contrast agents can be used to enhance the difference in density between the tissues of interest (Idee and Corot 1999; Idee et al. 2000; Muschick et al. 1995). Rather than scanning specific tissues within an animal, in vivo micro-CT wholebody scans can also be generated, typically at lower resolutions, to provide data on multiple tissues and their interactions using a systems biology approach. The goal of this chapter is to describe a basic methodology for the use of in vivo micro-CTa in small animal models.b

a

This protocol assumes the use of micro-CT to evaluate tissue quantity and quality in vivo. Micro–magnetic resonance imaging (micro-MRI) may serve as an alternative, albeit at lower resolutions (Ladinsky and Wehrli 2006). b For in vivo scanning of the distal forearm and distal tibia, a clinical micro-CT scanner is available for research purposes (XtremeCT; Scanco Medical, Switzerland).

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2. Materials • • • • • • • •

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In vivo micro-CT scanner (e.g. VivaCT 40; Scanco Medical, Switzerland)c Small animal model (mouse, rat, etc.) Anesthetic chamber d Isoflurane (e.g. USP; Halocarbon Products Corp., NJ, USA) Oxygen Isoflurane vaporizer that regulates the ratio of isoflurane to oxygen (e.g. EZAnesthesia; Euthanex Corp., PA, USA) Flowmeter that controls the flow of anesthetic mix (e.g. EZAnesthesia) Induction chamber that can be purged with an anesthetic and is used to induce anesthesia in the animal before scanning (e.g. EZAnesthesia) Holder to secure the animal within the scanner e Software interface of scanner to scan, segment, and evaluate the VOI

3. Methods 3.1. Prescan •

c

Scan phantoms with precisely known density values to allow for the determination of apparent tissue mineral density from the

Other suppliers of in vivo micro-CT devices include General Electric (Fairfield, CT, USA), Siemens (Knoxville, TN, USA), Micro Photonics (Allentown, PA, USA), and Echo Medical Systems (Houston, TX, USA). d An alternative method for anesthesia is intraperitoneal injection of a ketamine/xylazine solution. e Manufacturers may supply animal holders designed specifically for mice or rats. Many of these holders work well for scanning specific regions of an animal, but often suffer from a lack of versatility. To this end, we have found that many scan applications benefit from customized animal holders. Polystyrene foam is inexpensive, and can be readily cut and shaped to accommodate animals of very different sizes in different positions (Fig. 2); these holders can be secured within the scanning chamber with adhesive tape. Another advantage of polystyrene over the much denser plastics of premade animal holders is their very low density, which makes them seemingly invisible in micro-CT images and avoids interference with the measurement of soft tissues.

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linear attenuation values of the X-ray counts. The range of phantom densities should be selected to include the density of the tissue(s) of interest. For best results, calibration should be performed at least once a month. In preparation of the scan, create a control file that defines the in vivo micro-CT scan parameters. A direct link to this menu is typically provided by the software of the manufacturer. The variables within this control file include the energy settings, number of projections, scan dimensions, and integration time, all of which define or influence image variables such as the resolution, scan time, and signal-to-noise ratio. Use voltage and current as the basic energy settings to determine the X-ray characteristics. Inherently, the contrast between bone and soft tissue is excellent, and contrast optimization is not necessary; however, a precise spatial delineation of soft tissues may require adjustment of the tube voltage (for most applications, the highest current should be selected). Although increasing the voltage may enhance soft tissue contrast by increased X-ray penetration, it may take several trials to find the optimal settings. It should also be considered that an increase in energy translates to increased levels of radiation.f Determine the VOI for the scan (e.g. distal femur, hamstring, fracture callus, abdominal fat, whole body, etc.), and specify the boundaries that encompass the region.g To scan multiple regions within the same animal without user intervention, control files can be set up in batch mode.

Investigations into the effects of micro-CT–induced radiation on the tissue or animal have been ambivalent. Multiple scans at very high resolutions may have detrimental effects on bone morphology, particularly in young animals (Klinck and Boyd 2006), but such an effect was not observed in adult rats (Brouwers et al. 2007) or in mice subjected to disuse (Judex et al. 2005). Until precise radiation thresholds below which cellular metabolism is unchanged are identified (Boone et al. 2004), the number of longitudinal scans and the scan resolution need to be approached cautiously. g During the initial setup of the control file, it may be difficult to predefine the spatial scan range of the scout scan. To this end, a larger-than-necessary scout scan is performed with the animal in the chamber to decide on the optimal range. The radiation exposure to the scout scan is low; even multiple scout scans should not affect cell metabolism.

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Select an appropriate voxel resolution for the scan. The optimal resolution is heavily dependent on the size of the animal, the tissue of interest, and an acceptable scan time,h and will be determined by the isotropic increment size and the number of projections in the control file. Inherently, high-resolution scans will capture more detail of the tissue architecture, but will also increase scan time and hard disk requirements; in addition, the animal will be exposed to greater levels of isoflurane and radiation. Vice versa, low-resolution scans are fast, but may omit important tissue detail. To minimize the time and resources necessary to process and store data, the lowest resolution that will provide adequate detail should be selected. Select an appropriate integration time. Increasing the integration time will increase the signal-to-noise ratio at the expense of increased scan durations. To increase the integration time beyond the selectable maximal value, frame averaging can be applied. Save the optimal control settings as soon as they are determined. Parameters in this control file should not be altered throughout the study to avoid the introduction of potentially confounding variables.

3.2. Preparation of animal • •

h

Turn on the in vivo micro-CT scanner. Wait until the X-ray tube has warmed up (~20 minutes). Μaintain the animals under anesthesia for the entire duration of the scan. Verify that there is enough oxygen in the tank to maintain a constant flow rate of 0.1–0.5 L/min (depending on the animal size and condition).

The optimal scan resolution is dependent on the required detail. A fat pad of the order of centimeters in size could take more than 1 hour to scan at the highest resolution and, because of its homogeneous structure, the resulting image parameters would likely be indistinguishable from those of a lower-resolution scan. On the other hand, the morphology of cancellous bone in rodents may contain trabecular struts that are <60 µm thick and the scan resolution should be high enough to capture the intricate architecture. For example, typical in vivo scan resolutions are 10–20 µm for trabecular bone in mice, 20–60 µm for cortical bone, and 60–120 µm for fat and muscle.

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Purge the induction chamber with 5% isoflurane for 5–10 minutes, depending on the size of the chamber. If multiple animals are scanned, repurge the chamber periodically to maintain isoflurane concentration. Quickly place the animal into the induction chamber and secure the lid. If the animal is part of an unloading study (Squire et al. 2004; Morey-Holton and Globus 2002), place the animal in an unloaded position within the chamber to avoid loading of the hindlimbs prior to the induction of anesthesia. Monitor the animal in the induction chamber. An animal is properly anesthetized when its muscle tone is relaxed and its heart and respiration rates have slowed visibly. Remove the animal and transfer it to the scanning chamber. Secure the isoflurane nozzle to the head of the animal and assure that the flow has been diverted to the nozzle. Position the animal in the animal holder in a comfortable position, and ensure that the alignment is appropriate for scanning the VOI. For many applications, it is preferable for the image plane to be transverse to the longitudinal axis of the tissue. If necessary, the animal can be secured within the holder by using a combination of tape and rubber bands. Foam can be placed around the animal for a tighter fit. If the legs need to be fully extended, tension can be applied to their feet with tape (Fig. 2); do not apply tape to the fur of the animal. Position rubber bands outside the region(s) of interest [ROI(s)], as they are not transparent to the X-rays and may introduce image artifacts. Adjust the isoflurane level to the percentage necessary to keep the animal anesthetized. For an average-sized adult mouse, this range is 1.5%–2%. Larger animals and animals that have had repeated exposure to isoflurane may require more anesthetic. Smaller, younger, or chronically weak animals may require less anesthetic.

3.3. Scout view • • •

Select the predefined control file. Ensure that the settings in this file are appropriate and have not been changed. Load the control file. If required, perform a precalibration as specified by the manufacturer.

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Fig. 2. (a) Positioning of a mouse, whose distal femur will be scanned, in a customized polystyrene foam animal holder. The white arrow points to the manufacturer’s generic polymer rat holder. By shaping the polystyrene holder (yellow arrow), the femur can be readily aligned to result in image slices that are transverse to the longitudinal axis of the femur. (b) The sticky blue tape attached to the feet of the mouse can apply tension to the mouse hindlimbs in order to align the femur.

•

i

Use the scout-view function to visualize a preliminary plain X-ray image. The scout view should also be used to check on the proper alignment of the ROI(s).i To detect out-of-plane misalignments,

It is entirely possible to scan the tissue as is in its spatial position and to correct for any misalignments at postscanning. Image registration techniques are available to match the ROI(s) between different animals via anatomical landmarks or temporally within an animal (Boyd et al. 2006b). It should be noted, however, that image rotation may decrease the effective size of the region(s) that will be analyzed. This can be readily prevented by conservatively selecting the size of the original ROI(s). Smaller angles of rotation will require a lesser increase in the ROI(s).

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repeat the scout view at a distinct angle (e.g. rotated by 90°). If necessary, remove the animal from the chamber, adjust its position,j and repeat the scout view. It is recommended to save the scout-view image of the position in which the animal is scanned. Select the ROI by defining the boundaries. These reference lines should be highly reproducible between animals and are typically based on easily identifiable anatomical landmarks. Batch scans can be created if multiple regions are to be scanned. For longitudinal studies, it should be taken into account that the tissue of interest may have changed its size and morphology and, therefore, the relative position of the anatomical landmark may have changed. To address this issue, the operator may attempt to select either the identical region or the same landmark as in the previous scan. This choice may depend on the specific research question; generally, the former strategy is scientifically more relevant, while the latter is clinically more relevant. As an example, we have recently determined the longitudinal changes in the trabecular bone morphology of adult female mice which were subjected to 3 weeks of hindlimb unloading (disuse) followed by 3 weeks of reambulation. In this study, an identical anatomical landmark was used between the three different time points (baseline, completion of disuse, completion of reambulation) because the research question was aimed at quantifying the impact of altered levels of mechanical loading on the mechanical integrity of the distal femur (Fig. 3).

Physical adjustments that are required for the desired alignment generally include, but are not limited to, the following: (1) correcting the relative position of the legs, (2) aligning the bone of interest (e.g. tibia) in a given direction (e.g. proximal-distal), (3) adjusting the animal position for the scanning of vertebrae, and (4) reducing the torsional misalignments for whole-body scans. The lower extremities can be adjusted by changing the tension applied to the hindlimbs, while additional polystyrene layers can be inserted (or removed from) underneath the animal to alter its body position.

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Fig. 3. Response of (a) the trabecular bone (pink) of a female adult mouse to (b) 3 weeks of hindlimb unloading and (c) 3 weeks of subsequent reambulation. The region of interest (ROI) encompasses the distal metaphysis of femur. The loss in trabecular number and connectedness incurred during disuse is in part compensated for by the thicker trabeculae at the end of reambulation.

3.4. Postscan •

•

Αbout 5–10 minutes before the scan is complete, start preparing the next animal by placing it into the induction chamber. Even if only one anesthesia apparatus is available and the gaseous flow is directed to the animal in the scanner, there is typically enough residual isoflurane in the anesthesia chamber to induce an anesthetic state without the addition of fresh gas. Be careful not to shut off the flow to the animal in the scanner. Wait until the scan is complete and the X-ray indicator light on the scanner is off. Dismount the animal from the animal holder and immediately remove it from the scanner. The animal will normally recover from the anesthesia within 5–10 minutes. A heat lamp will aid in the recovery and keep the animal warm.

3.5. Scan evaluation •

During scanning, create a raw sequence (RSQ) file that includes all of the sinograms and X-ray counts from the scan. This raw file will be reconstructed into an ordered sequence of 2D sections

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of the scan region, the image sequence (ISQ) file; if necessary, this reconstruction can be manually performed using image processing language (IPL). The names of the files and the image software may vary between manufacturers. The final grayscale file will reflect the apparent density of each voxel, with denser tissues appearing lighter and less dense tissues appearing darker. Use evaluation software to open the ISQ file and view the 2D sections. Separate the tissue of interest from the surrounding environment (e.g. air, bone, and soft tissues) to define the region that will be evaluated. Contour lines are most commonly produced semiautomatically, with the operator drawing the descriptive contours for every x slices starting at the nth slice (n, n + x, n + 2x, … ). From these user-specified contour lines, the contour lines for the in-between slices are generated automatically by interpolation. As with all interactive data analysis techniques, the drawing of the contours should be consistent for all samples within the same study to avoid increased measurement variability and bias. For instance, drawing contour lines every 20 slices rather than every 10 slices can significantly affect the geometry of the morphed contour lines, in particular when geometrical features change substantially along the scan axis within the ROI. The optimal number of drawn contour lines depends entirely on the complexity of the tissue geometry. To this end, slices in which the contours were automatically morphed should be checked to ensure a close match between the morphed line and the actual tissue boundary. If necessary, the contour lines can be manually corrected. The entity of user-drawn and interpolated contour lines should be saved in a file format recognized by the evaluation software, such as geoworks object code (GOBJ).k The drawing of contour lines can be a very tedious

If multiple sets of contour lines are saved for the same measurement during the analysis, the software will typically select the contour definition that (1) is saved in the measurement directory, (2) is saved under the default name, and (3) is the latest version of the file.

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and time-consuming process, and automated segmentation algorithms have been suggested.l Use a Gaussian filter to reduce noise in the grayscale images. This filter has a characteristic Gaussian distribution with a range (support) and peak (sigma) that results in blurring of the image. These two parameters can substantially affect the quality of the image, and thus the selection of appropriate values is critical. A very small sigma will not reduce any noise within the image, whereas large sigma values reduce the effective resolution of the image and therefore tissue detail may disappear (Fig. 4). To preserve detail, the optimal sigma should be as small as possible. A rule of thumb to determine an appropriate support value, as used in several micro-CT laboratories, is to double the sigma value and round it up to the next integer. The easiest method for image segmentation is thresholding. Lower and upper thresholds should be selected in such a manner that the density of the tissue of interest falls within that range. Naturally, threshold ranges that are too large may not be specific enough, while a threshold range that is too small may not include the entire tissue of interest. Global thresholding refers to a technique by which the same threshold is used for multiple regions within a tomography; vice versa, local thresholding involves thresholds that are specific to a given region. The selection of the optimal threshold is anything but trivial, and differences in threshold can cause large data variability (Fig. 4). If the density of the tissue of interest and its spatial distribution are not known, a piece of tissue can be harvested from a dead animal and scanned fresh along with the anesthetized animal. The density value of the harvested tissue will be the first estimate of

Even though drawing contour lines is the current standard for determining the ROI, this procedure is ill-defined, cumbersome, and may lead to significant variations between users whose challenge is to stay close to the tissue boundary. With increasing sample size, the analysis of some tissues can become a prohibitively lengthy process; for instance, the manual drawing of contour lines to separate trabecular from cortical bone in ∼250 slices within the distal femoral metaphysis of a mouse can easily consume 1 hour. Recently, robust algorithms based on the detection of surfaces have been introduced to automate this contouring process, increase processing speed, and reduce variability (Buie and Boyd 2007; Lublinsky et al. 2007).

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Fig. 4. (a) Raw grayscale image of a metaphyseal slice of the distal femur in an adult mouse that was segmented with (b) appropriate filter and threshold settings; (c) a threshold that was too high, resulting in reduced trabecular connectedness and thickness; (d) a threshold that was too low, resulting in greater cortical and trabecular thickness as well as random speckled artifacts; and (e) an appropriate threshold but filtered with a sigma that was too high, resulting in loss of trabecular and cortical detail as well as excessive smoothing.

the mean of the threshold range. The specific range will then be determined iteratively by comparing the segmented image with the raw image. Sometimes, comparisons with a good histologic atlas are helpful in identifying the geometry and distribution of the tissue of interest (An and Martin 2003). Semiautomatic and fully automatic segmentation methods that only partially rely (or do not rely at all) on the selection of a threshold have been suggested (Laib and Ruegsegger 1999; Laib et al. 1998; Waarsing et al. 2004), but a fully standardized method is still elusive in this field. An appropriate threshold will produce a segmented image with a virtually identical appearance (in terms of thickness, connectedness, and porosity) compared to the nonsegmented image. Visual comparisons between

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raw and segmented images are typically more effective when performed in two rather than three dimensions. Once the Gaussian parameters and threshold values have been optimized, they should be maintained for all samples within the same study to avoid bias or increased data variability. Submit the grayscale images, including their contour lines, for segmentation. Using the manufacturer’s software, this step will automatically quantify volumetric parameters such as the volume of the segmented tissue or the mean density of the tissue. For tissues with a highly intricate architecture such as trabecular bone or vasculature, parameters such as the number of segments, mean segment thickness, mean segment separation, mean segment connectedness, and structural model index can be calculated. Export the final segmented 3D stack of images in different file formats for further analyses. For instance, the moment of inertia of a structure can be used to supplement force-displacement data from destructive mechanical testing in order to determine the bending strength or elastic modulus of the material (Bagi et al. 2006). The micro-CT–generated 3D geometry of a structure can also be directly converted into a finite element model to simulate its mechanical behavior in response to virtual loading (Judex et al. 2003a). Another option is to use rapid prototyping, which can automatically produce physical objects of the 3D scans from the segmented files (typically in .STL file format). The same ISQ image file can be analyzed repeatedly using different sigma, support, and threshold ranges to isolate and analyze different tissues. For example, to determine the bone volume, fat volume, and total volume of an animal, three different sets of contour lines and segmentation values are required (Fig. 5).

3.6. Statistics •

During the experimental design process, a sample-size (power) calculator can be used to determine the necessary sample sizes, based on either preliminary data or projected means and standard deviations.

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Fig. 5. (a) Grayscale images that are scaled to the maximal tissue density of the scanned region can be readily thresholded to (b) contrast calcified tissue (bone in white) from lean tissue (muscle and internal organs in red) and fat (adipose tissue in yellow). (c) Assembly of the segmented 2D images yields a 3D representation of the three types of tissues.

•

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Depending on the distribution of the data, appropriate parametric or nonparametric statistical tests will compare different groups of animals to each other or different time points within a group. Because micro-CT software will output several structurally relevant morphometric parameters, these parameters can be regressed against each other (e.g. multiple linear regression). For example, it can be tested whether differences in the trabecular bone volume fraction between two time points are associated with differences in trabecular thickness and trabecular number.

Acknowledgments Financial support by NASA, the Wallace Coulter Foundation, NSF, and the Whitaker Foundation is gratefully acknowledged.

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References An YH, Martin KL. Handbook of Histology Methods for Bone and Cartilage. Humana Press, Totowa, NJ, 2003. Bagi CM, Hanson N, Andresen C et al. The use of micro-CT to evaluate cortical bone geometry and strength in nude rats: correlation with mechanical testing, pQCT and DXA. Bone 38(1):136–144, 2006. Birkenhager-Frenkel DH, Courpron P, Hupscher EA et al. Age-related changes in cancellous bone structure. A two-dimensional study in the transiliac and iliac crest biopsy sites. Bone Miner 4(2):197–216, 1988. Boone JM, Velazquez O, Cherry SR. Small-animal X-ray dose from microCT. Mol Imaging 3(3):149–158, 2004. Boyd SK, Davison P, Muller R, Gasser JA. Monitoring individual morphological changes over time in ovariectomized rats by in vivo microcomputed tomography. Bone 39(4):854–862, 2006a. Boyd SK, Moser S, Kuhn M et al. Evaluation of three-dimensional image registration methodologies for in vivo micro-computed tomography. Ann Biomed Eng 34(10):1587–1599, 2006b. Brouwers J, Van Rietbergen B, Huiskes R. No effects of in vivo micro-CT radiation on structural parameters and bone marrow cells in proximal tibia of Wistar rats detected after eight weekly scans. J Orthop Res 25(10):1325–1332, 2007. Buie HR, Boyd SK. Automated segmentation based on a dual threshold for in vivo micro-CT bone analysis. Transactions of the 53rd Annual Meeting of the Orthopaedic Research Society, San Diego, CA, poster no. 1371, 2007. Cowan CM, Aghaloo T, Chou YF et al. MicroCT evaluation of threedimensional mineralization in response to BMP-2 doses in vitro and in critical sized rat calvarial defects. Tissue Eng 13(3):501–512, 2007. Feldkamp LA, Goldstein SA, Parfitt AM et al. The direct examination of three-dimensional bone architecture in vitro by computed tomography. J Bone Miner Res 4(1):3–11, 1989. Goldstein RZ, Alia-Klein N, Leskovjan AC et al. Anger and depression in cocaine addiction: association with the orbitofrontal cortex. Psychiatry Res 138(1):13–22, 2005. Idee JM, Corot C. Thrombotic risk associated with the use of iodinated contrast media in interventional cardiology: pathophysiology and clinical aspects. Fundam Clin Pharmacol 13(6):613–623, 1999.

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Idee JM, Lancelot E, Berthommier C et al. Effects of non-ionic monomeric and dimeric iodinated contrast media on renal and systemic haemodynamics in rats. Fundam Clin Pharmacol 14(1):11–18, 2000. Judex S, Boyd S, Qin YX et al. Adaptations of trabecular bone to low magnitude vibrations result in more uniform stress and strain under load. Ann Biomed Eng 31(1):12–20, 2003a. Judex S, Boyd SK, Qin YX et al. Combining high-resolution microCT with material composition to define the quality of bone tissue. Curr Osteoporos Rep 1(1):11–19, 2003b. Judex S, Chung H, Torab A et al. Micro-CT induced radiation does not exacerbate disuse related bone loss. Transactions of the 51st Annual Meeting of the Orthopaedic Research Society, Washington, DC, p. 1546, 2005. Klinck RJ, Boyd S. The effect of radiation on bone architecture for in vivo micro-computed tomography. Transactions of the 52nd Annual Meeting of the Orthopaedic Research Society, Chicago, IL, p. 1596, 2006. Ladinsky GA, Wehrli FW. Noninvasive assessment of bone microarchitecture by MRI. Curr Osteoporos Rep 4(4):140–147, 2006. Laib A, Hauselmann HJ, Ruegsegger P. In vivo high resolution 3D-QCT of the human forearm. Technol Health Care 6(5–6):329–337, 1998. Laib A, Ruegsegger P. Comparison of structure extraction methods for in vivo trabecular bone measurements. Comput Med Imaging Graph 23(2):69–74, 1999. Lublinsky S, Ozcivici E, Judex S. An automated algorithm to detect the trabecular-cortical bone interface in micro-computed tomographic images. Calcif Tissue Int 81(4):285–293, 2007. Mashiba T, Hirano T, Turner CH et al. Suppressed bone turnover by bisphosphonates increases microdamage accumulation and reduces some biomechanical properties in dog rib. J Bone Miner Res 15(4):613–620, 2000. McCormack EJ, Egnor MR, Wagshul ME. Improved cerebrospinal fluid flow measurements using phase contrast balanced steady-state free precession. Magn Reson Imaging 25(2):172–182, 2007. Morey-Holton ER, Globus RK. Hindlimb unloading rodent model: technical aspects. J Appl Physiol 92(4):1367–1377, 2002. Muller R, Hildebrand T, Ruegsegger P. Non-invasive bone biopsy: a new method to analyse and display the three-dimensional structure of trabecular bone. Phys Med Biol 39(1):145–164, 1994.

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Muller R, Ruegsegger P. Micro-tomographic imaging for the nondestructive evaluation of trabecular bone architecture. Stud Health Technol Inform 40:61–79, 1997. Muschick P, Wehrmann D, Schuhmann-Giampieri G, Krause W. Cardiac and hemodynamic tolerability of iodinated contrast media in the anesthetized rat. Invest Radiol 30(12):745–753, 1995. Rubin C, Recker R, Cullen D et al. Prevention of postmenopausal bone loss by a low-magnitude, high-frequency mechanical stimuli: a clinical trial assessing compliance, efficacy, and safety. J Bone Miner Res 19(3):343–351, 2004. Ruegsegger P, Koller B, Muller R. A microtomographic system for the nondestructive evaluation of bone architecture. Calcif Tissue Int 58(1): 24–29, 1996. Squire M, Donahue LR, Rubin C, Judex S. Genetic variations that regulate bone morphology in the male mouse skeleton do not define its susceptibility to mechanical unloading. Bone 35(6):1353–1360, 2004. Waarsing JH, Day JS, Weinans H. An improved segmentation method for in vivo microCT imaging. J Bone Miner Res 19(10):1640–1650, 2004. Watts NB. Fundamentals and pitfalls of bone densitometry using dualenergy X-ray absorptiometry (DXA). Osteoporos Int 15(11):847–854, 2004.

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Chapter 21

Positron Emission Tomography of Bone in Small Animals Erik Mittra, Shahriar S. Yaghoubi and Yi-Xian Qin

Molecular imaging is a relatively new field in which a variety of imaging modalities are used to evaluate cellular and molecular process in vivo. From within this multidisciplinary field, this chapter focuses on the methodology of positron emission tomography (PET) imaging of small animals. While clinical nuclear medicine imaging of the musculoskeletal system primarily encompasses MDP bone scans and FDG PET scans for the evaluation of osseous malignancy, the tracer of choice for research applications in small animals (usually mice or rats) is fluorine-18–fluoride ion (F−). This tracer is incorporated into the hydroxyapatite matrix, and is therefore a preferential bone-imaging agent. It is otherwise nonspecific and has been used for a variety of applications including imaging fractures, microdamages, and bone cancers. After detailing the materials and methods necessary to perform PET imaging of small animals using F−, a review of the current literature in this area is provided with comprehensive examples of the types of images that can be obtained for both visual and quantitative representations of various musculoskeletal processes. Pitfalls for this type of imaging are also discussed. Future applications of this powerful modality are expected to grow as the technology improves. Keywords:

Positron emission tomography; small animal PET; bone density; molecular imaging; fluorine; fluoride ion.

Corresponding author: Yi-Xian Qin. Tel: +1-631-6321481; fax: +1-631-6328577; E-mail: [email protected]

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1. Introduction Molecular imaging is an emerging multidisciplinary field in which various modalities are used with the goal of in vivo functional imaging. These modalities include single photon emission computed tomography (SPECT), positron emission tomography (PET), magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MRS), computed tomography (CT), ultrasound (US), fluorescence, and bioluminescence (Mayer-Kuckuk and Boskey 2006). The functional processes that are attempted to be visualized include gene expression, protein activity, cell trafficking, and metabolism. The ultimate applications of molecular imaging lie in oncology, cardiology, neurology, and the musculoskeletal system. These applications/modalities have found importance in both basic science research and clinical noninvasive diagnostics. For instance, in oncology, the primary clinical focus is on the accurate staging and restaging of different cancers using PET; meanwhile, basic science research is targeting the development of more specific tracers for individual cancers that can differentiate between infection, inflammation, and malignancy, and that may subsequently be used to deliver and image therapeutics (either nuclear- or viral-based). In current clinical practice, noninvasive functional imaging of the musculoskeletal system primarily employs three radiopharmaceuticals: technetium-99m bound to methylene diphosphonate (99mTc-MDP), fluorine-18 bound to fluorodeoxyglucose (18F-FDG), and free [18F] flouride ion (F−). 99mTc decays primarily by gamma emission and is therefore imaged using either planar or SPECT gamma cameras. 18F follows a positive beta (positron) decay scheme and is therefore imaged with a PET scanner (Ziessman et al. 2006). MDP is the most common diphosphonate compound used in clinical nuclear medicine and, like all diphosphonates, is bound to bone by chemoadsorption in the hydroxyapatite mineral and is therefore higher in areas of active bone formation (Ziessman et al. 2006). FDG, a glucose analog, is preferentially trapped in any hypermetabolic tissue and therefore is not specifically a bone-seeking agent. F− naturally incorporates into the bone matrix as the ions slowly exchange with hydroxyl groups in the hydroxyapatite crystal of bone

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to form fluoroapatite; as would be expected, then, the activity is greatest at the bone surface, where remodeling occurs. For all tracers, skeletal uptake is influenced by multiple factors including blood flow to bone, molecular size, net electric charge of the molecule, local pH, and metabolic activity of the bone (Berger et al. 2002). 99m Tc-MDP bone scintigraphy has been used for decades for a variety of applications, with the main ones being the evaluation of primary and metastatic diseases, occult fractures and traumas, infections (osteomyelitis), and metabolic diseases. 18F-FDG PET scanning is primarily used in oncology for the evaluation of carcinoma in all tissues including the skeleton. The bony lesions visualized by MDP bone scans versus 18F-FDG PET scans overlap; and several articles have tried to evaluate the relative sensitivity and specificity of one modality over the other for the evaluation of bone metastases of different cancer types (Ohta et al. 2001; Hsia et al. 2002; Nakamoto et al. 2003; Abe et al. 2005; Kato et al. 2005; Nakai et al. 2005; Even-Sapir et al. 2006; Fujimoto et al. 2006), with the general consensus that FDG PET is better for osteolytic lesions while MDP bone scans are better for osteoblastic lesions. F− is arguably the most useful tracer for bone, as it is a highly specific skeletal imaging agent. However, F− PET scans are not performed regularly because of the widespread availability, ease, and low cost of MDP bone scans. In humans, it is primarily used in research protocols. In small animal research, F− is the tracer of choice for functional imaging of the musculoskeletal system (Berger et al. 2002). Although attempts have been made to use the more widely available clinical PET/CT scanners for small animal imaging, the low resolution limits their applicability; therefore, a dedicated small animal PET scanner is recommended for research use (Tai et al. 2003; Seemann et al. 2006) (Fig. 1). Although preferentially bound in bone, F− is otherwise nonspecific and has therefore been used in a variety of studies on cancer models, fracture healing, fatigue loading, and microdamage (Berger et al. 2002; Li et al. 2005; Silva et al. 2006). The goal of this chapter is to describe the methodology of wholebody PET imaging of the skeleton in small animals using F−. The small animal PET imaging methods described by Yaghoubi and Gambhir (2006) are modified here for imaging with F−.

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Fig. 1. A 32-g mouse imaged with a dedicated small animal PET scanner (left) versus a clinical PET scanner (right) for 30 minutes each. Reproduced with permission from Tai and Laforest (2005).

2. Materials •

Small animal PET scanner

Figure 2 shows the two commercially available small animal PET scanners that are currently housed at the Small Animal Imaging Facility at Stanford University, Palo Alto, CA: one produced by Concorde Microsystems (MicroPET rodent R4), and the other by GE Healthcare (eXplore Vista). •

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Different strains of mice can be used, depending on the purpose of the research. Immunodeficient mice can be used for models with human tumors or metastases. Flourine-18 (18F)
Flouride ion is cyclotron-produced and has a half-life of 110 minutes. The production of this tracer is beyond the scope of this chapter, but requires full on-site cyclotron facilities.

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(a)

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Fig. 2. Examples of two commercially available small animal PET scanners, currently housed at the Small Animal Imaging Facility at Stanford University, Palo Alto, CA, USA. (a) A Concorde Microsystems MicroPET rodent R4 scanner. (b) A GE Healthcare (Suinsa) eXplore Vista small animal PET scanner. From mips.stanford.edu/public/instruments.adp/.

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Image analysis software It is provided by the manufacturer of the small animal PET scanner. Alternatively, AMIDE (A Medical Imaging Data Examiner) is freeware available online (Loening and Gambhir 2003) also for this purpose. Small animal anesthesia set up
It includes an induction/housing chamber and a working area with a nostril mask for performing the tail vein injection. Isoflurane is often used as the induction agent, although this may be adjusted based on the requirements of the individual small animal protocol at each institution. Figure 3 shows the setup at the Small Animal Imaging Facility at Stanford University, Palo Alto, CA, USA. Syringes
Insulin syringes can be used for tail vein injection of the PET tracer, with minimal residual left in the syringe. Alcohol pads Warming lamp or heat pads


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Fig. 3. Pictures of the small animal anesthesia setup, including the isoflurane pump, induction/housing chamber, and procedure bed at the Small Animal Imaging Facility at Stanford University.

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Gloves and masks, as appropriate Radiation safety equipment
It includes a shielded work area, shielded waste container, Geiger counter, etc.

3. Method Despite the relative novelty of this technology and procedure, the methodology for small animal PET imaging of mice is fairly straightforward. In general, it involves the injection of the tracer, a waiting period during the uptake phase, scanning with a small animal PET scanner, and image analysis using any number of commercially available software packages. While the methodology is uncomplicated, the power of the technique stems from the functional information available from the images and the experimental model being studied. The specifics of the methodology are as follows: • •

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Draw ∼750 µCi of [18F]fluoride ion into an insulin syringe. Record the exact dose and the time of measurement. Inject the entire amount of [18F]fluoride ion in the insulin syringe into the tail vein of the mouse. This procedure may require considerable practice to avoid injection of [18F]F− outside the vein. To dilate the vein, heat packs or lamps may be used. Record the injection time. Measure the residual activity in the emptied syringe. The optimal uptake time for [18F]F− has been previously determined to be about 1 hour. During the injection and waiting period, the mouse need not be anesthetized (as opposed to injection of 18F-FDG). For dynamic [18F]F− scans, image acquisition may start immediately after the [18F]F− injection. Start anesthetizing the mouse using the method described in your approved study protocol 5 minutes before scanning. In our laboratory, we used 2% isoflurane or a mixture of 80 mg/kg of ketamine and 20 mg/kg of xylazine. Position the unconscious mouse in the center of the field of view of the small animal PET scanner (either supine or prone position),

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and then move the bed into the camera. The micro-PET R4 scanner can usually scan an entire mouse in a single bed position. Refer to your scanner’s manual for instructions on image acquisition. For a whole-body static scan, a 10-minute acquisition time should be satisfactory. The quality of the images improves with longer acquisition times. Record the scan start time. Images are decay-corrected to the scan start time, which is necessary for calculating the injected dose at the scan start time. Be careful that the animal does not inadvertently wake up or move during the scan. Reconstruct the image. The manufacturers of the MicroPET R4 and eXplore Vista scanners provide software for the reconstruction of images with the filtered backprojection and ordered subsets expectation maximization (OSEM) algorithms (Hudson and Larkin 1994). The OSEM reconstruction algorithm usually yields images of better quality. Analyze the image. The manufacturer should have provided image analysis software. In our laboratory, we used the AMIDE software, freely available for download online (Loening and Gambhir 2003). This software allows the visualization and quantitative analysis of images acquired by a Concorde Microsystems MicroPET scanner. Display the images and perform quantitative calculations. Images can be displayed coronally, sagittally, or transaxially. By providing the injected dose and the cylinder calibration factor, the AMIDE program can calculate the percentage injected dose per gram or standard uptake values (SUVs) in the regions of interest (ROIs).

4. Results Figure 4 is an example of a normal F− PET scan on a mouse. The image shown was obtained 1 hour after the tail vein injection of ∼750 µCi of [18F]fluoride ion. As seen, the tracer binds preferentially to bone. There is very good uptake in the skull as well as the axial and

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Fig. 4. Whole-body mean coronal projection image from a PET fluoride ion scan in a healthy Swiss–Webster mouse. The regions of interest (ROIs) to obtain time–activity curves of bony structures like the humerus or femur are shown exemplarily. Reproduced with permission from Berger et al. (2002).

appendicular skeletons, with minimal uptake in soft tissues. Relatively small structures such as the fibula, which has a diameter of ∼350 µm in an adult mouse, are well visualized. These demonstrate the capability of such systems to acquire whole-body bone scans in mice with excellent quality. This uptake is graphically quantified in Fig. 5, showing that, over 2 hours (essentially equivalent to the half-life of 110 minutes of fluoride ion), there is an initially increasing and then stable uptake of the tracer in bone, with negligible washout seen. As mentioned previously, the nonspecific nature of F− uptake in bone has been utilized to study a variety of musculoskeletal processes including fracture, fatigue loading, microdamage, and, of course, cancer. Several examples of these applications are examined below. Figure 6 demonstrates the use of PET to temporally follow bone metabolism in a fracture model of the humerus in a Swiss–Webster

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Fig. 5. Time courses of decay-corrected activities from [18F]fluoride ion in bone tissue, obtained from the ROIs over bony regions in dynamic PET scans. The activity in the bony regions peaks and plateaus ∼60 minutes after tracer application. Data are expressed as mean counts/pixel per minute ± standard error. Reproduced with permission from Berger et al. (2002).

mouse. As seen, over the period from 5 days to 48 days postinjury, there is an increasing F− intensity during the initial phases of fracture healing, followed by a gradual decline as the rate of healing (and bone turnover) decreases. This is characterized by significantly increased normalized standardized uptake values (SUVs) on the affected humerus as compared to the contralateral control arm. Proof of the initial fracture and subsequent union is shown in the comparative radiographs at 1 and 42 days postfracture, respectively, consistent with the findings on PET. More subtle bone turnover can also be visualized and quantified, as shown in several fatigue loading models that induce microdamage in bone. Figure 7 shows PET, CT, and fused PET/CT images from a fatigue loading model of the humerus in a rat. These images were obtained 4 days after fatigue loading with 85% displacement. As seen, aside from the normal physiologic uptake in the remainder of the bone, there is a unilateral focus of tracer uptake in the right forelimb,

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Fig. 6. Temporal radiographic (left) and PET (right) evaluation of a fracture of the humerus in a mouse over 48 days. The radiographs demonstrate the initial fracture (top) followed by reunion 6 weeks postinjury (bottom). The [18F]fluoride ion scans show variable tracer uptake over an ROI over the humerus (normalized to whole-body ROI) in the affected limb (shown below animal) as the bone undergoes healing. The standardized uptake values (SUVs) of the contralateral control limb (shown below the bone) are stable and significantly (p = 0.001) lower than the SUVs obtained from the lesion. Reproduction with permission from Berger et al. (2002).

Fig. 7. Single-slice (a) PET, (b) micro-CT, and (c) coregistered PET/CT images of the cranial half of a rat 4 days after fatigue loading. Aside from diffuse physiologic bony uptake elsewhere, asymmetric uptake is seen in the central region of the right forelimb (arrow) in the region of loading. Reproduced with permission from Silva et al. (2006).

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Fig. 8. PET images of the cranial half of a mouse (a) 1 day after and (b) 12 days after fatigue loading of the right forelimb, showing higher intensity at the second time point secondary to more robust healing of the induced microcrack. Reproduced with permission from Li et al. (2005).

consistent with the region where the fatigue loading was performed. Negligible uptake is seen on the contralateral control forearm. As in the fracture model, a temporal increase in tracer intensity can also be seen in a fatigue loading model, as shown in Fig. 8. Here, increased tracer uptake is seen at 12 days postloading versus 1 day postloading, which is consistent with the increased bone turnover during the initial stages of healing. Presumably, this uptake would subsequently decrease over time, as in the fracture model. Confirmation of a microcrack in the region of increased uptake in the right forelimb is shown in Fig. 9 by autoradiography and microscopy. As mentioned, the most significant application of 18F-FDG PET in clinical practice is for the evaluation of primary or metastatic bone tumors. Of course, this application can also be examined in small animals. Figure 10 shows the comparative CT, F−, and FDG PET images of a mouse 2 months after intratibial injection of the androgen-independent prostate cancer cell line CL-1. At this time, the intratibial tumor measures 12 mm in diameter. The micro-computed axial tomography (microCAT) image shows the osteolytic nature of the lesion, the F− scan shows

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Fig. 9. (a) The increased tracer intensity seen in vivo in the right ulnar midshaft on PET is confirmed by (b) in vitro autoradiography which shows the enhanced accumulation of radioactive tracer, and with (c) microscopic evaluation showing a microcrack (arrows) in the longitudinal section. Reproduced with permission from Li et al. (2005).

high bone turnover in this region, and the FDG scan demonstrates high glucose uptake in this aggressive prostate cancer cell line. These examples show the sensitivity and general applicability of small animal PET for applications in bone. At the same time, the principal limitations of this technique are its lower resolution and lack of specificity. As the F− ion is incorporated in any remodeling bone, the resulting images are not specific to the underlying process (whether from injury, malignancy, or infection/inflammation); this may not be a concern in focused animal models, but remains a general limitation. Also, the resolution limit of the current iteration of small animal PET scanners is still not ideal for a very fine evaluation of bone remodeling,

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Fig. 10. Micro-CT, [18F]fluoride ion, and [18F]FDG PET images in a mouse 2 months after the intratibial injection of the androgen-independent prostate cancer cell line CL-1. The micro-computed axial tomography (micro-CAT) image demonstrates the osteolytic character of the lesion, while the [18F]fluoride ion PET scan shows increased bone turnover (with an increased SUV over the contralateral limb), and the [18F]FDG scan demonstrates high glucose uptake and metabolism in this very aggressive prostate cancer cell line. Tracer accumulation in both kidneys is seen in the [18F]FDG scan (horizontal arrows). Reproduced with permission from Berger et al. (2002).

such as at the trabecular level; with ongoing improvements in scanner technology, however, this issue will become less significant. Therefore, the future of this powerful modality is sure to grow as the equipment continues to improve and novel research applications are found. In particular, it is expected that future generations of scanners will have improved resolution and scan times, new tracers will be more specific to certain cell lines or physiological processes, and new fusion technologies such as PET/MRI will be made available for small animal research.

Acknowledgments This work was kindly supported by the National Space Biomedical Research Institute (TD00207 and TD00405 to Y.-X. Qin) through

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NASA Cooperative Agreement NCC 9-58, by the National Institutes of Health (R01 AR52379 and R01 AR49286 to Y.-X. Qin), and by the US Army Medical Research and Materiel Command (DAMD-1702-1-0218 to Y.-X. Qin).

References Abe K, Sasaki M, Kuwabara Y et al. Comparison of 18FDG-PET with 99mTcHMDP scintigraphy for the detection of bone metastases in patients with breast cancer. Ann Nucl Med 19(7):573–579, 2005. Berger F, Lee YP, Loening AM et al. Whole-body skeletal imaging in mice utilizing microPET: optimization of reproducibility and applications in animal models of bone disease. Eur J Nucl Med Mol Imaging 29(9):1225–1236, 2002. Even-Sapir E, Metser U, Mishani E et al. The detection of bone metastases in patients with high-risk prostate cancer: 99mTc-MDP planar bone scintigraphy, single- and multi-field-of-view SPECT, 18F-fluoride PET, and 18F-fluoride PET/CT. J Nucl Med 47(2):287–297, 2006. Fujimoto R, Higashi T, Nakamoto Y et al. Diagnostic accuracy of bone metastases detection in cancer patients: comparison between bone scintigraphy and whole-body FDG-PET. Ann Nucl Med 20(6):399–408, 2006. Hsia TC, Shen YY, Yen RF et al. Comparing whole body 18F-2-deoxyglucose positron emission tomography and technetium-99m methylene diphosphate bone scan to detect bone metastases in patients with non–small cell lung cancer. Neoplasma 49(4):267–271, 2002. Hudson HM, Larkin RS. Accelerated image reconstruction using ordered subsets of projection data. IEEE Trans Med Imaging 13(4):601–609, 1994. Kato H, Miyazaki T, Nakajima M et al. Comparison between whole-body positron emission tomography and bone scintigraphy in evaluating bony metastases of esophageal carcinomas. Anticancer Res 25(6C): 4439–4444, 2005. Li J, Miller MA, Hutchins GD, Burr DB. Imaging bone microdamage in vivo with positron emission tomography. Bone 37(6):819–824, 2005. Loening AM, Gambhir SS. AMIDE: a free software tool for multimodality medical image analysis. Mol Imaging 2(3):131–137, 2003. Mayer-Kuckuk P, Boskey AL. Molecular imaging promotes progress in orthopedic research. Bone 39(5):965–977, 2006.

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Nakai T, Okuyama C, Kubota T et al. Pitfalls of FDG-PET for the diagnosis of osteoblastic bone metastases in patients with breast cancer. Eur J Nucl Med Mol Imaging 32(11):1253–1258, 2005. Nakamoto Y, Osman M, Wahl RL. Prevalence and patterns of bone metastases detected with positron emission tomography using F-18 FDG. Clin Nucl Med 28(4):302–307, 2003. Ohta M, Tokuda Y, Suzuki Y et al. Whole body PET for the evaluation of bony metastases in patients with breast cancer: comparison with 99Tcm [sic]-MDP bone scintigraphy. Nucl Med Commun 22(8):875–879, 2001. Seemann MD, Beck R, Ziegler S. In vivo tumor imaging in mice using a state-of-the-art clinical PET/CT in comparison with a small animal PET and a small animal CT. Technol Cancer Res Treat 5(5):537–642, 2006. Silva MJ, Uthgenannt BA, Rutlin JR et al. In vivo skeletal imaging of 18Ffluoride with positron emission tomography reveals damage- and timedependent responses to fatigue loading in the rat ulna. Bone 39(2):229–236, 2006. Tai YC, Chatziioannou AF, Yang Y et al. MicroPET II: design, development and initial performance of an improved microPET scanner for small-animal imaging. Phys Med Biol 48(11):1519–1537, 2003. Tai YC, Laforest R. Instrumentation aspects of animal PET. Annu Rev Biomed Eng 7:255–285, 2005. Yaghoubi SS, Gambhir SS. PET imaging of herpes simplex virus type 1 thymidine kinase (HSV1-tk) or mutant HSV1-sr39tk reporter gene expression in mice and humans using [18F]FHBG. Nat Protoc 1(6):3069–3075, 2006. Ziessman HA, O’Malley JP, Thrall JH. Nuclear Medicine. Elsevier, Philadelphia, PA, 2006.

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Part IV Laboratory Animal Models

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Chapter 22

Surgical Anesthesia and Analgesia for Animals in Musculoskeletal Research Dewi K. Rowlands and Anthony E. James

In this chapter, the humane use of animals in surgical research is described, with reference to Russell and Burch’s The Principles of Humane Experimental Technique (1992) — commonly known as the 3R’s of replacement, reduction, and refinement — as well as the ethical need for researchers to justify the experiment and take responsibility for the well-being of animals in their care. The basic role of animal ethics committees is also discussed. The chapter then describes in practical terms the preparation of the experimental animal for surgery; the techniques for anesthesia, including knock-down, intubation, and maintenance; and the drugs used for premedication before anesthesia, maintenance of anesthesia, and, most importantly, pre-emptive and postoperative pain relief. The monitoring of the experimental animal under anesthesia and during recovery is also discussed. Keywords:

Ethics; anesthesia; analgesia; operative care; rats; rabbits; goats; surgery.

1. Introduction Over the past decade, a number of advances have been made in our knowledge of bone healing and fracture management, many of which Corresponding author: Dewi K. Rowlands. Tel: +852-26096042; fax: +852-26035723; E-mail: [email protected]

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would not have been achieved without the use of laboratory animal models in rodents, rabbits, and goats. In this chapter, a brief overview of the ethics of animal use as well as suitable analgesic and anesthetic protocols for use in these species are provided. This chapter serves as a guide to what is considered to be the most important principles in the anesthesia and surgical care of laboratory animals, including pain relief. It is by no means a comprehensive treatise to the very complex field of veterinary anesthesia, analgesia, and surgery. Detailed descriptions can be found in a number of excellent publications (van Zutphen et al. 2001; Flecknell 1996; Thurmon et al. 1996) and, as always, the advice of a veterinary surgeon and an experienced animal researcher is always recommended.

1.1. Ethics and law The argument between advocates supporting animal use — whether it is for research, food and fiber production, or companionship — and their opponents is not something new to the recent past, but an issue that has grown in importance since the early 19th century. The nowfamous words of Jeremy Bentham — “The question is not, Can they reason?, nor Can they talk? but, Can they suffer?” (Bentham 1789) — are one of the guiding principles used by many people concerned with animal welfare and the way in which animals should be used. Bentham’s utilitarian concept has been expanded upon by the philosopher Peter Singer in his book Animal Liberation (Singer 2001), although his utilitarian view on animal usage is not accepted by everyone. For the purpose of this chapter, however, it is not appropriate to explore the numerous arguments that have been put forward against and in defense of animals in research; instead, the reader is referred to the book by Dolan (1999) titled Ethics, Animals and Science, in which many alternative perspectives and counterarguments are suitably covered. In the majority of jurisdictions, although there may be doubts among members of the public about the morality of using animals in research, the fact is that the use of animals in research is not only permissible, but in some cases (e.g. toxicity testing) mandatory. Public opinion on the acceptability of animals in research is somewhat variable,

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depending on the survey methodology used (Hagelin et al. 2003). For example, in Britain, where research using animals is subject to strict legislative controls and where there are highly organized groups of antivivisection activists, the British public is generally accepting of research using animals, provided that the benefits for humankind are apparent and that the animals are treated humanely (UK Department of Trade and Industry 2006; Coalition for Medical Progress 2005). Russell and Burch’s book, The Principles of Humane Experimental Technique (“The Principles”) (1992), has been hugely influential on the humane use of animals in research. The Principles has somewhat simplistically been labeled as the 3R’s (replacement, reduction, and refinement), but in reality the treatise is far more significant than the simple acronym does justice. Nevertheless, it is by understanding at least the basic principles of Russell and Burch’s 3R’s that researchers can ensure and improve upon the humane care and use of animals in research. To quote the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, There are difficult ethical judgements to be made regarding the use of animals for scientific purposes. The Code requires A[nimal] E[thics] C[ommittees] to determine whether the case for animal use is justified and to ensure adherence to the principles of Replacement, Reduction and Refinement (3Rs). AECs apply a set of principles that are outlined in the Code and that govern the ethical conduct of people whose work involves the use of animals for scientific purposes. [Australian Government 2004]

Since The Principles was published, there has been much discussion about whether the 3R’s adequately cover the ethics of using animals in research. For the purposes of this book, it is fair to add two covenants to the 3R’s: the concept of responsibility (Dolan 1999) and the concept of justification (Dolan 1999). Researchers must take responsibility for all matters related to the welfare of their animals and must not abdicate this responsibility to their students, technical staff, or even vivarium staff. Even more significant is the concept of justification. Animals are sentient beings and are capable

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of suffering, but as “[p]ain and distress cannot be evaluated easily in animals investigators must assume that animals experience pain in a manner similar to humans. Decisions regarding their welfare in experimental activities must be based on this assumption unless there is evidence to the contrary” (Hong Kong Government 2004, p. 18). All matters associated with an experiment must therefore be justified by the researcher, whether it is the use of living animals, the choice of species, the protocol adopted, or the number of animals used. The National Research Council’s Guide for the Care and Use of Laboratory Animals states, “This edition of the Guide for the Care and Use of Laboratory Animals … strongly affirms the conviction that all who care for or use animals in research, teaching, or testing must assume responsibility for their well-being” (National Research Council 1996). For the 3R’s to successfully ensure the welfare of animals, researchers must not only justify their research to both peers and members of the public, but also accept their own responsibilities to the animals under their care. Many jurisdictions formalize the principles of humane animal care in legislation, where the responsibility of the researcher is codified and the research project justified in law via some form of licensing procedure (e.g. in the UK). Other jurisdictions require researchers to comply with a Code of Practice which has been mandated by legislation (e.g. Australia); while other jurisdictions have little or no legislation that mandates humane care of animals in research, although they may have a voluntary Code of Practice (e.g. China). No matter the case regarding legislation, researchers are morally obliged to have their research scrutinized by both peers and the public in order to have their research accepted by the international community. Dr Carol Newton suggested that this makes for not only good science, but also good sense and good sensibility (Dolan 1999). To this end, animal ethics committees (AECs) or institutional animal care and use committees (IACUCs) are incorporated into most, if not all, codes of practice. Furthermore, such committees are mandated in most, if not all, jurisdictions that have

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legislative protection for the welfare of animals in research. Legislation, codes of practice, and committees that oversee the welfare of animals in research go hand-in-hand with the principles of the 3R’s. AECs and IACUCs generally have a researcher (as a peer) to assess the scientific merits of the research project, a member of the public to present an independent lay opinion on the research project, a veterinary expert to provide advice on the physiological and behavioral well-being of the animals involved in the research project (National Research Council 1996; Australian Government 2004), and an individual with a commitment to animal welfare to provide an opinion on the humaneness or ethics of the research project (Australian Government 2004). This list is not exhaustive or mutually exclusive because such committees can and should include other individuals who can contribute to the assessment of the merit, justification, and humaneness of the proposed project. Hence, even in situations where legislative protection for the welfare of animals in research is poor or nonexistent, no reputable researcher can escape the responsibility that is incumbent upon them to justify the research and to carry it out in a humane manner according to the principles of the 3R’s (Australian Government 2004).

1.2. Preparation Prior to any surgical procedure, thorough preparation and planning are essential to reduce not only animal suffering, but also the chances of surgical variation which will inevitably affect results. The preparation should include an understanding of the basic principles and aims of what is to be achieved; the anatomy of the species; and the speciesspecific needs of preoperative, perioperative, and postoperative care. Preoperative preparation should include animals which are in good health, preferably with specific pathogen-free (SPF) status, and which ideally have acclimatized to their surroundings and fasted before surgery. The use of SPF rodents and disease-free rabbits and goats is of particular importance, since pathological changes are

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known to alter physiological parameters significantly (van Dongen et al. 1990); maintaining the rodent’s SPF status during and following surgery is also important. Therefore, an aseptic technique must be employed. Sufficient acclimatization of research animals prior to surgery or experimentation allows the animals to exhibit normal physiological parameters that may otherwise have been disturbed by transportation into a new environment. Indeed, numerous studies carried out in rodents have demonstrated significant changes in cardiovascular, gastric, neurophysiological, and circadian systems following transportation (Capdevila et al. 2007). Ideally, animals should be allowed a minimum of 7 days to adapt to a new environment prior to experimentation, and as long a duration as possible following surgery for sufficient recovery. Studies have shown that rodents take a minimum of 10 days to recover normal circadian rhythm following surgery (unpublished data), with other physiological parameters perhaps taking even longer to equilibrate. When conducting surgical procedures in ruminants such as goats, it is important to withdraw food, as goats may regurgitate rumen contents during or following anesthesia and may also suffer severe and life-threatening bloat. Food should be withdrawn for approximately 12–16 hours prior to surgery, but water should still be freely available. Food withdrawal is not as critical for rabbits and rodents, as they do not vomit. However, for all three species, preanesthetic treatment of an anticholinergic agent such as atropine will help reduce salivary/bronchial secretions and autonomic responses (Kohn et al. 1997). A note of warning, though: atropine in goats will reduce only the watery secretions that make up part of saliva and will not stop the mucoid salivary secretions; hence, goats continue to produce a more viscous type of saliva during surgery while under the influence of atropine. Other basic aspects of surgical preparation and planning should also include the obvious, but sometimes overlooked, need to arrange for sterilization of instruments, sufficient analgesic and anesthetic agents, and, most importantly, ethical/legal permission to perform the procedure.

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1.3. Anesthesia, analgesia, and perioperative care It is now recognized by many researchers that animals experience noxious stimuli in a comparable way to humans (van Zutphen et al. 2001; Hellebrekers 2000). Thus, procedures on animals that would likely result in pain or discomfort in humans would likely result in a similar level of discomfort or sensation of pain in animals (van Zutphen et al. 2001). Pain and distress in experimental animals must therefore be removed or reduced to such a level that would be acceptable to a human subject. This philosophy makes ethical as well as scientific sense, since pain and discomfort elicit a wide range of physiological responses that would lead to alteration and thus changes in the validity of the animal model.

1.3.1. Anesthesia In rodents and rabbits, anesthesia can be induced and maintained relatively simply using injectable anesthetic agents or a gaseous induction chamber; whereas goats need induction with a short-acting injectable agent followed by maintenance with either gaseous or injectable agents. The choice of whether to use inhalation or injectable anesthesia is largely practical, since vaporizers and scavenger systems are not always available or affordable to some researchers. However, there are distinct advantages in the use of inhalation agents such as isoflurane, especially in goats, as they can be easily controlled to rapidly manipulate the depths of anesthesia; whereas injectable agents tend to accumulate and are thought to prolong the recovery period. There is no absolute rule by which to determine the best anesthetic protocol or analgesic agent to use. The only criterion is that the following four major components of anesthesia are followed: a sufficient amount of mental sedation/loss of consciousness has been induced, skeletal muscle relaxation has been achieved, sensory analgesia has occurred, and the autonomic nervous system has been inhibited. Although simple general anesthesia can be attained by administration of a single drug such as isoflurane or propofol, these drugs alone may not suppress each component equally; therefore, it is

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desirable to administer different component drugs individually so that a balanced anesthesia can be produced. By providing a balanced anesthetic approach, lower doses of the general anesthetic may also be used, reducing the overall chance of overdose and the recovery time. 1.3.2. Analgesia Analgesia, or the prevention of pain, is an extremely important aspect of postoperative as well as perioperative care. It is a common misconception that analgesic relief is only needed during recovery, when in fact analgesia is much more effective when given pre-emptively. Exposing the patient to analgesia before noxious challenge prevents nociceptive receptor sensitization or analgesic wind-up, which if left untreated leads to hyperalgesia (inflammatory pain). The concept of pre-emptive analgesia is now therefore regarded as one of the most efficacious and cost-effective ways of preventing postoperative pain, and is encouraged for all surgical procedures (Perkowski and Wetmore 2006; Thurmon et al. 1999). There are two main classes of drugs that can be used for both perioperative and postoperative pain relief: opiates and nonsteroidal anti-inflammatory drugs (NSAIDs). Long-acting opiates such as buprenorphine are a good analgesic choice, and should be administered prior to the surgery and at subsequent 6–12-hour intervals after surgery to alleviate pain (Flecknell 1996; van Zutphen 2001). NSAIDs are considered a less effective choice, with carprofen currently considered the next best option if opiates are unsuitable, again being administered pre-emptively and for at least 48 hours after surgery. To be sure, both classes of drugs do have side-effects that may interfere with the experiment. Since NSAIDs may affect bone healing and prolong blood clotting, opiates may be better indicated for orthopedic pain relief, although opiates can also cause respiratory depression, lower blood pressure, and constipation (Simon and O’Connor 2007; Murnaghan et al. 2006). However, despite these concerns, providing pain relief is considered good science, good sense, and good sensibility (Dolan 1999), and is therefore essential.

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Analgesia should be administered pre-emptively as well as during and after all surgical procedures. In the clinical situation, humans are subject to various analgesic protocols, so the use of analgesics in animal models will only strengthen the orthopedic model by making it more clinically relevant. Where the supply of opiates or NSAIDs is seriously contraindicated, long-acting local anesthetics may be injected into the surgical wound or local anesthetics/analgesics may be delivered into the epidural space, and can be a substitute for systemic analgesia due to reduction in systemic effects. 1.3.3. Endotracheal intubation and artificial ventilation With rodents, endotracheal intubation (i.e. the insertion of a tube into the trachea) is rarely necessary, unless thoracic surgery is planned. However, in rabbits and goats, it is advisable that every anesthetized animal be intubated, even if artificial ventilation is not required, so that the airway is kept clear and so that the animal can be easily resuscitated in the event of respiratory depression. The technique varies from species to species, and requires an appropriatesized laryngoscope to be able to sight the epiglottis and vocal cords and to insert the tube into the trachea. In all cases, the animal should be sufficiently anesthetized and the use of local anesthetic is recommended to prevent laryngospasm in many species. A detailed description of intubation methods in a number of species may be found in a number of veterinary books (e.g. see Thurmon et al. 1996; Thurmon et al. 1999). Alternatively, if the experimental protocol does not require recovery of the animal, performing a tracheotomy is an option. The majority of animals under general anesthesia will continue to breathe spontaneously; thus, artificial ventilation may not always be required. However, during longer periods of anesthesia (more than 1–2 hours), when muscle relaxants are used or when thoracotomy is performed, the use of a ventilator is required to maintain adequate gaseous exchange. The settings vary between different species and types of ventilators, but as a guide rats need a tidal volume of 0.5–1 mL (50–80 breaths/min); rabbits, 40–60 mL

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(30–50 breaths/min); and goats, 120–300 mL (12–25 breaths/ min) (van Zutphen et al. 2001; Hikasa et al. 1998). If possible, medical oxygen should also be supplied when ventilating in order to maintain blood oxygen saturation, although not all models of ventilators will allow this. During ventilation, it may be necessary to inhibit spontaneous breathing by providing neuromuscular blocking agents such as vercuronium bromide, but be sure that the ventilator is set up and working properly before you administer the muscle relaxant. During artificial ventilation, there is also greater fluid loss from the lungs; therefore, fluid therapy is recommended. 1.3.4. Vital signs During anesthesia, it is important that the animal be closely monitored at all times for its vital signs. These include observations of the color of mucous membranes, pattern and rate of breathing, body temperature, and pulse rate. When animals are overdosed with anesthetic, they will likely develop respiratory and cardiac depression and will exhibit a blue hue due to lack of oxygenation and depressed cardiac rate. In small animals, body heat is lost rapidly under anesthesia and is a significant cause of mortality following surgery, so animals must be kept warm with the aid of a heat source or a form of insulation. However, a note of warning: as the thermoregulatory system of the animals is depressed, an improperly functioning heat source or a heat source not being monitored by an external thermometer can cause severe burns to the anesthetized animal. Although it is possible to monitor vital signs manually, electronic monitoring devices are relatively affordable and extremely useful for these purposes. At the very least, it is advisable to monitor the oxygen saturation and heart rate with a pulse oximeter. Prior to and during surgery, pain reflexes should be assessed as often as possible; the classical reflexes include pedal reflex, blink reflex, and assessment of deep needle penetration over the site of incision. If any brisk movement or response is detected during pain

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stimuli, the surgery should be stopped and anesthesia adjusted accordingly.

2. Materials 2.1. Drugs The following is a list of drugs that are required for any anesthetic/ analgesic protocol involving animals in musculoskeletal research. • •

•

• •

Preanesthetic: atropine sulphate (Weimer Pharma, Germany) Respiratory and/or cardiac stimulants: 0.1% adrenaline injection (Weimer Pharma, Germany), doxapram hydrochloride (Bomac Animal Health), reversine SA (Parnell Laboratories, Australia), naloxone HCl (Schering-Plough) Anesthetic agents (injectable and volatile): isoflurane (Halocarbon), ketamine (Alfasan, The Netherlands), propofol (B. Braun, Germany), sodium pentobarbital (Alfasan, The Netherlands), xylazine (Alfasan, The Netherlands) Analgesics: buprenorphrine (Schering-Plough, UK), long-acting local anesthetic, xylocaine spray (AstraZeneca) Others: heparin sodium (B. Braun, Germany), vecuronium bromide (Organon, The Netherlands), normal saline (as a minimum)

2.2. Equipment The following is a list of equipment that is required for any anesthetic/ analgesic protocol involving animals in musculoskeletal research. • • • • •

Anesthetic vaporizer Laryngoscope (Welch Allyn, USA) Pulse oximeter (Mindray, China) Syringe infusion pump (Terumo) Syringes, needles, catheters (Terumo)

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3. Methods 3.1. Anesthetic induction 3.1.1. Rats •

•

•

•

• •

Following appropriate acclimatization and food withdrawal (min. 6 hours), restrain the rat by picking it up by the base of the tail and placing it on your forearm. Maintain a grip on the base of the tail with one hand, and restrain the body and head by scruffing the loose skin behind the head and shoulders with your free hand. Invert the rat so that its head is pointing downward, and identify the point of intraperitoneal (i.p.) injection away to the side of the bladder. Inject a mixture of ketamine (75 mg/kg) and xylazine (10 mg/kg) into the peritoneal cavity, ensuring that the mixture is not injected into the viscera or intravenously (i.v.) by briefly retracting the needle plunger (van Zutphen et al. 2001; Flecknell 1996). Place the rat back into its cage until it loses consciousness, as indicated by the loss of righting reflex (i.e. the ability to return to an upright position after being placed on its back). Proceed to shave or cleanse the surgical area. This induction procedure will provide approximately 30 minutes of unconsciousness.

3.1.2. Rabbits •

•

•

Following appropriate acclimatization and food withdrawal (min. 6 hours), remove the rabbit from its cage by grasping the skin on the back and neck with one hand and supporting the hind legs and underbelly with the other hand. Once placed on a table, restrain the rabbit by either wrapping it in a large towel or using a rabbit restraining box, ensuring that the rabbit does not struggle and cause itself injury. Administer a mixture of ketamine (35 mg/kg) and xylazine (5 mg/kg) intramuscularly (i.m.) into the hind leg to induce anesthesia (Suckow and Douglas 1997).

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Once unconscious, proceed to intubate and prepare the surgical wound area, followed by initiation of an injectable or gaseous anesthesia protocol (see below). This induction procedure will provide approximately 30 minutes of unconsciousness.

3.1.3. Goats • •

•

•

•

•

Following appropriate acclimatization and food withdrawal (overnight), separate the goat in a small enclosure. As goats are large and strong animals, it is advisable to use a restraining device. Such a device is called a “crush”, and designs are readily available from websites and textbooks. Using a syringe with an extension tube, inject a mixture of ketamine (11 mg/kg, i.m.), xylazine (0.22 mg/kg, i.m.), and atropine (0.13 mg/kg, i.m.) into one of the large muscles of the neck or thigh (Allen and Borkowski 1999; Kohn et al. 1997). The extension tube gives flexibility in the event that the goat moves while restrained, and therefore prevents the needle from accidentally coming out while the anesthetic drugs are being given, minimizing the risk of injury to the human handlers and to the goat. Wait until complete loss of consciousness before attempting to move the animal. This induction procedure will provide approximately 20 minutes of unconsciousness. Once unconscious, give a small dose of i.v. anesthetic such as barbiturate or propofol to increase the depth of anesthesia, reduce jaw tone, and suppress laryngeal reflexes. The animal is now ready to intubate, connect to the anesthetic vaporizer, and have the surgical wound area prepared.

3.2. Pre-emptive analgesia Pre-emptive analgesia is by far the most effective method of pain relief, and should be administered prior to every surgical procedure (Perkowski and Wetmore 2006) for all frequently used animals (i.e. rats, rabbits, and goats).

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Following anesthetic induction (see above), inject buprenorphrine at doses of 0.01–0.05 mg/kg subcutaneous (s.c.)/i.v. in rats, 0.01–0.05 mg/kg i.m./s.c. in rabbits, and 0.01 mg/kg i.m. in goats at least 15 minutes prior to the first painful stimulus (van Zutphen et al. 2001; Suckow and Douglas 1997; Allen and Borkowski 1999). Repeat administration every 6–12 hours for 2 days following surgery or as needed.

3.3. Injectable anesthesia 3.3.1. Rats The induction protocol combined with the pre-emptive analgesia method described above is often sufficient for short procedures in rats. However, if longer periods of anesthesia are needed following induction, it is possible to top up the ketamine dose for up to 1 hour. If longer periods are necessary, it is advisable to intubate and use gaseous anesthesia. •

•

•

Following induction with a mixture of ketamine and xylazine (see Sec. 3.1.1) as well as pre-emptive analgesia with buprenorphrine (see Sec. 3.2), anesthesia may be extended using ketamine (75 mg/kg, i.p.) and a lower dose of xylazine (<5 mg/kg, i.p.) for up to 1 hour. During anesthesia, monitor the respiratory rate and the color of mucous membranes continuously to ensure adequate gaseous exchange. Observe for signs of pain such as flinching, and maintain surgical levels of anesthesia by monitoring responses to tail-pinch and pedal-reflex tests.

3.3.2. Rabbits and goats • •

Induce anesthesia and intubate the animal as previously described (see Secs. 3.1.2 and 3.1.3). Provide pre-emptive analgesia with an injection of buprenorphrine as described above (see Sec. 3.2).

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•

•

•

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Identify the marginal ear vein in the rabbit or the jugular vein in the goat. Clean the area with 70% ethanol, which will also aid vasodilatation. Catheterize the vein using a 22G i.v. needle catheter for rabbit or a 20G i.v. needle catheter for goat. Attach a three-way valve and secure the catheter with surgical tape. Flush the catheter with sodium heparin solution. Fill a 20-mL syringe with propofol and attach to a syringe pump. Infuse at a rate of 12–36 mg/kg/h for rabbit or 24 mg/kg/h for goat (note: values vary greatly between individual animals) (Suckow and Douglas 1997; Allen and Borkowski 1999). If thoracic surgery is needed or muscle relaxant is indicated, connect the endotracheal tube to a ventilator and attach an additional line of vercuronium bromide (or similar) to the three-way valve. Administer at approximately 3 µg/kg/min. During anesthesia, monitor the respiratory rate and the color of mucous membranes continuously to ensure adequate gaseous exchange. Observe for signs of pain, and maintain surgical levels of anesthesia by monitoring vital signs and responses to painful stimuli.

3.4. Gaseous anesthesia Gaseous anesthesia can be effectively used in all species, but does require some specialist equipment such as an anesthetic vaporizer. • • •

Induce anesthesia and intubate the animal with an appropriatesized endotracheal tube. Provide pre-emptive analgesia with an injection of buprenorphrine as described above. Connect the endotracheal tube to the anesthetic machine, and set isoflurane and air/oxygen supply to a level that provides surgical anesthesia. The volumes and rates required vary considerably among individual animals and depend on which other drugs are coadministered. As a guide, one or two times the minimal

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alveolar concentration (MAC) of isoflurane — 1.0% in rats (Criado et al. 2000), 1.0% in rabbits (Marano et al. 1996), and 1.3% in goats (Hikasa et al. 1998) — should produce adequate anesthesia when coadministered with buprenorphine. Ensure that the ventilator is set to the appropriate tidal volume and rate for that individual animal in case artificial ventilation is required. During anesthesia, monitor the respiratory rate and the color of mucous membranes continuously to ensure adequate gaseous exchange. Observe for signs of pain, and maintain surgical levels of anesthesia by monitoring vital signs and responses to painful stimuli.

3.5. Complication prevention and recovery To reduce the possibility of complications during and following surgery, the following precautions should be taken: • • •

•

• • •

Thorough preparation (e.g. equipment, drugs, procedure, knowledge). Food withdrawal and sufficient acclimatization. Preparation of emergency drugs such as adrenalin for cardiac depression, doxapram hydrochloride for respiratory depression, reversine SA for ketamine/xylazine overdose, and naloxone for opiate reversal (Perkowski and Wetmore 2006). Use of aseptic technique whenever possible to prevent infection. Instruments and drapes should be sterile and a surgical mask should be worn. If proper aseptic technique is not possible, prophylactic broad-spectrum antibiotic treatment should be considered. Continuous monitoring of vital signs (e.g. respiratory rate, heart rate, color of mucous membranes) during surgery. Frequent monitoring of food consumption, body weight, and signs of distress following surgery. Provision of warmth to prevent hypothermia in the form of a heating mat, water bath, or heating lamp during and following surgery.

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Fluid therapy with sterile saline, 5% dextrose, or lactated Ringer’s solution during and following surgery, administered either i.v. or s.c.

4. Summary In this chapter, the authors have described protocols that may be used on rodents, rabbits, and goats employed in musculoskeletal research. They have also described the need for researchers to be responsible for the animals used in such research and to be able to justify the use of animals and species used, the numbers used, and the protocol used. The authors cannot emphasize enough the importance of quiet and competent handling of animals to minimize stress, as well as the importance of technical competence to ensure that all procedures from induction to intubation to maintenance of anesthesia and ultimately to recovery and nursing do not cause cruelty or render the data being collected unusable. Surgical competence has not been discussed in this chapter, but it too is of high priority in the humane handling of animals in order to minimize tissue damage and the occurrence of adverse consequences. Finally, the use of an appropriate analgesic protocol ensures that good science is performed while the researchers fulfill their responsibility to the animals under their care.

References Allen M, Borkowski GL. The Laboratory Small Ruminant. Laboratory Animal Reference Series. CRC Press, Boca Raton, FL, pp. 81–84, 1999. Australian Government. Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, 7th ed. pp. 1, 11, 20, 2004. Bentham J. An Introduction to the Principles of Morals and Legislation. Available at http://www.econlib.org/library/Bentham/bnthPML.html/, 1789. Capdevila S, Giral M, Ruiz de la Torre JL et al. Acclimatization of rats after ground transportation to a new animal facility. Lab Anim 41:255–261, 2007. Coalition for Medical Progress. Use of Animals in Medical Research: January 2005 Study. Research Study Conducted for the Coalition for Medical

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Progress. Available at http://www.medicalprogress.org/uploads/docs/ CMP_MORI_2005_Report.pdf/, 2005. Criado AB, Gomez de Segura I, Tendillo FJ, Marsico F. Reduction of isoflurane MAC with buprenorphine and morphine in rats. Lab Anim 34: 252–259, 2000. Dolan K. Ethics, Animals and Science. Blackwell, Oxford, UK, pp. 39–65, 113–143, 206, 207, 212–214, 1999. Flecknell PA. Laboratory Animal Anaesthesia: A Practical Introduction for Research Workers and Technicians, 2nd ed. Academic Press, San Diego, CA, 1996. Hagelin J, Hans-Erik C, Hau J. An overview of surveys on how people view animal experimentation: some factors that may influence outcome. Public Underst Sci 12:67–81, 2003. Hellebrekers LJ. Animal Pain: A Practice-Oriented Approach to Effective Pain Control in Animals. Van Der Wees, Utrecht, The Netherlands, pp. 11–13, 2000. Hikasa Y, Okuyama K, Kakuta T et al. Anesthetic potency and cardiopulmonary effects of sevoflurane in goats: comparison with isoflurane and halothane. Can J Vet Res 62:299–306, 1998. Hong Kong Government. Code of Practice: Care and Use of Animals for Experimental Purposes. Available at http://www.afcd.gov.hk/english/ publications/publications_qua/files/code.pdf/, p. 18, 2004. Kohn DF, Wixson SK, White WJ, Benson GJ. Anesthesia and Analgesia in Laboratory Animals. Academic Press, San Diego, CA, pp. 291–293, 1997. Marano G, Grigioni M, Tiburzi F et al. Effects of isoflurane on cardiovascular system and sympathovagal balance in New Zealand white rabbits. J Cardiovasc Pharmacol 28:513–518, 1996. Murnaghan M, Li G, Marsh DR. Nonsteroidal anti-inflammatory druginduced fracture nonunion: an inhibition of angiogenesis? J Bone Joint Surg Am 88(Suppl 3):140–147, 2006. National Research Council. Guide for the Care and Use of Laboratory Animals. National Academy Press, Washington, DC, pp. 1, 9, 1996. Perkowski SZ, Wetmore LA. The science and art of analgesia. In: Gleed RD, Ludders JW (eds.), Recent Advances in Veterinary Anesthesia and Analgesia: Companion Animals, Document no. A1405.1006, International Veterinary Information Service (www.ivis.org), Ithaca, NY, 2006. Russell WMS, Burch RL. The Principles of Humane Experimental Technique, special ed. Universities Federation for Animal Welfare, Potters Bar, UK, 1992.

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Simon AM, O’Connor JP. Dose and time-dependent effects of cyclooxygenase-2 inhibition on fracture-healing. J Bone Joint Surg Am 89:500–511, 2007. Singer P. Animal Liberation. Harper Perennial, New York, NY, 2001. Suckow M, Douglas F. The Laboratory Rabbit. Laboratory Animal Reference Series. CRC Press, Boca Raton, FL, pp. 56–58, 1997. Thurmon JC, Tranquilli WJ, Benson GJ. Lumb and Jones’ Veterinary Anesthesia, 3rd ed. Williams and Wilkins, Baltimore, MD, 1996. Thurmon JC, Tranquilli WJ, Benson GJ. Essentials of Small Animal Anesthesia and Analgesia. Williams and Wilkins, Philadelphia, PA, pp. 28–60, 292–325, 1999. UK Department of Trade and Industry. Views on Animal Experimentation: Research Study Conducted for the Department of Trade and Industry. Available at http://www.ipsos-mori.com/polls/2006/pdf/dti.pdf/, 2006. van Dongen JJ, Remie R, Rensema JW. Manual of Microsurgery on the Laboratory Rat. Techniques in the Behavioral and Neural Sciences Series. Elsevier, Amsterdam, The Netherlands, pp. 3–16, 1990. van Zutphen LFM, Baumans V, Beynen AC. Principles of Laboratory Animal Science: A Contribution to the Humane Use and Care of Animals and to the Quality of Experimental Results, revised ed. Elsevier, New York, NY, pp. 277–311, 2001.

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Chapter 23

Mouse Model of Calvarial Osteolysis Chao Zhang and Ting-Ting Tang

Wear debris–induced periprosthetic disease is a major concern after total joint replacement. The wear debris stimulates a cascade of inflammation, resulting in peri-implant osteoclastogenesis and osteolysis. Although in vitro studies have contributed considerably to our understanding of wear debris–induced adverse biological reactions, animal experiments are necessary to understand the more complex mechanisms in vivo. In this chapter, we describe a mouse model that allows the quantification of osteoclastogenesis and osteolysis. Ultrahigh molecular weight polyethylene (UHMWPE) particles are implanted onto calvariae in C57BL/J6 mice, which then develop greater levels of active inflammatory osteolysis than do the control species. The particles are washed in ethanol to remove surface-adherent endotoxin, thus reducing endotoxin interference. Osteolysis can be found in the middle sagittal suture and the adjacent region in mouse calvaria 1 week after implantation. Decalcified hematoxylin and eosin (H&E)stained sections are used to quantify the osteolysis area. Osteoclastogenesis regions are identified and quantified in tartrate-resistant acid phosphatase (TRAP)-stained sections. Larger areas of osteolysis and TRAP-stained osteoclastic activity are found to be induced by UHMWPE particles than developed by the sham group. Keywords:

Wear debris; osteolysis; calvarium; osteoclastogenesis; mouse model; middle suture.

Corresponding author: Ting-Ting Tang. Tel: +86-21-63137020; fax: +86-21-63137020; E-mail: [email protected]

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1. Introduction Wear debris–induced osteolysis is a major problem affecting the longterm results of joint arthroplasty (Berry et al. 2002). Much effort has been made to understand the mechanism of wear debris–induced osteolysis, and to develop methods of treatment and prevention with drugs or gene therapy (Merkel et al. 1999; Childs et al. 2001; Epstein et al. 2005b; von Knoch et al. 2005). Although in vitro studies have contributed considerably to our understanding of wear debris–induced adverse biological reactions, animal experiments remain a necessary approach to understand more complex mechanisms in vivo. Some rodent models have been designed to study wear debris–induced osteolysis, including the rat intramedullary model (Iwase et al. 2002), the mouse intramedullary model (Epstein et al. 2005a), the mouse calvarial osteolysis model (Merkel et al. 1999; Schwarz et al. 2000), the mouse air pouch model (Wooley et al. 2002; Ren et al. 2006), and the rabbit osteolysis model (Ma et al. 2006). Large animal models such as dog or goat have also been reported (Rahbek et al. 2005; Malkani et al. 2005); however, small animal models are more convenient, inexpensive, time-sparing, and easy to duplicate. The mouse calvarial osteolysis model as induced by wear debris was first developed by Merkel et al. (1999) and later advocated by Schwarz et al. (2000). This model can easily reflect the relationship between particle-induced inflammation and resulting osteolysis. It has the advantages of being easy to perform, analyze, and duplicate. Although there are the disadvantages that a prosthesis implant is absent and that the calvarium is a flat bone (which cannot truly reflect the relationship between a prosthesis and the long bone medullary canal), this model can quantify osteolysis and osteoclastogenesis, thus providing a reliable way to evaluate the effects of drugs or biotherapies. In this chapter, we introduce the detailed steps to establish this model and report some preliminary data concerning osteolysis and osteoclastogenesis.

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2. Materials • •

• • •

Mouse: preferably C57BL/J6 male, 8-week-old healthy micea Wear particles: preferably ultrahigh molecular weight polyethylene (UHMWPE) particles, poly(methyl methacrylate) (PMMA) particles, or titanium particles with a mean diameter smaller than 10 µm, as they are most active in inducing inflammatory reactions b Ethanol: 100% ethanol — for washing wear particles for the removal of particle-adherent endotoxin Phosphate buffered saline (PBS): sterile PBS — for suspending wear particles and dissolving drugs 1% pentobarbital sodium



• • •

Hematoxylin and eosin (H&E) reagent (Shanghai Rainbow Medical Reagent Research Co. Ltd, Shanghai, China) Tartrate-resistant acid phosphatase (TRAP) staining kit (Shanghai Rainbow Medical Reagent Research Co. Ltd, Shanghai, China) Fixative: 4% glutaraldehyde in PBS (pH 7.4)


• a

Dissolve pentobarbital sodium in PBS at 1% concentration. To anesthetize 8-week-old C57BL/J6 mice, we usually use a dosage of 45 mg/kg.c

Prepare before use.

Delcalcification solution: 10% EDTA in PBS (pH 7.4)d

For this study, the C57BL/J6 mouse is preferred because it is more sensitive to wear particle–induced inflammatory osteolysis than other mice, as observed from our preliminary data (Fig. 1). b For wear particles, UHMWPE or PMMA particles are preferred as they are more active than titanium particles (Warashina et al. 2003). Particles should be washed in ethanol for 48 hours in a shaker to remove surface-adherent endotoxin and reduce endotoxin interference. For each mouse, 30 mg of particles with a mean diameter smaller than 10 µm should be applied to induce calvarial osteolysis and osteoclastogenesis. For decalcification and paraffin sectioning, UHMWPE or PMMA particles are easier to handle than titanium particles, as difficulties occur during sectioning with titanium particles adhering to the calvaria. c To anesthetize small animals such as mice, 1% pentobarbital sodium at a dosage of 45 mg/kg is preferred. This dosage and concentration allows optimal anesthesia, rarely causing animal death. d Add NaOH to the solution to set the pH value at 8.0. This allows the EDTA to dissolve completely, and the pH can then be adjusted to 7.4.

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Fig. 1. Areas of development of UHMWPE particle-induced mouse calvarial osteolysis are found to be significantly greater in C57BL/J6 mice than in Balb/c mice or Kunming mice (n = 6, P < 0.01).

3. Methods 3.1. Animal surgery • • •

•

•

Prepare sterile instruments such as eye scissors, small forceps, and suture material. Prepare sterile pipettes with 200-µL tips, and cut the tips for particle suspension aspiration. Prepare wear debris. Immerse particles in ethanol in a test tube at 100 mg particles/50 mL ethanol, and rock the tube for 48 hours to remove surface endotoxin. Anesthetize the mouse with 1% pentobarbital sodium at a dosage of 45 mg/kg. The weight of an 8-week-old C57BL/J6 mouse is usually 23 g ± 3 g, so approximately 100 µL of 1% pentobarbital sodium is usually needed. After shaving the hair, sterilize the skin. An incision is made between the two external ears. A 1-cm middle sagittal incision is made, the subcutaneous bursa is entered, and an approximately 1-cm2 area is exposed, with care not to disrupt the periosteum. Thirty milligrams of PMMA, UHMWPE, or titanium particles in a 100-µL sterile phosphate buffer solution are uniformly spread

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over the exposed periosteum. The skin is closed in a simple disrupted suture. In the control group, the mice receive sham operations and 100 µL of sterile phosphate buffer solution without particles. After the operation, the mice are warmed up, allowed to recover, and sent back to the animal maintenance facility.

3.2. Fixation and decalcification • • • •

Euthanize the mice 1 week after surgery. Dissect out the calvarial bone and fix in 4% glutaraldehyde at 4°C for 12 hours. Rinse the calvaria and decalcify in 10% EDTA at 4°C for about 2 weeks, changing the EDTA solution every 3 days. Determine the completion of decalcification using X-ray photography. Once the decalcification is complete, paraffin embedding can proceed.

3.3. Embedding of samples in paraffin • •

Flush the decalcified samples in water for 12 hours. Immerse the sample in an ethanol gradient as follows: 75% ethanol for 12 hours 80% ethanol for 12 hours 85% ethanol for 6 hours 95% ethanol for 4 hours, repeat two times 100% ethanol for 2 hours, repeat three times

• •

Immerse the sample in xylene for 21 minutes to clarify. Immerse the samples in paraffin at 60°C for 36 hours, and then embed.e

3.4. Sectioning •

e

Section the samples in a continuous manner at 5–8-µm thickness, producing at least five sections for each specimen.

Embed the samples in paraffin with a sagittal suture perpendicular to the plane for sectioning.

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3.5. H&E staining of sections •

Perform H&E staining according to the methods described by Helen and Jane (2003).

3.6. TRAP staining of sections • • •

• • •

Remove the paraffin in xylene, and rehydrate the sections in an ethanol gradient. Prepare the TRAP staining working solution according to the methods described by Helen and Jane (2003). Immerse the rehydrated sections in a water bath at 37°C. The TRAP working solution is added to cover the samples. Incubation continues for 90 minutes. Soak the sections in water for 2 minutes. Counterstain with 0.5% methyl green to show cell nuclei. Mount the sections in neutral balsam.

3.7. Osteolysis and osteoclastogenesis analysis 3.7.1. Osteolysis •

•

Take photographs of H&E sections at 10× magnification with the middle suture in the center for calculating osteolysis areas using Image Pro-Plus 5.0 (Media Cybernetics, USA) or a similar image analysis software. Select the middle sagittal suture and adjacent area to measure the osteolysis area (Fig. 2). Five sections of each sample should be used and the mean calculated to represent one sample (Figs. 3 and 4).

3.7.2. Osteoclastogenesis •

Count TRAP-stained cells in the middle suture and adjacent area under a light microscope (Figs. 5 and 6). For osteoclastogenesis analysis, five sections of each sample should be used and the mean calculated to represent one sample.

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Fig. 2. An outline of the middle suture and adjacent area selected for measuring the osteolysis area induced by UHMWPE particles (H&E staining, 10×).

Fig. 3. (a) There is no obvious osteolysis in the sham-operated mouse. (b) Calvarial osteolysis in a C57BL/J6 mouse can be seen 1 week after UHMWPE particle implantation. H&E staining, 10×.

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Fig. 4. The mice with implanted UHMWPE particles induced more significant bone destruction compared with the sham-operated mice (n = 6, p < 0.001).

Fig. 5. Less TRAP-stained osteoclasts are observed in the middle suture and adjacent area in (a) the sham-operated mouse than (b) the UHMWPE particle-implanted mouse. TRAP staining, 10×.

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Fig. 6. Significantly more TRAP-stained osteoclasts are observed in the middle suture and adjacent area in the UHMWPE particle-implanted group than in the sham-operated group (n = 6, p < 0.001).

3.8. Statistical analysis •

Depending upon the data and test grouping, we routinely use one-way analysis of variance (ANOVA) or Student’s t-test to determine the significance.

References Berry DJ, Harmsen WS, Cabanela ME, Morrey BF. Twenty-five-year survivorship of two thousand consecutive primary Charnley total hip replacements: factors affecting survivorship of acetabular and femoral components. J Bone Joint Surg Am 84-A:171–177, 2002. Childs LM, Goater JJ, O’Keefe RJ, Schwarz EM. Efficacy of etanercept for wear debris–induced osteolysis. J Bone Miner Res 16:338–347, 2001.

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Epstein NJ, Bragg WE, Ma T et al. UHMWPE wear debris upregulates mononuclear cell proinflammatory gene expression in a novel murine model of intramedullary particle disease. Acta Orthop 76(3):412–420, 2005a. Epstein NJ, Warme BA, Spanogle J et al. Interleukin-1 modulates periprosthetic tissue formation in an intramedullary model of particle-induced inflammation. J Orthop Res 23:501–510, 2005b. Helen EG, Jane AI. Basic staining and immunohistochemical localization using bone sections. In: An YH, Martin KL (eds.), Handbook of Histology Methods for Bone and Cartilage, Humana Press, Totowa, NJ, pp. 281–286, 2003. Iwase M, Kim KJ, Kobayashi Y et al. A novel bisphosphonate inhibits inflammatory bone resorption in a rat osteolysis model with continuous infusion of polyethylene particles. J Orthop Res 20(3): 499–505, 2002. Ma T, Nelson ER, Mawatari T et al. Effects of local infusion of OP-1 on particle-induced and NSAID-induced inhibition of bone ingrowth in vivo. J Biomed Mater Res A 79(3):740–746, 2006. Malkani AL, Voor MJ, Hellman EJ et al. Histologic and mechanical evaluation of impaction grafting for femoral component revision in a goat model. Orthopedics 28(1): 49–58, 2005. Merkel KD, Erdmann JM, McHugh KP et al. Tumor necrosis factor-alpha mediates orthopedic implant osteolysis. Am J Pathol 154:203–210, 1999. Rahbek O, Kold S, Bendix K et al. Superior sealing effect of hydroxyapatite in porous-coated implants: experimental studies on the migration of polyethylene particles around stable and unstable implants in dogs. Acta Orthop 76(3):375–385, 2005. Ren W, Wu B, Peng X et al. Erythromycin inhibits wear debris–induced inflammatory osteolysis in a murine model. J Orthop Res 24(2):280–290, 2006. Schwarz EM, Benz EB, Lu AP et al. Quantitative small-animal surrogate to evaluate drug efficacy in preventing wear debris–induced osteolysis. J Orthop Res 18(6):849–855, 2000. von Knoch F, Heckelei A, Wedemeyer C et al. Suppression of polyethylene particle-induced osteolysis by exogenous osteoprotegerin. J Biomed Mater Res 75A:288–294, 2005.

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Warashina H, Sakano S, Kitamura S et al. Biological reaction to alumina, zirconia, titanium and polyethylene particles implanted onto murine calvaria. Biomaterials 24(21):3655–3661, 2003. Wooley PH, Morren R, Andary J et al. Inflammatory responses to orthopaedic biomaterials in the murine air pouch. Biomaterials 23(2):517–526, 2002.

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Chapter 24

Distraction Osteogenesis Model Chun-Wai Chan and Kwok-Sui Leung

Distraction osteogenesis refers to bone regeneration under tensile stress with axial rhythmic distraction after osteotomy. It is applied on limb lengthening to treat dwarfism, bone transport, correction of limb deformity, and arthrodiastasis. However, clinical complications have been reported, including delayed consolidation, pin tract infection, and muscle wasting. It is essential to generate animal models for investigating the enhancement of bone formation, the design of bone lengthening fixation, and the distraction protocol. This chapter describes and discusses a rabbit tibial distraction osteogenesis model established by the authors for various applications. Keywords:

Distraction osteogenesis; callotasis; bone lengthening; DXA; pQCT; mechanical testing.

1. Introduction Distraction osteogenesis is a complex process that involves the spatial and temporal orchestration of bone regeneration under tensile stress with axial rhythmic distraction after osteotomy. It is also known as callotasis (Ozerdem et al. 1988), callus distraction (Mader et al. 2003), or bone lengthening (Mizuta et al. 2003). Ilizarov, a pioneer who discovered and investigated distraction osteogenesis, first applied this Corresponding author: Chun-Wai Chan. Tel: +852-26323309; fax: +852-26377889; E-mail: [email protected]

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method on fracture and nonunion to treat chronic osteomyelitis in 1951 (Ilizarov 1989a; Ilizarov 1989b; Ilizarov 1990). Since then, distraction osteogenesis has been widely applied on limb lengthening (Dal Monte and Donzelli 1987; Paley et al. 1988; Zarzycki et al. 2002), bone transport (Martini and Castaman 1987; Aronson and Harp 1992; Mekhail et al. 2004), deformity correction (Saldanha et al. 2004), and arthrodiastasis (Grill and Franke 1987; Ozger et al. 2003). Distraction osteogenesis can be stratified into three stages: latency, distraction, and consolidation. The period of latency varies with different distraction models; for example, the rabbit mandible distraction model has a 2–5-day latency (Aida et al. 2003), whereas the rat model has a 0-day latency (Aronson et al. 1997). A 7-day latency is commonly used in the rabbit tibia model (Chan et al. 2006a; Chan et al. 2006b; Machen et al. 2002; Sakurakichi et al. 2004). In the distraction stage, the mechanical tensile stress exerts on callus. Cells in callus are activated to proliferate and/or differentiate with the aid of cyclic loading by weight bearing (Meyer et al. 2001). Bone formation takes place in the endocortical region and the periosteal region. In clinical practice, the standard distraction protocol is 0.25-mm distraction four times a day (Ilizarov 1989a; Ilizarov 1989b; Ilizarov 1990). In the rabbit tibia model, a distraction rate of 0.7–1.3 mm has been reported as the optimal rate for angiogenesis (Li et al. 1999), and 0.7 mm for proliferation (Li et al. 1997) and collagen synthesis (Li et al. 2000). Thus, the optimal distraction rate may depend not only on the animal models, but also on the parameters for investigation. In the consolidation stage, distraction ceases and the tensile forces gradually decrease. The distraction gap only receives cyclic weight bearing. The consolidation of the distraction gap is a very slow process. The amount of osseous tissue increases continuously through intramembranous and endochondral ossification. Finally, the newly formed bone remodels into a high-mechanical-strength lamellar bone (Aronson 1994). The distraction osteogenesis operation on rabbit tibia introduced below is an open osteotomy on bone, fixed by a special bone

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lengthening external fixator, that has been established and modified for our recent studies (Chan et al. 2006a; Chan et al. 2006b). The bone lengthening external fixator is designed to stabilize the lengthening distance during animal mobility. The fixator is fixed on the anteromedial side to serve an animal model for biophysical intervention studies, such as those using low-intensity pulsed ultrasound (Chan et al. 2006a; Chan et al. 2006b).

2. Materials and Methods 2.1. Materials • • • • • • •

Operation surgery set (Synthes) Hair shaver Hibatine in 70% ethanol Operation drapes Mini-air drill for hand surgery, maxillofacial surgery, and neurosurgery (Synthes/Mathys Medical Ltd, Bettlach, Switzerland) 2-mm and 2.5-mm drill bits 3-mm half-pin

2.2. Method • • • • • •

Autoclave the surgical instruments before operation or dip into diluted hibatine for at least 30 minutes for sterilization. After general anesthesia, shave the rabbit hair using a hair shaver to expose skin. Place the rabbit in a supine position on a special custom-made plastic operation table, exposing the tibia only. Make an anteromedial incision in the middiaphyseal region of the tibia [Fig. 1(a)]. Carefully lift up the periosteum using a periosteal elevator, preserving it as intact as possible. Make the first drill hole on the anteromedial flattened side of the tibia using a mini-air drill with 1.5-mm drill bit. Then, use a 2.5-mm drill bit in the second drilling to widen the drill hole [Fig. 1(a)].

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(a)

(b)

Fig. 1. Operation procedure of rabbit tibia distraction osteogenesis. A mini-air drill is used to create a pin hole (a) for insertion (b). After four holes are drilled, the pins are inserted (c). The osteotomy is performed by an oscillating saw transversely (d) to make a fracture site (e). The bone lengthening fixator is assembled (f ). The wound is closed by layers.

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• •

• • • • •

•

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Insert the first 3-mm half-pin [Fig. 1(b)]. Use the fixator as a template to drill a hole in the fourth position (as in step 6), and insert the pin parallel and in the same plane as the first pin. Repeat to drill the second and third holes and insert the remaining two pins [Fig. 1(c)]. Insert a small metal plate on the lateral side of the bone to prevent damage to the surrounding soft tissue and periosteum during sawing [Fig. 1(d)]. Use an oscillating saw to make a cross-sectional fracture site between the second and third pins [Fig. 1(e)]. Assemble the lengthening external fixator [Fig. 1(f )]. Close the periosteum using vicryl 6-0. Close the wounds by layers. Inject the animal intramuscularly with Temgesic® (0.1 mL/kg body weight) (0.3 mg/mL buprenorphine hydrochloride) once after operation for analgesic purposes. Make sure that all animals receive conventional care, feeding, and ambulation in the rabbit holding cages. Keep warm before recovery from anesthesia.

3. Results A successful distraction osteogenesis operation can be monitored by X-ray radiography in prone position. The radiolucent osseous tissue can be examined in the distraction gap, usually after 1 week of distraction, and increases after cessation of distraction. The final outcome of distraction callus is subject to mechanical testing, e.g. torsional testing (Chan et al. 2006b). Different distraction rates were studied in this model. We found that the maximum length of acute distraction should be less than 2 mm because soft tissue restrains the distraction; it also causes the rabbit to suffer pain. Provided that a distraction rate of 3 mm/day is attained, a distraction rate of 1.5 mm/day two times will be applied. Longitudinal radiographical monitoring is suggested. It helps in finding loosening of pins and misalignment of distracting callus.

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Moreover, it can serve as a quantitative measurement for bone mineral changes during distraction osteogenesis. Three types of radiographical monitoring are used: (1) plain X-ray, (2) dual-energy X-ray absorptiometry (DXA), and (3) peripheral quantitative computed tomography (pQCT). For plain X-ray, rabbits are put on shelf position 6 (76 cm away from the X-ray source) under general anesthesia. In prone position, the tibia contacts the X-ray cassette closely to avoid blur images or magnification effect. Plain X-ray is taken by an X-ray machine (e.g. Faxitron X-ray System Model 43855C; Faxitron X-Ray Corp., Wheeling, IL, USA) [Fig. 2(a)] after operation and subsequently on a weekly basis in the anteroposterior view. The X-ray output voltage is set at 60 kV, 0.3 mA for 5 seconds. A standard aluminum stepwedge is placed near the tibia. The relative bone density of distraction callus can be measured in units of millimeters thick of the aluminum stepwedge [Fig. 2(b)] (Chan et al. 2006b). The radiolucent area of mineralized tissue is visualized in the distraction gap region of serial X-ray radiographs [Fig. 2(c)]. For DXA, the rabbits are put in supine position under general anesthesia [Fig. 3(a)]. The tibia is fixed by a custom stand in horizontal position. The actual bone mineral density (BMD) can be measured in units of g/cm2 [Fig. 3(b)]. Both plain X-ray and DXA measurements are projected assessments in the anteroposterior view; however, BMD only provides the regional measurement of proximal and distal or lateral and medial, not anterior and posterior. It will hinder the regional analysis of some localized treatment, e.g. low-intensity pulsed ultrasound (LIPUS) on the anterior side. Thus, volumetric BMD measurement can solve this problem. For pQCT measurement, the rabbits are put in supine position under general anesthesia [Fig. 4(a)]. The metal fixator is replaced by a custom-made plastic jig (Fig. 5). The tibia is fixed by a custom stand in horizontal position. The actual BMD can be measured in units of g/cm3 [Fig 4(b)]. Moreover, bone mineral content (BMC), volume, and moment of inertia can be generated. Regional

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Fig. 2. (a) Plain X-ray is taken weekly using an X-ray machine (Faxitron X-ray System Model 43855C; Faxitron X-Ray Corp., Wheeling, IL, USA). (b) Representiave X-ray radiography of distraction tibia with aluminum stepwedge. Newly formed bone is found in the proximal region of the distraction gap. The relative bone density of distraction callus can be measured in units of millimeters thick of the aluminum stepwedge. (c) Serial representative lateral plain X-rays of the rabbit tibial distraction osteogenesis model during the consolidation stage. The radiolucent area of mineralized tissue in the distraction gap increases in intensity and area over time (W: week).

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analysis in all directions can also be assessed (Fig. 6), even anterior and posterior. The fixator can be kept in position during both plain X-ray and DXA measurements; however, the custom-made plastic jig should be used to prevent metal interference in pQCT images. The temporary plastic jig fixation should be kept stable; otherwise, the muscle contraction will lead to a shortening of the distraction gap. Besides bone mineral changes, the outcome of distraction osteogenesis can be the mechanical testing. Torsional testing is a good method for measuring mechanical strength and torsional stiffness, and can be correlated to the moment of inertia measured by pQCT. The tibia after harvest is then embedded with performance polymers (UREOL 5202-1A, UREOL 5202-1B, and Filler DT082; Ciba, Basel, Switzerland) and mounted onto a custom-made testing jig

(a)

Fig. 3. (a) A dual-energy X-ray absorptiometer (DXA) XR-36 (Norland Corp., Fort Arkinson, WI, USA) measures the bone mineral content (BMC) and hard callus area of rabbit tibia under general anesthesia of rabbit. (b) A typical DXA pseudocolour image, with results displayed in the datasheet. The newly formed bone in the distraction gap (left arrow) is measured using pins as a reference point (right arrow).

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(b)

Fig. 3.

(Continued )

[Figs. 7(a)–7(c)], where the callus (18 mm long) is exposed with a distance of 5 mm in proximal and distal fragments [Fig. 7(d)]. A rotational speed of 10° per minute is selected for testing until failure by a servohydraulic biaxial universal material testing machine (Bionix 858; MTS Systems, Minneapolis, MN, USA) [Fig. 8(d)]. The ultimate failure torque of specimens is recorded. The stiffness is calculated by the torque versus the angular displacement curve. The percentage ratio of ultimate failure torque and stiffness of the distracted side is compared with that obtained from the contralateral control side.

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(a)

(b)

Fig. 4. (a) The rabbit is in place for peripheral quantitative computed tomography (pQCT) scanning (Densiscan 2000; Scanco Medical, Bassersdorf, Switzerland) under general anesthesia. (b) A typical tomography of the rabbit bilateral tibiae is shown. LIPUS treatment is applied on the anterior side of the distracted tibia. The distracted tibia with soft tissue is fixed by a custom-made plastic jig during quantitative CT scanning. The contralateral control is also measured. The rabbit tail and pQCT holding stand are visualized without interference of the X-ray irradiation on tibia for quantitative measurement.

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(a)

(b)

Fig. 5. A plastic jig, which is made of acrylic plate (a), is used to replace the metallic bone lengthening device during pQCT scanning (b).

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Fig. 6. Tomograms produced by pQCT scanning from a proximal to distal region of the distraction gap in tibia. (a) Plain X-ray of lengthened tibia at week 4 of consolidation. (b) X-rays of proximal, middle, and distal regions, respectively.

4. Remarks •

•

•

For anatomy of rabbit tibia, the fibula is fused within the nearly middle region of the tibia shaft. This is known as syndesmosis. The fracture site should be below syndesmosis; otherwise, when the tibia is under distraction, the fibula will affect the distracted callus of tibia and make it not along the longitudinal axis of the tibia. The growth plate of fibula may also be distracted. Rabbit bone is relatively brittle. The standard 3-mm half-pin should be applied on skeletally matured rabbits (about 30 weeks old or more than 4 kg in weight); otherwise, the tibia will be easily broken during the insertion of pins. Provided that a young rabbit is used (18–22 weeks old, 3–3.6 kg), a custom-made pin with a thinner threaded shaft (2.5 mm) can be applied. Latent period after osteotomy is essential for the re-establishment of vascularization before distraction. It is usually 5–7 days for the rabbit tibia model.

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(a)

(b)

Fig. 7. (a) The distracted portion of the tibia is wrapped with saline gauze and then fixed by a calibrated stand. (b) The performance polymer is mixed together and poured into the mold. The embedded samples are removed until the performance polymer is solidified. (c) The other end of the bone is re-embedded using the above method. (d) The only distracted portion of the tibia is exposed for mechanical testing. G: gauze with saline; P: solidified performance polymer.

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(c)

(d)

Fig. 7.

(Continued )

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Fig. 8. The custom-made fixation jig for torsional test [(a), proximal part; (b), distal part]. The performance-polymer-embedded tibia (c) is fixed in the servohydraulic universal testing machine (Bionix 858; MTS Systems, USA) (d).

•

For the distraction period, 7-day, 10-day, and 21-day bone lengthening periods can be found in the literature. The study period of the consolidation stage is usually the same as or double the duration of the lengthening period.

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Pin tract infection happens sometimes. It will cause loosening of pins from the insertion site. Antibiotic spray is applied after operation. The rabbit is monitored regularly for any chronic inflammation or infection on wound, animal health and behavior, eating and drinking habits, etc.

5. Summary Experimental distraction osteogenesis is established to promote the study of enhancement of consolidation by both biological and biophysical interventions such as LIPUS. The beneficiary treatment can be translated to clinical application. Moreover, the unstable microenvironment during distraction shows the importance of mechanical stimulation on bone regeneration. Readers may follow the step-bystep descriptions to establish this model for their own institutional applications.

References Aida T, Yoshioka I, Tominaga K, Fukuda J. Effect of latency period in a rabbit mandibular distraction osteogenesis. Int J Oral Maxillofac Surg 32:54–62, 2003. Aronson J. Experimental and clinical experience with distraction osteogenesis. Cleft Palate Craniofac J 31(6):473–481, 1994. Aronson J, Harp J. Cavitary osteomyelitis treated by fragmentary cortical bone transportation. Clin Orthop 280:153–159, 1992. Aronson J, Shen XC, Skinner RA et al. Rat model of distraction osteogenesis. J Orthop Res 15(2):221–226, 1997. Chan CW, Qin L, Lee KM et al. Dose-dependent effect of low intensity pulsed ultrasound on callus formation during rapid distraction osteogenesis. J Orthop Res 24(11):2072–2079, 2006a. Chan CW, Qin L, Lee KM et al. Low intensity pulsed ultrasound accelerated bone remodeling during consolidation stage of distraction osteogenesis. J Orthop Res 24(2):263–270, 2006b. Dal Monte A, Donzelli O. Tibial lengthening according to Ilizarov in congenital hypoplasia of the leg. J Pediatr Orthop 7:135–138, 1987.

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Grill F, Franke J. The Ilizarov distractor for the correction of relapsed or neglected clubfoot. J Bone Joint Surg Br 69:593–597, 1987. Ilizarov GA. The tension-stress effect on the genesis and growth of tissues: part I. The influence of fixation and soft-tissue preservation. Clin Orthop 238:249–281, 1989a. Ilizarov GA. The tension-stress effect on the genesis and growth of tissues: part II. The influence of the rate and frequency of distraction. Clin Orthop 239:263–285, 1989b. Ilizarov GA. Clinical application of the tension-stress effect for limb lengthening. Clin Orthop 250:8–26, 1990. Li G, Simpson AH, Kenwright J, Triffitt JT. Assessment of cell proliferation in regenerating bone during distraction osteogenesis at different distraction rates. J Orthop Res 15(5):765–772, 1997. Li G, Simpson AH, Kenwright J, Triffitt JT. Effect of lengthening rate on angiogenesis during distraction osteogenesis. J Orthop Res 17(3): 362–367, 1999. Li G, Virdi AS, Ashhurst DE et al. Tissues formed during distraction osteogenesis in the rabbit are determined by the distraction rate: localization of the cells that express the mRNAs and the distribution of types I and II collagens. Cell Biol Int 24(1):25–33, 2000. Machen MS, Tis JE, Inoue N et al. The effect of low intensity pulsed ultrasound on regenerate bone in a less-than-rigid biomechanical environment. Biomed Mater Eng 12(3):239–247, 2002. Mader K, Gausepohl T, Pennig D. Shortening and deformity of radius and ulna in children: correction of axis and length by callus distraction. J Pediatr Orthop B 12(3):183–191, 2003. Mekhail AO, Abraham E, Gruber B, Gonzalez M. Bone transport in the management of posttraumatic bone defects in the lower extremity. J Trauma 56(2):368–378, 2004. Meyer U, Meyer T, Schlegel W et al. Tissue differentiation and cytokine synthesis during strain-related bone formation in distraction osteogenesis. Br J Oral Maxillofac Surg 39(1):22–29, 2001. Mizuta H, Nakamura E, Mizumoto Y et al. Effect of distraction frequency on bone formation during bone lengthening. A study in chickens. Acta Orthop Scand 74(6):709–713, 2003. Ozerdem OR, Kivanc O, Tuncer I et al. Callotasis in nonvascularized periosteal bone grafts and the role of periosteum: a new contribution to the concept of distraction osteogenesis. Ann Plast Surg 41(2):148–155, 1988.

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Ozger H, Eralp L, Atalar AC. Articulated distraction of the hip joint in the treatment of benign aggressive tumors located around the hip joint. Arch Orthop Trauma Surg 123(8):399–403, 2003. Sakurakichi K, Tsuchiya H, Uehara K et al. Effects of timing of low-intensity pulsed ultrasound on distraction osteogenesis. J Orthop Res 22(2): 395–403, 2004. Saldanha KA, Saleh M, Bell MJ, Fernandes JA. Limb lengthening and correction of deformity in the lower limbs of children with osteogenesis imperfecta. J Bone Joint Surg Br 86(2):259–265, 2004.

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Chapter 25

Fracture Nonunion Animal Model Xia Guo, Mu-Qing Liu, Chi-Cheung Hui and Zheng Guo

Nonunion of fracture has presented an overall therapeutic challenge in clinical practice. Selection of an adequate nonunion model is the basis for testing effective prevention or treatment of nonunion by new intervention techniques. This chapter describes two atrophic nonunion models in rabbits for simulating the clinical conditions that cause nonunion: by the creation of a critical-sized bony defect, and by the interposition of soft tissue. Both nonunion models are evaluated radiographically and histomorphologically at the end of the experiment. Atrophic nonunion characteristics are clearly present in all animals in the defect and interposition of soft tissue models 12 weeks after operation, and persist until 22 weeks. Both the critical-size defect and the tissue interposition techniques are therefore regarded as effective methods to develop atrophic nonunion models. Keywords:

Nonunion; fracture; atrophic nonunion; periosteum; rabbit.

1. Introduction Bone is a unique tissue that is able to regenerate with normally predictable results when compared with any other tissue in the body. It has been reported that approximately 6.2 million fractures occur annually in the United States, with 5% to 10% nonunion or delayed Corresponding author: Xia Guo. Tel: +852-27666720; fax: +852-23308656; E-mail: [email protected]

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union rates (Einhorn 1995). It is known from clinical experience that all types of fractures can be repaired within a certain period of time after injury. Nonunion of fracture is defined as the cessation of all reparative processes of healing without bone union (Crenshaw 1987; Gerstenfeld et al. 2003; Megas 2005). According to the AO Principles of Fracture Management (Ruedi et al. 2007), a delayed union describes the situation in which a fracture fails to heal within the time usually required. A nonunion is usually declared as the failure of a fracture to heal, as evidenced by radiography of the absence of progressive repair between 6 months and 8 months following fracture (Hernigou et al. 2005). Fracture nonunion occurs when the reparative sequence of fracture healing is interrupted. Aseptic nonunion of fractures can be classified into atrophic and hypertrophic categories according to the radiographic appearance (Bruder et al. 1994). Atrophic nonunion is the one with little callus to bridge the fracture gap; while hypertrophic nonunion is presented with abundant callus, yet is not able to bridge the fracture gap (Megas 2005). The pathophysiology of these two types remains unclear, although under certain circumstances a nonunion can be predicted based on a known inciting cause. The risk factors that impair fracture healing can generally be divided into biological factors and mechanical factors (Simmons 1985; Einhorn 1995; Einhorn and Trippel 1997). The use of animal models to study fracture healing is designed to answer questions that relate to the most efficient and effective way to treat fractures in humans. Much of the information on fracture healing in humans has been inferred from animal models, and has been supported by human clinical and epidemiologic studies. In order to develop potential means for the prevention and treatment of fracture nonunion, the establishment of a relevant animal nonunion model (as well as the evaluation of its efficacy) is an important step before clinical trials and finally systemic application to patients. Thus, experimental models of fracture healing, delayed healing, and nonunion have been developed (Aro et al. 1985; Bostrom et al. 1996; Choi et al. 2004; Einhorn et al. 1984; Hietaniemi et al. 1995; Hollinger and Kleinschmidt 1990; Reed et al. 2003; dos Santos Neto and Volpon

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1984; Schmitz and Hollinger 1986; Utvag et al. 1998). Some of these models are extremely useful for answering specific questions related to the biology and pathophysiology of bone repair or the regeneration of bone with biological or biophysical enhancement. Previous attempts to establish animal fracture nonunion models were generally based on the elimination of osteoconductive and osteoinductive factors in the fracture focus. Models that are currently available for experimental nonunion include those which produce a critical-size defect (Hollinger and Kleinschmidt 1990; Schmitz and Hollinger 1986) and those with attempts to prevent healing (Choi et al. 2004; Reed et al. 2003; Wallace et al. 1991; Yoo and Johnstone 1998). A critical-size defect is defined as the smallest intraosseus wound that would not heal by bone formation in the lifetime of the animal (Schmitz and Hollinger 1986); in essence, it involves removing enough bone so that two fracture fragments do not heal because of an inability of the fracture gap of that size to be bridged by callus. Other models of nonunion would be more applicable to clinical situations where some known or unknown factor(s) other than loss of bone tissue itself caused failure of bony union. The objectives of this chapter are to demonstrate two atrophic nonunion models using rabbit tibia for simulating clinical conditions that cause nonunion, namely, bone defect and interposition of soft tissues. It also suggests ways of creating and evaluating the nonunion models.

2. Materials • •

Animals: mature New Zealand white rabbits 18 weeks of age (see also Sec. 5.2), with an average body weight of 3.2 kg (range, 2.7–3.8 kg) Chemicals or drugs for anesthesia and antisepsis








Ketamine (e.g. Pfizer, Ballerup, Denmark), xylacaine (e.g. Pfizer, Ballerup, Denmark), and sodium pentobarbital (e.g. Sigma Chemicals Co., St. Louis, MO, USA) for anesthesia Buprenorphine (e.g. Temgesic; Reckitt & Colman, Hull, UK) for controlling pain after surgery Antibiotic spray (e.g. Nebacetin; Byk Gulden Konstanz, Germany) for antisepsis

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Faxitron cabinet X-ray system (e.g. Model 43855C; Faxitron X-Ray Corp., Wheeling, IL, USA) High-resolution X-ray films (e.g. Struct D4 Pb Vacupac; Agfa, Japan)

Saw microtome for sectioning bone sample without decalcification





•

Stainless-steel external fixator (e.g. modified from Orthofix M103) K-wires with 1-mm diameter (Arista Surgical Supply Co., MA, USA)

Silastic tube for creating tissue interposition model (Silastic Corp., USA) X-ray system


•

Oscillating saw of air-powered drill, with a saw blade of 0.4 mm in thickness and drill tips (e.g. Synthes/Mathys AG, Bettlach, Switzerland) Suture (e.g. 3/0 Mersilk; Ethicon Ltd, Edinburgh, Scotland)

External fixator and K-wire


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Saw microtome (e.g. Leica SP1600; Leica, Germany) for creating thick sections Polycut E microtome (e.g. Leica SM 2500E; Leica, Germany) for thin sections

Chemicals for histomorphology






Ethanol solutions (70%, 80%, 90%, and 100%) for tissue dehydration Hematoxylin and eosin (H&E) for tissue staining Polymethylmethacrylate (PMMA) for embedding undecalcified bone

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3. Methods 3.1. Grouping Twelve animals were randomly assigned into two groups of six rabbits each to create different nonunion models. In group A, a critical-size defect on the tibia was performed. In group B, insertion of the silastic tube was applied to create a nonunion model.

3.2. Anesthesia The animals were anesthetized with intramuscular injection of ketamine (25 mg/kg) and xylacaine (5 mg/kg), and then intravenous injection of 2.5% sodium pentobarbital (0.8 mL/kg). Buprenorphine (0.1 mg/kg Temgesic) was used for postoperative pain relief for 2–3 days.

3.3. Surgical procedure 3.3.1. Tibial critical-size defect model (group A) Osteotomy was conducted under general anesthesia and sterile conditions. An incision was made along the tibia at the medial side of the leg. The periosteum was separated carefully from the surrounding muscles. A 5-mm defect (see Sec. 5.4) located about 12 mm proximal to the tibial tuberosity was created by using an oscillating saw of air-powered drill, with a saw blade of 0.4 mm in thickness (Synthes/Mathys AG). The defect was left empty and a stainless-steel external fixator (modified from Orthofix M103) was used for bony fixation (Fig. 1). The transected ends were stripped of periosteum for 5 mm, and the intramedullary canal was curetted for 5 mm. The surgical incision was closed by suture (3/0 Mersilk). Antibiotic spray (Nebacetin) was used for disinfection.

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(a)

(b)

Fig. 1. Operative procedure for creating a critical-size defect nonunion model. (a) The bone defect; (b) the external fixation.

3.3.2. Fracture gap interposition of soft tissues (group B) (see Sec. 5.7) The right lower limb was scrubbed and draped in a surgical sterile manner. An anteromedial incision was made along the middle third of the diaphysis of the right tibia. A transverse fracture was created by osteotomy using an air-powered dental burr at the middle third of the tibial shaft. The periosteum on either side of the fracture site was excised, and the marrow was removed through curettage. A stainlesssteel external fixator (modified from Orthofix M103) was applied to

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fix the fracture, and a gap of 2 mm was maintained between the two ends of the fracture. Both ends of the fracture were covered by a 1-cm silastic tube each. After the surgery was finished, the wound was closed in a routine fashion following irrigation with normal saline solution. Antibiotic spray (Nebacetin) was used for disinfection. Eight weeks after the operation, the rabbits were sedated with intramuscular injection of ketamine (25 mg/kg) and xylacaine (5 mg/kg) and then the silastic tube was removed through the previously approaching direction. Unprotected weight bearing was allowed immediately after operation. The animals were kept in individual metal cages with free cage activity immediately after operation, until the rabbits were sacrificed with an intravenous pentobarbital injection (Mebumal, 200 mg/mL; Sygehus Apotekerne, Aarhus, Denmark) at 22 weeks after the osteotomy.

3.4. Assessment of the nonunion 3.4.1. Radiographic evaluation An anterior-posterior (AP) radiograph of the fractured hind limb was taken using a Faxitron cabinet X-ray system after operation at a 2-week interval (Fig. 2). 3.4.2. Histomorphological evaluation The histomorphology of the fracture nonunion can be evaluated by using undecalcifying or decalcifying bone sections. For the preparation of undecalcifying bone sections, tibiae were fixed and dehydrated in a series of graded ethanol solutions (70%, 80%, 90%, and 100%), embedded in PMMA, and then sectioned at a thickness of 100 µm (Schoellner et al. 2002) along the longitudinal axis of the tibia using a saw microtome (Leica SP1600). A microradiograph of each bone section was taken by using highresolution X-ray films (Struct D4 Pb VacuPac) and a radiograph machine (Faxitron cabinet X-ray system) under identical exposure

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Fig. 2. Position of the rabbit for taking an anterior-posterior (AP) radiograph of the fractured hind limb using a Faxitron cabinet X-ray system.

conditions: at 45 kV, 2 mA and with an X-ray source-to-object distance of 40 cm. PMMA-embedded samples were also sectioned with a Polycut E microtome to a thickness of 8 µm. The sections were stained with H&E (Wang et al. 2001) for descriptive histology under light microscopy.

4. Results At week 12 after the osteotomy, a typical atrophic nonunion was confirmed radiographically in all 12 rabbits in both groups, characterized by a persistent fracture gap, very little callus formation, and sclerosis of the transected cortices [Fig. 3(a)]. These characteristics of atrophic nonunion could be observed until 22 weeks after surgery [Figs. 3(b) and 3(c)]. Light microscopic findings from H&E-stained bone sections are shown in Fig. 4. At the end of week 12, the interfragmental zone was mostly filled with loose, hypocellular fibrous tissue mixed with small islands of hyaline cartilage [Figs. 4(a) and (b)]. There was only

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(a)

(b)

Fig. 3. AP radiographs of the nonunion site at (a) week 12, (b) week 16, and (c) week 22.

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(c)

Fig. 3.

(Continued )

occasional periosteal bone formation with its surface covered by fibrous tissue. The intramedullary canal was sealed by endosteal bone, and the cortical ends showed a sharp surface with small osteoclastic resorption regions [Figs. 4(a) and 4(c)]. The transected cortical ends were necrotic with numerous empty lacunae [Fig. 4(d)]. These histological characteristics of rabbit nonunion models are quite similar to those of clinical nonunion.

5. Discussion 5.1. Animals used as fracture nonunion models Animal species that have been used in experimental fracture studies range in size from as small as a mouse (Hiltunen et al. 1993) to as large as a horse (Nunamaker et al. 1986); and in between are rat (Allen et al. 1980), rabbit (Brighton et al. 1985), cat (Henry et al. 1985), dog (dos Santos Neto and Volpon 1984), and sheep (Cheal et al.

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Fig. 4. Microscopic illustration of the histomorphology of the nonunion at week 12 (H&E staining, longitudinal section).

1991). Because studies of fractures are used to predict and explain fracture healing in humans, the differences in the anatomy and biology of fracture healing for various animal species and models must be considered when designing the experiments and interpreting the results (Rhinelander 1974). Rabbit fracture nonunion models have been used in our previous research projects. This was based on both cost-effectiveness as well as the biophysiological and anatomical charateristics of rabbit bone. Small rodents (mice and rats) are disadvantageous because of a more primitive bone structure that does not include Haversian systems (Nunamaker 1998). Small rodents remodel bone at the fracture site using resorption cavities that form near the fracture surface, and osteoblasts fill in the resorption cavities as the bone heals. Although similar to full Haversian remodeling, there is no secondary Haversian

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system in place when the process is finished and there is little understanding of the importance of this anatomic difference between humans and rodents. Progressing up the phylogenetic scale, Haversian bone appears in the rabbit, cat, and dog. Cortical plexiform bone in sheep remains in place for long periods of time, with secondary Haversian systems being stimulated by trauma such as fracture or osteotomy (Newman et al. 1995). All of these differences in the presentation of Haversian bone between species may play an important role in the study of fracture healing as it applies to humans. The rabbit has been a popular animal model for the study of fracture healing over the years (Ashhurst et al. 1982; Strong et al. 1992; Stafford et al. 1994). Its size lends itself to various studies, from biophysical experiments to fixation methods such as external skeletal fixation, plate and screws, or intramedullary fixation (Brighton et al. 1985; Danckwardt-Lilliestrom et al. 1970; Deibert et al. 1994; Kaplan et al. 1985; Pilla et al. 1990; Terjesen and Johnson 1986). The animal’s size still allows for the systemic use of expensive drugs or chemical agents (Critchlow et al. 1995).

5.2. Age of animals The age of animals for establishing a fracture nonunion model is also an important consideration. Most laboratory animals are young and often still at the growth stage with open growth plates. Fracture healing appears age-sensitive, with young, growing animals achieving healing of their fractures in a more rapid and reproducible manner than older animals do. Radiographic closure of the growth plate is commonly used to determine adulthood, except in the rat (Nunamaker 1998). According to the study of Eggli et al. (1988), rabbits are mature by the age of 6 months and can be considered adults; whereas Rudert (2002) stated that epiphyseal growth plates could still be detected as persistent in 6-month-old animals (3.5–5.5 kg), so the mature age of rabbit may be older than 6 months.

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5.3. Grouping for comparison The success of a fracture, delayed union, or nonunion model can be judged by the sensitivity and specificity of the results that support the hypothesis. Statistical significance of the results from a particular experiment is an important outcome in most studies. Uniformity of the data with respect to biological variance within the experiment should be kept as tight as possible. As compared with human studies, animal studies are cost-effective because of reasons including short duration and rather small sample size. Reasonable effort should be made to optimize the model and minimize group sizes.

5.4. About the critical-size defect (CSD) The literature is replete with reports that the length of bone defects, which lead to nonunion, range from 3 mm to 25 mm in animal models (Bostrom et al. 1996; Bruder et al. 1998a; Bruder et al. 1998b; Cook et al. 1994; Einhorn 1995; Einhorn et al. 1984; Gerhart et al. 1993; Kawaguchi et al. 1994; Radomsky et al. 1998; Yasko et al. 1992). A critical-size defect (CSD) is defined as the smallest intraosseus wound that does not heal by bone formation in the lifetime of the animal (Schmitz and Hollinger 1986). In essence, it involves removing enough bone such that a normal skeleton cannot heal it because of an inability to bridge a gap of that size. This concept must be distinguished from that of a true nonunion, in which the gap defect is small enough for a normal skeleton to bridge it but some pathologic process exists to prevent healing. The CSD is dependent on animal size. Yasko et al. (1992) established a CSD in the rat femur by testing several sizes, and concluded that 5 mm is the smallest defect that would not heal unless a bone graft or osteoinductive substance were to be implanted. Le Guehennec et al. (2005) indicated that the CSD of rat femur was 3 mm, and that CSDs of 6 mm or 4 mm should be used in rabbit femur; this size corresponded to half of the diameter of the smallest bone (rabbit tibia, approximately 8 mm). Other investigators pointed out that the CSD in Copenhagen white rabbit tibia

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should be larger than 8 mm because control defects 8 mm in diameter healed spontaneously (Aaboe et al. 1994).

5.5. Technique of external fixation The cortical bone of the rabbit tibia is thin and crisp, unlike that of human, and its diameter is only 4–5 mm in some sites. In order to provide credible support to the weight loading, four 2.5-mmdiameter stainless steel pins which seemed rather thick for the rabbit tibia were used to produce the fixation. To avoid splitting of the bone, a thinner drill tip with a diameter of 1.5 mm was first used and then the pin holes were renamed with a 2.0-mm drill tip. The sequence of the pin insertion was as follows: first, fourth, second, and then third. Osteotomy was performed after the four pins were screwed. Assemblage of the external fixator was the final step. This procedure could reduce the bone split caused by shear force. A thin stainless steel wire was on hand to fix the bone in case a split occurred.

5.6. Techniques for preventing osteogenesis Because there are a large number of bone healing enhancing factors in the periosteum and bone marrow, the periosteum on either side of the fracture site shall be excised and the marrow shall be removed through curettage or reaming with a drill, as conducted by our research group. However, the distance of the stripped periosteum remained indeterminate. To devitalize the bone next to the fracture gap, freezing at −20°C has been recommended (An et al. 1999).

5.7. Nonunion caused by direct impairment of healing at the fracture site Apart from the procedure of nonunion model with CSD concept, there are some other models of nonunion that are established by means of preventing the normal healing of bone. These models would be more applicable to clinical situations where loss of bone tissue is

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not the problem, but rather where some other known or unknown cause of failure of bony union exists. Patients who sustain severe injuries to an extremity often present with open skeletal injuries involving extensive tissue necrosis. These injuries are prime settings for the development of nonunion. To model these systems, investigators have attempted to impair the healing process at the site of injury. In the study by Baltzer et al. (1999), nonunion was produced in the tibiae of New Zealand white rabbits by creating a fracture with a dental burr, stripping the periosteum for an indeterminate distance distal and proximal to the fracture site, reaming the bone marrow, and placing silastic tubing over the fracture site to prevent healing. After 16 weeks, an atrophic nonunion was observed. In our study, the modified isolation technique was used (Guo et al. 2004).

5.8. Nonunion models caused by mechanical manipulation Few studies have succeeded in producing nonunion in lower-order animals by mechanical manipulation. Utvag et al. (1998) produced nonunion in male Wistar rats by performing standard osteotomy in the femoral diaphysis, reaming the endosteum to accommodate a 1.5-mm soft polyethylene nail, and then mechanically manipulating the fracture site in bending and rotation for 5 weeks; when healing was assessed radiographically at 12 weeks after osteotomy, a hypertrophic nonunion was observed. In a similar study, middiaphyseal fractures were produced by creating a partial osteotomy in the rat femur and completing the fracture manually (Hietaniemi et al. 1995). Fractures thus produced were fixed with a loose-fitting 7-mm steel wire to permit rotational instability. In addition, the periosteum 2 mm proximal and 2 mm distal to the fracture site was cauterized. By 3 weeks after fracture, a visible hypertrophic callus had formed adjacent to the fracture site; however, by 5 weeks after fracture, there was no bridging callus. By 7 to 9 weeks, there was a diminution in the formation of callus; and by 1 year after fracture, the appearance of the fracture site was that of an atrophic nonunion.

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In our study, the preliminary results were unsatisfactory. The radiological and histological evidences displayed an appearance of delayed union of fracture in all cases. In this model, it is difficult to control the time and amount of passive and active relative motion in each day. The degree of motion at the fracture site may be the key point to reproduce a nonunion model of the rabbit tibial diaphysis. Therefore, it is necessary to plan the mechanical mobilization for producing a nonunion model in rabbits in great detail.

6. Summary This chapter describes two atrophic nonunion models in rabbits, namely, a critical-sized bony defect and interposition of soft tissue. Atrophic nonunion characteristics were clearly present in all animals in both models 12 weeks after operation, and persisted until 22 weeks. These two models can be used for effective evaluation of the efficacy of both biophysical and pharmaceutical interventions for the treatment of atrophic nonunion.

References Aaboe M, Pinholt EM, Hjorting-Hansen E. Unicortical critical size defect of rabbit tibia is larger than 8 mm. J Craniofac Surg 5:201–203, 1994. Allen HL, Wase A, Bear WT. Indomethacin and aspirin: effect of nonsteroidal anti-inflammatory agents on the rate of fracture repair in the rat. Acta Orthop Scand 51:595–600, 1980. An YH, Friedman RJ, Draughn RA. Animal models of fracture or osteotomy. In: An YH, Friedman RJ (eds.), Animal Models in Orthopaedic Research, CRC Press, Boca Raton, FL, pp. 197–218, 1999. Aro H, Eerola E, Aho AJ. Development of nonunions in the rat fibula after removal of periosteal neural mechanoreceptors. Clin Orthop 199:292–299, 1985. Ashhurst DE, Hogg J, Perren SM. A method for making reproducible experimental fractures of the rabbit tibia. Injury 14:236–242, 1982. Baltzer AW, Lattermann C, Whalen JD et al. A gene therapy approach to accelerating bone healing. Evaluation of gene expression in a New

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Zealand white rabbit model. Knee Surg Sports Traumatol Arthrosc 7:197–202, 1999. Bostrom M, Lane JM, Tomin E et al. Use of bone morphogenetic protein2 in the rabbit ulnar nonunion model. Clin Orthop 327:272–282, 1996. Brighton CT, Hozack WJ, Brager MD et al. Fracture healing in the rabbit fibula when subjected to various capacitively coupled electrical fields. J Orthop Res 3:331–340, 1985. Bruder SP, Fink DJ, Caplan AL. Mesenchymal stem cells in bone development, bone repair and skeletal regeneration therapy. J Cell Biochem 56: 283–294, 1994. Bruder SP, Kraus KH, Goldberg VM, Kadiyala S. The effect of autologous mesenchymal stem cell implants on the healing canine segmental bone defects. J Bone Joint Surg 80A:985–996, 1998a. Bruder SP, Kurth AA, Shea M et al. Bone regeneration by implanting of purified, culture-expanded human mesenchymal stem cells. J Orthop Res 16:155–162, 1998b. Cheal EJ, Mansmann KA, DiGioia AM et al. Role of interfragmentary strain in fracture healing: ovine model of a healing osteotomy. J Orthop Res 9:131–142, 1991. Choi P, Ogilvie C, Thompson Z et al. Cellular and molecular characterization of a murine non-union model. J Orthop Res 22:1100–1107, 2004. Cook SD, Baffes GC, Wolfe MW et al. The effect of recombinant human osteogenic protein-1 on healing of large segmental bone defects. J Bone Joint Surg 76A:827–838, 1994. Crenshaw H. Delayed union and nonunion of fractures. In: Crenshaw AH (ed.), Campbell’s Operative Orthopaedics, Vol. 3, CV Mosby, St. Louis, MO, pp. 2053–2118, 1987. Critchlow MA, Bland YS, Ashhurst DE. The effect of exogenous transforming growth factor-beta 2 on healing fractures in the rabbit. Bone 16:521–527, 1995. Danckwardt-Lilliestrom G, Lorenzi GL, Olerud S. Intramedullary nailing after reaming. An investigation on the healing process in osteotomized rabbit tibias. Acta Orthop Scand 134S:S1–S7, 1970. Deibert MC, Mcleod BR, Smith SD, Liboff AR. Ion resonance electromagnetic field stimulation of fracture healing in rabbits with a fibular ostectomy. J Orthop Res 12:878–885, 1994. dos Santos Neto FL, Volpon JB. Experimental nonunion in dogs. Clin Orthop 187:260–271, 1984.

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Eggli PS, Hunziker EB, Schenk RK. Quantitation of structural features characterizing weight- and less-weight-bearing regions in articular cartilage: a stereological analysis of medial femoral condyles in young adult rabbits. Anat Rec 222:217–227, 1988. Einhorn TA. Enhancement of fracture healing. J Bone Joint Surg 77A: 940–956, 1995. Einhorn TA, Lane JM, Burstein AH et al. The healing of segmental bone defects induced by demineralized bone matrix. A radiographic and biomechanical study. J Bone Joint Surg 66A:274–279, 1984. Einhorn TA, Trippel SB. Growth factor treatment of fractures. Instr Course Lect 46:483–486, 1997. Gerhart TN, Kirker-Head CA, Kriz MJ et al. Healing segmental femoral defects in sheep using recombinant human bone morphogenetic protein. Clin Orthop 293:317–326, 1993. Gerstenfeld LC, Culliname DM, Barnes GL, Einhorn TA. Fracture healing as a post-natal developmental process: molecular, spatial and temporal aspects of its regulation. J Cell Biochem 88:873–874, 2003. Guo Z, Guo X, Zheng ZY. Application of tissue isolation technique and mechanical mobilization in developing non-union models of the rabbit tibial diaphysis. Chin J Clin Rehabil 15:961–963, 2004. Henry WB Jr, Schachar NS, Wadsworth PL et al. Feline model for the study of frozen osteoarticular hemijoint transplantation: qualitative and quantitative assessment of bone healing. Am J Vet Res 46: 1714–1720, 1985. Hernigou P, Poignard A, Beaujean RH. Percutaneous autologous bone marrow grafting for nonunion. J Bone Joint Surg 87A:1430–1437, 2005. Hietaniemi K, Peltonen J, Paavolainen P. An experimental model for nonunion in rats. Injury 26:681–686, 1995. Hiltunen A, Vuorio E, Aro HT. A standardized experimental fracture in the mouse tibia. J Orthop Res 11:305–312, 1993. Hollinger JO, Kleinschmidt JP. The critical sized defect as an experimental model to test bone repair materials. Craniofac Surg 1:60–68, 1990. Kaplan SJ, Hayes WC, Mudan P et al. Monitoring the healing of a tibial osteotomy in the rabbit treated with external fixation. J Orthop Res 3:325–330, 1985.

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Kawaguchi H, Kurokawa T, Hanada K et al. Stimulation of fracture repair by recombinant human basic fibroblast growth factor in normal and streptozotocin-diabetic rats. Endocrinology 135:774–781, 1994. Le Guehennec L, Goyenvalle E Aguado E et al. Small-animal models for testing macroporous ceramic bone substitutes. J Biomed Mater Res B Appl Biomater 15:69–78, 2005. Megas P. Classification of non-union. Injury 36S:S30–S37, 2005. Newman E, Turner AS, Wark JD. The potential of sheep for the study of osteopenia: current status and comparison with other animal models. Bone 16S:S277–S284, 1995. Nunamaker DM. Experimental models of fracture repair. Clin Orthop 355 (Suppl):S56–S65, 1998. Nunamaker DM, Richardson DW, Buttenveck DM et al. A new external skeletal fixation device that allows immediate full weightbearing: application in the horse. Vet Surg 151:345–355, 1986. Pilla AA, Mont MA, Nasser PR et al. Non-invasive low-intensity pulsed ultrasound accelerates bone healing in the rabbit. J Orthop Trauma 4:246–253, 1990. Radomsky ML, Spiro R, Poser J. Augmentation of fracture healing with fibroblast growth factor in a hyaluronan gel. Abstracts from the Sixth Meeting of the International Society for Fracture Repair, Strasbourg, France, pp. 41–42, 1998. Reed AAC, Joyner CJ, Brownlow HC, Simpson AHRW. Vascularity in a new model of atrophic nonunion. J Bone Joint Surg 85B:604–610, 2003. Rhinelander FW. Tibial blood supply in relation to fracture healing. Clin Orthop 105:34–81, 1974. Rudert M. Histological evaluation of osteochondral defects: consideration of animal models with emphasis on the rabbit, experimental setup, follow-up and applied methods. Cells Tissues Organs 171:229–240, 2002. Ruedi TP, Buckley RE, Moran CG. AO Principles of Fracture Management, 2nd ed. Thieme Medical Publishers, Stuttgart, Germany, 2007. Schmitz JP, Hollinger JO. The critical size defect as an experimental model for craniomandibulofacial nonunions. Clin Orthop 205:299–308, 1986. Schoellner C, Rompe JD, Decking J, Heine J. High-energy ESWT for pseudarthrosis. Orthopade 31:658–662, 2002. Simmons DJ. Fracture healing perspectives. Clin Orthop 200:100–113, 1985.

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Stafford HJ, Roberts MT, Oni OO et al. Localization of bone-forming cells during fracture healing by osteocalcin immunocytochemistry: an experimental study of the rabbit tibia. J Orthop Res 12:29–39, 1994. Strong ML, Wong-Chung J, Babikian G, Brody A. Rotational remodeling of malrotated femoral fractures: a model in the rabbit. J Pediatr Orthop 12:173–176, 1992. Terjesen T, Johnson E. Effects of fixation stiffness on fracture healing. External fixation of tibial osteotomy in the rabbit. Acta Orthop Scand 57:146–148, 1986. Utvag SE, Grundes O, Reikeras O. Graded exchange reaming and nailing of non-unions. Arch Orthop Trauma Surg 5:1–6, 1998. Wallace AL, Draper ER, Strachan RK et al. The effect of devascularisation upon early bone healing in dynamic external fixation. J Bone Joint Surg 73B:819–825, 1991. Wang CJ, Chen HS, Chen CE, Yang KD. Treatment of nonunions of long bone fractures with shock waves. Clin Orthop 387:95–101, 2001. Yasko AW, Lane JM, Fellinger EJ et al. The healing of segmental bone defects induced by recombinant human bone morphogenetic protein (rhBMP-2). A radiographic, histological and biomechanical study in rats. J Bone Joint Surg 74A:659–670, 1992. Yoo JU, Johnstone B. The role of osteochondral progenitor cells in fracture repair. Clin Orthop 355S:S73–S78, 1998.

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Chapter 26

Establishment of Osteoporosis Model in Goats Wing-Sum Siu, Ling Qin and Kwok-Sui Leung

Fixation of osteoporotic fracture is always a challenging procedure in orthopedic surgery. Loosening of fixation implants from osteoporotic bone is not uncommon. A large osteoporotic animal model that resembles human osteoporotic changes is therefore essential in osteoporosis research. It can be used to test for better fixation techniques and biomaterials developed for the enhancement of osteoporotic fractures. However, many factors should be considered in developing osteoporotic animal models; these include the comparability to human skeletal physiology, the risk of zoonotic disease, the ease of handling, as well as the possibility of regional and climatic accommodation of the animals. Among the large animals, goat is the most suitable one to fulfill the requirements in the authors’ institution and region. This chapter describes the methodologies developed and adopted for developing an osteoporotic goat model. Ovariectomy (OVX), one of the common methods to induce osteoporosis in animals, is systemically described. The importance of a low-calcium diet for accelerating bone loss in OVX goats is incorporated and discussed. The chapter also illustrates different methods of monitoring the development of osteoporosis as well as the relative results. These include the monitoring of serum estradiol concentration, monitoring of changes in bone mineral density (BMD) using peripheral quantitative computed tomography (pQCT) on iliac crest biopsies and calcanei, analysis of the trabecular microarchitecture of iliac crest biopsies using Corresponding author: Kwok-Sui Leung. Tel: +852-26322724; fax: +852-26377889; E-mail: [email protected]

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Animal model; goat; osteoporosis; ovariectomy; bone mineral density (BMD); trabecular microarchitecture; biomechanical test.

1. Introduction Osteoporotic fractures of the hip and spine may cause serious complications including loss of mobility and independence, and even death. These fractures are difficult to heal. Loosening of implants from osteoporotic bone after fracture fixation is common (Leung et al. 2006; Zink 1996). Hence, osteoporotic fracture fixation remains a major challenge for orthopedic surgeons. With the increase in the aging population worldwide, healthcare expenditures — especially those used to treat osteoporosis and osteoporotic fractures — are increasing annually (Orsini et al. 2005; Rousculp et al. 2007). In order to develop better surgical implants and biomaterials for the enhancement of osteoporotic fractures, it is highly desirable to establish a large osteoporotic animal model for preclinical research (Leung et al. 2006; Lill et al. 2000; Siu et al. 2004). When considering a large osteoporotic animal model, nonhuman primates, dogs, and pigs are large enough to receive prosthetic implants. They can withstand repetitive bone biopsies and largevolume blood sampling (Egermann et al. 2005). However, the acquisition of primates and dogs is difficult and very costly. Many primates are facing extinction, while dogs are one of the most popular pets among humans. They are therefore less acceptable by society as an animal model for scientific or preclinical research (Egermann et al. 2005; Newman et al. 1995). More critically, the risk of zoonotic disease transmission, including HIV, from primates is relatively high (Newman et al. 1995). Some studies have also reported insignificant bone loss in dogs after the cessation of ovarian function (Kimmel 1991; Shen et al. 1992) due to their relatively low levels of estrogen and the semiannual estrous cycle. Ovariectomy (OVX) is difficult to be performed on pigs because the blood supply to their uterus is more friable. All of these animals are also aggressive and therefore give rise to handling problems.

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Sheep and goats often serve as models for human conditions (Egermann et al. 2005; Leung et al. 2006; Siu et al. 2004). Similarity in iliac crests and hormonal profiles between the sheep and the human has been reported (Goodman 1994; Turner and Villanueva 1993). Most recently, many in vivo experimental studies used bones of goat to study bone mineral density (BMD), microarchitecture, and mechanical properties (Sigrist et al. 2007; Siu et al. 2003; Siu et al. 2004; Turner 2006). Sheep and goats are docile and compliant flock animals that can be handled easily. They suffer the least stress when housed in groups compared to the large animals mentioned previously. This chapter describes methodologies developed and adopted for establishing an osteoporotic goat model. OVX is the most common method to induce osteoporosis in animals.a

2. Materials •

Animal: skeletal mature Chinese mountain goat with radiographically confirmed closure of growth plate at the proximal tibia


•

a

It would be ideal to know the exact age of the animals in order to minimize the variations in animal age for comparative studies.

Diet: (1) Low-calcium dietb — food pellet with 0.2% calcium (e.g. Glen Forrest Stockfeeders, Glen Forrest, Australia),

OVX is a surgical operation to excise ovaries from females. Some studies reported that the prevalence of OVX among osteoporotic patients was higher than expected (Drozdzowska 2006; Stpan et al. 1987). It has also been recognized as a well-established method to induce osteoporosis in animals. It was reported to be successful in rat (Frost and Jee 1992; Mathey et al. 2007; Zhang et al. 2006a; Zhang et al. 2006b), rabbit (Castaneda et al. 2006), goat (Siu et al. 2004; Leung et al. 2006), sheep (Jiang et al. 2005; Phillips et al. 2006; Turner et al. 1995), and monkey (Fox et al. 2007; Legrand et al. 2003). b Since estrogen deficiency induces a focal imbalance at remodeling sites, metabolic changes that increase the remodeling rate of bone will accelerate skeletal loss even further. Therefore, it is not uncommon for a restricted- or low-calcium diet to be introduced together with OVX in order to develop an osteoporotic animal (Jiang et al. 1997; Yoshida et al. 1998). There is considerable evidence — accumulated over the past several decades from cross-sectional (Sentipal et al. 1991), longitudinal (Dawson-Hughes et al. 1987), and intervention studies (Smith et al. 1989) — indicating that dietary calcium intake is an important determinant of bone mass. During calcium depletion, the serum calcium level tends to fall, which stimulates the secretion of PTH and the synthesis of calcitriol, which in turn increase the activation of bone remodeling and induce a decrease in bone mass.

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wheaten chaff with 0.3% calcium (e.g. O’Driscoll, Greerock, Australia); (2) normal diet — food pellet with 0.85% calcium (e.g. Glen Forrest Stockfeeders, Glen Forrest, Australia), lucerne chaff (e.g. Southern Cross Grains Pty Ltd, Victoria, Australia) Anesthetic drug: 5% halothane (e.g. Fluothane; Zeneca, Cheshire, UK), together with a mixture of nitrous oxide (1 L/min) and oxygen (2 L/min) Antibiotic: Amoxicillin (e.g. AlfaMedic Ltd, Hong Kong) Analgesic: Temgesic (e.g. Reckitt & Colman Products Ltd, Hull, UK) Euthanasia: 25% pentobarbital (e.g. Sigma Chemical, St. Louis, MO, USA) overdose (50 mg/kg), intravenous injection ELISA kit for serum estradiol-17β analysis (e.g. Fertigenix E2-EASIA; BioSource Europe S.A., Nivelles, Belgium) X-ray machine for confirmation of skeletal maturity of goats (e.g. SFR-510; Shower X-ray Co. Ltd, Tokyo, Japan) Imaging analysis system (e.g. PACE System DiagnostiX 2048; PACE Medical, Freiberg, Germany) Peripheral quantitative computed tomography (pQCT) (e.g. Densiscan 2000; Scanco Medical AG, Bassersdorf, Switzerland) Micro-computed tomography (micro-CT) (e.g. µCT40; Scanco Medical AG, Bassersdorf, Switzerland) Solutions for hard tissue processing: ethanol (75%, 95%, and 100%), xylene, methylmethacrylate (MMA) (e.g. Sigma-Aldrich, St. Louis, USA) Saw microtome (e.g. Leica SP1600; Leica Instruments, Nussloch, Germany)

b (Continued) Feeding with a low-calcium diet increases the severity of bone loss in OVX goats; this is also demonstrated by comparing our goat model (Leung et al. 2001) with the osteopenic ewe model reported by Turner et al. (1995). Another popular method tested to accelerate OVX-induced bone loss is the use of corticosteroid (Klopfenstein Bregger et al. 2007). A lowcalcium diet helps to reduce the animal holding time for intervention studies, and is especially relevant for studying implants and biomaterials developed to enhance osteoporotic fractures (Leung et al. 2006).

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High-resolution X-ray film (e.g. Structurix D4 Pb Vacupac; Agfa, Belgium) X-ray machine for microradiography (e.g. Faxitron Cabinet X-ray System Model 43855C; Faxitron X-Ray Corp., Wheeling, IL, USA) Low-speed saw (e.g. Isomet, IL, USA) Material testing machine (e.g. KS 25; Hounsfield Test Equipment Ltd, Redhill, Surrey, UK)

3. Methods 3.1. Ovariectomy (OVX) • • •

• • • • • •

Fast the goat for 1 day before the operation. Place it in left recumbency on an operation table with the surgical side facing upward. Anesthetize it via inhalation of 5% halothane (e.g. Fluothane; Zeneca, Cheshire, UK), together with a mixture of nitrous oxide (1 L/min) and oxygen (2 L/min), until the animal is unconscious and its corneal reflex is inhibited. Keep it anesthetized by intubation with a 7.0-mm or 7.5-mm endotracheal tube using a laryngoscope. Monitor its breathing pattern using an electronic respiration monitor throughout the whole operation. Introduce a nasogastric tube through the nostril to drain the excessive fluid from the rumen in order to prevent the development of ruminal tympany. Shave the hair of the left lumbar region of the goat using an electric clipper. Clean the skin with 30% habitant and sterilize it with 75% ethanol. Cover the body of the goat with sterilized drapes, exposing only the operation site. Make a 10-cm incision over the flank. Dissect and retract the external and internal abdominal obliques as well as the transversus abdominis to expose the peritoneum. Incise the peritoneum, and expose the ovaries and suspensory ligaments.

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Fig. 1. A 10-cm incision is made over the flank. The peritoneum is exposed and incised to expose the ovaries (within the white circles) and suspensory ligaments. After ligating the ovary arteries, the ovaries are excised bilaterally from the suspensory ligaments.

• • • • •

Ligate the ovary arteries, and excise the ovaries bilaterally from the suspensory ligaments (Fig. 1). Suture to close the peritoneum and abdominal muscles. Apply subcutaneous antibiotic (Amoxicillin, 10 mg/kg/day) injections for 4 consecutive days to prevent wound inflammation. Apply intramuscular analgesic (Temgesic, 0.5 mL every 6 hours) injections for 2 days for pain release. For sham control, repeat all of the above steps except ligation and excision of the ovaries.

3.2. Serving of low-calcium diet • • •

Feed all of the goats with lucerne chaff in the first week. Gradually change the diet of OVX goats to 50% food pellet plus 50% wheaten chaff with calcium 0.2% and 0.3%, respectively. Feed the goats in the sham group with a normal diet containing 1.1% calcium on average (50% food pellet with 0.85% calcium plus 50% lucerne chaff ).

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3.3. Iliac crest biopsy To harvest two biopsies alternately from the left and right iliac crests and to collect the first one before the OVX/sham operation and the second one from another side after 6 months, the surgical procedure is as follows: • • • • •

Anesthetize the goat in the same way as for OVX. Shave, clean, and sterilize the iliac crest. Make a 3-cm incision to expose the tuber coxae. Cut out a transverse biopsy, approximately 1 cm × 0.8 cm × 0.4 cm. Fix the biopsy in 75% ethanol immediately.

3.4. Bone autopsy • • •

Euthanize the goat by an overdose intravenous injection of 25% pentobarbital. Collect and wrap the humeri and calcanei of both legs by gauze soaked with 0.9% saline. Preserve all of the specimens at −20°C before use.

3.5. Monitoring of changes in serum estradiol (E2) level • • • • •

Collect the blood in the morning before the operation (OVX/ sham) as baseline and monthly after the operation. Obtain the serum by centrifuging the blood samples at 3000 rpm and 4°C for 10 minutes. Aliquot the samples and store them at −80°C. Measure the serum E2 using an ELISA kit (e.g. Fertigenix E2EASIA; BioSource). Determine the serum E2 level by a microplate reader with a filter at 450 nm and a reference filter at 630 nm.

3.6. BMD measurement •

Fix all of the bone specimens vertically in custom-made plastic holders for pQCT (Densiscan 2000) measurement.

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Fig. 2. (a) pQCT tomograph (Densiscan 2000) of an iliac crest biopsy used for measuring the mean volumetric trabecular BMD from the central 50% of core volume within the entire cross-sectional area using the built-in software. (b) Micro-CT tomograph scanned at a spatial resolution of 20 µm with the same region of interest (ROI) of pQCT for the quantification of trabecular microarchitecture.

• • •

Scan the whole iliac crest biopsy continuously (consecutive multislices). Scan the middle region of the calcaneus with 6 mm in total length consecutively. Measure the mean volumetric BMD (the central 50% core volume within the entire cross-sectional area of the bicortical scan) using built-in software of the pQCT (Ruegsegger et al. 1996) (Fig. 2).

3.7. Analysis of trabecular microarchitecture • • •

Place the iliac crest biopsies horizontally into a cylindrical sample holder provided by the manufacturer of micro-CT. Stabilize them in the scan tube with foam. Fill with 75% ethanol.

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Fig. 3. 3D reconstructions of micro-CT bone biopsies used for structural analysis. (a) Trabecular bone at baseline. (b) Trabecular bone 6 months after OVX.

• • •

• •

Set the spatial resolution for scanning to 20 µm (Siu et al. 2004). Scan each biopsy consecutively with thickness and increment at 20 µm for 120 slices. Define the region of interest (ROI) as the central 50% of the whole biopsy with reference to the BMD measurement using pQCT (Figs. 2 and 3). Set the threshold at 122 to obtain the best coverage of all cancellous bone. Exclude the cortical bone manually. Evaluate the architectural parameters of trabeculae by the built-in method in the system.

3.8. Microradiography • •

Dehydrate the iliac crest biopsies with increasing concentrations of ethanol (75%, 95%, and 100%) and finally with xylene. Embed the biopsies in a series of a mixture of xylene and unpolymerized MMA (UMMA), UMMA, and finally in polymerized MMA (PMMA) (Schenk et al. 1984).

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Fig. 4. Microradiograph of sectioned iliac crest biopsies. Obvious less number and connection of the trabeculae are shown in the iliac crest biopsy of OVX goats after 6 months of operation (upper right) as compared with the biopsy from Sham goats (lower right) (Siu et al. 2004).

• • •

•

Put the specimens in UMMA and PMMA into a vacuum desiccator for degassing.c Section the embedded specimens into 500-µm thickness using a saw microtome (Leica Instruments, Germany). Put the sections onto a high-resolution X-ray film (e.g. Agfa), and expose under X-ray at 30 kVp for 40 minutes (Faxitron Cabinet X-ray System). Analyze the images of the X-ray films (Fig. 4), e.g. using the abovementioned imaging analysis system.

3.9. Biomechanical indentation test d •

Thaw the calcanei and humeri overnight at room temperature.

c Specimens immersed in solutions with UMMA or PMMA should be stored at 4°C after degassing. Abundant gas bubbles will be formed or “boiling” may occur if they are kept at room temperature overnight. d During the indentation test, preventing the specimens from dehydration by moisturizing them with 0.9% saline frequently is important. The biomechanical properties of the trabeculae of cancellous bone will be seriously affected if it becomes dehydrated, and the result will not be consistent (Qin and Zhang 2005).

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Cut away the first 5 mm and 8 mm (measured from the apex) of the calcaneum and the humeral head, respectively, to expose the cancellous bone using a low-speed saw (Isomet). Cut the following 8 mm of the specimen parallel to the first cut to obtain a cylindrical sample specimen for indentation test. Indent the center of the specimen using the material testing machine (Hounsfield) with a 1000-N load cell at room temperature. Set a constant loading speed of 2 mm/min for a total displacement of 3 mm. The diameters of the stainless steel rod for the calcaneum and the humeral head are 2.5 mm and 4.0 mm, respectively (Fig. 5). Keep all the specimens moist with 0.9 % saline during the whole preparation and testing procedure. Calculate the total indentation work done by summating the area under the load–displacement curve (from displacement of 0 mm to 3 mm).

4. Results 4.1. Change in serum estrogen level The serum estradiol (E2) concentration of the OVX goats decreased significantly after 6 months of OVX as compared with the sham group (Table 1), suggesting successful OVX-induced estrogen depletion.

4.2. Change in BMD At 6 months postoperation, the BMD of the iliac crest of OVX goats decreased by 25% significantly. When compared with the sham groups, the BMD at the iliac crest and the calcaneus of the OVX was 40.3% and 32.8%, respectively, lower (Table 2).

4.3. Microarchitectural analysis of trabeculae The bone volume density (BV/TV), trabecular number (Tb.N), and connectivity density (Conn.D) measured using micro-CT decreased

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(a)

(b)

(c)

Fig. 5. Preparation of calcaneum for indentation test. (a) A low-speed saw (Isomet) is used to cut away the first 5 mm (measured from the apex) of the calcaneum along the calcaneus shaft to expose the cancellous bone. (b) The following 8 mm of the specimen is cut parallel to the first cut to obtain a cylindrical specimen, which is fixed in a jig. (c) The center of the specimen (premarked with a paper circle) is aligned with the indenter before the indentation test using a material testing machine.

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Table 1. Median percentage change of serum E2 of goats from baseline to 6 months after ovariectomy (OVX) (data in median percentage change from the baseline). Month(s) Group after OVX OVX Sham

0

1

2

3

4

5

6

Median(%) 0.00 −42.30a −42.38a −46.78a −47.28a −49.56a −52.39a,b n 10 8 7 7 7 7 7 Median(%) 0.00 14.40 17.18 6.05 −15.73 −21.98 −24.06 n 4 3 3 3 3 3 3

n: Sample size of the goats in the respective month of the group. a Significant difference between the two groups in the same month; p = 0.017. b Significant difference between the sixth month and the baseline within the same group; p = 0.018.

Table 2.

Region Iliac crest biopsy

Calcaneus

Differences of BMD in goats after ovariectomy (OVX).

Group

% change after 6 months of operation

% difference between OVX and sham

p-value

OVX Sham OVX − Sham OVX − Sham

−25.0% 15.4% NA NA

NA NA −40.3% −32.8%

0.006 0.310 0.028 0.001

NA: not applicable.

significantly after 6 months of OVX when the trabecular plate separation (Tb.Sp) was accordingly greater than the baseline (Table 3). All of these parameters showed that OVX-induced bone loss was attributed by a deterioration of trabecular microarchitecture.

4.4. Microradiography Similar to the results of microarchitectural analysis using micro-CT, measurements on high-resolution X-ray film showed an obvious decrease in the number and connection of trabeculae in the iliac crest biopsy of OVX goats after 6 months of operation, while such an observation was not revealed in the biopsy of sham goats (Fig. 4).

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Table 3. Median percentage differences of microarchitectural parameters of the iliac crest biopsies compared between baseline and 6 months after ovariectomy (OVX).

Parameters BV/TV Tb.N Tb.Sp Conn.D

Baseline (median)

6 months after OVX (median)

Median Difference (%)

p-value

17.31% 1.761 mm−1 0.580 mm 7.838 mm−3

15.99% 1.537 mm−1 0.653 mm 6.465 mm−3

−8.34 −8.51 8.26 −18.52

0.045a 0.022a 0.022a 0.049a

p < 0.05; n = 10 for baseline and 6 months postoperation.

a

Table 4. Percentage difference of work done in indentation test between OVX and sham goats 6 months after OVX. Region Calcaneum Humeral head

% difference (OVX − Sham)

p-value

−52.1% −54.3%

0.001 0.006

4.5. Biomechanical indentation test The energy required for making a 3-mm indentation in the cancellous region of the calcaneum and humeral head from OVX goats was significantly less than that from sham goats (Table 4). This test indicated that the ability of the bone of goats after 6 months of OVX to sustain load was decreased.

5. Discussion and Summary This chapter describes an OVX goat model that was evaluated using multiple approaches. Each method may have its advantages and disadvantages (e.g. invasive vs. noninvasive, general vs. specific, more accurate vs. less sensitive). Unlike clinical osteoporosis research, where dual-energy X-ray absorptiometry (DXA) is still the current gold standard for diagnosing osteoporosis as recommended by the World Health Organization (Kanis and Klüer 2000; World Health

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Organization 1994), there is no gold standard or threshold window to define the degree of osteoporosis in animal models. As not all research laboratories may have all of the abovementioned assessment tools, we recommend using at least one of the abovementioned endpoint measurements to demonstrate statistically significant bone loss, e.g. on average more than 10% bone loss in the highturnover trabecular bone region. Bone loss can also be confirmed biomechanically. As the main aim of establishing animal models is to evaluate potential pharmaceutical and nonpharmaceutical means for the prevention and treatment of osteoporosis and osteoporotic fractures, it is desirable to establish noninvasive or nondestructive monitoring methods, such as using DXA or pQCT. The recent development of micro-CT (XtremeCT) for human and large animal applications will help advance our studies efficaciously (Dambacher et al. 2007). In summary, this chapter describes the technique to establish an osteoporotic goat model. The procedure of OVX and the ingredients of a low-calcium diet are listed in detail. It also illustrates the tests and their results, which prove the successful establishment of osteoporosis in OVX goats.

Acknowledgments The authors deeply appreciate Dr Edmund Cheung’s help and suggestions in the surgical procedures. We also want to thank Dr Anthony James for his help in looking for the low-calcium diet for the OVX goats, and Ms Vivian Hung for her help in BMD measurement using pQCT. This study was funded by the Earmarked Grant CUHK 4270/98M, Research Grants Council, Hong Kong.

References Castaneda S, Largo R, Calvo E et al. Bone mineral measurements of subchondral and trabecular bone in healthy and osteoporotic rabbits. Skeletal Radiol 35(1):34–41, 2006.

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Dambacher MA, Neff M, Radspieler R et al. In vivo bone mineral density and structures in humans — from Isotom over Densiscan to Xtreme-CT. In: Qin L, Genant HK, Griffith JF, Leung KS (eds.), Advanced Bioimaging Technologies in Assessment of the Quality of Bone and Scaffold Materials, Springer Verlag, Berlin, pp. 65–72, 2007. Dawson-Hughes B, Jacgues P, Shipp C. Dietary calcium intake and bone loss from the spine in healthy postmenopausal women. Am J Clin Nutr 46:685–687, 1987. Drozdzowska B. Quantitative ultrasound measurements at the calcaneus in natural and surgically induced menopause. Maturitas 53(1):107–113, 2006. Egermann M, Goldhahn J, Schneider E. Animal models for fracture treatment in osteoporosis (review). Osteoporos Int 16(2):S129–S138, 2005. Fox J, Miller MA, Newman MK et al. Treatment of skeletally mature ovariectomized rhesus monkeys with PTH(1–84) for 16 months increases bone formation and density and improves trabecular architecture and biomechanical properties at the lumbar spine. J Bone Miner Res 2(2):260–273, 2007. Frost HM, Jee WS. On the rat model of human osteopenia and osteoporosis. Bone Miner 18:227–236, 1992. Goodman RL. Neuroendocrine control of the ovine estrous cycle. In: Knobil E, Neill J (eds.), Physiology of Reproduction, Raven Press, New York, pp. 659–709, 1994. Jiang Y, Zhao J, Aberman HM et al. Long-term changes in bone mineral and biomechanical properties of vertebrae and femur in aging, dietary calcium restricted, and/or estrogen-deprived/-replaced rats. J Bone Miner Res 12(5):820–831, 1997. Jiang Y, Zhao J, Geusens P et al. Femoral neck trabecular microstructure in ovariectomized ewes treated with calcitonin: MRI microscopic evaluation. J Bone Miner Res 20(1):125–130, 2005. Kanis JA, Glüer C. An update on the diagnosis and assessment of osteoporosis with densitometry. Osteoporos Int 11:192–202, 2000. Kimmel DB. The oophorectomized beagle as an experimental model for estrogen-depletion bone loss in the adult human. Cells Mater 5(Suppl): 75–84, 1991.

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Klopfenstein Bregger MD, Schawalder P, Rahn B et al. Optimization of corticosteroid induced osteoporosis in ovariectomized sheep. A bone histomorphometric study. Vet Comp Orthop Traumatol 20(1):18–23, 2007. Legrand JJ, Fisch C, Guillaumat PO et al. Use of biochemical markers to monitor changes in bone turnover in cynomolgus monkeys. Biomarkers 8(1):63–77, 2003. Leung KS, Siu WS, Cheung NM et al. Goats as an osteopenic animal model. J Bone Miner Res 16(12):2348–2355, 2001. Leung KS, Siu WS, Li SF et al. An in vitro optimized injectable calcium phosphate cement for augmenting screw fixation in osteoporotic goats. J Biomed Mater Res B Appl Biomater 78(1):153–160, 2006. Lill CA, Fluegel AK, Schneider E. Sheep model for fracture treatment in osteoporotic bone: a pilot study about different induction regimens. J Orthop Trauma 14(8):559–565, 2000. Mathey J, Mardon J, Fokialakis N et al. Modulation of soy isoflavones bioavailability and subsequent effects on bone health in ovariectomized rats: the case for equol. Osteoporos Int 18(5):671–679, 2007. Newman E, Turner AS, Wark JK. The potential of sheep for the study of osteopenia: current status and comparision with other animal models. Bone 16(4 Suppl):277S–284S, 1995. Orsini LS, Rousculp MD, Long SR, Wang S. Health care utilization and expenditures in the United States: a study of osteoporosis-related fractures. Osteoporos Int 16(4):359–371, 2005. Phillips FM, Turner AS, Seim 3rd HB et al. In vivo BMP-7 (OP-1) enhancement of osteoporotic vertebral bodies in an ovine model. Spine J 6(5):500–506, 2006. Qin L, Zhang M. Mechanical testing for bone specimens. In: Deng HW, Liu YZ (eds.), Current Topics of Bone Biology, World Scientific, Singapore, pp. 177–212, 2005. Rousculp MD, Long SR, Wang S et al. Economic burden of osteoporosisrelated fractures in Medicaid. Value Health 10(2):144–152, 2007. Ruegsegger P, Elsasser U, Anliker M et al. Quantification of bone mineralization using computed tomography. Radiology 121:93–97, 1996. Schenk RK, Olah AJ, Herrmann W. Preparation of calcified tissues for light microscopy. In: Dickson GR (ed.), Methods of Calcified Tissue Preparation, Elsevier Science, Amsterdam, pp. 4–10, 1984.

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Sentipal JM, Wardlaw GM, Mahan J, Matkvoic V. Influence of calcium intake and growth indexes on vertebral bone mineral density in young females. Am J Clin Nutr 54:425–428, 1991. Shen V, Dempster DW, Birchman R et al. Lack of changes in histomorphometric, bone mass, and biochemical parameters in ovariohysterectomized dogs. Bone 13:311–316, 1992. Sigrist IM, Gerhardt C, Alini M et al. The long-term effects of ovariectomy on bone metabolism in sheep. J Bone Miner Metab 25(1):28–35, 2007. Siu WS, Qin L, Cheung WH, Leung KS. A study of trabecular bones in ovariectomized goats with micro-computed tomography and peripheral quantitative computed tomography. Bone 35:21–26, 2004. Siu WS, Qin L, Leung KS. pQCT bone strength index may serve as a better predictor than bone mineral density for long bone breaking strength. J Bone Miner Metab 21(5):316–322, 2003. Smith EL, Giligan C, Smith PE, Sempos CT. Calcium supplementation and bone loss in middle-aged women. Am J Clin Nutr 50:833–842, 1989. Stpan JJ, Pospichal J, Presl J, Pacovsky V. Bone loss and biochemical indices of bone remodeling in surgically induced postmenopausal women. Bone 8(5):279–284, 1987. Turner AS. Seasonal changes in bone metabolism in sheep: further characterization of an animal model for human osteoporosis. Vet J 174(3): 460–461, 2007. Turner AS, Alvis M, Myers W et al. Changes in bone mineral density and bone-specific alkaline phosphatase in ovariectomised ewes. Bone 17(Suppl 4):395S–402S, 1995. Turner AS, Villanueva AR. Histomorphometry of the iliac crest: 9–11 year old ewes. Proc Vet Surg 22:413, 1993. World Health Organization (WHO). Assessment of fracture risk and its application to screening for postmenopausal osteoporosis. World Health Organ Tech Rep Ser, No. 843. WHO, Geneva, 1994. Yoshida Y, Moriya A, Kitamura K et al. Responses of trabecular and cortical bone turnover and bone mass and strength to bisphosphonate YH529 in ovariohysterectomized beagles with calcium restriction. J Bone Miner Res 13(6): 1011–1022, 1998. Zhang G, Qin L, Hung WY et al. Flavonoids derived from herbal Epimedium Brevicornum Maxim prevent OVX-induced osteoporosis in

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rats independent of its enhancement in intestinal calcium absorption. Bone 38(6):818–825, 2006a. Zhang Y, Lai WP, Leung PC et al. Effects of Fructus Ligustri Lucidi extract on bone turnover and calcium balance in ovariectomized rats. Biol Pharm Bull 29(2):291–296, 2006b. Zink PM. Performance of ventral spondylodesis screws in cervical vertebrae of varying bone mineral density. Spine 21:45–52, 1996.

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Chapter 27

Posterior Spinal Fusion Model Chun-Wai Chan and Jack Chun-Yiu Cheng

Spinal fusion is commonly used in spinal surgery to treat spinal deformity and degenerative disease. In animal models, two commonly used posterior spinal fusion surgeries are intertransverse process and interbody spinal fusion. These experimental spinal fusions could provide clues for the medical treatment of patients by stem cell therapy, tissue engineering, or gene therapy. They also serve as study models for testing new biomaterials and their derived composites with bioactive factors or cells. In this chapter, the procedure for rabbit experimental posterior intertransverse process spinal fusion and the method to assess the success of this animal model are described. Keywords:

Posterior spinal fusion; tissue engineering; biomaterials; stem cell; manual palpation; pQCT; micro-CT.

1. Introduction Spinal fusion is commonly used in spinal surgery to treat spinal deformity and degenerative disease. The standard surgical technique combines bone grafting on transverse processes with decortication and instrumentation, i.e. posterior intertransverse process spinal Corresponding author: Chun-Wai Chan. Tel: +852-26323309; fax: +852-26377889; E-mail: [email protected]

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fusion. Another approach is to perform fusion between vertebral bodies with stabilization of disk space, i.e. interbody fusion (Kruyt et al. 2004). However, even with the gold standard of using an autograft, there is a relatively high failure rate of pseudoarthrosis with high morbidity. Efforts have been made to promote the spinal fusion rate by osteoinductive factors such as BMP-4 (Cheng et al. 2002; Guo et al. 2002), BMP-2 (Ohyama et al. 2004), and osteogenic protein-1 (White et al. 2004); gene therapy (Lee et al. 2006); and biophysical interventions such as low-intensity pulsed ultrasound (Glazer et al. 1998), pulsed electromagnetic fields (Glazer et al. 1997), and direct current (Fredericks et al. 2007). Bone substitution research, which is essential given the limited supply of autografts, has also been done using allografts (Chan et al. 2005), biomaterials (Minamide et al. 2004), and a cell–biomaterials hybrid construct (Chan et al. 2007a; Chan et al. 2007b; Chan et al. 2007c). To carry out extensive research, an appropriate spinal fusion model is required. One of the most commonly used animal models is the noninstrumented rabbit posterior spinal fusion model (Boden et al. 1995). This chapter introduces the procedure for rabbit posterior spinal fusion that is established at the authors’ institution for some of the abovementioned applications.

2. Materials and Methods 2.1. Materials • • • •

Operation surgery set Hair shaver Hibatine in 70% ethanol Operation drapes

2.2. Methods •

Autoclave the surgical instruments before operation or dip them into diluted hibatine for at least 30 minutes for sterilization.

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•

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After general anesthesia, shave the rabbit hair using a hair shaver to expose the back skin. Usually, the intertransverse process fusion is done on L4–L5 or L5–L6 for single-level fusion. Remove the hair to expose one more spinal segment proximally and distally. Sterilize the skin by hibatine in ethanol. Sterilize the operation table by hibatine in ethanol as well. Put the animal in prone position on the operation table [Fig. 1(a)]. Make a dorsal midline skin incision and then two paramedian fascial incisions [Fig. 1(b)]. Dissect and penetrate the multifidus and longissimus muscles using artery forceps to expose the transverse processes [Figs. 1(c) and 1(d)]. Use a periosteal elevator to expose the dorsal surface of the transverse processes carefully without damaging the nearby ligament and facet joint. Use a specially designed retractor to open the operation side [Fig. 1(e)]. Use an air-driven power drill and drill bit (2.5 mm in diameter) to make a hole on the dorsal surface of the transverse processes monolaterally for decortication [Fig. 1(f)]. Use a blur (3.0 mm in diameter) to enlarge the hole up to the width of the implant [Fig. 1(g)]. Sterilize the implant (e.g. autograft, allograft, biomaterials) in advance. The cell–biomaterials composite should be kept in an aseptic culture environment before operation. Remove all soft tissues of the autograft or allograft. Bony tissue (preferably cancellous bone) is exposed by the blur. Insert the implant over the decorticated area [Fig. 1(h)]. Use a blunt-ended instrument (e.g. artery forceps) to press on the implant. Gently overlay the paraspinal muscle on top of the implant for noninstrumented fixation. Close the soft tissue by layers using vicryl 3-0 sutures. Close the skin using monocryl 4-0 sutures. Keep warm before recovery from anesthesia.

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(a)

(b)

Fig. 1. The rabbit posterior spinal fusion procedure. (a) The rabbit is put in prone position. (b–d) The midline insertion is made down to the paraspinal muscle. (e) The dorsal surface of transverse processes is exposed by a retractor. (f, g) The air-driven drill and then the blur are used for decorticating the transverse processes. (h) The graft or biomaterial is implanted onto the transverse processes. Finally, the wound is closed by layers.

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Fig. 1.

(Continued )

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Fig. 1.

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3. Results Successful posterior intertransverse process fusion can be monitored by X-ray radiography after operation in vivo. Plain X-ray radiography is suggested periodically. It can help to notice any mobility of implant for the implantation of less radiolucent autografts, allografts, or polymeric-type implants (e.g. polylactic acid). The X-ray radiograph of rabbit posterior spinal fusion can demonstrate the bony bridge between two processes [Figs. 2(a) and 2(b)] (Cheng et al. 2002; Guo et al. 2002). Recently, numerous biomaterials studies have been conducted in spinal fusion. The regenerated bone is formed underneath the biomaterial implant. However, due to the high radiolucent property of calcium phosphate–based implants (e.g. beta-tricalcium phosphate and hydroxyapatite), the newly formed bone from transverse processes cannot be easily distinguished because it is masked by the implant in the anterior-posterior view of plain X-ray radiography. The progress of regenerated bone thus cannot be examined during spinal fusion [Figs. 2(c) and 2(d)]. Peripheral quantitative computed tomography (pQCT) is used to monitor trabecular architecture in vivo. The bony tissue of transverse processes can be examined through a cross-sectional scanning approach (Fig. 3). The pQCT measurement is suitable for extensive bone formation in spinal fusion. However, the low resolution of pQCT images only assesses bone mineral density (BMD) and the volume of regenerating transverse processes (Chan et al. 2007a; Chan et al. 2007b; Chan et al. 2007c). High-resolution micro-computed tomography (micro-CT) is used to generate three-dimensional (3D) reconstructive images (Fig. 4). The vertebral segments are harvested after the animal is euthanized at a particular time-point. The samples are excised into two halves saggitally. The samples are put in a scanning holder with 70% ethanol. The samples are scanned by a micro-CT machine (e.g. µCT40; Scanco Medical, Bassersdorf, Switzerland) in a cross-sectional manner. The resolution is set at 36 µm per voxel. The 3D spinal segment is reconstructed with a standardized segmentation parameter (sigma, 1.2; support, 1; threshold, 143). Although the detailed structural changes of

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(a)

(b)

Fig. 2. (a) X-ray radiograph of rabbit posterior spinal fusion implanted with autograft after operation. The arrow shows the iliac crest graft, which participated in fusion mass regeneration to show bony fusion with a continuous bony bridge between two processes at week 7 postoperation (b). (c) X-ray of spinal fusion with the beta-tricalcium phosphate implant. Even though the transverse processes underneath the calcium phosphate implant are visualized, the newly formed bone cannot be easily distinguished at week 7 due to the high radiolucent property of the calcium phosphate block (d).

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(d)

Fig. 2.

(Continued )

fusion mass are measured, the samples should be collected after animal euthanasia because the animal (except mouse) cannot be put in a scanning holder. The final outcome of spinal fusion is solid fusion. Manual palpation of the harvested spinal segment is simple and effective for

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(a)

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(c)

(d)

Fig. 3. Cross-sectional images of rabbit spinal fusion implanted with betatricalcium phosphate. The newly formed bone is found underneath the implant.

demonstrating solid fusion (Cheng et al. 2002; Guo et al. 2002). After harvesting vertebral segments, the soft tissue is removed. The mobility of the vertebral joint and implant-to-transverse processes can be assessed by manual palpation. Further bone mineral density assessment and histological analysis can be performed for detailed analysis (Chan et al. 2005; Chan et al. 2007a; Chan et al. 2007b; Chan et al. 2007c). Even tissue is collected for gene expression study (Hisamitsu et al. 2006; Fredericks et al. 2007).

4. Remarks •

Sterilization for animal operation is essential for good animal operation procedure and handling. It also reduces complications and pain in animals.

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(a)

(b)

Fig. 4. The anterior view of micro-CT reconstructive images of rabbit spinal fusion implanted with (a) autograft and (b) beta-tricalcium phosphate. (a) The autograft enhances the bone regeneration of transverse processes, which grow towards each other and fuse together (arrow). (b) In contrast, the porous beta-tricalcium phosphate (dotted arrow) is osteoconductive on the regenerated bone. Since there is no osteoinductive effect, the limited bone is formed to leave a fusion gap (G) between transverse processes.

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•

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This is a noninstrumented implantation surgery. The fixation of implant is basically by the paraspinal muscle. The muscle is exposed around the length of the implant. It can facilitate the fixation of the implant without shifting in a proximal or distal direction. After insertion of the implant and temporary fixation by blunt-ended forceps, the paraspinal muscle is uplifted and overlaid on the implant. Although noninstrumentation is applied, the implant (e.g. autograft, allograft, biomaterials block) can be fixed in position without mobility (Boden et al. 1995; Cheng et al. 2002; Guo et al. 2002; Chan et al. 2005; Chan et al. 2007a; Chan et al. 2007b; Chan et al. 2007c). The rabbit is monitored regularly for any chronic inflammation or infection of the wound, animal health and behavior, eating and drinking habits, etc.

5. Summary Experimental posterior spinal fusion in the rabbit is a clinically relevant model for advancing the technology of the enhancement and acceleration of the fusion rate. It can also help in studying the biology of bone regeneration with grafting and implant interface biology. Moreover, it can assess the inductive or conductive effect of novel biomaterials, the bioactivity of osteoinductive factors in vivo, and novel biophysical interventions. In the future, studies involving in vivo XtremeCT can be adopted as a powerful in vivo monitoring method for evaluating fusion dynamics (Dambacher et al. 2007).

References Boden SD, Schimandle JH, Hutton WC. An experimental lumbar intertransverse process spinal fusion model. Radiographic, histologic, and biomechanical healing characteristics. Spine 20(4):412–420, 1995. Chan CW, Lee KM, Qin L et al. Mesenchymal stem cell derived osteogenic cells is superior to bone marrow aspirate impregnated biomaterial complex in posterior spinal fusion. Key Eng Mater 330–332:1149–1152, 2007a.

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Chan CW, Qin L, Lee KM et al. Bio-engineered mesenchymal stem cell– tricalcium phosphate ceramics composite augmented bone regeneration in posterior spinal fusion. Key Eng Mater 334–335:1201–1203, 2007b. Chan CW, Wong KHK, Lee KM et al. Can basic fibroblast growth factor pre-treatment enhance mesenchymal stem cell therapy in undecorticated posterior spinal fusion? Key Eng Mater 330–332:1137–1140, 2007c. Chan CW, Yeung HY, Lee KM et al. Temporal and spatial expression pattern of VEGF and VEGF receptor in the posterior spinal fusion with allograft. Key Eng Mater 288–289:491–494, 2005. Cheng JCY, Guo X, Law LP et al. How does recombinant human bone morphogenetic protein-4 enhance posterior spinal fusion? Spine 27(5): 467–474, 2002. Dambacher MA, Neff M, Radspieler HT et al. In vivo bone mineral density and structures in humans: from Isotom over Densiscan to Xtreme-CT. In: Qin L, Genant HK, Griffith J, Leung KS (eds.), Advanced Bioimaging Technologies in Assessment of the Quality of Bone and Scaffold Materials, Springer Verlag, Berlin, pp. 65–78, 2007. Fredericks DC, Smucker J, Petersen EB et al. Effects of direct current electrical stimulation on gene expression of osteopromotive factors in a posterolateral spinal fusion model. Spine 32(2):174–181, 2007. Glazer PA, Heilmann MR, Lotz JC, Bradford DS. Use of electromagnetic fields in a spinal fusion. A rabbit model. Spine 22(20):2351–2356, 1997. Glazer PA, Heilmann MR, Lotz JC, Bradford DS. Use of ultrasound in spinal arthrodesis. A rabbit model. Spine 23(10):1142–1148, 1998. Guo X, Lee KM, Law LP et al. Recombinant human bone morphogenetic protein-4 (RhBMP-4) enhanced posterior spinal fusion without decortication. J Orthop Res 20:740–746, 2002. Hisamitsu J, Yamazaki M, Suzuki H et al. Gene expression for type-specific collagens in osteogenic protein-1 (rhBMP-7)-induced lumbar intertransverse process fusion in rabbits. Connect Tissue Res 47(5):256–263, 2006. Kruyt MC, van Gaalen SM, Oner FC et al. Bone tissue engineering and spinal fusion: the potential of hybrid constructs by combining osteoprogenitor cells and scaffolds. Biomaterials 25(9):1463–1473, 2004.

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Lee TC, Ho JT, Hung KS et al. Bone morphogenetic protein gene therapy using a fibrin scaffold for a rabbit spinal-fusion experiment. Neurosurgery 58(2):373–380, 2006. Minamide A, Kawakami M, Hashizume H et al. Experimental study of carriers of bone morphogenetic protein used for spinal fusion. J Orthop Sci 9(2):142–151, 2004. Ohyama T, Kubo Y, Iwata H, Taki W. Beta-tricalcium phosphate combined with recombinant human bone morphogenetic protein-2: a substitute for autograft, used for packing interbody fusion cages in the canine lumbar spine. Neurol Med Chir (Tokyo) 44(5):234–240, 2004. White AP, Weinstein MA, Patel TCh et al. The 2002 Marshall Urist Young Investigator Award Paper. Lumbar arthrodesis gene expression: a comparison of autograft with osteogenic protein-1. Clin Orthop Relat Res 429:330–337, 2004.

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Chapter 28

Functional Disuse Model for Musculoskeletal Adaptation Ho-Yan Lam and Yi-Xian Qin

Musculoskeletal diseases, especially osteoporosis, are serious health threats to many individuals including the aging elderly, astronauts, and longduration-functional-resting (e.g. spinal cord injury) patients. The degree of bone loss and muscle atrophy due to disuse is closely associated with increased fracture risk, affecting the morbidity and mortality of the population. Many appropriate animal models have been developed to fully investigate the mechanisms responsible for musculoskeletal adaptations under disuse environment; more importantly, these models are the key in discovering new interventions for osteoporosis. Hindlimb suspension (HLS) is a well-accepted functional disuse model employed on rodents. In this model, the animal’s hindlimbs are lifted and suspended for a period of time (days to weeks), thus removing daily weight-bearing activities to the hindlimbs. This chapter will mainly focus on the technical aspects of HLS as a functional disuse model for studying musculoskeletal tissues. Detailed materials and methods are provided for investigators to easily design and efficiently set up a HLS study. The limitations of HLS and other alternative functional disuse models (i.e. casting and neurectomy) are also discussed for further consideration. Keywords:

Bone loss; muscle atrophy; fracture risk; hindlimb suspension; disuse model; casting; neurectomy.

Corresponding author: Yi-Xian Qin. Tel: +1-631-6321481; fax: +1-631-6328577; E-mail: [email protected]

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1. Introduction The quantity and quality of our bones and skeletal muscles are closely interrelated and play important roles in daily physical functions. The deterioration of musculoskeletal tissues, i.e. bone loss and muscle atrophy, is the result of functional disuse osteoporosis. This disease is a threat for millions of individuals, with the increased fracture risk due to bone and muscle fragility increasing the morbidity and mortality of the population and imposing a financial burden on the community (Bikle and Haloran 1999; Garnero and Delmas 2004). The etiology of functional disuse osteoporosis is apparent, yet the detailed mechanism leading to the loss and deterioration of bone and muscle is not fully understood. Common countermeasures for osteoporosis include exercise and the use of anabolic agents. Osteopenia and osteoporosis are characterized by low bone mass and deterioration of the microarchitectural network. Functional mechanical loading significantly influences bone mass and morphology (Wolff 1986). The removal of weight-bearing activity results in disuse osteoporosis. Disuse osteoporosis is experienced mainly by astronauts on long-term space missions and patients with spinal cord injury (SCI). It has been established that significant and rapid bone loss often occurs in the lower extremities, i.e. the femoral neck and pelvis. It is estimated that 1%–2% of site-specific bone mineral density (BMD) is lost per month during exposure to microgravity (Iwamoto et al. 2005; Lang et al. 2004). In a 6-month spaceflight, cosmonauts experienced up to 6% and 3% decreases in the tibia trabecular bone mass and cortical bone mass, respectively, and a 13.2% loss of calcaneus broadband ultrasound attenuation (Collet et al. 1997; Vico et al. 2000). Others have demonstrated that trabecular volumetric BMD was lost at a rate of 2.2%–2.7% per month at the hip and 0.7% per month at the spine (Lang et al. 2004). The lack of physical activity following SCI leads to a dramatic reduction in bone mass. In a study with 41 SCI patients, 61% was diagnosed with osteoporosis, 20% with osteopenia, and 34% with fracture incidence (Lazo et al. 2001). The quantity of bone loss in humans subjected to microgravity and SCI is comparable,

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yet the mechanisms may not be equivalent. Reambulation after disuse allows some site-specific bone mass to be restored, but it may not completely return to its normal quantity and quality (Vico et al. 2000; Wilmet et al. 1995). Thus, interventions are continuously being developed to investigate the physiological mechanisms underlying disuse osteoporosis. The interrelationship between muscular and skeletal tissues is an important topic in studying a major skeletal disease like osteoporosis. The structural and functional adaptations of skeletal muscle to microgravity and disuse after SCI have been studied at various levels, and have been linked to the reduction of skeletal integrity. In terms of lower extremity muscle volume, studies reported a decrease of 10% in the quadriceps and 19% in the gastrocnemius and soleus after a 6-month space mission (LeBlanc et al. 2000). Computed tomography measurements of the muscle cross-sectional area (CSA) indicated a decrease of 10% in the gastrocnemius and 10%–15% in the quadriceps after short-term missions (Narici et al. 1997). Similar results were concluded after SCI, where patients suffered significant 21%, 28%, and 39% reductions of the CSA in the quadriceps femoris, soleus, and gastrocnemius muscles, respectively (Gorgey and Dudley 2007; Shah et al. 2006). In addition to the whole muscle size, muscle fiber characteristics are also modified due to inactivity. Muscle fibers can be mainly classified into two types: slow (type I) fibers play an important role in maintaining body posture, while fast (type II) fibers respond to physical activity. Under disuse condition, all fiber types were found to decrease in size: 16% for type I, and 23%–36% for type II (Edgerton et al. 1995). The atrophied soleus muscles also underwent a shift from type I (−8% in fiber number) to type II fibers (Stewart et al. 2004). The development of functional disuse animal models to mimick conditions such as microgravity or temporary paralysis, as experienced by astronauts and SCI patients, is an essential step toward biomedical research and new interventions. These models are critical for elucidating the mechanisms regulating the musculoskeletal adaptive responses to a functional disuse environment. Investigators have used various animal models, from rodents to primates, to study disuse osteoporosis. In this chapter, a rodent hindlimb suspension model

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will be described in-depth, including a detailed technical protocol to successfully design a functional disuse rat model. The advantages and limitations of the hindlimb suspension (HLS) model, and other alternative methods, will also be discussed later in the chapter.

2. Materials •

Hindlimb suspension cage: stainless steel cage with dimensions 18′′ (length) × 18′′ (width) × 24′′ (height)



•

Aluminum flashing


•

A fishline swivel is connected through a small hole drilled in the middle of a ∼20′′ metal rod.

Isoflurane, ketamine, and xylazine — for anesthetizing the animals 70% ethanol — for cleaning the animal’s tail Tincture of benzoin — adhesive for attaching the skin tape to the tail Paperclip-looped plastic tab





•

Cover the lower half of the HLS cage interior to prevent the animal from climbing the sides of the cage and standing on its hindlimb.

Metal rod with fishline swivel


• • • •

The cages should have a water bottle and food holder. The dimensions of the cage may vary based on the size of the animal.

A paperclip is inserted through small holes drilled in a piece of plastic. The size of the tab should vary depending on the size of the animal (e.g. 1′′ × ½′′ plastic tab for a ∼250–300-g rat).

Surgical tape





There are multiple types of surgical tapes that can be used for HLS. One suggestion is the Dermiform hypoallergenic knitted tape (Johnson & Johnson). The size of each tape should be ∼1/4′′ (width) × 12′′ (length) for a ∼250–300-g rat.

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•

Adhesive bandage





•

461

There are multiple types of adhesive bandages available. One suggestion is the elastic adhesive bandage (Elastoplast). The bandage should be cut to the size of ∼ ½′′ (width) × 4′′ (length).

Gauze


Any type of thin gauze pad will be sufficient for wrapping the tail, if needed.

3. Methods 3.1. Preparation for the suspension cage (Fig. 1) • • •

Clean the stainless steel cages before setting up. Cut panels of aluminum flashing, 18′′ (width) × 12′′ (height), to cover the interior sides of HLS cages. Fill the cage bottom with standard bedding.

Fig. 1. A representative image of the stainless steel HLS cage [18′′ (length) × 18′′ (weight) × 24′′ (height)] and the basic setup of the metal rod with the fishline swivel.

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Set up the water bottle and place in the holder for each cage. Place ∼50 g of food inside the cage.

3.2. Preparation for the hindlimb suspension •

• • •

•

• •

•

•

For anesthesia, place the animal under a light anesthetic by inhaling a low dose of isoflurane or by injecting a mixture of ketamine (50 mg/kg) and xylazine (10 mg/kg) intraperitoneally.a Clean the tail of the animal with 70% ethanol and leave to air-dry. Once the tail is dried, apply a thin coat of tincture of benzoin and allow the adhesive to dry for 2–3 minutes. Thread a piece of surgical tape (its width and length determined by the size of the tail) through the paperclip loop of the tail harness.b When the thin coat of benzoin becomes “sticky”, adhere the surgical tape to the sides of the tail. The surgical tape should be attached from the base of the tail and cover ¾ of its length. Use two strips of elastic adhesive bandage, one strip to secure the surgical tape over the ends and the other about halfway up the tail.c To avoid the animal from “picking” on the tapes, wrap a thin layer of gauze around the tape area. Ensure that the gauze is used only if necessary because the tail is a critical location for thermoregulation. Once the animal is awake from the anesthesia, attach the papercliplooped plastic tab, which is now adhered to the animal’s tail, to the fishline swivel that hangs from the top portion of the HLS cage. Adjust the fishline swivel to ensure the following details:








a

The animal’s hindlimbs (extended) should not touch the cage bottom. The body of the animal should be in a head-down position with a 30° angle between the midbelly and the cage bottom. The forelimbs of the animals should have full access to the entire cage bottom.

For experienced researchers, anesthesia of the animal is not a necessary step and may be avoided for HLS setup. b Cover the plastic tab, especially the edges, with soft fabric or tape because irritation and/or inflammation to the tail may occur due to daily movement of the tail against the plastic. c The elastic adhesive bandage should be adhered loosely to allow normal blood circulation of the tail.

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3.3. Daily maintenance •

The animals should be cleaned and inspected daily for the following:







•

Signs of stress in overall appearance and activity; Irritation and/or inflammation of the tail; Change in or addition to food and water consumption, if necessary; and Maintenance of room temperature at ∼70°F.

Body weight should be monitored closely throughout the experimental period.d

4. Results Hindlimb suspension (HLS) is a functional disuse model, where the animal’s hindlimbs are lifted and suspended for the duration of the study, resulting in unloading of the hindlimbs which normally have a weightbearing function. There are similarities when comparing musculoskeletal adaptations between animal and human disuse models. Site specificity is one such factor: bone loss after space missions is mainly at the metaphysis and epiphysis regions of the lower extremities (Collet et al. 1997; Lafage-Proust et al. 1998); while bones subject to HLS are also sitespecific, where only weight-bearing bone is negatively affected. In a 4-week study, we have demonstrated the different effects of HLS on the femoral cortical and trabecular bones. The initial body weight of the animals on day 0 of the experiment was not significantly different between the control and HLS groups (317 g ± 28 g and 324 g ± 30 g, respectively). After 4 weeks, the body mass of the control group decreased slightly by 3%, while the body weight of the HLS rats decreased by 10%. The femur length was measured and showed no difference between the control and HLS groups (37.1 mm ± 0.62 mm vs. 37.0 mm ± 0.78 mm, respectively). The quantity and quality of the femoral cortical and trabecular bones were assessed using micro-CT. The middiaphysis cortex area was not different

d

Body weight usually decreases dramatically in the first 2 weeks (within 15% of the original weight), but stabilizes by the fourth week.

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(a)

7

Ct.Ar (mm^2)

6 5 4 3 2 1 0

(b)

Baseline

Age-matched

HLS

Baseline

Age-matched

HLS

12

Ps.En (mm^2)

10 8 6 4 2 0

(c)

6

*

Ec.En (mm^2)

5 4 3 2 1 0 Baseline

Age-matched

HLS

Fig. 2. Micro-CT analysis of the middiaphyseal region of 6-month-old rat femurs. Each graph shows three animal groups: baseline control, 4-week age-matched control, and 4-week HLS. (a) Cortical bone area (mm2); (b) periosteal envelope (mm2); (c) endosteal envelope (mm2). Values are mean ± SD. A significant difference is considered as *p < 0.05 vs. agematched control and baseline control.

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(a)

(b)

Fig. 3. Representative images of the trabecular network at the metaphyseal region of 6-month-old rat distal femur for (a) 4-week age-matched control and (b) 4-week HLS.

between the age-matched control and HLS animals, but the periosteal and endosteal envelopes were 13% and 37% higher in the HLS animals (Fig. 2). Figure 3 illustrates the microarchitecture of distal metaphysis trabecular bone between the age-matched control and HLS animals at the femur. HLS animals were able to induce significant trabecular bone loss and structural deterioration (Fig. 4). The graphs show a 50% decrease in bone volume fraction, a 77% decrease in connectivity, a 29% reduction in trabecular number, and a 45% increase in trabecular spacing. Other HLS studies with skeletally mature adult rats have resulted in up to 20% reduction of trabecular BMD at the femoral neck and proximal tibia (Bloomfield et al. 2002). Cortical BMD reduction was not affected by the HLS as much as the trabecular bone site (Allen and Bloomfield 2003). HLS reduced calcium content by 7%–12% at

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(a)

0.3 0.25 BV/TV (%)

0.2

**

0.15 0.1 0.05 0 Baseline

(b)

Age-matched

HLS

120

Conn.D (1/mm^3)

100 80 60 40

**

20 0 Baseline

(c)

Age-matched

HLS

5

Tb.N (1/mm)

4.5 4

**

3.5 3 2.5 2 1.5 1 0.5 0 Baseline

Age-matched

HLS

Fig. 4. Micro-CT analysis of the distal metaphyseal region of 6-month-old rat femurs. Each graph shows three animal groups: baseline control, 4-week age-matched control, and 4-week HLS. (a) Bone volume fraction (BV/TV, %); (b) connectivity (Conn.D, 1/mm3); (c) trabecular number (Tb.N, 1/mm). Values are mean ± SD. A significant difference is considered as **p < 0.05 vs. age-matched control and baseline control.

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the tibia and 11% at the femur (Sessions et al. 1989; Vico et al. 1995). Bone resorption biochemical markers increased in both human and rat HLS disuse models. However, reductions in bone formation were not often mentioned in human spaceflight studies. Histomorphometric analyses in HLS experiments indicated a decrease in the bone formation rate at various sites, i.e. 34% at the tibiofibular junction (Sessions et al. 1989), 65%–88% at the tibial middiaphysis periosteal surface (Bloomfield et al. 2002; Hefferan et al. 2003), and 19% at the distal femur metaphyseal trabecular bone surface (Allen and Bloomfield 2003). Furthermore, hindlimb-suspended muscles, i.e. soleus and gastrocnemius, undergo atrophy and a slow-to-fast phenotype transition similar to humans in spaceflight and after SCI. Soleus atrophy was demonstrated in various HLS studies, reducing the soleus-to-bodyweight ratio by 30% and the individual fiber CSA by 66% (Wang et al. 2006). HLS induces muscular adaptation via regulating cellular activities. Satellite cells, myogenic precursor cells, have been shown to serve as a source of new myonuclei during regeneration (Schultz et al. 1994). Sixteen days of HLS diminished satellite cell mitotic activity, yet a period of reloading returned its mitotic activities to normal (Wang et al. 2006). Other cellular activities, such as apoptosis, have also activated in response to muscle disuse (Siu et al. 2005).

5. Remarks 5.1. Age of animals The age of the rodents is an important element in designing experiments. For HLS, age plays a significant role in determining the body weight of the animals, the skeletal maturity, and the ability to handle stress throughout the experiment. Young rats less than 3 months of age appear to adapt more readily to HLS than aged animals. These animals (<200 g) are rapidly growing rats and may not lose weight during HLS. However, the rate of growth may decrease in HLS animals when compared to age-matched control animals (Matsumoto et al. 1998; Morey-Holton and Globus 1998). Although the growth

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of rats continues to slow down gradually throughout their lifespan, it is widely agreed that these rodents reach skeletal maturity at approximately 6 months of age (Bloomfield et al. 2002; Vico et al. 1995). During HLS, skeletally mature animals will most likely lose weight during the initial period of unloading and stabilize by the second week. Aged adult rats (>400 g) are a fitting model for studying agerelated diseases, but may have difficulty adapting to the HLS environment (Morey-Holton and Globus 2002). Thus, additional maintenance and monitoring may be required when designing a HLS experiment with aged animals.

5.2. Expansion of HLS This chapter provides an in-depth HLS protocol primarily for the rat. However, this model has long been adapted for use on mice to study the musculoskeletal adaptive responses due to disuse. Results have indicated that musculoskeletal adaptation to unloading and mechanical stimulus is dependent on the genetic background of the mouse strain (Judex et al. 2004; Zhong et al. 2005). With the increased availability of transgenic strains of mice, functional disuse research has expanded tremendously to investigate molecular mechanisms (e.g. BMP-2 and IGF-I) that mediate responses to the deterioration of bone and muscle. However, there are several considerations to bear in mind when using mouse HLS models: • •

•

Stress response in mice is less tolerated than in rats. Significant decreases in body mass and hair loss are often the two indicators. The materials listed above must be downsized to accommodate the smaller body size. These include the HLS cage, the metal rod, and the paperclip-looped plastic tab.e Mice tend to be more active and flexible due to their small body size. To prevent mice from climbing onto their own tail and

e The conventional plastic cage for normal rat housing can be substituted for an 18′′ × 18′′ × 24′′ stainless steel cage for mouse HLS.

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“picking” on the tape, an additional device should be considered to replace the gauze wrapped around the skin tape.f

5.3. Limitations of rodent HLS Aside from the apparent advantages in using rodents (i.e. inexpensive, fast generation time, and easy to handle compared to larger models) for functional disuse research, there are several limitations to be cautious of: •

•

There is a skeletally physiological difference between rodents and humans. Although trabecular bone loss due to disuse (i.e. site specificity) is similar in both species, rat is a poor animal model to study the effects on cortical bone because of the lack of Haversian systems, which can alter the coupling of the bone remodeling mechanisms and attenuate cortical porosity (Huttunen et al. 2007). The effects of HLS are limited to the musculoskeletal system. The head-tilt position has been shown to induce fluid shift and generate pressure gradients in the vasculature, producing structural alterations in basilar and mesenteric resistance arteries (Colleran et al. 2000; Wilkerson et al. 2005).

5.4. Alternative functional disuse model — casting Various types of casts, i.e. plaster and fiberglass casts, have been used on small animals for immobilization studies. This method is relatively effective in generating muscle atrophy and osteopenia. A 2-week cast immobilization study resulted in a 37% decrease in soleus wet weight and a significant reduction in type I muscle fiber (Sakakima 2004). Similarly, cast immobilization led to a decrease of 13% in tibia ash weight, 23% in bone volume, and 42% in bone formation rate (Tuukkanen et al. 1991) However, the technique is expensive and is often associated with other f

Using a 3-cc syringe, cut off the ends and attach the additional device to the paperclip-looped plastic tab before attaching it to the animal’s tail. Be careful with any shape edge of the plastic that might damage the tail due to daily movement.

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medical complications of the hindlimb, such as severe weight loss, swelling, and skin ulceration at the site of casting.

5.5. Alternative functional disuse model — neurectomy Sciatic neurectomy is another commonly used method to induce disuse osteopenia and sarcopenia. Briefly, an incision is made to the upper hindlimb, posterior to the femoral trochanteric region, of the anesthetized rat. The sciatic nerve can be exposed with a blunt dissection, and a 5-mm length of nerve is excised. The incision can be closed with suture or staples. This is certainly a more invasive method compared to HLS. It is critical to perform any surgical procedure in a sterilized environment to avoid infection to the wound. The outcomes of this procedure are very similar to those of HLS animals. Neurectomy resulted in a 46%–57% reduction in trabecular bone volume, but no change in the cortical bone area (Zeng et al. 1996); the trabecular bone loss was associated with a decrease in bone formation and an increase in osteoclastic activities. In addition, microarchitectural analysis showed significant deterioration of trabecular bone, with a 40% decrease in trabecular number and a 28% decrease in thickness (Ito et al. 2002). Other studies have also demonstrated the effects of sciatic neurectomy on hindlimb skeletal muscle wet weight (63% and 75% weight loss for soleus and gastrocnemius, respectively) and protein expression levels, i.e. myostatin and collagen (Savolainen et al. 1988; Zeng et al. 1996; Zhang et al. 2006).

5.6. Alternative functional disuse model — Botox A recently developed model has demonstrated paralysis of the animal’s hindlimb by injecting Botulinum toxin (Botox). In this localized disuse model (Warner et al. 2006), toxin was injected into the quadriceps and posterior muscles of the calf. The treatment was able to significantly reduce muscle mass (−47% to −60%). In addition, the interrelationship between skeletal muscle and bone was illustrated with this study. Muscle paralysis induced bone degradation, reducing

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hindlimb trabecular bone volume by 43%–54% and trabecular thickness by 25%. It is important to point out some limitations to the model, however. The mechanism in which Botulinum toxin paralyzes the surrounding skeletal muscular tissue is not well understood. The injected chemical may have a direct consequence on the skeleton, affecting its adaptive responses; thus, bone loss may not be a pure result of functional disuse. Using a similar toxin, others have shown mixed results, indicating no significant change in DEXA analysis between the paralysis and the control tibia (Chappard et al. 2001).

5.7. General guidelines to animal studies Before considering conducting animal research, investigators should review the guidelines for the care and use of laboratory animals set by the National Advisory Committee for Laboratory Animal Research (NACLAR) and the Office of Laboratory Animal Welfare (OLAW). In addition to the approval of the Institutional Animal Care and Use Committee (IACUC), researchers need to ensure that animals are observed daily, if not more frequently, and that records are clearly kept to include husbandry details and animals’ health status. In HLS, the animal’s behavior (indicating stress) and body weight loss are the most important issues. The consumption of food and water should also be carefully monitored.

Acknowledgments This work was kindly supported by the National Institutes of Health (R01 AR52379 and R01 AR49286 to Y.-X. Qin) and the U.S. Army Medical Research and Materiel Command (DAMD-17-02-1-0218 to Y.-X. Qin).

References Allen MR, Bloomfield SA. Hindlimb unloading has a greater effect on cortical compared with cancellous bone in mature female rats. J Appl Physiol 94(2):642–650, 2003.

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Bikle DD, Haloran BP. The response of bone to unloading. J Bone Miner Metab 17(4):233–244, 1999. Bloomfield SA, Allen MR, Hogan HA, Delp MD. Site- and compartmentspecific changes in bone with hindlimb unloading in mature adult rats. Bone 31(1):149–157, 2002. Chappard D, Chennebault A, Moreau M et al. Texture analysis of X-ray radiographs is a more reliable descriptor of bone loss than mineral content in a rat model of localized disuse induced by the Clostridium botulinum toxin. Bone 28(1):72–79, 2001. Colleran PN, Wilkerson MK, Bloomfield SA et al. Alterations in skeletal perfusion with simulated microgravity: a possible mechanism for bone remodeling. J Appl Physiol 89(3):1046–1054, 2000. Collet P, Uebelhart D, Vico L et al. Effect of 1- and 6-month spaceflight on bone mass and biochemistry in two humans. Bone 20(6):547–551, 1997. Edgerton VR, Zhou MY, Ohira Y et al. Human fiber size and enzymatic properties after 5 and 11 days of spaceflight. J Appl Physiol 78(5):1733–1739, 1995. Garnero P, Delmas PD. Contribution of bone mineral density and bone turnover markers to the estimation of risk of osteoporotic fracture in postmenopausal women. J Musculoskelet Neuronal Interact 4(1):50–63, 2004. Gorgey AS, Dudley GA. Skeletal muscle atrophy and increased intramuscular fat after incomplete spinal cord injury. Spinal Cord 45(4):304–309, 2007. Hefferan TE, Evans GL, Lotinun S et al. Effect of gender on bone turnover in adult rats during simulated weightlessness. J Appl Physiol 95(5):1775–1780, 2003. Huttunen MM, Tillman I, Viljakainen HT et al. High dietary phosphate intake reduces bone strength in the growing rat skeleton. J Bone Miner Res 22(1):83–92, 2007. Ito M, Nishida A, Nakamura T et al. Differences of three-dimensional trabecular microstructure in osteopenic rat models caused by ovariectomy and neurectomy. Bone 30(4):594–598, 2002. Iwamoto J, Takeda T, Sato Y. Interventions to prevent bone loss in astronauts during space flight. Keio J Med 54(2):55–59, 2005.

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Judex S, Garman R, Squire M et al. Genetically linked site-specificity of disuse osteoporosis. J Bone Miner Res 19(4):607–613, 2004. Lafage-Proust MH, Collet P, Dubost JM et al. Space-related bone mineral redistribution and lack of bone mass recovery after reambulation in young rats. Am J Physiol 274(2 Pt 2): R324–R334, 1998. Lang T, LeBlanc A, Evans H et al. Cortical and trabecular bone mineral loss from the spine and hip in long-duration spaceflight. J Bone Miner Res 19(6):1006–1012, 2004. Lazo MG, Shirazi P, Sam M et al. Osteoporosis and risk of fracture in men with spinal cord injury. Spinal Cord 39(4):208–214, 2001. LeBlanc A, Lin C, Shackelford L et al. Muscle volume, MRI relaxation times (T2), and bone composition after spaceflight. J Appl Physiol 89(6):2158–2164, 2000. Matsumoto T, Nakayama K, Kodama Y et al. Effect of mechanical loading and reloading on periosteal bone formation and gene expression in tail-suspended rapidly growing rats. Bone 22(5 Suppl):89S–93S, 1998. Morey-Holton ER, Globus RD. Hindlimb unloading rodent model: technical aspects. J Appl Physiol 92(4):1367–1377, 2002. Morey-Holton ER, Globus RK. Hindlimb unloading of growing rats: a model for predicting skeletal changes during space flight. Bone 22(2 Suppl): 83S–88S, 1998. Narici MV, Kayser B, Barattini P, Cerretelli P. Changes in electrically evoked skeletal muscle contractions during 17-day spaceflight and bed rest. Int J Sports Med 18(Suppl 4):S290–S292, 1997. Sakakima H. Effect of immobilization and subsequent low and high frequency treadmill running on rat soleus muscle and ankle joint movement. J Jpn Phys Ther Assoc 16:43–48, 2004. Savolainen J, Myllyla V, Myllyla R et al. Effects of denervation and immobilization on collagen synthesis in rat skeletal mscle and tendon. Am J Physiol 254(6 Pt 2):R897–R902, 1988. Schultz E, Darr KC, Macius A. Acute effects of hindlimb unweighting on satellite cells of growing skeletal muscle. J Appl Physiol 76(1):266–270, 1994. Sessions ND, Halloran BP, Bikle DD et al. Bone response to normal weight bearing after a period of skeletal unloading. Am J Physiol 257(4 Pt 1): E606–E610, 1989.

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Shah PK, Stevens JE, Gregory CM et al. Lower-extremity muscle crosssectional area after incomplete spinal cord injury. Arch Phys Med Rehabil 87(6):772–778, 2006. Siu PM, Pistilli EE, Always SE. Apoptotic responses to hindlimb suspension in gastrocnemius muscles from young adult and aged rats. Am J Physiol Regul Integr Comp Physiol 289(4):R1015–R1026, 2005. Stewart BG, Tarnopolsky MA, Hicks AL et al. Treadmill training-induced adaptations in muscle phenotype in persons with incomplete spinal cord injury. Muscle Nerve 30(1):61–68, 2004. Tuukkanen J, Wallmark B, Jalovaara P et al. Changes induced in growing rat bone by immobilization and remobilization. Bone 12(2):113–118, 1991. Vico L, Bourrin S, Very JM et al. Bone changes in 6-mo-old rats after headdown suspension and a reambulation period. J Appl Physiol 79(5):1426–1433, 1995. Vico L, Collet P, Guignandon A et al. Effects of long-term microgravity exposure on cancellous and cortical weight-bearing bones of cosmonauts. Lancet 355(9215):1607–1611, 2000. Wang XD, Kawano F, Matsuoka Y et al. Mechanical load-dependent regulation of satellite cell and fiber size in rat soleus muscle. Am J Physiol Cell Physiol 290(4):C981–C989, 2006. Warner SE, Sanford DA, Becker BA et al. Botox induced muscle paralysis rapidly degrades bone. Bone 38(2):257–264, 2006. Wilkerson MK, Lesniewski LA, Golding EM et al. Simulated microgravity enhances cerebral artery vasoconstriction and vascular resistance through endothelial nitric oxide mechanism. Am J Physiol Heart Circ Physiol 288(4):H1652–H1661, 2005. Wilmet E, Ismail AA, Heilporn A et al. Longitudinal study of the bone mineral content and of soft tissue composition after spinal cord section. Paraplegia 33(11):674–677, 1995. Wolff J. The Law of Bone Remodeling. Springer, Berlin, 1986. Zeng QQ, Jee WS, Bigornia AE et al. Time responses of cancellous and cortical bones to sciatic neurectomy in growing female rats. Bone 19(1):13–21, 1996.

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Zhang D, Liu M, Ding F, Gu X. Expression of myostatin RNA transcript and protein in gastrocnemius muscle of rats after sciatic nerve resection. J Muscle Res Cell Motil 27(1):37–44, 2006. Zhong N, Garman RA, Squire ME et al. Gene expression patterns in bone after 4 days of hind-limb unloading in two inbred strains of mice. Aviat Space Environ Med 76(6):530–535, 2005.

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Chapter 29

Neurogenic Limb Disuse Animal Models Xia Guo, Xiao-Yun Wang and Wai-Ling Lam

Since disuse can be induced by or associated with nerve injury in human beings, it is necessary to develop a neurogenic disuse model for studying the potential beneficial effects of both pharmaceutical and nonpharmaceutical interventions on the prevention and treatment of disuse-related problems, such as disuse-induced osteoporosis. Bone innervation plays an important role in the local modulation of bone metabolism in both intact bone and fracture healing. The establishment of an animal fracture model associated with denervation will facilitate further study of the effect of reinnervation on bone healing. The aim of this chapter is to describe the method for establishing and evaluating (1) the neurogenic disuse model by sciatic nerve resection and (2) the fracture model associated with sciatic nerve resection. The effects of sciatic nerve resection on the responses of both trabecular and cortical bones are usually studied in the tibia of the rat. Peripheral quantitative computed tomography (pQCT) and bone histomorphometry are frequently used methods for evaluating decrease in bone mass and deterioration of the bone microarchitecture in disused bone. The process of fracture healing can be evaluated by radiography, mechanical testing, pQCT, and histological methods. The related key findings are also included in this chapter, and will help in our understanding of the strengths and weaknesses or limitations of these methods for creating and evaluating the two relevant animal models. Practical tips for performing the experiments Corresponding author: Xia Guo. Tel: +852-27666720; fax: +852-23308656; E-mail: [email protected]

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A Practical Manual for Musculoskeletal Research are provided, illuminating potential confounding factors and enhancing our awareness of potential limitations. Keywords:

Bone; innervation; denervation; disuse osteoporosis; fracture healing.

1. Introduction It is well known that disuse of weight-bearing bone, such as limbs, results in suspended mechanical stimuli to the affected bone and thus leads to bone loss. Since many disuse conditions are induced by or associated with nerve injury in human beings, it is necessary to develop a neurogenic disuse model for experimental studies related to disuse bone loss. Neurogenic limb disuse models are created mainly through methods such as neurotomy, neurectomy, and hemicordotomy (Tuukkanen et al. 1991; Yeh et al. 1993; Zeng et al. 1996). Among them, unilateral sciatic neurotomy and neurectomy are the most simple surgical procedures and are therefore widely used. The sciatic nerve contains both motor and sensory nerves originating in the sacral plexus and running through the pelvis and upper leg. In neurectomized limbs, some movements of the hindlimbs (e.g. knee joint) persist, but movements of the lower hindlimb and ankle joint are completely abolished. The effects of sciatic nerve resection on the responses of both trabecular and cortical bones are usually studied in the tibia of the rat. Peripheral quantitative computed tomography (pQCT) and bone histomorphometry analysis are performed to detect decrease in bone mass and deterioration of the bone microarchitecture in two dimensions. Micro-CT is a new, available approach for objective analysis of three-dimensional (3D) bone microarchitecture (Beaupied et al. 2006). Sciatic nerve resection is a denervation method, as the sciatic nerve also contains sensory nerves. It has been reported that the periosteum and bone are richly innervated by sensory and sympathetic fibers (Bjurholm et al. 1988; Hukkanen et al. 1992a). Several neuropeptides have been implicated as local modulators of bone metabolism because of their direct effects on osteoblasts and osteoclasts (Hohmann et al. 1983; Hukkanen et al. 1992b). In fracture models, the decrease in or

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absence of innervation has been observed in delayed union or nonunion, raising the possibility that lack of neural control may lead to delayed fracture healing (Aro 2001; Dyck et al. 1983; Zaidi et al. 1988). Therefore, the establishment of an animal fracture model associated with denervation will facilitate further studies to investigate the effects of innervation on fracture healing. The process of fracture healing can be conventionally evaluated by plain radiography, mechanical testing, pQCT, or histological methods. This chapter describes the establishment and related evaluation methods of (1) the neurogenic disuse model by sciatic nerve resection and (2) the fracture model associated with sciatic nerve resection.

2. Materials •

•

•

• • •

Animals: Sparague–Dawley rats, aged 3 monthsa
The number of animals to be used depends on the number of treatment groups in the experiment. Chemicals: ketamine and xylazine, normal saline, 4% phosphate buffered paraformaldehyde, 10% ethylenediaminetetraacetic acid (EDTA), 100% ethanol, 90% ethanol, 80% ethanol, 70% ethanol, xylene, paraffin wax, hematoxylin and eosin (H&E)b Surgical instruments: shaver, dissecting scissors, surgical knife handle and scalpel, dissecting forceps, needle holder, hemostatic forceps, stainless steel wound clip, 21-gauge needle, tampon, gauze, disinfectant, resorbable sutures Three-point impact bending device — for creating fracture model Surgical drill — for creating a hole in the tibial condyle (see Sec. 3.2) X-ray machine (e.g. Faxitron Cabinet X-ray System Model 43855C; Faxitron X-Ray Corp., Wheeling, IL, USA) and X-ray

a Mature animals are usually used for disuse and fracture models. Alternatively, pubertal animals can be used for disuse osteoporosis models that aim to study the effects of treatment on limb growth. b The pH value of 4% phosphate buffered paraformaldehyde and 10% EDTA should be fixed at 7.2–7.6.

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developer (e.g. Okamoto X3; Okamoto Manufacturing Co. Ltd., Taiwan) Mechanical testing machines (e.g. Hounsfield material testing machine, Model H10KM; Hounsfield Test Equipment, UK) — for testing the quality and strength of fracture healing Peripheral quantitative computed tomography (pQCT) (e.g. Stratec XCT 2000; Norland Stratec, Germany) Automatic tissue processor (e.g. Shadon, England) Semimotorized rotary microtome (e.g. Leica SP1600; Leica, Germany) Microscope (e.g. Eclipse 80i; Nikon, Japan)

3. Methods 3.1. Neurogenic disuse model • • • • • •

•

Rats are anesthetized with an intraperitoneal injection of a mixture of ketamine (50 mg/kg) and xylazine (10 mg/kg). The hindlimb is shaved and sterilized. An incision is made on the upper thigh just posterior to the femoral trochanteric region. Following blunt dissection, the sciatic nerve is mobilized and a 5-mm section is then excised (Fig. 1). The skin incision is closed with stainless steel wound clips. After operation, all of the rats are bred for 28 days and kept individually in wire-top plastic cages with free access to tap water and standard laboratory rodent chow in a 12-hour light/12-hour dark cycle. The nonoperated contralateral tibia serves as a control.

3.2. Fracture model associated with sciatic nerve resection • •

Rats are anesthetized with an intraperitoneal injection of a mixture of ketamine (50 mg/kg) and xylazine (10 mg/kg). Through a 1-cm longitudinal incision, the medial tibial condyle just below the medial part of the knee joint is exposed. Using a surgical drill, a hole of 3 mm in diameter is created.

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Fig. 1.

• • •

• •

•

481

Surgical resection of sciatic nerve.

A 21-gauge needle is inserted into the medullary canal. The soft tissue and the skin are closed with resorbable sutures. The stabilized tibia is then placed in a three-point bending device, and the diaphysis of the fixed tibia is fractured at the midshaft by the force of a 500-g weight dropped from 35 cm (Fig. 2). The sciatic nerve of the fractured leg is dissected (the method is the same as for the neurogenic disuse model described above). After completion of the surgical procedure, all of the rats are bred for 21 days and kept individually in wire-top plastic cages with free access to tap water and standard laboratory rodent chow in a 12-hour light/12-hour dark cycle. The fractured tibia with intact sciatic nerve and cast immobilization serves as a control.

3.3. Radiographic evaluation for fracture model •

Plain lateral radiographs of the operated limb for each animal are taken on postoperation days 1, 7, 14, and 21 with the use of a Faxitron X-ray machine (Model 43855C; Faxitron

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Fig. 2.

• •

•

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Fracture-creating method.

X-Ray Corp., Wheeling, IL, USA) at 60 kV for 5 seconds (Fig. 3).c The films are processed using an automatic X-ray developer. The diameters of the original bone shaft at the same level as the fracture created on the fractured limb (D1) and the maximum diameter of the callus (D2) are measured from the X-ray photograph taken on postoperation day 21 using image analysis software (e.g. Image J 1.29×; Wayne Rasband, National Institutes of Health, USA). The callus index is caculated as D2/D1. The fracture union rate is also evaluated at 21 days postoperation.

3.4. Mechanical testing for fracture model •

c

A four-point bending test is usually selected and conducted on the bilateral tibiae of those animals assigned for mechanical testing using a Hounsfield material testing machine (H10KM;

The parameters must be set consistently for each time.

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Fig. 3. Lateral tibial radiograph for evaluating the position and orientation of the fracture.

• •

• •

Hounsfield Test Equipment, UK) with a 1000-N load cell (Fig. 4).d A constant span length of 20 mm is set between the two lower supports for each specimen. The tibia is positioned horizontally with the anterior surface downward, centered on the supports, and the pressing force is directed vertically to the midshaft of the bone (Fig. 5).e Loading is applied at a constant speed of 2 mm/min until failure (Bak and Andreassen 1988; Bak and Jensen 1992). The load–displacement curve generated by the computer system is used for analysis (Fig. 6).

d Specimens are wrapped by gauze soaked with 0.9% saline and stored at −20°C. They are thawed at room temperature a few hours before mechanical testing. e In order to minimize the effect of deep freezing that might be introduced to the specimen, each specimen is preconditioned with five oscillation cycles of a preload (20 N and 50 N for fractured limb and contralateral limb, respectively) at a constant rate of 2 mm/min. Saline is sprayed on the specimens during testing to avoid dehydration.

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Fig. 4. Hounsfield material testing machine (H10KM; Hounsfield Test Equipment, UK) with a 1000-N load cell.

Fig. 5.

Setup and orientation of the specimen for the four-point bending test.

3.5. Perfusion and fixation •

The animals are anesthetized with an intraperitoneal injection of a mixture of ketamine (50 mg/kg) and xylazine (10 mg/kg) and

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Fig. 6. Load–displacement curve generated by the computer system of the mechanical testing machine.

•

then perfused with a total of 300 mL of normal saline, followed by 4% phosphate buffered paraformaldehyde in vivo via cannulation of the ascending aorta through the left cardiac ventricle (Guo 1994). The bilateral tibiae are removed, cleaned of all soft tissues, and immersed in 4% phosphate buffered paraformaldehyde at 4°C overnight.

3.6. pQCT evaluation •

• •

A high-resolution, multislice pQCT machine (XCT 2000; Norland Stratec, Germany) can be used to evaluate the bone mineral density (BMD), bone mineral content (BMC), cross-sectional bone area (BA), and cross-sectional moment of inertia of the tibia (Fig. 7). Specimens are kept in a plastic tube during the whole process to avoid dehydration. Both the operated tibia and its contralateral tibia are suggested to be scanned together (Fig. 8).f

3.7. Decalcification and staining •

The tibia is decalcified with 10% EDTA for 3 to 4 weeks.

f A standard phantom measurement should be performed daily to detect the reproducibility of pQCT. The tibiae must be measured at the same position for each specimen.

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XCT 2000; Norland Stratec, Germany.

Data retrieval by the pQCT machine.

Specimens are put into tissue cassettes and then undergo a series of processes of dehydration with graded ethanol solutions, are cleared with xylene, and are finally embedded in paraffin wax with the use of an automatic tissue processor (Shadon, England). Paraffin sections 8 µm in thickness are cut parallel to the longitudinal axis by using a semimotorized rotary microtome (RM2145; Leica, Germany). Sections are stained with H&E.

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3.8. Histomorphometric analysis for disuse model •

•

For the proximal tibia, trabecular bone morphometry measurements are performed on the epiphysis area beginning 1 mm proximal to the growth plate metaphyseal junction, and on the metaphyseal area beginning 1 mm distal from the growth plate metaphyseal junction to 4 mm. Total tissue area, trabecular area, and perimeter are measured. These endpoints are used to calculate the trabecular bone volume (BV/TV, %), trabecular width (Tb.Wi, µm), trabecular number (Tb.N, mm), and trabecular separation (Tb.Sp, µm) according to the methods of Parfitt et al. (1983 and 1987). Micro-CT can provide 3D histomorphometric data of undecalcified bone specimens, and is a powerful objective approach for histomorphometric analysis (Parfitt et al. 1987). For the tibial shaft, cortical bone volume measurements include total tissue area and marrow cavity area, which are used to calculate the cortical bone area (Parfitt et al. 1987).

3.9. Statistics • •

Statistical analysis is performed using SPSS data analysis software. The t-test is used to compare values such as BMD, BMC, BA, and the like between the experimental and control tibia. An α level of 0.05 is set for all statistical analyses.

4. Results The results of our studies are illustrated as follows.

4.1. Radiographic evaluation for fracture model At day 21 postoperation, the callus index was 1.13 ± 0.16 (n = 8) and the union rate was 52% (n = 8) in the operated tibia with sciatic nerve resection. The callus index and union rate were 1.05 ± 0.01 (n = 8) and 62% (n = 8), respectively, in the operated tibia with intact sciatic nerve.

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A Practical Manual for Musculoskeletal Research Table 1.

pQCT measurements in the fracture model.

Groups

n

Total BMD (mg/cm3)

Total BMC (mg)

Total BA (mm2)

Fracture with intact nerve Fracture with neurectomy

20

654.81 ± 81.43

8.56 ± 1.94

13.32 ± 3.80

19

637.91 ± 104.56

8.52 ± 2.15

13.69 ± 4.19

Note : Data are shown as mean ± SD.

4.2. Mechanical testing for fracture model At day 21 postoperation, the ultimate load was 39.39 N ± 30.45 N (n = 5) and the stiffness was 103.68 N ± 85.43 N (n = 5) from the four-point bending test in the operated tibia with sciatic nerve resection. The ultimate load and the stiffness were 33.91 N ± 33.21 N (n = 5) and 115.38 N ± 96.79 N (n = 5), respectively, in the operated tibia with intact sciatic nerve.

4.3. pQCT results for disuse model and fracture model Table 1 shows the BMD, BMC, and BA measured by pQCT for the fracture model. Figure 9 shows significant differences in the average BMD between the operated and nonoperated tibiae (p < 0.01) in the epiphysis at 28 days postoperation for the disuse model. No significant difference was observed in the diaphysis of the tibiae.g

4.4. Bone histomorphometry of disuse model Table 2 shows the results of bone histomorphometry of the epiphysis in rats from the disuse model. At 28 days postoperation, BV/TV g

The average BMD decreased significantly in the epiphysis of operated tibiae when compared with the controls; however, no difference was detected in the diaphysis. This is probably because the diaphysis is mainly composed of cortical bone, while more cancellous bone constitutes the epiphysis. Previous experiments suggested that cancellous and cortical bones differ in their anatomic, biomechanical, and metabolic properties (Sander et al. 1992). Frost (1969) further demonstrated that cancellous bone is approximately eight times as metabolically active as cortical bone.

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Fig. 9. Average BMD in the epiphysis and diaphysis of tibiae in the disuse model (**p < 0.01).

Table 2. Group

n

Bone histomorphometry of tibial epiphysis.

BV/TV (%)

Operated 6 23.56 ± 7.17a Nonoperated 6 34.17 ± 7.95

Tb.Wi (µm)

Tb.N (1/mm)

Tb.Sp (µm)

53.31 ± 11.12b 4.41 ± 0.96b 256.94 ± 93.42b 62.46 ± 10.80 5.48 ± 0.88 179.07 ± 45.82

Note : Data are shown as mean ± SD. a p < 0.01. b 0.01 < p < 0.05.

decreased very significantly (p < 0.01) in the operated group. Significant differences were also observed in Tb.Wi, Tb.N, and Tb.Sp between the two groups (p < 0.05).

4.5. H&E staining of disuse and fracture models H&E staining results showed changes in the microstructure of the epiphysis and metaphysis of the proximal tibia between experimental and control groups in the disuse model (Fig. 10). The trabecular

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Fig. 10. H&E staining of epiphysis and metaphysis in the tibias of rats. (a) Epiphysis in the control group; (b) metaphysis in the control group; (c) epiphysis in the operated group; and (d) metaphysis in the operated group. Magnification, 100×; scale bar, 200 µm.

bone was visibly thinner and less in the operated group when compared with the control group. In the fracture model, the results from 7, 14, and 21 days postfracture showed that the callus in the group with intact nerves healed

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Fig. 11. H&E staining of fracture model. (a) Intact nerve group at 7 days postfracture. Fibrous tissue (F) and small amount of cartilage (C) are found in the fracture site. (b) Neurectomy group at 7 days postfracture. Fibrous tissue (F) are mainly found in the fracture site. (c) Intact nerve group at 14 days postfracture. A mixture of different types of bone tissue — fibrous (F), cartilage (C), and woven bone (W) — is found in the fracture site. (d) Neurectomy group at 14 days postfracture. Fibrous tissue (F) are mainly found in the fracture site. (e) Intact nerve group at 21 days postfracture. Woven bone (W) and lamellar bone (L) are mainly found in the fracture site. (f) Neurectomy group at 21 days postfracture. A mixture of fibrous tissue (F), cartilage (C), and woven bone (W) is found in the fracture site. Magnification, 100×; scale bar, 100 µm.

faster and better than in the group without (Fig. 11). At 7 days postfracture, the callus was mainly filled with fibrous tissue in both groups. However, the cartilage as well as woven and lamellar bones (more mature bone types) were observed in the fracture callus at 14 days

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postfracture in the neurally intact groups, while mainly fibrous tissue was found in the fracture callus in the neurectomy group. At day 21 postfracture, more mature callus was found in the group with intact sciatic nerves when compared with the H&E-stained sections from the different-treatment group.

5. Summary The results of our studies prove that bone innervation plays an important role in the local modulation of bone metabolism in both intact bone and fracture healing. This chapter introduced the method for establishing and evaluating the neurogenic disuse model including sciatic nerve resection and the fracture model associated with sciatic nerve resection. These denervation models can help facilitate further studies on the effect of innervation on bone healing.

References Aro H. Development of nonunions in the rat fibula after removal of periosteal neural mechanoreceptors. Clin Orthop Relat Res 199: 292–299, 2001. Bak B, Andreassen TT. Reduced energy absorption of healed fracture in the rat. Acta Orthop Scand 59:548–551, 1988. Bak B, Jensen KS. Standardization of tibial fractures in the rat. Bone 13: 289–295, 1992. Beaupied H, Chappard C, Basillais A et al. Effect of specimen conditioning on the microarchitectural parameters of trabecular bone assessed by micro-computed tomography. Phys Med Biol 51:4621–4634, 2006. Bjurholm A, Kreicbergs A, Terenius L. Neuropeptide Y-, tyrosine hydroxylaseand vasoactive intestinal polypeptide-immunoreactive nerves in bone and surrounding tissues. J Auton Nerv Syst 25:119–125, 1988. Dyck PJ, Stevens JC, O’Brien PC et al. Neurogenic arthropathy and recurring fractures with subclinical inherited neuropathy. Neurology 33:357–367, 1983. Frost HM. Tetracycline-based histological analysis of bone remodeling. Calcif Tissue Res 3:211–237, 1969.

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Guo X. Histomorphological study of interface between Schanz screw and cortical bone in a sheep tibia fracture model. MD thesis, University of Essen Medical School, Essen, Germany, 1994. Hohmann EL, Levine L, Tashjian AH. Vasoactive intestinal peptide stimulates bone resorption via a cyclic adenosine 3′5′-monophosphatedependent mechanism. Endocrinology 112:1233–1239, 1983. Hukkanen M, Konttinen YT, Rees RG et al. Innervation of bone from healthy and arthritic rats by substance P and calcitonin gene related peptide containing fibers. J Rheumatol 19:1252–1259, 1992a. Hukkanen M, Konttinen YT, Rees RG et al. Distribution of nerve endings and sensory neuropeptides in rat synovium, meniscus and bone. Int J Tissue React 14:1–10, 1992b. Parfitt AM, Drezner MK, Glorieux FH et al. Bone histomorphometry: standardization of nomenclature, symbols, and units. J Bone Miner Res 2:595–610, 1987. Parfitt AM, Mathews CHE, Villanueva AR et al. Relationships between surface and thickness of iliac trabecular bone in aging and osteoporosis: implications for the macroanatomic and cellular mechanism of bone loss. J Clin Invest 72:1396–1409, 1983. Sander T, Felsenberg D, Kalender WA et al. Compact and trabecular components of the spine using quantitative computed tomography. Calcif Tissue Int 50:502–506, 1992. Tuukkanen J, Wallmark B, Jalovaara P et al. Changes induced in growing rat bone by immobilization and remobilization. Bone 12:113–118, 1991. Yeh J, Liu CC, Aloia JF. Effects of exercise and immobilization on bone formation and resorption in young rats. Am J Physiol 264:E182–E189, 1993. Zaidi M, Chambers TJ, Bevis PJ et al. Effects of peptides from the calcitonin genes on bones and bone cells. Q J Exp Physiol 73:471–485, 1988. Zeng QQ, Jee WSS, Bigornia AE et al. Time responses of cancellous and cortical bones to sciatic neurectomy in growing female rats. Bone 19:13–21, 1996.

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Chapter 30

Establishment of Steroid-Associated Osteonecrosis Rabbit Model Ge Zhang, Ling Qin and Hui Sheng

Steroid-associated osteonecrosis (ON), also known as avascular necrosis (AVN), and subsequent subchondral joint collapse are clinically reported, since pulsed steroids are frequently prescribed as a life-saving agent for serious infectious diseases and chronic autoimmune diseases. However, the postsurgical prognosis of total joint replacement is especially poor in steroid-associated ON patients. It is necessary to establish an appropriate animal model(s) in order to test the efficacy of agents developed for clinical applications. This chapter mentions three classical, published induction protocols for steroid-induced or steroid-associated ON and their drawbacks, leading to a new approach for establishing a more effective model with a detailed description of the induction protocol. Multiple bioimaging methods are established for the evaluation of the pathogenic pathway related to decreased blood flow to bone and the endpoint of ON, i.e. histopathological evidences of ON lesion formation. Applications of this model are proposed for future applications involving efficacy studies of agents developed for the prevention of steroid-associated ON. Keywords:

Steroid-associated osteonecrosis (ON); avascular necrosis (AVN); prethrombotic disorders; dynamic MRI; perfusion function; peak enhancement percentage; micro-CT; microangiography.

Corresponding author: Ge Zhang. Tel: +852-26323308; fax: +852-26324618; E-mail: [email protected]

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1. Introduction Pulsed steroids are frequently prescribed as a life-saving agent for serious infectious diseases such as severe acute respiratory syndrome (SARS) (So et al. 2003) and acquired immune deficiency syndrome (AIDS) (Scribner et al. 2000), or as a disease-modifying drug for chronic autoimmune diseases such as systemic lupus erythematosus (SLE) (Garin et al. 1986) and rheumatoid arthritis (RA) (Adebajo and Hall 1998). Inevitably, steroid-associated osteonecrosis (ON), also known as avascular necrosis (AVN), with either moderate or high incidence has been reported (Griffith et al. 2005; Nagasawa et al. 2005; Penzak et al. 2005; Zabinski et al. 1998). Total joint replacement is the last option for treatment of ON, yet the postsurgical prognosis is poor in steroid-associated ON patients (Saito et al. 1989). Therefore, intervention of the development or progress of steroidassociated ON lesions is highly desirable (Lieberman et al. 2002; Wang et al. 2000). An appropriate steroid-associated ON animal model should be established to test hypothesized intervention stratagems. One must consider that the primary disease prescribed with pulsed steroids often differs among steroid-associated ON patients, thus endowing complexity in etiopathogenesis. If we accept the fact that we lack an exact grasp of the etiopathogenesis of steroid-associated ON, we can simplify the problem by extracting two common features in steroid-associated ON patients: prethrombotic disorders (Cheras et al. 1997; Jones 1999) and pulsed steroid administration (Assouline-Dayan et al. 2002). There are three published classical induction protocols of steroid-induced or steroid-associated ON in rabbits (Table 1; protocols a–c) that show a close linkage between the two common features and steroid-associated ON development, even though the etiopathogenesis remains unclear. One protocol is to use a single injection of high-dose steroid administration; however, a low incidence of ON (43%) is reported (Yamamoto et al. 1997). The other two protocols are both a combination of prethrombosis induction and subsequent pulsed steroid administration; however, they have a high mortality (50%) in spite of moderate to high ON incidence (70%–85%) (Matsui et al. 1992; Yamamoto et al. 1995). For the

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Table 1. Four published protocols of steroid-induced or steroid-associated osteonecrosis (ON).

Authors Yamamoto et al. (1997) Yamamoto et al. (1995) Matsui et al. (1992) Qin et al. (2006)

Prethrombosis induction

Induction protocol

ON Postinduction incidence (%) mortality (%)

No

H-MPS × 1a

43

0

Yes

H-LPS × 2 + H-MPS × 3b S × 2 + H-MPS × 3c

85

50

70

0

93

0

L-LPS × 1 + H-MPS × 3d

a

A single injection of methylprednisolone (20 mg/kg). Two intravenous injections of lipopolysaccharide (20 µg/kg for each injection) at an interval of 24 hours, and three intramuscular injections of methylprednisolone (20 mg/kg for each injection) at an interval of 24 hours after the second lipopolysaccharide injection. c Two intravenous injections of sterile horse serum (10 mL/kg for each injection) at a 3-week interval and three intraperitoneal injections of methylprednisolone (40 mg/kg for each injection) at 2 weeks after the second serum injection. d One intravenous injection of lipopolysaccharide (20 µg/kg for each injection) and three intramuscular injections of methylprednisolone (20 mg/kg for each injection) at an interval of 24 hours after the lipopolysaccharide injection. b

former single-injection protocol, the low incidence of ON could be explained by the absence of prethrombotic induction before steroid administration. For the latter combinational injection protocols, the high mortality could be attributed to the Shwartzman reaction induced by a high dose of the prethrombosis induction agent endotoxin. Therefore, to test intervention strategies for steroid-associated ON, it is necessary to develop an alternative and convenient induction protocol for establishing ON lesions with a high incidence but with low or no mortality. Recently, we have established a practical induction protocol for the steroid-associated ON rabbit model by employing multiple bioimaging modalities (Qin et al. 2006) (Table 1; protocol d). Using this model, we have also examined the prevention effect of Chinese herbal Epimedium-derived flavonoid fractions on steroid-associated

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osteonecrosis development (Zhang et al. 2007a). Below is a detailed description of the establishment of the induction protocol for establishing ON lesions in rabbits with a high incidence but low or even no mortality.

2. Induction Protocol of Steroid-Associated ON Model 2.1. Materials •

Animals: 28-week-old New Zealand white male rabbits



• •

The growth plate in rabbit skeleton is closed at 28 weeks old. Male animals are used, as steroid-associated ON is frequently found in male patients.

Steroid: lipopolysaccharide (LPS) (Escherichia coli 0111:B4; Sigma-Aldrich, Inc., USA) Endotoxin: methylprednisolone (MPS) (Pharmacia & Upjohn, USA)

2.2. Method • •

The rabbit is intravenously injected with 10 µg/kg body weight of LPS. Twenty-four hours later, three injections of 20 mg/kg body weight of MPS are given intramuscularly at a time interval of 24 hours.

3. Assessment Protocol of Steroid-Associated ON Model A consensus pathophysiological pathway has been established whereby an impaired structure-function of the intraosseous blood supply system decreases blood flow to bone, leading to ischemia and subsequent osteocyte death (Wang et al. 2000). Therefore, perfusion function of the intraosseous vasculature, three-dimensional (3D)

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architecture of the intraosseous vasculature, and histopathological features of bone lesion are recommended for integration into the critical assessment protocol.

3.1. Perfusion function evaluated by dynamic MRI 3.1.1. Materials • • • • • •

Magnetic resonance imaging (MRI): 1.5 T or higher superconducting system (e.g. ACS-NT Intera; Philips, The Netherlands) Extremity coil (transmit-receive surface coil) Automatic injection pump (e.g. Philips, The Netherlands) 21-gauge catheter Anesthetic: general anesthesia, e.g. using sodium pentobarbital (0.8 mL/kg, intravenous injection; Sigma Chemical Co., USA) Contrast: gadopentetate dimeglumine (Magnevist; Schering, Berlin, Germany)

3.1.2. Method •

• •

•

The rabbit is anesthetized and placed in a supine position with the lower limb flexed by fixation using adhesive tape. This is for a dynamic MRI-derived vasculature perfusion function index of bilateral proximal femurs by a 1.5 T superconducting system. An extremity coil (transmit-receive surface coil) is used on either the right or left proximal thigh. To define both optimal coronal planes and the region of interest (ROI) before dynamic MRI scan, a T1-weighted sequence (TR = 425 ms; TE = 13 ms) is employed with the following scanning parameters: section thickness, 3 mm; intersection gap, 1 mm; and imaging matrix, 256 × 128. An ellipse-like femoral head is accordingly marked as the ROI in obtained midcoronal planes (Fig. 1). A bolus of gadopentetate dimeglumine (0.1 mmol/kg/body weight) is rapidly injected by an automatic pump linked to a previously placed 21-gauge catheter into an auditory vein.

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Fig. 1. A T1-weighted coronal MRI image of the rabbit proximal femur after Gd-DTPA administration. The region of interest (ROI) in the central part of the femoral head with a size of 8–10 pixels (64–80 mm2) is defined for analysis of local intraosseous perfusion.

•

•

Dynamic MRI scan with ultrafast T1-weighted gradient-echo sequence (TR = 2.2 ms; TE = 0.92 ms) starts as soon as contrast medium injection commences. A series of dynamic images is obtained in 600 seconds. For the vascularization index, signal intensity (SI) values derived from the ROI are plotted against time values as the time–intensity curve (TIC) using built-in software. The baseline SI value (SIbase) in a TIC is calculated as the mean SI value in the first three images. The maximum SI value (SImax) is defined as the peak enhancement in the first pass of the injected contrast medium (Chen et al. 2002). The vascularization index, i.e. the peak enhancement percentage (PEP), is defined as the maximum percentage increase from the baseline in SI. The average bilateral proximal femur PEP is recorded (Fig. 2).

PEP =

(SI max - SIbase ) ¥ 100%. SIbase

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Fig. 2. Representative time–intensity curves derived from contrastenhanced dynamic MRI on the proximal femur. The peak enhancement percentage (PEP) shows a significant decrease in local perfusion from the baseline after induction.

3.2. Microangiography evaluated by micro-CT 3.2.1. Materials (Duvall et al. 2004; Qin et al. 2006; Zhang et al. 2007a) • • • • • • •

Micro-CT system: µCT40 or vivaCT40 (Scanco Medical, Bassersdorf, Switzerland), or other commercially available systems Cabinet X-ray system (e.g. specimen radiography system, Faxitron 43855C; Faxitron X-Ray Corp., Wheeling, IL, USA) Automatic pump apparatus (e.g. PHD 22/2000; Harvard Apparatus, USA) 25-mm syringe Conventional surgical instruments Anesthetic: sodium pentobarbital for intravenous injection (0.8 mL/kg; Sigma Chemical Co., USA) Package of perfused radiopaque contrasts (Microfil; Flow Tech, Inc., Carver, MA, USA): (1) MV-Diluent, (2) MV-117 Orange, (3) MV Curing Agent

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Preconditional solution before perfusion of radiopaque contrasts: (1) heparinized normal saline (50 U/mL), (2) 10% neutral buffered formalin Decalcification solution: 10% ethylenediaminetetraacetic acid (EDTA) (pH 7.4) Tissue fixation solution: paraformaldehyde (4%)

3.2.2. Method (Qin et al. 2006; Zhang et al. 2007a; Duvall et al. 2004) •

•

•

•

•

•

•

Under general anesthesia (0.8 mL/kg for sodium pentobarbital) with 2.5% sodium pentobarbital (0.4 mL/kg), the abdominal cavity of the animals is opened and a scurf-needle with a 25-mm syringe is inserted into the abdominal aorta distal to the heart with ligation of that proximal to the heart. The vasculature is flushed with heparinized normal saline at 37°C and at a flow speed of 20 mm/min (Qin et al. 1999) via an automatic pump apparatus linked to the 25-mm syringe. As soon as the outflow from an incision of the abdominal vein is limpid, 10% neutral buffered formalin (37°C) should be pumped into the vasculature to fix the nourished skeletal specimen. The formalin is flushed from the vasculature using the heparinized normal saline and then the vasculature is injected with Microfil, a lead chromate-containing confected radiopaque silicone rubber compound, based on the manufacturer’s protocol (Flow Tech; Carver, MA, USA) (Fig. 3). Animals are euthanized with an overdose of sodium pentobarbital and stored at 4°C for 1 hour to ensure polymerization of the contrast agent before microangiography. Bilateral femoral samples are harvested and fixed in paraformaldehyde (4%) for 3 days, and then decalcified with 0.5 M EDTA for 28–42 days at 4°C. Success of complete decalcification is confirmed by anteroposteriorview radiographs taken using a cabinet X-ray system under an exposure condition of 40 kV/5 s (Fig. 4). Both the proximal 1/3 and distal 1/3 of each femur are dissected for micro-CT scan.

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Fig. 3. Perfusion with radiopaque substance. Under general anesthesia, the abdominal cavity of the animal is opened and a scurf-needle with a 25-mm syringe is inserted into the abdominal aorta. The vasculature is flushed with heparinized normal saline at 37°C and at a flow speed of 20 mm/min via an automatic pump apparatus linked to the 25-mm syringe.

Fig. 4. X-ray examination for the success of decalcification. Before decalcification, the ‘Bone’ signal and ‘Microfil’ signal are not differentiable. After the completion of decalcification, which excludes the ‘Bone’ signal, the ‘Microfil’ signal shown represents the angiographic structure. The intraosseous vasculature is well preserved at network in normal bone, but this is not the case in necrotic bone.

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Fig. 5. Positioning of the rabbit proximal femur for micro-CT scanning. The radiograph is used for illustration and the ROI is defined for studying the local vasculature.

•

•

•

•

•

Either the proximal or distal part of each femur is placed with the proximal or distal end into a polymethyl methacrylate (PMMA) sample tube. The femoral shaft is fixed in the tube with its long axis perpendicular to the bottom of the tube for micro-CT scanning (Fig. 5). The scan is then perpendicular to the shaft and initiated from a reference line 10 mm away from the bottom with an entire scan length of 10 mm, which is performed at a resolution of 36 µm per voxel with a 1024 × 1024 pixel image matrix. For segmentation of blood vessels from the background, noise is removed using a low-pass Gaussian filter (sigma = 1.2; support = 2) and blood vessels are then defined at a threshold of 85. In order to reconstruct the 3D angiographic structure of the decalcified sample, the perfused Microfil radiopaque substance is included with a semiautomatically drawn contour at each 2D section by a built-in “Contouring Program”. A histogram is subsequently generated to display the size distribution of the angiographic structure, and a color-coded scale is mapped to the surface of the 3D images to produce a visual representation (Figs. 6 and 7).

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Fig. 6. Representative images of micro-CT–reconstructed 3D microangiography of the proximal femur from necrotic bone (right) and normal bone (left). A typical angiogram of osteonecrotic bone shows a few dilated vessel-like structural units surrounded by both several disconnected vessel-like structural units and numerous disseminated leakageparticle-like structural units when compared to the angiogram of normal bone. Less large blood vessels are revealed in the osteonecrotic bone than in the normal bone.

3.3. Histopathology evaluated by decalcified histological technique 3.3.1. Materials • • • •

Tissue processor (e.g. Shandon Pathcenter; Shandon, USA) Tissue embedding unit (e.g. Histocenter II-N; Thermolyne, USA) with paraffin Rotary microtome (e.g. Leica RM2165; Leica, Germany) Conventional reagents for hematoxylin and eosin (H&E) staining

3.3.2. Method •

The decalcified samples are embedded in paraffin by using a tissue embedding unit.

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Fig. 7. Representative histogram with size distribution of angiographic structure in both normal bone (from normal rabbit) and necrotic bone (found in osteonecrosis lesion). Compared with the normal bone, the necrotic bone demonstrates a redundancy of 601–1000-µm-sized angiographic structures and an absence of 401–600-µm-sized angiographic structures. It also shows numerous disconnected angiographic structures and disseminated formless angiographic structures 200–400 µm in size. The absence of 401–600-µmsized angiographic structures in necrotic bone indicates blocked blood vessels, while the redundant 601–1000-µm-sized angiographic structures indicate upstream dilated vessels due to downstream blocked vessels. The numerous disconnected angiographic structures in necrotic bone indicate hypoxiainduced neovasculature, while the disseminated formless angiographic structures indicate leaking radiopaque substances from the neovasculature.

•

• •

The paraffin blocks are cut into 6-µm-thick sections along the coronal plane for the proximal femoral parts and along the axial plane for the distal femoral parts. Sections are stained with H&E. Histopathological identification of osteonecrosis: Entire areas of each dissected part of bilateral femoral samples are examined for the presence of ON, which shall be judged blindly by two pathologists. The consensus histopathological features of osteonecrosis are characterized as the diffuse presence of empty lacunae or pyknotic nuclei of osteocytes in the trabeculae, accompanied by surrounding necrotic bone marrow (Yamamoto et al. 1995;

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Fig. 8. Histopathological features in necrotic bone by H&E staining. Compared to normal bone (a), lamellar trabeculae with numerous typical empty lacunae are present in necrotic bone (b) and are surrounded by large adipocyte-rich marrow containing amorphous substance (articular cartilage). Marrow space is lost by increased lipid deposition. Limited repair, i.e. appositional bone formation with lining cells (osteoblasts) (indicated with arrows), around the necrotic bone is shown in (c). Destructive repair, i.e. granulation tissue creep (pointed by arrows), linked to necrotic bone resorption is shown in (d).

•

Yamamoto et al. 1997). A rabbit that has at least one osteonecrotic lesion in the areas examined is defined as ON+, while one with no osteonecrotic lesion is defined as ON−(Fig. 8). Calculation of osteonecrosis incidence: It is defined as the number of ON+ rabbits divided by the number of total rabbits in a certain experimental group (Qin et al. 2006; Zhang et al. 2007a).

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Calculation of osteonecrosis extension: It is defined as the number of ON lesions per affected rabbit (Qin et al. 2006; Zhang et al. 2007a).

4. Discussion The etiopathogenesis and pathophysiology of steroid-associated osteonecrosis still remain unclear. The model with the employed bioimaging modalities described in this chapter may provide a research platform to examine our hypothesis regarding etiopathogenesis and pathophysiology. This induction protocol is also essential in examining systemic prevention efficacy of candidate drugs for the prevention of steroid-associated ON (Zhang et al. 2007a). Recently, we have employed this model to examine the local osteogenic efficacy of implanted marrow mononuclear cells on local osteonecrosis lesions after surgical core decompression (Sheng et al. 2007; Zhang et al. 2007b). Potential biophysical interventions (e.g. ultrasound, shockwave, vibration) may be locally or systemically employed on this model for preclinical research and development, even though there is no such published study.

5. Summary This chapter presented an induction protocol for establishing ON lesions in rabbits with a high ON incidence, but without mortality.

References Adebajo AO, Hall MA. The use of intravenous pulsed methylprednisolone in the treatment of systemic-onset juvenile chronic arthritis. Br J Rheumatol 37:1240–1242, 1998. Assouline-Dayan Y, Chang C, Greenspan A et al. Pathogenesis and natural history of osteonecrosis. Semin Arthritis Rheum 32:94–124, 2002. Chen WT, Shih TT, Chen RC et al. Blood perfusion of vertebral lesions evaluated with gadolinium-enhanced dynamic MRI: in comparison with compression fracture and metastasis. J Magn Reson Imaging 15:308–314, 2002.

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Cheras PA, Whitaker AN, Blackwell EA et al. Hypercoagulability and hypofibrinolysis in primary osteoarthritis. Clin Orthop Relat Res 334:57–67, 1997. Duvall CL, Robert Taylor W, Weiss D, Guldberg RE. Quantitative microcomputed tomography analysis of collateral vessel development after ischemic injury. Am J Physiol Heart Circ Physiol 287:H302–H310, 2004. Garin EH, Sleasman JW, Richard GA et al. Pulsed methylprednisolone therapy compared to high dose prednisone in systemic lupus erythematosus nephritis. Eur J Pediatr 145:380–383, 1986. Griffith JF, Antonio GE, Kumta SM et al. Osteonecrosis of hip and knee in patients with severe acute respiratory syndrome treated with steroids. Radiology 235:168–175, 2005. Jones JP Jr. Coagulopathies and osteonecrosis (review). Acta Orthop Belg 65(Suppl 1):5–8, 1999. Lieberman JR, Berry DJ, Monty MA et al. Osteonecrosis of the hip: management in the 21st century. J Bone Joint Surg Am 84:834–853, 2002. Matsui M, Saito S, Ohzono K et al. Experimental steroid-induced osteonecrosis in adult rabbits with hypersensitivity vasculitis. Clin Orthop Relat Res 277:61–72, 1992. Nagasawa K, Tada Y, Koarada S et al. Very early development of steroidassociated osteonecrosis of femoral head in systemic lupus erythematosus: prospective study by MRI. Lupus 14:385–390, 2005. Penzak SR, Formentini E, Alfaro RM et al. Prednisolone pharmacokinetics in the presence and absence of ritonavir after oral prednisone administration to healthy volunteers. J Acquir Immune Defic Syndr 40:573–580, 2005. Qin L, Mak ATF, Cheng CW et al. Histomorphological study on pattern of fluid movement in cortical bone in goats. Anat Rec 255:380–387, 1999. Qin L, Zhang G, Sheng H et al. Multiple bioimaging modalities in evaluation of an experimental osteonecrosis induced by a combination of lipopolysaccharide and methylprednisolone. Bone 39:863–871, 2006. Saito S, Saito M, Nishina T et al. Long-term results of total hip arthroplasty for osteonecrosis of the femoral head. A comparison with osteoarthritis. Clin Orthop Relat Res 244:198–207, 1989. Scribner AN, Troia-Cancio PV, Cox BA et al. Osteonecrosis in HIV: a casecontrol study. J Acquir Immune Defic Syndr 25:19–25, 2000. Sheng H, Qin L, Zhang G et al. Cryopreserved mononuclear cells activity in steroid-associated osteonecrosis rabbit model. Bone 40(Suppl):s281–s282, 2007.

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So LK, Lau AC, Yam LY et al. Development of a standard treatment protocol for severe acute respiratory syndrome. Lancet 361:1615–1617, 2003. Wang GJ, Cui Q, Balian G. The pathogenesis and prevention of steroidinduced osteonecrosis. Clin Orthop Relat Res (370):295–310, 2000. Yamamoto T, Hirano K, Tsutsui H et al. Corticosteroid enhances the experimental induction of osteonecrosis in rabbits with Shwartzman reaction. Clin Orthop Relat Res 316:235–243, 1995. Yamamoto T, Irisa T, Sugioka Y, Sueishi K. Effects of pulse methylprednisolone on bone and marrow tissues: corticosteroid-induced osteonecrosis in rabbits. Arthritis Rheum 40:2055–2064, 1997. Zabinski SJ, Sculco TP, Dicarlo EF, Rivelis M. Osteonecrosis in the rheumatoid femoral head. J Rheumatol 25:1674–1680, 1998. Zhang G, Qin L, Sheng H et al. Epimedium-derived phytoestrogen exert beneficial effect on preventing steroid-associated osteonecrosis in rabbits with inhibition of both thrombosis and lipid-deposition. Bone 40:685–692, 2007a. Zhang G, Sheng H, Qin L et al. Local implantation of autologous-marrowmononuclear-cell cryopreserved prior to pulsed-steroid-administration exerted beneficial effect on restoration of perfusion function in rabbit steroid-associated osteonecrosis lesion. Bone 40(Suppl):s282, 2007b.

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Chapter 31

Establishment of Anterior Cruciate Ligament Reconstruction Model in Rabbit Chun-Yi Wen and Kai-Ming Chan

Anterior cruciate ligament (ACL) reconstruction is a significant clinical problem. Clinical studies reveal that 11%–32% of patients show an unsatisfactory prognosis and that up to 10% may require surgical revision. Graft–tunnel healing is one of the major factors affecting the outcome of ACL reconstruction. Given that there is no analog like tendon insertion in the bone tunnel in animal or human, it is necessary to establish an appropriate animal model for a better understanding of the biology of graft–tunnel healing; in particular, the structure of the tendon insertion site in the bone tunnel should be known in prior. Recently, a protocol for establishing ACL reconstruction animal models has been developed after a critical review of the literature in the past decades. The assessment protocol consists of three-dimensional (3D) structural analysis of bone ingrowth by micro-computed tomography (micro-CT), two-dimensional (2D) structural analysis of newly formed tendon insertion in the bone tunnel by routine histology, and mechanical testing of the strength of the graft–tunnel complex as the endpoint evaluation. Densitometric evaluation is also used to assess the changes of pre-existing bone with

Corresponding author: Kai-Ming Chan. Tel: +852-26323311; fax: +852-26324618; E-mail: [email protected]

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A Practical Manual for Musculoskeletal Research graft–tunnel healing by peripheral quantitative computed tomography (pQCT). In this chapter, we also discuss several aspects on the application of this experimental model for developing therapeutic strategies to enhance graft–tunnel healing. Keywords:

ACL reconstruction; long digital extensor tendon; bone ingrowth; micro-CT; pQCT.

1. Introduction Anterior cruciate ligament (ACL) reconstruction remains a significant clinical problem to date. Over the past decades, short- and long-term clinical outcome studies have revealed that 11%–32% of patients show unsatisfactory treatment results and that up to 10% may require surgical revision after initial ACL reconstruction (Anderson et al. 2001; Aune et al. 2001; Herrington et al. 2005). Graft–tunnel healing is one of the major factors affecting the outcome of ACL reconstruction. The graft–tunnel interface is regarded as the “weak link” of the graft–tunnel construct, necessitating a delay back to the field (Grana et al. 1994; Magen 1999). Recently, issues relating to donor site morbidity, such as kneeling/ patellofemoral pain, quadriceps weakness, and arthrofibrosis, for bonepatella-bone grafts have caused a paradigm shift from 86.9% to 21.2% between 2000 to 2004 to hamstring tendon (semitendinosus and gracilis tendon) autografts (Forssblad et al. 2006; Freedman et al. 2003). However, hamstring-tendon–to–bone healing is slower and weaker than bone-patella-bone-graft–to–bone healing (Tomita et al. 2001). Furthermore, tunnel widening and considerable bone loss are not uncommon after ACL surgery (Leppala et al. 1999; Clatworthy et al. 1999; Webster et al. 2001). Given that there is no analog like tendon insertion in the bone tunnel in animal or human, it is necessary to establish an appropriate animal model for a better understanding of the biology of graft–tunnel healing; in particular, the structure of the newly regenerated tendon insertion site in bone tunnel should be known in prior. Literature review shows that ACL reconstruction rabbit models were often used in past decades to study the biology of graft–tunnel

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healing (Table 1). However, the description of model establishment is very brief in most of these studies. Even in more recent studies, basic information of rabbit like gender and skeletal maturity is not well clarified. Some key information in the surgical procedures is always missing, including graft size, graft tensioning, and tunnel placement, which directly affect the outcome of ACL surgery (Segawa et al. 2003; Tohyama and Yasuda 1998; Yamazaki et al. 2002). Recently, we have developed a practical protocol for establishing ACL reconstruction models in rabbit (Wen et al. 2006). It essentially duplicates the procedure used in humans. This chapter provides a practical, technical description of this protocol as well as relevant evaluations on both the structure and function of the reconstructed ACL insertion in bone tunnel.

2. Protocol for Establishing ACL Reconstruction Model in Rabbit 2.1. Materials •

Animals: female 26-week-old New Zealand white rabbits (3.5–4.0 kg) The growth plate in rabbit skeleton is closed at 26 weeks old. Reconstruction of ACL using a long digital extensor tendon autograft has often been recommended for female athletes. It was reported that the clinical failure rate was 23% (9 of 39) for female patients and 4% (1 of 26) for male patients, and there was also a trend toward increased laxity in female patients (Noojin et al. 2000).
Bilateral ACL reconstruction can be successfully performed on rabbits (Wang et al. 2005). Because the semitendinous tendon of rabbit is too thin compared with intact ACL, the long digital extensor tendon is selected for grafting (Wang et al. 2005). Anesthesia: 10% ketamine/2% xylazine, sodium phenobarbital (Sigma Chemical Co., St. Louis, MO, USA)



•

Species

Gender

Age/Weight Unilateral/

Rabbit

/

/

/

Descriptive

Rabbit

/

/

/

Descriptive

Rabbit

/

9–10 kg

Bi

Descriptive

Rabbit

/

2.4–3.5 kg

Uni

Anderson (2001) Martinek (2002) Mutsuzaki (2004)

Controlled (bone growth factors) Controlled (BMP-2 gene transfer) Controlled (CaP hybrid)

Rabbit

/

/

Bi

Rabbit

Female

/

Rabbit

Male

Tien (2004) Lim (2004)

Controlled (calcium-phosphate) Controlled (MSC)

Rabbit

Male

Skeletal mature Skeletal mature, 3.5 kg Adult, 4.0 kg

Rabbit

/

Demirag (2005) Wang (2005)

Controlled (Blockage of MMP) Controlled (shockwave)

Rabbit

/

Rabbit

Male

Ma (2007)

Controlled (BMP-2 and noggin)

Rabbit

Male

Note: “/” represents no relevant information.

Bi

Bi

Skeletal mature, 2.0– 2.5 kg Adult, 3.5– 4.0 kg 12 months, 2.79–3.65 kg

Bi

Bi

/

Bi

/

Semitendinosus tendon Semitendinosus tendon Semitendinosus tendon Semitendinosus tendon Semitendinosus tendon Semitendinosus tendon Flexor digital longus tendon Semitendinosus tendon Hamstring tendon Semitendinosus tendon Long digital extensor tendon Semitendinosus tendon

Graft

Graft

Tunnel

Tunnel

Post-op

size

fixation

tensioning

size

placement

(immobilization)

/

/

/

/

/

/

/

Suture

/

/

/

/

/

Suture

No

/

/

No

/

Suture

Tightened in extension

2 mm for each tunnel (2 tunnels)

/

No

/

Suture

1.7 mm

/

/

/

Suture to metal button Suture to metal button

Slight tension /

2.00 mm

/

No

/

3.2 mm

/

No

/

/

Suture

/

2.4 mm

/

No

/

Suture

/

2.0 mm

/

No

/

Suture

/

1.7 mm

/

No

/

Suture

30° in flexion

Graft size match

/

No

/

Suture

/

2.4 mm

/

No

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Summary of ACL reconstruction in rabbit models using soft tissue graft (published between 1980 and 2007). Subjects

Author/Year

514

Table 1.

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Air-driven drill: drill bit of 2.7 mm in diameter (Synthes Medical Device Co., USA) Suture fixation: PDS II (polydioxanone) suture (Johnson & Johnson Co., USA)
PDS II suture has the longest tensile strength retention profile. Sutures smoothly pass through tissue with great pliability and low tissue reactivity, which are ideal for slow-healing or compromised tissue.

2.2. Method (Wen et al. 2006) •

•

For general anesthesia, 10% ketamine/2% xylazine is injected intramuscularly (Ketalar, 1 mL:1 mL), and sedation is maintained with 2.5% sodium phenobarbital intravenous injection (Sigma Chemical Co., St. Louis, MO, USA). A medial incision is made, and then a long digital extensor tendon graft of 3 mm in length is harvested and the graft size is recorded. Το prepare the graft, the attached muscle is removed and the holding sutures are passed through each end of the tendon graft in an interdigitating whipstitch fashion [Fig. 1(a)].

Fig. 1. Operative procedure of ACL reconstruction in rabbit. (a) Preparation of graft: the attached muscle is removed and the holding sutures are passed through each end of the tendon graft in an interdigitating whipstitch fashion. (b) Parapatellar arthrotomy is done to expose the knee joint. The patella is dislocated and the subpatella fat pad is removed to expose the joint cavity. (c) The tibial tunnel is created into the articular cavity through the footprint of the original ACL at an angle of 55° to the articular surface. (d) The graft is then inserted and routed through the bone tunnels via the holding sutures. The graft is fixed to the neighboring soft tissue at the extra-articular tunnel exit by secure knots. (e) Postoperative X-ray shows the placement of the bone tunnel.

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•

•

•

•

• • •

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Parapatellar arthrotomy is done to expose the knee joint. The patella is dislocated and the subpatella fat pad is removed to expose the joint cavity. Then, the ACL is lacerated. The ACL stumps and transverse geniculate ligament should be shaved [Fig. 1(b)]. The anterior/medial tibial periosteum is cut longitudinally 1 cm below the articular surface. The tibial tunnel is created into the articular cavity through the footprint of the original ACL at an angle of 55° to the articular surface [Fig. 1(c)]. The femoral tunnel is created inside the articular cavity through the footprint of the knee joint at a flexion angle of 120°. The exit of the femoral tunnel should be well planed before drilling. It should be located at the lateral surface of the distal femur and 1 mm above the articular surface. A drill bit of 2.7 mm in diameter is used. The graft is then inserted and routed through the bone tunnels via the holding sutures. The graft at one end of the femoral tunnel extra-articular exit is fixed to the neighboring soft tissue by secure knots [Fig. 1(d)]. The periosteum on the anterior/medial tibia is sutured to cover the exit of the tibial tunnel. Maximal tension is applied to the graft, with the knee flexed at 30°. Then, the other end of the graft is fixed to the surrounding periosteum. The wound is closed in layers and wrapped with dressing. Postoperative X-ray shows the placement of the bone tunnel [Fig. 1(e)]. The rabbits are allowed for free cage movement immediately without immobilization.

3. Assessment Protocol for ACL Reconstruction Model in Rabbit 3.1. Functional evaluation Knee stability is evaluated by Lachman test (Papachristou et al. 1998). Intra-articular graft appearance and length or articular surface appearance is used to identify osteoarthritis lesions.

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3.1.1. Materials • • •

Samples: knee joint which is harvested with the femoral and tibial shafts 8 cm in length and wrapped with saline-soaked gauzes Lachman test platform — for quantifying the anterior translation of tibia when performing the Lachman test Caliper (Shenji International Co. Ltd, China)

3.1.2. Method •

• •

The shafts of femur and tibia are fixed onto the platform with a knee joint flexion angle of 30°. The elevation of tibial tuberosity is recorded when performing the Lachman test; this measurement is affected by the status of soft tissue when harvesting the knee joint. It should be noted that the posterior cruciate ligament (PCL), collateral ligament, posterior joint capsule, and muscle attachment are kept intact as well as the reconstructed ACL. The intra-articular graft appearance is examined and its length is measured by a caliper. Osteoarthritis lesions are searched for by eyeballing.

3.2. Three-dimensional (3D) structure analysis of bone ingrowth by micro-CT Even though grafted tendon healing in bone tunnel has not been fully explored, a consensus has been reached that bone ingrowth conducts the mineralized collagen fibers to reconnect the grafted tendon to tunneled bone (Rodeo et al. 1993). Bone ingrowth is identified as woven bone under polarized microscopy between the grafted tendon and the pre-existing lamellar bone in animal studies and clinical biopsies (Ma et al. 2007; Robert et al. 2003; Nebelung et al. 2003; Peterson and Laprell 2000). It serves as an insertion site for tendon collagen fiber anchorage on bone (Ma et al. 2007; Robert et al. 2003; Nebelung et al. 2003; Oguma et al. 2001; Petersen and Laprell 2000).

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Graft–tunnel healing is actually three-dimensional (3D); thus, we recommend micro-computed tomography (micro-CT) to provide a 3D structure analysis of bone ingrowth. 3.2.1. Material •

Micro-CT system: µCT40 or vivaCT40 (Scanco Medical, Bassersdorf, Switzerland) (http://www.scanco.ch/cgi-bin/scanco.pl), or other commercial systems

3.2.2. Method •

The samples are placed with their long axes in the vertical position and immobilized with a foam pad in a cylindrical sample holder, which is filled with 70% ethanol [Fig. 2(a)]. For each sample, bone tunnels on the femoral side and the tibial side are scanned [Fig. 2(b)]. For each part, continuous scans are prescribed that are perpendicular to the long axis of the limb at an isotropic resolution of 30 µm3.

Fig. 2. Micro-CT scan and analysis. (a) Photograph showing the positioning of rabbit knee fixed by foam in a radiolucent tube of 3 mm in diameter for scanning. (b) X-ray showing the scout view of scanning, covering the whole tunnel on both femoral and tibial sides. (c) 2D cross-sectional images defining the region of interest (ROI) based on the samples at time zero. It is turned along the long axis of the bone tunnel for further analysis.

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•

•

•

•

•

The region of interest (ROI) is defined as a circular region of 3 mm in diameter based on the images of tunnel void at time zero [Fig. 2(c)]. The ROI can be turned along the long axis of the bone tunnel in 3D reconstruction for further analysis. Tendon insertion in bone tunnel is formed in the trabecular bone environment instead of the marrow environment (Grassman et al. 2002). Thus, the ROI is set from the first slice, with a complete circle of the bone tunnel wall made up of trabecular bone, to the 150th slice to cover the entire region with the tunnel surrounded by trabecular bone. The acquired 3D data set is first convoluted with a 3D Gaussian filter with a width and support equal to 1.2 and 2, respectively, to remove the noise. Bone tissue is segmented from the marrow and soft tissue using a global thresholding procedure. A threshold equal to or above 210 signifies bone tissue; whereas a threshold below 210 represents the marrow and soft tissue. The cut-off point is defined by users based on the raw images acquired by micro-CT (Dufresne 1998). This produces a cylindrical ROI of 31 mm3. The same volume of interest (VOI) is used in all of the bone samples for 3D analysis. The volume and microarchitecture of mineralized tissue are automatically evaluated using the built-in program of the micro-CT (Fig. 3). Five parameters are applied to characterize bone ingrowth, namely, bone volume (BV), trabecular bone thickness (Tb.Th), degree of anisotropy (DA), connectivity density (Conn.D), and structure model index (SMI) (Butz et al. 2006; Muller and Ruegsegger 1997). Bone volume represents the volume of bone ingrowth; while Tb.Th, DA, Conn.D, and SMI are used to signify the structural maturity.

3.3. Two-dimensional (2D) structural analysis of tendon insertion site in bone tunnel by routine histology 3.3.1. Materials •

519

Tissue processor (e.g. Shandon Pathcenter, USA)

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Fig. 3. Upper row: representative 3D images showing bone ingrowth in the tibial tunnel at weeks 2, 6, and 12 after surgery. Lower row: charts showing the temporal changes of bone ingrowth in the material bone mineral density (BMD), bone volume, trabecular thickness, degree of anisotropy, connectivity density, and structure morphology index.

• • • •

Tissue embedding unit (e.g. Histocenter II-N; Thermolyne, USA) with paraffin Rotary microtome (e.g. Leica RM2165; Leica, Germany) Conventional reagents for hematoxylin & eosin (H&E) staining and Safranin O staining Microscopic image analysis system (Leica Q500MC; Leica Cambridge Ltd, Cambridge, UK)

3.3.2. Method •

The decalcified samples are embedded in paraffin by using a tissue embedding unit.

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Fig. 4. The autografts are not separated in the joint. They are of a white color with densely packed fiber bundles without the pearly appearance like in intact ACL. No articular fibrillation or cartilage erosions are noticed on the joint surface. The samples are processed for subsequent histological examination on the graft–tunnel healing interface.

• • •

•

The paraffin blocks are cut into 7-µm-thick sections along the long axis of the bone tunnel (Fig. 4). Sections are batch-stained with H&E and Safranin O/fast green. Sharpey’s fibers (Fig. 5) are defined as penetrating collagen fibers that directly connect the noncalcified zone with the calcified zone at normal tendon insertion to bone (Benjamin et al. 2002). The collagen fiber connection is examined under polarized microscopy. Fibrocartilage (Fig. 5) is composed of a cluster of chondrocytes embedded in the fibrous matrix. It is present in the normal skeletal attachment of tendon or ligament (Benjamin et al. 2002). At the tendon–bone healing interface, fibrocartilage formation can be detected by Safranin O staining (Wong et al. 2004).

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Fig. 5. Representative micrographs demonstrating bone ingrowth with Sharpey’s fiber formation. They show mildly positive signals after Safranin O (SO) staining at week 2 after surgery. The cartilage zone becomes distinct at week 6 with aligned chondrocyte-like cells and proteoglycan deposition. Bone progressively grows toward the grafted tendon with diminished cartilaginous interface and Sharpey’s fiber (SF) formation (arrow). Bone ingrowth is identified as woven bone under polarized microscopy. The process of bone ingrowth resembles endochondral ossification. B, bone; C, cartilage; T, tendon (original magnification, 100×).

3.4. Mechanical testing for the strength of the tendon insertion site in bone tunnel 3.4.1. Materials • •

Hounsfield testing machine (H25K-S; Hounsfield Test Equipment Ltd, Surrey, UK) Custom-made mechanical jig (Fig. 6)

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Fig. 6. A custom-made testing jig mounted with the femur-graft-tibia complex, allowing for tensile loading along the long axis of the graft.

3.4.2. Method •

•

•

•

The samples are harvested with both femoral and tibial shafts of 8 cm in length. They are stored at −20°C within 1 month until biomechanical testing. The samples are thawed overnight at room temperature before testing. The sample is carefully dissected of surrounding soft tissue until the only physical connection between the two bones is the ACL tendon graft. The femur-reconstructed ACL graft–tibia complex is fixed in the custom-made mechanical jig, allowing tensile loading along the long axis of the graft in a Hounsfield testing machine. A load cell of 1 kN is used. With a preload of 1 N and a load displacement rate of 50 mm/min, tensile force is applied to the graft–tunnel complex until failure. The failure mode, the ultimate load to failure (N), and stiffness (N/mm) are recorded with the load–displacement curve. After testing, the broken graft–tunnel complex is also processed for histological examination (Fig. 7).

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Fig. 7. Left : A custom-made testing jig is designed for pull-out testing of femur–ACL graft–tibia complexes. Right : The failure mode and the load– displacement curve are recorded.

3.5. Densitometric analysis on pre-existing bone by pQCT 3.5.1. Materials •

pQCT (Densiscan 2000; Scanco Medical, Bassersdorf, Switzerland)

3.5.2. Method (Chan et al. 2006; Qin et al. 2006; Wen et al. 2006) •

•

After anesthesia, the rabbits are secured in a supine position in a suitable radiolucent tunnel to fix the knee joint in the position of full extension for scanning (Fig. 8). An anteroposterior (AP) scout view is obtained with the joint line as the reference, and cross-sectional slices are then taken perpendicular to the long axis of the limb with a slice thickness of 1 mm.

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Fig. 8. The rabbits are secured in a supine position in a suitable radiolucent tunnel to fix the knee joint in the position of full extension for pQCT scanning. The AP scout view is obtained with the joint line as the reference, and crosssectional slices are then taken perpendicular to the long axis of the limb with a slice thickness of 1 mm. The volumetric BMD (mg/mm3) at the distal femur, proximal tibia, and calcaneus are measured. The tunnel void area — referred to as the radiolucent region in the bone tunnel and its area — is measured.

•

The volumetric BMD (mg/mm3) at the distal femur, proximal tibia, and calcaneus is measured (Fig. 8). The pQCT scanner acquires continuous images at the distal femur, proximal tibia, and calcaneus. A total of two slices with images of the calcaneus are selected for analysis. At the distal femur or proximal tibia, five consecutive slices of 1 mm above or beneath the intra-articular aperture, respectively, are selected as the representative slices for subsequent measurement. On each slice, two parameters — the tunnel void area and BMD — are

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Fig. 9. Curves showing the trend of significant decrease in BMD on both the femoral and tibial sides after surgery. The volumetric BMD (mg/mm3) on the tibial side decreases significantly (up to 15% of time zero), whereas there is a slight decrease in the calcaneus.

Fig. 10. Temporal changes of six bone tunnels on both the femoral and tibial sides after surgery. There is no statistically significant tunnel widening during the observation period.

measured. The average BMD is taken from these five consecutive slices, and the changes in BMD are computed as a percentage of time zero. The tunnel void area — referred to as the radiolucent region in the bone tunnel and its area — is measured (Figs. 9 and 10).

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A standard IBT (Institut fur Biomedizinische Technik, of the Eidgenossische Technische Hochschule and the University of Zurich) phantom measurement is performed weekly with three measurement values within the given reference ranges provided by the manufacturer. This gives a precision error measurement of 5% for repeated measurement of the samples.

4. Results 4.1. Successful restoration of ACL function to restrict anterior-posterior translation The Lachman test showed 6.0 mm ± 1.0 mm on average after the original ACL removal and 1.0 mm ± 0.5 mm after the surgical procedure. The intact ACL had a white pearly appearance with densely packed fiber bundles. A synovial membrane covered the ACL, 8.0 mm ± 0.7 mm in average length; the graft was not separated in the joint. The autografts were of a white color with densely packed fiber bundles without the pearly appearance (Fig. 4). The length of the grafts was 7.8 mm ± 0.8 mm on average. No graft laxity was noted at sacrifice. No articular fibrillation or cartilage erosions were noticed on the joint surface after ACL reconstruction. All of these findings indicated that the surgical procedures are successful in reproducing the function of ACL to restrict anteriorposterior translation and prevent subsequent cartilage injury during the observation period.

4.2. Progressive bone ingrowth with structural immaturity shown by micro-CT It was observed that bone ingrowth progressively formed in the bone tunnel with time. However, the bone ingrowth was composed of much more plate-like trabecula (higher structure model index) with smaller thickness, and a higher degree of anisotropy (Fig. 3).

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It indicated the structural immaturity of bone ingrowth during the period of observation.

4.3. Bone ingrowth serving as grafted tendon insertion on tunneled bone Bone ingrowth was identified as woven bone at the graft–tunnel healing interface. It conducted Sharpey’s fiber formation to reconnect the grafted tendon with the tunneled bone (Fig. 5). Transient fibrocartilage was noted at the graft–tunnel healing interface, but the classic four-zone structure of intact ACL bony insertion was not regenerated during the period of observation.

4.4. The healing interface remaining as one of the weak points of graft–tunnel complexes with progressive bone ingrowth The failure mode of eight samples of the graft–tunnel complex varied at week 12 postsurgery, including rupture of the intra-articular graft midsubstance (n = 3) and graft pulled out of the bone tunnel (n = 5, 2 from the femoral side and 3 from the tibial side). The graft was pulled out of the bone tunnel with bony attachment (Fig. 7). It indicated that the healing interface remains one of the weak points of graft–tunnel complexes, even with bone ingrowth at 12 weeks after surgery.

4.5. Pre-existing bone mineral loss concomitant with bone ingrowth There was up to 15% bone loss at the operated site and less than 5% bone loss at the nonoperated site (Fig. 9). No statistically significant tunnel widening was noted during the observation period (Fig. 10).

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5. Discussion 5.1. New aspects in the current ACL reconstruction rabbit model Factors including graft/tunnel size, graft tensioning, and tunnel placement have been identified to be associated with the outcome of ACL reconstruction (Segawa et al. 2003; Tohyama and Yasuda 1998; Yamazaki et al. 2002). These factors have been taken into consideration in clinical practice (Fu et al. 1999). As shown in Table 1, such important information was always missing in the description of surgical procedures in almost all of the previous studies. The current protocol for establishing an ACL reconstruction rabbit model pays attention to control those factors and essentially duplicates the procedures used in human beings. The newly regenerated tendon insertion in bone tunnel is 3D and routine histological examination has limitations to evaluate it. In previous studies, it was known that bone ingrowth conducted mineralized collagen fiber reconnect with grafted tendon and served as newly regenerated tendon insertion in bone tunnel. Thus, micro-CT was introduced in our study to evaluate bone ingrowth three-dimensionally. It can also help us to better understand the quality of the newly regenerated tendon insertion site in terms of the architecture of bone ingrowth. In addition, pQCT was also successfully applied to longitudinally monitor changes in the BMD of host bone after surgery. These powerful imaging tools help us better understand graft–tunnel healing, particularly the changes in bone tissue.

5.2. Limitations of the current ACL reconstruction rabbit model As shown in our study, graft fixation by suture provides an acceptable functional outcome of ACL reconstruction. Such a fixation method is also widely accepted in the establishment of the rabbit model (Table 1). However, this suture fixation method is only to mimick suspensory

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fixation by endobutton or washer, which is not used in the current clinical practice. In current clinical practice, suspensory fixations by endobutton and washer are often combined with other fixation methods including interference screw, cross-pin, etc. Therefore, it should be noted that the current ACL reconstruction rabbit model is only used to understand the biology of graft–tunnel healing by one kind of fixation. The information may be helpful for an explanation of the clinical phenomenon, but cannot be directly applied back to clinical practice.

5.3. Future applications of the current ACL reconstruction rabbit model As shown in this chapter, the structure of the calcified zone of tendon insertion in bone tunnel is still immature and easily broken at week 12 after ACL surgery. Osteoconductive biomaterial may be helpful to consolidate the calcified zone of tendon insertion in bone tunnel. The model established in this chapter will be used to test this hypothesis. The current ACL reconstruction rabbit model can be further modified by applying other fixation methods like interference screw, crosspin, etc. It is known that the fixation strength by such devices is related to BMD (Nyland et al. 2004). The current rabbit model can be used to examine the effect of bone mineral loss on the initial fixation strength and outcome of ACL surgery (Wen et al. 2006). It is clinically oriented.

6. Summary The model with multiple bioimaging modalities described in this chapter may provide a research platform to investigate the biological behavior of tendon healing in bone tunnel. Furthermore, it will be helpful to investigate the potential prevention or treatment efficacy of systemic or local protocols such as drug, cytokine, and/or stem cell combined with biomaterial, ultrasound, shockwave, vibration, etc.

Acknowledgments This study was supported by the Hong Kong Research Grant Council Earmarked Grant 06-07 (4497/06M).

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References Anderson AF, Snyder RB, Lipscomb AB Jr. Anterior cruciate ligament reconstruction. A prospective randomized study of three surgical methods. Am J Sports Med 29(3):272–279, 2001. Aune AK, Holm I, Risberg MA et al. Four-strand hamstring tendon autograft compared with patellar tendon-bone autograft for anterior cruciate ligament reconstruction. A randomized study with two-year follow-up. Am J Sports Med 29(6):722–728, 2001. Benjamin M, Kumai T, Milz S et al. The skeletal attachment of tendons — tendon “entheses”. Comp Biochem Physiol A Mol Integr Physiol 133(4):931–945, 2002. Butz F, Ogawa T, Chang TL, Nishimura I. Three-dimensional bone–implant integration profiling using micro-computed tomography. Int J Oral Maxillofac Implants 21(5):687–695, 2006. Chan CW, Qin L, Lee KM et al. Dose-dependent effect of low-intensity pulsed ultrasound on callus formation during rapid distraction osteogenesis. J Orthop Res 24(11):2072–2079, 2006. Clatworthy MG, Annear P, Bulow JU, Bartlett RJ. Tunnel widening in anterior cruciate ligament reconstruction: a prospective evaluation of hamstring and patella tendon grafts. Knee Surg Sports Traumatol Arthrosc 7(3):138–145, 1999. Dufresne T. Segmentation techniques for analysis of bone by three-dimensional computed tomographic imaging. Technol Health Care 6(5–6):351–359, 1998. Forssblad M, Valentin A, Engstrom B, Werner S. ACL reconstruction: patellar tendon versus hamstring grafts — economical aspects. Knee Surg Sports Traumatol Arthrosc 14(6):536–541, 2006. Freedman KB, D’Amato MJ, Nedeff DD et al. Arthroscopic anterior cruciate ligament reconstruction: a metaanalysis comparing patellar tendon and hamstring tendon autografts. Am J Sports Med 31(1): 2–11, 2003. Fu FH, Bennett CH, Lattermann C, Ma CB. Current trends in anterior cruciate ligament reconstruction. Part 1: biology and biomechanics of reconstruction. Am J Sports Med 27(6):821–830, 1999. Grana WA, Egle DM, Mahnken R, Goodhart CW. An analysis of autograft fixation after anterior cruciate ligament reconstruction in a rabbit model. Am J Sports Med 22(3):344–351, 1994.

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Grassman SR, McDonald DB, Thornton GM et al. Early healing processes of free tendon grafts within bone tunnels is bone-specific: a morphological study in a rabbit model. Knee 9(1):21–26, 2002. Herrington L, Wrapson C, Matthews M, Matthews H. Anterior cruciate ligament reconstruction, hamstring versus bone-patella tendon-bone grafts: a systematic literature review of outcome from surgery. Knee 12(1):41–50, 2005. Leppala J, Kannus P, Natri A et al. Effect of anterior cruciate ligament injury of the knee on bone mineral density of the spine and affected lower extremity: a prospective one-year follow-up study. Calcif Tissue Int 64(4):357–363, 1999. Ma CB, Kawamura S, Deng XH et al. Bone morphogenetic proteins-signaling plays a role in tendon-to-bone healing: a study of rhBMP-2 and noggin. Am J Sports Med 35(4):597–604, 2007. Magen HE, Howell SM, Hull ML. Structural properties of six tibial fixation methods for anterior cruciate ligament soft tissue grafts. Am J Sports Med 27(1):35–43, 1999. Muller R, Ruegsegger P. Micro-tomographic imaging for the nondestructive evaluation of trabecular bone architecture. Stud Health Technol Inform 40:61–79, 1997. Nebelung W, Becker R, Urbach D et al. Histological findings of tendonbone healing following anterior cruciate ligament reconstruction with hamstring grafts. Arch Orthop Trauma Surg 123(4):158–163, 2003. Noojin FK, Barrett GR, Hartzog CW, Nash CR. Clinical comparison of intraarticular anterior cruciate ligament reconstruction using autogenous semitendinosus and gracilis tendons in men versus women. Am J Sports Med 28(6):783–789, 2000. Nyland J, Kocabey Y, Caborn DN. Insertion torque pullout strength relationship of soft tissue tendon graft tibia tunnel fixation with a bioabsorbable interference screw. Arthroscopy 20(4):379–384, 2004. Oguma H, Murakami G, Takahashi Iwanaga H et al. Early anchoring collagen fibers at the bone–tendon interface are conducted by woven bone formation: light microscope and scanning electron microscope observation using a canine model. J Orthop Res 19(5):873–880, 2001. Papachristou G, Tilentzoglou A, Efstathopoulos N, Khaldi L. Reconstruction of anterior cruciate ligament using the doubled tendon graft technique: an

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experimental study in rabbits. Knee Surg Sports Traumatol Arthrosc 6(4): 246–252, 1998. Petersen W, Laprell H. Insertion of autologous tendon grafts to the bone: a histological and immunohistochemical study of hamstring and patellar tendon grafts. Knee Surg Sports Traumatol Arthrosc 8(1):26–31, 2000. Qin L, Lu H, Fok P et al. Low-intensity pulsed ultrasound accelerates osteogenesis at bone–tendon healing junction. Ultrasound Med Biol 32(12):1905–1911, 2006. Robert H, Es-Sayeh J, Heymann D et al. Hamstring insertion site healing after anterior cruciate ligament reconstruction in patients with symptomatic hardware or repeat rupture: a histologic study in 12 patients. Arthroscopy 19(9):948–954, 2003. Rodeo SA, Arnoczky SP, Torzilli PA et al. Tendon-healing in a bone tunnel. A biomechanical and histological study in the dog. J Bone Joint Surg Am 75(12):1795–1803, 1993. Segawa H, Koga Y, Omori G et al. Influence of the femoral tunnel location and angle on the contact pressure in the femoral tunnel in anterior cruciate ligament reconstruction. Am J Sports Med 31(3): 444–448, 2003. Tohyama H, Yasuda K. Significance of graft tension in anterior cruciate ligament reconstruction. Basic background and clinical outcome. Knee Surg Sports Traumatol Arthrosc 6(Suppl 1):S30–S37, 1998. Tomita F, Yasuda K, Mikami S et al. Comparisons of intraosseous graft healing between the doubled flexor tendon graft and the bone-patellar tendon-bone graft in anterior cruciate ligament reconstruction. Arthroscopy 17(5):461–476, 2001. Wang CJ, Wang FS, Yang KD et al. The effect of shock wave treatment at the tendon–bone interface — an histomorphological and biomechanical study in rabbits. J Orthop Res 23:274–280, 2005. Webster KE, Feller JA, Hameister KA. Bone tunnel enlargement following anterior cruciate ligament reconstruction: a randomised comparison of hamstring and patellar tendon grafts with 2-year follow-up. Knee Surg Sports Traumatol Arthrosc 9(2):86–91, 2001. Wen CY, Lui PY, Wong MWN et al. Local bone loss after anterior cruciate ligament reconstruction — a peripheral quantitative computed tomographic study in a rabbit model. J Orthop Surg (Hong Kong) 10(Suppl): 24, 2006.

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Wong MW, Qin L, Tai JK et al. Engineered allogeneic chondrocyte pellet for reconstruction of fibrocartilage zone at bone-tendon junction — a preliminary histological observation. J Biomed Mater Res B Appl Biomater 70(2):362–367, 2004. Yamazaki S, Yasuda K, Tomita F et al. The effect of graft-tunnel diameter disparity on intraosseous healing of the flexor tendon graft in anterior cruciate ligament reconstruction. Am J Sports Med 30(4):498–505, 2002.

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Chapter 32

Establishment of Normal and Delayed Bone–Tendon Junction Repair Models Kwok-Sui Leung and Ling Qin

The bone-to-tendon (B-T) or osteotendinous junction is a unique structure within the musculoskeletal system that connects both bone and tendon through the transitional fibrocartilage zone. Injuries to the B-T junction occur as a result of trauma, sports injury, or local chronic inflammation. B-T repair is, however, inferior compared to repair taking place within homogeneous tissues such as bone fracture or tendon repair, as B-T repair involves regeneration of the transitional fibrocartilage zone. Delay in B-T junction healing may often occur, thus preventing early mobilization and rehabilitation. How to accelerate B-T healing is challenging. This chapter describes an experimental partial patellectomy model established for studying both normal and delayed B-T junction healing, with the aim of providing a platform in order to evaluate potential biological and biophysical interventions developed or to be developed for acceleration and/or enhancement of B-T junction repair. Keywords:

Bone–tendon (B-T) junction; partial patellectomy; patella– patellar tendon complex; rabbits; normal and delayed healing; osteogenesis; endochondral ossification; bone mineral density; fibrocartilage; tensile strength.

Corresponding author: Ling Qin. Tel: +852-26323071; fax: +852-26324618; E-mail: [email protected]

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1. Introduction Injuries to the bone–tendon (B-T) or osteotendinous junction are not uncommon due to trauma, overloading, or chronic stress disorders (Hung et al. 1993; Liu et al. 1997; Wang et al. 2006a; Wang et al. 2006b). The affected B-T junction regions are located around dynamic joints such as the hand, foot, knee, ankle, shoulder, etc. Repair of the B-T junction is difficult and delayed due to the limited regeneration capacity of its interpositional or interface fibrocartilage zone experimentally (Leung et al. 2002; Qin et al. 1999) and clinically (Liu et al. 1997) as compared with bone fracture or tendon healing, which takes place within homogeneous tissues (Galatz et al. 2005; Leung et al. 2002; Markel et al. 1995). This suggests that a longer period of immobilization is needed for B-T postoperative healing. The associated adverse effects of immobilization are well known, especially in the case of delay in B-T junction healing that prevents early mobilization and rehabilitation (Galatz et al. 2005). How to accelerate B-T healing is therefore a challenging topic within musculoskeletal research. Early studies concentrated on characteristics of the intact B-T junction in both animals and humans (Clark and Stechschulte 1998; Rufai et al. 1995). Recent experimental studies focus on the healing of B-T junction injuries (Galatz et al. 2005; Galatz et al. 2006; Lu 2006; Qin et al. 2006a; Qin et al. 2006b; Wang et al. 2007; Wang et al. 2008). Because of poor healing capacity in B-T repair, how to accelerate its healing process has therefore become a focus of musculoskeletal research. This includes studies using the rotator cuff model in dogs (Markel et al. 1995) and rats (Galatz et al. 2006), as well as studies from the authors’ group where we have been using both the calcaneus– Achilles tendon model in goats (Wilson 2000) and the partial patellectomy model in goats (Wong et al. 2003) and rabbits (Lu 2006; Qin et al. 2006a; Qin et al. 2006b; Wang et al. 2007; Wang et al. 2008). As B-T injury to the patella–patellar tendon (PPT) complex of the knee joint is quite common, this chapter describes an experimental

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partial patellectomy model in rabbits that we have established for studying both normal and delayed B-T junction healing (Leung et al. 1999; Leung et al. 2002; Lu 2006; Qin et al. 1999; Qin et al. 2006a; Qin et al. 2006b). Such an experimental model provides a useful platform to evaluate potential biological and biophysical interventions developed or to be developed for acceleration and/or enhancement of B-T junction repair.

2. Materials and Methods 2.1. Materials •

• • •

Animals: male and female mature New Zealand white rabbits (around 32 weeks old) with a body weight of around 4.0–4.5 kg, which mimics physically active young adultsa Anesthesia and pain relief drugs: sodium pentobarbital and Temgesic Surgical instruments and fixation materials: hair shaver, surgical scalpel, saw, driller and drill bits, suture, wire, plastic casts Evaluation devices and chemicals: specified in the corresponding sections below

2.2. Methods 2.2.1. Surgery for establishing a normal B-T junction healing model •

•

a

Anesthesia: General anesthesia is required, e.g. using sodium pentobarbital (0.8 mL/kg, intravenous injection; Sigma Chemical Co., USA). Skin preparation: Under aseptic condition, one of the knees is shaved using an animal hair shaver. A surgical scalpel is used to

Other animal models can also be used, especially those with knee biomechanics close to humans, such as goats (Wong et al. 2003). The age of animals may vary depending on the study background.

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Fig. 1. Surgical procedures of partial patellectomy. (a) Standard transverse osteotomy is performed between the proximal 2/3 and the distal 1/3 of the patella using an oscillating saw (Synthes/Mathys AG, Bettlach, Switzerland). (b, c) The distal 1/3 of the patella is removed and two longitudinal holes are drilled into the proximal patella. (d) The patellar tendon is reattached back to the remaining proximal patella via two longitudinal drill holes using nonabsorbable suture. (e) The patella–patellar tendon (PPT) complex is then protected with a “figureof-eight” tension band wire proximally by inserting it into the quadriceps tendon next to the upper pole of the patella and distally by drilling a frontal hole in the tibial tuberosity. (f) The fixation is confirmed with plain X-ray.

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(e)

(f )

Fig. 1.

•

make an anterolateral skin incision to explore the patella–patellar tendon (PPT) complex.b Partial patellectomy (Fig. 1)


b

(Continued)

Transverse osteotomy: This is performed between the proximal 2/3 and the distal 1/3 of the patella using an oscillating hand saw with a saw blade around 0.4 mm in thickness (e.g.

Anterolateral skin incision is highly recommended instead of pure anterior skin incision, as it can avoid compression between the skin incision and the immobilization cast postoperatively. In addition, anterolateral incision is particularly important in studying the treatment effects of biophysical interventions, e.g. for placement of ultrasound probe during immobilization (Fig. 2).

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hand saw from Synthes/Mathys AG, Bettlach, Switzerland).c The distal 1/3 of the patella is then excised [(Fig. 1(a)]. Suturing of patellar tendon to proximal patella: Two holes of 0.8 mm in diameter are drilled vertically along the patella. The patellar tendon is directly sutured to the proximal 2/3 of the patella at the resting angle of the knee joint via two vertically drilled holes that are prepared along the patellar long axis with nonabsorbable suture (e.g. 3/0 Mersilk from Ethicon Ltd., Edinburgh, Scotland) [(Figs. 1(b)–1(d)]. Protection of suture fixation: The suture fixation is protected using a “figure-of-eight” tension band wire (e.g. using 0.4 mm from Biomet Ltd, Waterton, UK), which is drawn around the superior pole of the patella to the tibial tuberosity at the resting angle of the knee joint [(Figs. 1(e) and 1(f)].d Closing of skin incision and disinfection: 4-0 absorbable polyglycolic acid suture (Mersilk; Ethicon Ltd, Edinburgh, UK), for example, is used to close the skin incision. Antibiotic spray (e.g. Nebactin; Byk Gulden Konstanz, Germany) is used for disinfection. Immobilization of the operated knee: The knee is wrapped with a layer of soft cotton pad and then immobilized with a long leg cast (e.g. Scotchcast, Orthopedic Products; 3M Health Care, MN, USA) at a resting knee joint angle for 3 weeks.e If the study is designed to evaluate the effects of early biophysical interventions — e.g. low-intensity pulsed ultrasound (LIPUS) — for accelerating PPT healing, an open window is created on the upper cast, which allows the placement of an ultrasound transducer directly on the surface of the patella for LIPUS treatment (Fig. 2).

It is difficult to perform a precise transverse osteotomy between the proximal 2/3 and the distal 1/3 of the patella. We use a ruler to measure the entire length of the patella, and then use a scalpel to create a bony marker between the proximal 2/3 and the distal 1/3 of the patella before sawing. d The fixation at the proximal tibia should be 2–3 mm distal to the growth plate in order to achieve better fixation for “figure-of-eight” (Figs. 1 and 2). e The knee fixation angle for experimental studies in rabbits is reported at 90°–120° flexion. Most importantly, the fixation angle for the operated knee should be consistent during fixation and immobilization.

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Fig. 2. Cast immobilization of the rabbit knee joint after partial patellectomy. (a) An open window is created onto the upper cast where the patella–patellar tendon is surgically reattached. (b) This window is used to place an ultrasound transducer directly on the surface of the patella for postoperative treatment during the immobilization period.




Postoperative pain relief: Pain relief drugs (e.g. Temgesic; Reckitt & Colman Pharmaceuticals, Hull, UK) are given subcutaneously at a dose of 0.01 mg/kg for 3 consecutive days after operation.

2.2.2. Surgery for establishing a delayed B-T junction healing model All preparations and postsurgical events are the same as those described above in Sec. 2.2.1 for the normal B-T healing model, except for the following three aspects: •

•

Shielding for B-T junction initial healing: This step comes after removing the distal 1/3 of the patella and prior to suturing the patellar tendon to the proximal patella. A 2 mm × 3 mm rectangular latex sheet with a thickness of 1 mm (e.g. from Ansell Medical, Victoria, Australia) is prepared and interposed between the patellar tendon and the proximal patella before suturing together and protection with figure-of-eight tension band wiring and cast immobilization [(Fig. 3(a)]. Duration of immobilization: A 4-week duration is recommended, i.e. 1 week more compared with the 3 weeks mentioned

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Fig. 3. Delayed B-T healing model and treatment. (a) A latex slice (left arrow) is interposed to shield the bridge of tendon and patella (right arrow) for the first 4 postoperative weeks, and is then removed surgically to facilitate patellar tendon–proximal patella healing at the initial osteotomy side in delayed healing condition. (b) The B-T junction in delayed healing is treated with extracorporeal shock wave therapy (ESWT) 4 weeks after partial patellectomy; the shockwave is delivered perpendicular to the healing B-T junction of the operated rabbit knee via contact gel (arrow).

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in Sec. 2.2.1. Delayed PPT junction healing may be treated biologically and biophysically, e.g. by extracorporeal shock wave therapy (ESWT) a few days after removal of the shielding latex sheet [(Fig. 3(b)]. Removal of B-T healing shielding: Under general anesthesia, the immobilization cast is removed using a hand saw, and hair over the operated knee is shaved for preparation of a surgical incision at the anterolateral side (refer above). The initial osteotomy side is explored to identify the interposed latex for removal using a surgical clamper, yet without cutting the initial suture and figure-ofeight wire used for fixation of the PPT complex.

3. Postoperative Evaluation of B-T Junction Healing This section presents the routine evaluations we have established for our research group based on study hypotheses and objectives, expertise, and available research facilities. The readers may like to modify and supplement these further based on their own study hypotheses, objectives, and available methodologies.

3.1. Postoperative monitoring and treatment 3.1.1. Radiological confirmation of surgical fixation quality Lateral X-ray on the operated knee is taken immediately after surgery and before cast immobilization for inspection [(Fig. 1(f )].

3.1.2. Regular inspection of cast fixation quality This is important, as less experienced staff may not always perform adequate cast fixation and the rabbits may also bite the cast from time to time; these may result in loosening of the cast and/or induce skin injury and infection. Cast replacement for further immobilization may therefore be needed.

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3.1.3. Injection of sequential fluorescence If bone dynamic remodeling is one of the study objectives, sequential fluorescence labeling is recommended by intramuscular injection of calcein green (10 mg/kg) and xylenol orange (30 mg/kg) (e.g. from Sigma Chemical Co., St. Louis, MO, USA) at 4 and 2 weeks, respectively, prior to euthanasia (Qin et al. 2006b; Lu 2006).

3.2. Sampling and sample preservation After euthanizing the animals with an overdose of sodium pentobarbital at a given time point(s),f the hind limb is surgically harvested. As it is most likely impossible to have all animal experiments completed on the same day, it is recommended to wrap the hind limbs with 0.9% saline-soaked gauze, seal in airtight plastic freezer bags, and store in a −20°C freezer before further preparation and evaluation.

3.3. B-T healing model evaluations The freezer-stored hindlimbs are thawed in a room overnight before preparation of the PPT complex for radiological, bone densitometric, histological, and biomechanical evaluations. 3.3.1. Radiological evaluation High-resolution plain X-ray of the PPT complex is taken under identical exposure conditions using X-ray films (e.g. from Fujiphoto Film Co. Ltd, Japan) and an X-ray machine (e.g. Faxitron cabinet X-ray system model; Faxitron X-Ray Corp., NJ, USA). An anteriorposterior (A-P) X-ray of the PPT complex is taken under identical conditions (e.g. at 45 kV, 2 mA and with an X-ray source–object f

The points for sampling are mainly defined based on study objectives, e.g. if it is mainly related to investigation of early healing events with an emphasis on molecular or cellular changes or to healing quality in the long term (e.g. up to 6 months or even over 12 months, where the tensile strength of the integral PPT healing complex would be one of the major endpoints for evaluation).

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distance of 40 cm). X-ray films are developed under identical conditions. After digitizing the X-ray films into an image analysis system (e.g. MetaMorph image analysis system; Universal Imaging Corp., PA, USA), ostogenesis or new bone formation at the PPT healing junction in terms of newly formed bone area or an enlarged bony part from the proximal remaining patella is quantified by a single examiner for either normal healing (Fig. 4) or delayed healing with or without treatment (Fig. 5). In addition, a phantom (aluminum step wedge) is recommended for taking X-ray so that the optical density of the new bone can be calculated based on its mean optical density (Li and Murnaghan 2007; Qin et al. 1999; Wang et al. 2006b). 3.3.2. Bone mineral density (BMD) and structure measurement Dual-energy X-ray absorptiometry (DXA) is a popular method to measure aerial (two-dimensional) BMD. Because of its relatively low imaging resolution, its accuracy is problematic for rabbit patella. We employ multislice peripheral quantitative computed tomography (pQCT) (Scanco Medical, Bassersdorf, Switzerland) with a slice thickness of 1 mm to scan the new bone and the residual proximal patella for quantification of its volumetric (threedimensional) BMD (vBMD) and bone mineral content (BMC) for studying osteogenesis and bone remodeling at the B-T healing junction (Lu 2006; Wang et al. 2007; Wang et al. 2008). A new micro-CT is also available for studying osteogenesis at the PPT healing junction interface, which demonstrates not only new bone formation in a 3D manner but also provides quantitative data of volume and structural parameters (Fig. 6). 3.3.3. Histology and histomorphometry For histology, the PPT complex is halved using a saw with thin blade (e.g. not thicker than 1 mm) along its midsagittal plane, half for decalcification and another half for undecalcification using conventional histological protocols (Recker 1983) and those adopted by the

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(b)

(c)

Fig. 4. Representative anteroposterior radiographs of patella. (a) Intact patella. (b) Proximal patella with new bone formation (white arrow) at the healing interface 8 weeks after partial patellectomy. (c) Proximal patella with new bone formation (white arrow) at the healing interface 16 weeks after partial patellectomy.

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Fig. 5. Representative anteroposterior radiographs of the remaining proximal patella after partial patellectomy with newly formed bone at the healing interface (bony region below the white dotted line, i.e. the initial osteotomy site). (a) Specimens of week 8 with delayed healing. (b) Week 8 specimens with delayed healing, but treated with shockwave. (c) Specimens of week 12 with delayed healing. (d) Week 12 specimens with delayed healing, but treated with shockwave.

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(a)

(b)

(c)

Fig. 6. Representative micro-CT images of the PPT healing junction. (a) Remaining proximal patella with two drilled holes of a sample 4 weeks after partial patellectomy. (b) Remaining proximal patella of a week 16 sample after partial patellectomy with new bone formation at the PPT healing interface of the proximal patella in antero-posterior (3D) view. (c) Midsagittal (2D) view of (b).

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authors of this chapter (Leung et al. 2002; Lu 2006; Qin et al. 1999; Qin et al. 2006a; Qin et al. 2006b).g For decalcified histology, conventional paraffin sections are prepared for general histomorphology stained with hematoxylin and eosin (H&E) [(Figs. 7 and 8(b)], and for cartilage-like tissues stained with Safranin O [(Fig. 8(a)]. For undecalcified histology, the PPT complex is embedded in methyl methacrylate (MMA) without decalcification. Thin sagittal sections with a thickness of 10 µm are prepared using a heavy-duty microtome (e.g. Polycut; Leica Instruments, Nussloch, Germany); or thick sections around 200 µm are prepared using a microtome (e.g. Leica Sp1600; Leica Instruments, Nussloch, Germany), polished to a thickness of 150 µm with a grinding machine (e.g. Phoenix 4000; Wirtz Buehler, Germany) to study osteogenesis and bone dynamic remodeling under fluorescence microscopy (Lu 2006; Qin et al. 2006b) as well as collagen alignment under polarized microscopy (e.g. using Leica Q500MC system; Leica Cambridge Ltd, Cambridge, UK) (Lu 2006; Qin et al. 1999) [(Fig. 8(c)]. 3.3.4. Mechanical testing Hind limbs are thawed overnight at room temperature for preparation of the quadriceps–patella–patellar-tendon–tibia (QPPTT) complex after removing all periarticular connective soft tissues around the knee, suture material, and metal tension band wire, and by disconnecting the femur. The QPPTT complex is then mounted on a custom-made fixator to measure the cross-sectional area (CSA) of the PPT junction using a fine caliper by a single examiner before fixation onto a custom-made tensile testing jig, which consists of one upper clamp and one lower clamp to fix the distal quadriceps and tibia, respectively (Leung et al. 2002; Lu 2006; g

Because of the rather smaller rabbit patella, to halve the patella at the midsagittal plane would sacrifice the midsagittal sections that are mote representative for the study of PPT junction healing histologically or histomorphologically.

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(b)

(c)

Fig. 7. Histology of midsagittal sections of the representative PPT healing complex after partial patellectomy. The PPT healing complex postoperatively with new bone formation at different healing time points (the portion right from the line where the initial osteotomy side is), i.e. at (a) postoperative week 4, (b) postoperative week 8, and (c) postoperative week 12. H&E staining (magnification, 16×).

Wang et al. 2007; Wang et al. 2008). The distal quadriceps, its tendon, and the proximal patella are clamped directly in line with the axis of loading (Fig. 9). Standard material testing machines, e.g. a uniaxial one such as the Hounsfield test machine (Hounsfield H25KM; Hounsfield Test

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Fig. 8. Serial sections of interpositional fibrocartilage zone with four typical zones between the bone and tendon at the PPT complex in rabbit. (a) Section stained with Safranin O/fast green, used for assessment of the proteoglycan content in terms of stain intensity and distribution. (b) Section stained with H&E, used for quantitative evaluation of the thickness of the fibrocartilage zone (zone I, subchondral bone [SCB] for anchoring foundation; zone II, calcified fibrocartilage [CFC] for dispersing load transmission; zone III, uncalcified fibrocartilage [UCFC] for attaching tendon and allowing flexibility; and zone IV, tendon fiber [TF] for carrying tensile load). (c) The collagen alignment of fibrocartilage zone observed under polarized light microscopy, where the tidemark (dotted line) separates the uncalcified fibrocartilage zone and the calcified fibrocartilage zone (magnification, 200×).

Equipment Ltd, UK), are used for tensile testing of the QPPTT complex. A load cell of 2000 N or 1000 N is recommended for testing, as our previous test recorded an average 500 N maximal tensile force of intact QPPTT complex. The testing speed selected may have a wide range, e.g. 20–100 mm/min; however, for direct comparison, we have to use the same testing speed. The failure mode is also documented to study the mechanisms of failure, i.e. the weakest region within the QPPTT complex (Leung et al. 2002; Lu 2006;

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Fig. 9. Tensile testing for the quadriceps–patella–patellar-tendon–tibia (QPPTT) complex. The QPPTT complex mounted on a custom-made tensile testing jig that consists of (a) one lower fixation holder and (b) an upper clamp (upper portion) to fix the distal quadriceps and tibia, respectively.

Wang et al. 2006b).h The maximum tensile force and energy at failure are recorded and calculated using built-in software. The tensile strength is calculated by normalizing the tensile force over the CSA of the PPT healing junction.i h

Failure modes of the PPT complex can be recorded during and after mechanical testing. They can be further confirmed histologically. Understanding the mechanism of the failure mode will help us to work out adequate strategies for B-T junction healing enhancement (Lu 2006; Wang et al. 2007; Wang et al. 2008). i The CSA of the B-T healing junction can be measured using a fine caliper by assuming that the healing junction is a square one, and its width and thickness are then directly measured using pQCT (Qin et al. 1999; Leung et al. 2002; Lu 2006; Wang et al. 2007; Wang et al. 2008). Micro-CT is an even better tool for measuring the CSA of the rabbit PPT healing interface.

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4. Discussion 4.1. Normal healing Our studies reported healing characteristics of the PPT healing junction after partial patellectomy, with an emphasis on osteogenesis and its remodeling with healing over time (Lu 2006; Qin et al. 1999; Qin et al. 2006a; Qin et al. 2006b; Wang et al. 2006b), where we found that the new bone formation and its size predicted the quality of its postoperative healing quality (Wang et al. 2006b). In addition, our studies also suggested that more new bone formation at the PPT healing interface was associated with better regeneration of interpositional fibrocartilage. This is an important index for future studies on potential interventions to be used for studying their treatment efficacy. Whether the findings generated from the PTT healing complex may also be generalized for radiographic prediction of direct B-T junction healing quality in regions like the Achilles tendon– calcaneus and rotator cuff requires further experimental and clinical investigations. Tendon cells are known to be able to undergo metaplasia around regions subjected to compressive forces. This phenomenon is also found in the current partial patellectomy model, as the PPT junction allows limited flexibility and transmission of large tensile and compressive forces (Chan et al. 2000; Leung et al. 1999). We also observed that the scar tissue next to the articular surface of the healing interface underwent cartilaginous metaplasia over time. Evaluation of its development and size would also be helpful as a functional index to predict PPT junction healing quality. These complicated biological and dynamic healing processes, e.g. the formation and remodeling of scarring tissues between adjunct healing tendon and bone, remain for further investigations, including the following: • • •

Cell recruitment and differentiation; Mechanical forces and cell anchorage to matrix; Timing of structural maturation and functional adaptation in conjunction with cell apoptosis; and

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Regeneration of interpositional fibrocartilage, especially the origins of fibrocartilage cells (e.g. differentiated from bone marrow cells and/or healing tendon cells and/or remodeling within the healing scar?).

4.2. Delayed healing Certainly, there are alternative or better approaches as piloted in the present study using latex to shield PPT healing for creating delayed B-T junction healing. Initially, we also tried mimicking delayed fracture healing using the “critical gap” concept for establishing delayed B-T healing, but it was not successful in our pilot study. This was attributed to technical difficulties in fixing both patellar tendon (soft tissue) and remaining proximal patella (bone) after partial patellectomy as compared with stable fixation for two bony fragments in establishing delayed fracture union. Delayed B-T healing is a new and not-well-established area for scientific exploration and clinical investigations. Our recent histological and histomorphometrical evaluations provided evidence of typical delay in B-T junction healing after 4 weeks’ shielding of the PPT healing interface in rabbits (Wang et al. 2007; Wang et al. 2008), which is similar to biological and histomorphological characteristics of delayed B-T healing in rats (Galatz et al. 2005) and delayed fracture union in rabbits (Rijal et al. 1994). This model can be used to evaluate both biological and biophysical interventions for their treatment effects that have been partially evaluated for the enhancement of B-T junction repair (Lu 2006; Markel et al. 1995; Pluhar et al. 2006; Qin et al. 2006a; Qin et al. 2006b; Wong et al. 2004; Wang et al. 2007; Wang et al. 2008).

5. Summary B-T healing is complex. Many biological and biomechanical aspects, especially those related to regeneration of the fibrocartilage zone, remain largely unexplained. The established normal and delayed rabbit B-T healing models provide us with a research platform or foundation to evaluate the potential biological and biophysical

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interventions developed or to be developed for acceleration and/or enhancement of B-T junction repair before they can be further validated and adopted into our orthopedic rehabilitation clinics.

Acknowledgments Financial support received for the related work from the Research Grants Council, Hong Kong SAR, China (CUHK4155/02M, CUHK4342/03M), and AO Research Grant (AO/ASIF 97-L15, AO/ASIF 03-Q26) is herewith acknowledged.

References Chan KM, Qin L, Hung LK et al. Alteration of patellofemoral contact during healing of canine patellar tendon after removal of its central third. J Biomech 33(11):1441–1451, 2000. Clark J, Stechschulte DJ Jr. The interface between bone and tendon at an insertion site: a study of the quadriceps tendon insertion. J Anat 192(4):605–616, 1998. Galatz LM, Rothermich SY, Zaegel M et al. Delayed repair of tendon to bone injuries leads to decreased biomechanical properties and bone loss. J Orthop Res 23:1441–1447, 2005. Galatz LM, Sandell LJ, Rothermich SY et al. Characteristics of the rat supraspinatus tendon during tendon-to-bone healing after acute injury. J Orthop Res 24:541–550, 2006. Hung LK, Lee SY, Leung KS et al. Partial patellectomy for patellar fracture: tension band wiring and early mobilization. J Orthop Trauma 7(3): 252–260, 1993. Leung KS, Qin L, Fu LLK, Chan CW. Bone to bone repair is superior to bone to tendon healing in patella–patellar tendon complex — an experimental study in rabbits. J Clin Biomech 17(8):594–602, 2002. Leung KS, Qin L, Leung MCT et al. Decrease in proteoglycans content of the remaining patellar articular cartilage after partial patellectomy in rabbits. J Clin Exp Rheumatol 17:597–600, 1999. Li G, Murnaghan M. Monitoring fracture healing using digital radiographies. In: Qin L, Genant HK, Griffith JF, Leung KS (eds.), Advanced Bioimaging Technologies in Assessment of the Quality of Bone and Scaffold Biomaterials, Springer Verlag, Berlin, pp. 537–546, 2007.

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Liu SH, Panossian V, al-Shaikh R et al. Morphology and matrix composition during early tendon to bone healing. Clin Orthop Relat Res 339:253–260, 1997. Lu HB. Low intensity pulsed ultrasound for accelerating bone–tendon junction healing. PhD thesis, The Chinese University of Hong Kong, Hong Kong, 2006. Markel DM, Wood SA, Bogdanske JJ et al. Comparison of allograft/ endoprosthetic composites with three types of gluteus medius attachment. J Orthop Res 13(1):105–114, 1995. Pluhar GE, Manley PA, Heiner JP et al. The effect of recombinant human bone morphogenetic protein-2 on femoral reconstruction with an intercalary allograft in a dog model. J Orthop Res 19(2):308–317, 2006. Qin L, Fok PK, Leng Y et al. Low intensity pulsed ultrasound increases the matrix hardness of the healing complex of bone–tendon junction healing — acoustic microscopic and micro-indentation study. Clin Biomech 21(4):387–394, 2006a. Qin L, Leung KS, Chan CW et al. Enlargement of remaining patella after partial patellectomy in rabbits. Med Sci Sports Exerc 31(4):502–506, 1999. Qin L, Lu HB, Fok PK et al. Low-intensity pulsed ultrasound accelerates osteogenesis at bone–tendon healing junction. Ultrasound Med Biol 32(12):1905–1911, 2006b. Recker RR (ed.). Bone Histomorphometry: Techniques and Interpretation. CRC Press, Boca Raton, FL, 1983. Rijal KP, Kashimoto O, Sakurai M. Effect of capacitively coupled electric fields on an experimental model of delayed union of fracture. J Orthop Res 12:262–267, 1994. Rufai A, Ralphs JR, Benjamin M. Structure and histopathology of the insertional region of the human Achilles tendon. J Orthop Res 13(4): 585–593, 1995. Wang L, Hu Y, Qin L, Chan KM. Recent development in bone–tendon junction repair and extracorporeal shockwave therapy. Chin J Sports Med [Chinese] 25(4):459–462, 2006a. Wang L, Lu HB, Fok PK et al. New bone formation and its size predict the repair at patella–patellar tendon healing complex in rabbits. Clin Biomech [Chinese] 21(4):291–297, 2006b.

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Wang L, Qin L, Lu HB et al. Extracorporeal shock wave therapy in treatment of delayed bone–tendon healing. Am J Sports Med 36(2):340–347, 2008. Wang W, Chen HH, Yang XH et al. Postoperative programmed muscle tension augmented osteotendinous junction repair. Int J Sports Med 28: 691–696, 2007. Wong MW, Qin L, Tai JK et al. Engineered allogeneic chondrocyte pellet for reconstruction of fibrocartilage zone at bone–tendon junction — a preliminary histological observation. J Biomed Mater Res 70B(2):362–367, 2004. Wong NW, Qin L, Lee KM et al. Healing of bone tendon junction in a bone trough: a goat partial patellectomy model. Clin Orthop Relat Res 413:291–302, 2003.

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Chapter 33

Anterior Cruciate Ligament Transection (ACLT)-Induced Osteoarthritis in Rats Ya-Feng Zhang, Jun-Fei Wang and Ge Zhang

There are no gold standard experimental models for osteoarthritis (OA). In recent years, around 25 different OA models have been reported, including surgically induced, enzymatically or chemically induced, spontaneous, genetically modified, and drug- or supplement-induced models using different animal species. Each model has its advantages and disadvantages. This chapter introduces an anterior cruciate ligament transection (ACLT)induced OA model in rats, and the relevant histological evaluation methods for confirmation of the successful establishment of this model. Keywords:

Osteoarthritis (OA); anterior cruciate ligament transection (ACLT); animal model; knee; rat; histology.

1. Introduction Osteoarthritis (OA) is the most common form of arthritis, and is recognized as one of the most important health problems in modern industrial societies (Badley and Wang 1998). The Chinese University of Hong Kong surveyed 426 adult men and 621 adult women in Hong Kong in 2000. The study found that, among men aged 50 years and older, 17% had persistent knee pain and 7% were diagnosed with Corresponding author: Ya-Feng Zhang. E-mail: [email protected]

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osteoarthritis of the knee. Among women aged 50 years and older, 24% had persistent knee pain and 13% were diagnosed with osteoarthritis of the knee (from university website, not published). These findings are very similar to the Framingham study in America (Kopec et al. 2007). Establishing OA animal models is important to evaluate and improve various diagnostic techniques and treatment methods for later clinical applications. OA animal models may include spontaneous OA models using aging animals (Jallali et al. 2005); the genetically modified OA mice model (Glasson et al. 2005); and surgically (Kamekura et al. 2005), enzymatically (Blom et al. 2004), or chemically (Bove et al. 2003) induced animal models. Among the surgically induced OA animal models, the anterior cruciate ligament (ACL) transection model in dogs (DeGroot et al. 2004) and the partial meniscus resection model in rabbits (Choi et al. 2007) have been widely used. These models continue to be investigated in these species and in other animals such as sheep (Funakoshi et al. 2007) and cat (Boyd et al. 2005), or in small animals such as rat (Hayami et al. 2006) and recently mouse (Clements et al. 2003). Compared with other animals, the rat provides a cost-effective and repeatable OA animal model (Hayami et al. 2006). This chapter describes the OA model in rats induced by anterior cruciate ligament transection (ACLT) that can be used as a platform for the evaluation of both pharmacological and nonpharmacological interventions.

2. Induction Protocol of ACLT-Induced Osteoarthritis Model 2.1. Materials • • •

Animals: 3-month-old Sprague–Dawley rats Anesthesia and pain relief drugs: sodium pentobarbital and Temgesic Surgical instruments and fixation materials: hair shaver, surgical scalpel, surgical clamp, microscissors, suture, rat operation table

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Evaluation devices and chemicals: specified in the corresponding sections below

2.2. Method (Stoop et al. 2000) • •

• • •

• •

• • • • •

The rat is kept fasting overnight before surgical operation. The rat is anesthetized with 2.5% sodium pentobarbital (60 mg/kg) intraperitoneal injection, and then fixed supinely on a rat operation table. The stifle joint is shaved using an animal hair shaver. 0.5% iodophor is used to disinfect the operating field. With the leg in extension, a medial parapatellar skin incision is made. An incision on the medial side of the patellar tendon provides access to the joint cavity. The patella is dislocated laterally when the knee is found in hyperextension. The knee joint is then placed at full flexion and an ACL is transected with microscissors. An anterior drawer test is performed to ensure complete transection of the ligament, and the joint surface is washed with sterile saline solution. The knee joint is extended and the patella is relocated. The capsule and subcutaneous tissue are closed with absorbable suture, e.g. Vicryl 4-0 braided absorbable suture. The skin is closed with monofilament 4-0 Nylon threads. In sham-operated animals, the wounds are closed after luxation of the patella and normal saline washing. Buprenorphine hydrochloride (0.1 mL/kg subcutaneously) is given as an analgesic immediately after the operation.

3. Assessment Protocol of ACLT-Induced Osteoarthritis Model 3.1. Gross appearance of the distal femur (Hayami et al. 2006) 3.1.1. Materials and facilities •

Anesthesia and pain relief drugs: sodium pentobarbital and Temgesic

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Surgical instruments: hair shaver, surgical scalpel, surgical clamp, scissors, saw, rat operation table Evaluation devices and chemicals: normal sodium and digital camera

3.1.2. Method • • • • •

The rat is fasted overnight before operation. The rat is sacrificed by an overdosage of 20% sodium pentobarbital. The stifle joint is shaved using an animal hair shaver. The knee is disarticulated, and then both the femur and tibia are dissected free of muscle and washed with sterile saline solution. The gross appearance of femoral condyles and tibial plateaux is recorded by a digital camera to evaluate the cartilage erosion.

3.2. Ink staining (Kobayashi et al. 2000) • •

•

Materials and facilities: normal saline, ink, digital camera Histology: The femoral condyles and tibial plateaux are stained with ink, then washed with normal saline, and finally recorded with a digital camera. Digitalization: After recording by digital camera, the femoral condyles and tibial plateaux are fixed using formalin for further processing.

3.3. Histological evalution (Kleemann et al. 2005) 3.3.1. Materials and facilities •

•

Materials: band saw, 0.01 M phosphate buffered saline (PBS), 10% EDTA (conventional reagents of hematoxylin and eosin [H&E] staining), Safranin O stain Facilities: tissue processor (Histokinette 2000; Reichert-Jung GmbH, Nussloch, Germany), tissue embedding unit (Thermolyne Sybron, Dubuque, IA, USA), rotary microtome (Reichert-Jung GmbH)

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3.3.2. Method •

• • • •

After fixation for 24 hours with formalin, the tibia is put in 10% EDTA to decalcify for 1 month. The EDTA solution is changed every week. After decalcification, the tibia is halved at the center of the articular surface along the medial collateral ligament in the frontal section. Then, the samples are embedded in paraffin using a tissue embedding unit. The paraffin blocks are cut into 6-µm-thick sections for further staining. The sections are stained with H&E stain solution and Safranin O stain solution using standard protocols.

3.4. Vascular invasion into calcified cartilage (Hayami et al. 2004; O’Connor 1997) Vascular invasion into the calcified cartilage can be quantified by counting the number of contacts of the calcified cartilage to the subchondral marrow space.

3.5. Histopathological scores •

• •

Semiquantitative histopathological grading is performed according to the Mankin scoring system established for grading OA changes (Cake et al. 2000). Three sections from each sample with 100 µm apart are scored, as suggested by two independent observers. Mankin scoring system (Mankin et al. 1971): Table 1 summarizes the Mankin scoring system for data comparison. Data analysis: The Mankin scores between different groups are compared according to the study designs of researchers.

4. Discussion Animal models have played an important role in improving the understanding of early events occurring during OA disease progression,

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A Practical Manual for Musculoskeletal Research Table 1. Mankin scoring system (Mankin et al. 1971). Grade

I Structure a. Normal b. Surface irregularities c. Pannus and surface irregularities d. Clefts to transitional zone e. Clefts to radial zone f. Clefts to calcified zone g. Complete disorganization II Cells a. Normal b. Diffuse hypercellularity c. Cloning d. Hypocellularity

0 1 2 3 4 5 6 0 1 2 3

Grade III Safranin O staining a. Normal b. Slight reduction c. Moderate reduction d. Severe reduction e. No dye noted

IV Tidemark integrity a. Intact b. Crossed by blood vessels

0 1 2 3 4

0 1

since available human joint tissues usually represent advanced OA. There are many methods to establish animal models for OA, for example, spontaneous OA models; genetically modified animal models; and surgically, enzymatically, or chemically induced animal models. However, up to now, no consensus currently exists regarding which model and species is the most relevant for human OA. The lack of a gold standard animal model of OA originates from a poor understanding of the disease etiology. Although no consensus currently exists regarding the most relevant animal model for OA, it is clear that each model has its advantages and disadvantages. Spontaneous models best mimick the slow progression of the human disease, but their reproducibility is poor and the variation between individuals is big; moreover, spontaneous models are quite time-consuming. Genetically modified mice models constitute the best tool for mechanistic studies aimed at understanding the functional role of specific molecules in cartilage homeostasis and OA pathology, but their physiologic relevance to the human disease is questionable.

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Surgically and enzymatically induced models develop rapid and reproducible damage, but they are more relevant to traumatic forms of OA than the classical degenerative form of OA. Chemically induced models develop even more rapid damage, but their physiological relevance is debatable. Among animals, as far as the same model is concerned, the rate of disease progression increases as the size and lifespan of the animal decreases (Ameye and Young 2006). Because there is no gold standard model of OA, even the same treatment can cause different results; for example, vitamin C increased the severity of spontaneous knee OA in guinea pig (Kraus et al. 2004), but the same dosage decreased the severity of surgically induced knee OA (Schwartz et al. 1981). Therefore, it is important to choose suitable models for different purposes. The early-onset and severe models are more economical and would probably lead to a lower number of false positives, but they could also cause false-negative results of treatment. In contrast, the slow-onset models are more time-consuming and expensive, and the variations between individuals are big; however, because they closely mimick the human disease, they would probably lead to a lower number of false-negative treatment results. Two points should be highlighted regarding the technique employed in the ACLT model. First, after cutting the ACL, perform an anterior drawer test to make sure that the ACL is fully cut. Second, when cutting the ACL, be careful not to injure the cartilage.

5. Summary As reviewed by Ameye and Young (2006), too many different models of OA have been reported. One should choose a suitable model for a particular research purpose. Although the ACLT-induced OA model in rat is a traumatic form of OA rather than a classical degenerative form of OA, it has some advantages such as being cheap, quick, reliable, and reproducible; therefore, it is a reliable model for OA basic research.

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References Ameye LG, Young MF. Animal models of osteoarthritis: lessons learned while seeking the “Holy Grail”. Curr Opin Rheumatol 18(5):537–547, 2006. Badley EM, Wang PP. Arthritis and the aging population: projections of arthritis prevalence in Canada 1991 to 2031. J Rheumatol 25:138–144, 1998. Blom AB, van Lent PL, Holthuysen AE et al. Synovial lining macrophages mediate osteophyte formation during experimental osteoarthritis. Osteoarthritis Cartilage 12(8):627–635, 2004. Bove SE, Calcaterra SL, Brooker RM et al. Weight bearing as a measure of disease progression and efficacy of anti-inflammatory compounds in a model of monosodium iodoacetate-induced osteoarthritis. Osteoarthritis Cartilage 11(11):821–830, 2003. Boyd SK, Muller R, Leonard T, Herzog W. Long-term periarticular bone adaptation in a feline knee injury model for post-traumatic experimental osteoarthritis. Osteoarthritis Cartilage 13(3):235–242, 2005. Cake MA, Read RA, Guillou B, Ghosh P. Modification of articular cartilage and subchondral bone pathology in an ovine meniscectomy model of osteoarthritis by avocado and soya unsaponifiables (ASU). Osteoarthritis Cartilage 8(6):404–411, 2000. Choi SI, Heo TR, Min BH et al. Alleviation of osteoarthritis by calycosin7-O -beta-D-glucopyranoside (CG) isolated from Astragali radix (AR) in rabbit osteoarthritis (OA) model. Osteoarthritis Cartilage 15(9):1086–1092, 2007. Clements KM, Price JS, Chambers MG et al. Gene deletion of either interleukin-1beta, interleukin-1beta-converting enzyme, inducible nitric oxide synthase, or stromelysin 1 accelerates the development of knee osteoarthritis in mice after surgical transection of the medial collateral ligament and partial medial meniscectomy. Arthritis Rheum 48(12): 3452–3463, 2003. DeGroot J, Verzijl N, Wenting-van Wijk MJ et al. Accumulation of advanced glycation end products as a molecular mechanism for aging as a risk factor in osteoarthritis. Arthritis Rheum 50(4):1207–1215, 2004. Funakoshi Y, Hariu M, Tapper JE et al. Periarticular ligament changes following ACL/MCL transection in an ovine stifle joint model of osteoarthritis. J Orthop Res 25(8):997–1006, 2007.

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Glasson SS, Askew R, Sheppard B et al. Deletion of active ADAMTS5 prevents cartilage degradation in a murine model of osteoarthritis. Nature 434(7033):644–648, 2005. Hayami T, Pickarski M, Wesolowski GA et al. The role of subchondral bone remodeling in osteoarthritis: reduction of cartilage degeneration and prevention of osteophyte formation by alendronate in the rat anterior cruciate ligament transection model. Arthritis Rheum 50(4): 1193–1206, 2004. Hayami T, Pickarski M, Zhuo Y et al. Characterization of articular cartilage and subchondral bone changes in the rat anterior cruciate ligament transection and meniscectomized models of osteoarthritis. Bone 38(2): 234–243, 2006. Jallali N, Ridha H, Thrasivoulou C et al. Vulnerability to ROS-induced cell death in ageing articular cartilage: the role of antioxidant enzyme activity. Osteoarthritis Cartilage 13(7):614–622, 2005. Kamekura S, Hoshi K, Shimoaka T et al. Osteoarthritis development in novel experimental mouse models induced by knee joint instability. Osteoarthritis Cartilage 13(7):632–641, 2005. Kleemann RU, Krocker D, Cedraro A et al. Altered cartilage mechanics and histology in knee osteoarthritis: relation to clinical assessment (ICRS Grade). Osteoarthritis Cartilage 13(11):958–963, 2005. Kobayashi K, Amiel M, Harwood FL et al. The long-term effects of hyaluronan during development of osteoarthritis following partial meniscectomy in a rabbit model. Osteoarthritis Cartilage 8(5):359–365, 2000. Kopec JA, Rahman MM, Berthelot JM et al. Descriptive epidemiology of osteoarthritis in British Columbia, Canada. J Rheumatol 34(2): 386–393, 2007. Kraus VB, Huebner JL, Stabler T et al. Ascorbic acid increases the severity of spontaneous knee osteoarthritis in a guinea pig model. Arthritis Rheum 50(6):1822–1831, 2004. Mankin HJ, Dorfman H, Lippiello L, Zarins A. Biochemical and metabolic abnormalities in articular cartilage from osteo-arthritic human hips. II. Correlation of morphology with biochemical and metabolic data. J Bone Joint Surg Am 53(3):523–537, 1971. O’Connor KM. Unweighting accelerates tidemark advancement in articular cartilage at the knee joint of rats. J Bone Miner Res 12(4):580–589, 1997.

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Schwartz ER, Oh WH, Leveille CR. Experimentally induced osteoarthritis in guinea pigs: metabolic responses in articular cartilage to developing pathology. Arthritis Rheum 24(11):1345–1355, 1981. Stoop R, Buma P, van der Kraan PM et al. Differences in type II collagen degradation between peripheral and central cartilage of rat stifle joints after cranial cruciate ligament transection. Arthritis Rheum 43(9):2121–2131, 2000.

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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

Physeal injury is not uncommon in pediatric orthopedics, with Salter– Harris type II (SH II) fracture being the most common type that may lead to growth arrest and eventually limb shortening. Therefore, research on SH II fracture will hold great potential to benefit children with such an injury. This chapter outlines the creation of a partial growth plate defect model in rabbits that mimicks a SH II fracture for applications in various growth plate or articular cartilage research topics. The establishment of an SH II rabbit model described in this chapter provides some relevant and applicable evaluation methods. This model will be helpful for research on the biology of premature physeal closure during injuries or the exploration of new biomaterials for physeal reconstruction. Keywords:

Growth plate; osteotomy; physeal closure; physeal injury; Salter–Harris fracture.

1. Introduction Growth plate fracture is a common pediatric orthopedic condition that results from traumatic incident, athletic stress, or overuse. If the patient does not receive immediate and proper medical attention, it Corresponding author: Kwoon-Ho Chow. Tel: +852-26323312; E-mail: [email protected]. edu.hk

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may well cause premature physeal closure and deformity, eventually requiring reconstructive or corrective surgery (Rohmiller et al. 2006). The Salter–Harris (SH) classification is the most accepted system of classifying fractures involving the growth plate. A Salter–Harris type II (SH II) fracture has the highest occurrence amongst all of the five types (Salter and Harris 1963; Czitrom et al. 1981; Kawamoto et al. 2006). Such a fracture typically involves a fracture in part of the growth plate and the metaphysis, creating a triangular fragment of the metaphysis attached to the intact epiphysis. SH II fractures are considered to be lower-risk SH fractures and require no open reduction (Salter and Harris 1963; Weber et al. 1980); however, some suggest that an open reduction could reduce the incidence of premature physeal closure (Rohmiller et al. 2006). An emerging branch of reconstructive orthopedics is also suggesting the application of bioengineered biomaterials to restore the growth of fractures involving the growth plate (Lee et al. 2003; Jin et al. 2006). Some of these groups have introduced physeal defect animal models, mostly by the creation of a slit, by saw osteotomy directly at the physeal plate (Johnstone et al. 2002). However, their models not only destroy the cartilage at the growth plate; they often involve the destruction of cortical surfaces of the growth plate, immediately underneath the growth plate cartilage on the contacting surfaces between the metaphysis and epiphysis, and thus cannot provide a direct comparison to the real clinical situation or simulate clinical outcomes. Hence, the purpose of establishing this animal model is to supplement additional details in the biology of this type of fracture healing, and to provide a model to investigate the treatment efficacies of various biophysical interventions in healing this type of fracture. The following advantages could be provided by this model: • •

•

At the site of defect, bony surfaces of the growth plate remain intact on the contacting surfaces between the metaphysis and epiphysis. The defective growth plate can be compared to the normal growth plate in the exact same bone, at the same site, on the same histological section. The model provides an easily accessible site for the transplantation of various bioengineered materials for parallel comparison studies.

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2. Materials and Methods 2.1. Materials • • •

•

Animals: young rabbits at 10 weeks of age, either male or female a Anesthesia and pain relief drugs: sodium pentobarbital and Temgesic Autoclaved surgical instruments and fixation materials: hair shaver, scalpel, forceps, oscillating saw, stainless steel wire, power drill with drill bit, curette, resorbable and nonresorbable sutures, needle holder (Fig. 1) Evaluation devices: specified in Sec. 3

2.2. Methods •

• •

General anesthesia by 2.5% pentobarbital (Sigma Chemicals Co., USA) is administered intravenously at 1.0 mg/kg. The entire procedure for creating the defect will normally take 1 hour to complete. An X-ray radiograph is taken preoperatively to confirm the unclosed growth plate at the distal femur (Chan et al. 2006). After shaving and disinfecting the surgical site, an anteromedial incision of approximately 4 cm is made to the skin at the distal femur longitudinally towards the proximal direction. A further incision through the muscle of the vastus medialis and rectus femoris is made to expose the distal femur.b

a New Zealand white rabbits typically reach skeletal maturity at 20–22 weeks of age (Masoud et al. 1986; Ross and Zionts 1997). Ten-week-old rabbits are used because it will grant 10–12 weeks of an active growth period to monitor growth and to observe differences in growth patterns. The creation of the defect involves general anesthesia and invasive procedures; therefore, using rabbits under the age of 8 weeks is not suggested, due to their lower tolerance to anesthetic drugs as well as their inadequacy in femur stiffness and size for surgery. b It is important to be careful not to damage the femoral artery and vein that are running between the vastus medialis and gracillus, as it would cause an excessive amount of blood loss during the operation. To minimize bleeding during the operation, an elastic band at the upper thigh might be considered to temporarily restrict blood flow to the area.

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(a)

(b)

(c)

Fig. 1. Some of the instruments required to establish the model. (a) A curette is used to completely remove the growth plate cartilage at the defective site. (b) A pneumatic-powered drill is used to make 1.0-mm-diameter holes for fixation. (c) A pneumatic-powered oscillating saw is used to create the triangular fragment.

•

•

•

Two holes each with a 0.5-mm diameter are predrilled on the diaphysis bicortically in an anterior-posterior direction for wire fixation later. Once the distal femur is exposed, the patella is laterally displaced to expose the articular surface of the distal femur. An oscillating saw is used to make a transverse resection from the midpoint of the growth plate diagonally towards the anterior surface of the diaphysis. A triangular fragment of the defect is created (Fig. 2). A small No. 3 scalpel is used to cut along the growth plate cartilage on the anterior side, leaving the posterior half of the growth plate untouched and intact. Force is applied horizontally on the

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Fig. 2. Schematic representation of the partial physeal defect model. (a) A lateral view of the distal femur is shown with the epiphysis, metaphysis, and diaphysis. (b) After osteotomy by an oscillating saw, force is applied sideways in the transverse plane of the animal to dislocate the fragment. (c) A triangular metaphyseal fragment displacement.

triangular fragment at the diaphyseal end until the fragment is dislocated [Fig. 3(a)].c c

After sufficient scalpel cutting along the exposed line of the growth plate, a horizontal force is applied along the transverse plane, perpendicular to the sagittal plane, using the epiphysis as the pivoting point. Be careful not to damage any articular cartilage that might induce unwanted pain and functional disturbance to the animal.

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(a)

(b)

(c)

(d)

(e)

Fig. 3. Operational photos demonstrating the creation of the defect. (a) The fragment is shown displaced, a typical characteristic of the Salter–Harris type II fracture. (b) The growth plate cartilage is removed to ensure the occurrence of premature physeal closure and growth arrest. (c) The fragment is fixed with a thin stainless steel wire. (d) The muscular incision is closed. (e) Postoperative radiography is used as a reference point to monitor the healing process.

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• •

•

•

575

After exposing the physis, a curette is used to scrape off all cartilage tissue on both the metaphyseal surface and the epiphyseal surface of the fragment until the bone is exposed [Fig. 3(b)]. The bone fragment is replaced to its original position and fixed with a 0.5-mm-diameter stainless steel wire [Fig. 3(c)]. The wound is closed with 3-0 nonabsorbable suture (Ethilon; Johnson & Johnson, USA). Antibiotic spray (Nebactic; Byk Gulden Konstanz, Germany) is used to treat the wound for disinfection [Fig. 3(d)]. For postoperative pain control, Temgesic® (Reckitt & Colman Pharmaceuticals, Hull, UK) is given to relieve pain subcutaneously at a dose of 0.01 mg/kg for 3 consecutive days. Cast immobilization is not recommended in this animal model.d X-ray is taken postoperatively to monitor the healing process [Fig. 3(e)].

3. Postoperative Evaluation This animal model is suitable for the study of different methods that are intended to correct a Salter–Harris type II fracture. The length of the long bone, morphology of the bone in both size and shape, and calcification of the physeal region are evaluated with respect to the contralateral control limb. Other parameters or evaluation technologies shall also be employed to answer specific research questions, depending on the study hypothesis and/or objectives. • •

d

Euthanasia of rabbit is carried out with an intravenous injection of overdosed pentobarbital at 20% m/v, 1.0 mL/kg. Femurs on both sides are harvested at the time of euthanasia. After removing soft tissues surrounding the distal femur, general morphology is recorded by digital photography and any visible deformity is recorded [Fig. 4(a)].

At the proximal end of the metaphyseal defect, there exists a mechanically weakest point. A cast on the femur will limit the range of motion but create a pivot, and so increase the chances of a fracture occurring at the weakness point.

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Fig. 4. Possible evaluation methods applicable to this rabbit model. (a) Gross morphology of an operated (L) and nonoperated (R) distal femur. The operated distal femur shows deformity due to a premature growth plate closure. (b) The rabbit is euthanized after 8 weeks of postoperative followup. Image analysis software is used to measure the length of the long bones. (c) Sagittal section of a normal rabbit distal femur. The sample is harvested at the age of 18 weeks, 8 weeks after sham operation. H&E staining, 1.6×. (d) Distal femur shown with anterior growth plate destroyed and signs of calcification after 8 weeks, whereas the posterior growth plate is left intact. H&E staining, 1.6×.

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The length of long bones is measured at the endpoint by X-ray radiography [Fig. 4(b)] and an image analysis system (MetaMorph image analysis system; Universal Imaging Corp., PA, USA) (Leung et al. 2004).e Histological analysis


e

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The distal femurs are dehydrated and fixed with formalin and sequential ethanol baths. The specimens are then decalcified

Anteroposterior (AP) views of the digitized radiographs are used to measure the femoral lengths. The longest distance between the greater trochanter and the femoral condyle is measured by an image analysis software of choice. At least three repeated measurements are made and averaged.

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in 9% formic acid, changed once every 2 days, for 14–21 days.f They are then subjected to an automatic tissue processing program (Shandon Tissue Processor; Thermo Electron Corp., MA, USA). After removing the stainless steel wire, the processed specimens are embedded in paraffin wax. Seven-micrometer sections are made with a microtome (Leica Microsystems GmbH, Wetzlar, Germany). Standard staining of hematoxylin and eosin (H&E) and Safranin O is used to study the histological sections (Lee et al. 2003) [Figs. 4(c) and 4(d)].g

Acknowledgment The establishment of this animal model was supported by the RGC Earmarked Grant, Research Grant Council of Hong Kong (CUHK 4510/05).

References Chan CW, Qin L, Lee KM et al. Dose-dependent effect of low-intensity pulsed ultrasound on callus formation during rapid distraction osteogenesis. J Orthop Res 24(11):2072–2079, 2006. Czitrom AA, Salter RB, Willis RB. Fractures involving the distal epiphyseal plate of the femur. Int Orthop 4(4):269–277, 1981. Jin XB, Luo ZJ, Wang J. Treatment of rabbit growth plate injuries with an autologous tissue-engineered composite: an experimental study. Cells Tissues Organs 183(2):62–67, 2006.

f

The decalcification period varies, depending on the size and volume of the bone tissue. Usually, after 14 days of decalcification, X-ray radiographs can be taken to confirm completion of the process. If calcified mineral still remains, extend the decalcification period in 9% formic acid for 1 extra week with repeated confirmation by X-ray. g Potentially suitable repairing methods related to Salter–Harris fractures can be investigated by using this model. Many reports have suggested that either scaffold or scaffold-free methods, for example, a conventional chondrocyte pellet or various biomaterials (e.g. collagen, alginate, demineralized bone matrix), carry high restoring potential in this type of physeal damage (Lee et al. 2003; Jin et al. 2006).

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Johnstone EW, McArthur M, Solly PB et al. The effect of osteogenic protein-1 in an in vivo physeal injury model. Clin Orthop Relat Res (395): 234–240, 2002. Kawamoto K, Kim WC, Tsuchida Y et al. Incidence of physeal injuries in Japanese children. J Pediatr Orthop B 15(2):126–130, 2006. Lee KM, Cheng AS, Cheung WH et al. Bioengineering and characterization of physeal transplant with physeal reconstruction potential. Tissue Eng 9(4):703–711, 2003. Leung KS, Cheung WH, Yeung HY et al. Effect of weightbearing on bone formation during distraction osteogenesis. Clin Orthop Relat Res 419: 251–257, 2004. Masoud I, Shapiro F, Kent R, Moses A. A longitudinal study of the growth of the New Zealand white rabbit: cumulative and biweekly incremental growth rates for body length, body weight, femoral length, and tibial length. J Orthop Res 4(2):221–231, 1986. Rohmiller MT, Gaynor TP, Pawelek J, Mubarak SJ. Salter–Harris I and II fractures of the distal tibia: does mechanism of injury relate to premature physeal closure? J Pediatr Orthop 26(3):322–328, 2006. Ross TK, Zionts LE. Comparison of different methods used to inhibit physeal growth in a rabbit model. Clin Orthop Relat Res 340:236–243, 1997. Salter RB, Harris WR. Injuries involving the epiphyseal plate. J Bone Joint Surg Am 45:587–622, 1963. Weber BG, Brunner CH, Freuler F. Treatment of Fractures in Children and Adolescents. Springer, Berlin, 1980.

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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

Nondestructive three-dimensional (3D) micro-computed tomography (CT) image analysis used in orthopedic research needs to be accompanied by adequate tools for the numerical assessment of experimental systems. Such quantitative tools should be user-friendly and intuitive, not too complex for the orthopedic researcher to implement, as well as accurate and repeatable in order to be suitable for laboratory application. Here, two experimental systems are examined and straightforward micro-CT analysis methods are described, allowing the experimental outcomes to be accurately quantified in a flexible and multidimensional manner. These systems include the study of osteointegration around a metal implant in bone, and the study of porosity of a biocompatible scaffold matrix for tissue engineering (specifically, the study of scaffolds for permeability to cellular ingrowth) Both studies involve a number of standard image analysis techniques applied in a 3D manner, such as erosion and dilation (applied flexibly to both the image and the region of interest), distance transforms, and novel techniques like “shrink wrap”. Applied in combination in an easily programmable “task list” (otherwise known Corresponding author: Phil Salmon. E-mail: [email protected]

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A Practical Manual for Musculoskeletal Research as “scripting”), these functions provide a powerful and versatile range of 3D structural analyses. Keywords:

Micro-CT; bone; implant; scaffold; osteointegration; porosity; methods.

1. Introduction Orthopedic preclinical research is proving to be a fertile ground for applications of the technique of micro-computed tomography (micro-CT). High-quality three-dimensional (3D) X-ray image data can be obtained from bones of rabbits, rats, and mice, as well as from synthetic biomaterials such as bone-like cements and scaffolds for tissue engineering. With the increasing ability to generate high-resolution 3D image data for orthopedic research, however, follows the requirement to analyze this image data in order to obtain quantitative measurement endpoints and realize experimental objectives. There are three principal requirements for the measurement of quantitative parameters from micro-CT datasets in experimental studies, orthopedic or otherwise: (1) the measurements are repeatable (using standardized protocols and referenced volumes of interest for analysis); (2) they are accurate; and (3) they are reasonably practicable, not excessively complex or time-consuming, and suitable for the necessary experimental throughput rate. Out of the many and increasing experimental applications of microCT in the orthopedic field, two examples will be examined here: (1) the study of the contact and integration of bone around a metallic implant, and (2) the study of the porosity of a synthetic biomaterial. This will include both micro-CT imaging and the subsequent quantitative image analysis. These are two of the most widely used orthopedic micro-CT applications (or potential applications); interestingly, they represent opposite extremes in terms of the physical characteristics and X-ray density of samples for micro-CT analysis. Metals such as titanium and steel are a challenge for micro-CT imaging due to their high X-ray density; while bioscaffold materials such as poly(lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA), and particularly collagen in dry form, are at the low end of material densities scannable by micro-CT.

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Anyone beginning to apply micro-CT to the imaging of bones with metal implants soon confronts the reality of the imaging difficulties and artifacts caused by the very high X-ray absorption of metals. For high spatial resolution in the X-ray imaging of biological tissues, and indeed of many other materials where micron-level resolution is required, relatively low X-ray energies are necessary for appropriate transmission and contrast in the sample. Over the range of X-ray energies used for micro-CT (about 10–50 keV), X-ray absorption is approximately proportional to the fourth power of the atomic number (Z ) of the chemical elements in absorbing material. This acute sensitivity of X-ray absorption to variations in elemental composition is fortunate, of course, since it makes X-ray imaging possible in the first place; it provides, for example, excellent contrast between calcium in bones (Z = 20) and other soft tissue (Z < 10). However, the problems begin when a metal is added to the picture. The high Z of most metals — combined with their physical density — results in very strong absorption of the X-rays used in micro-CT, causing imaging artifacts following Feldkamp tomographic reconstruction. Titanium (Ti) — from which many orthopedic implants are constructed — has a Z of 22, only two higher than that of calcium, although titanium metal is monoelemental; while calcium (Ca) atoms comprise only a small percentage of all the atoms in bone, the rest being low-Z biological elements such as hydrogen, carbon, oxygen, and nitrogen. As a result, titanium metal has severalfold higher X-ray absorption in micro-CT scanners compared to bone. However, adequate X-ray transmission through Ti is possible in micro-CT by the use of a high applied X-ray voltage such as 100 kV and a strong X-ray filter containing copper, as illustrated in Fig. 1(a). The imaging results are better if the titanium insert is not too large. But if you move just four places higher in the periodic table to iron (Fe, Z = 26), the chief constituent of steel, the picture changes dramatically, and the shadow image and other tomographic artifacts of high density become very much worse than for Ti. This means that, in practice, accurate analysis of bone surrounding a Ti implant by micro-CT is practicable, while for steel implants it is much more difficult and at the margin of what is achievable. Figure 1(b) shows the more-severe-density artifacts associated with a steel implant in a canine bone.

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Fig. 1. (a) Coronal section from a micro-CT scan of a titanium screw implant (2.2 mm in diameter) in a rat distal femur (SkyScan 1172; 7.8 micron voxels). Scanning at maximum X-ray energy at over 360° helps to minimize X-ray density artifacts associated with the metal, allowing analysis of bone close to the implant surface. (b) A cross-section through a canine femur containing a steel pin with a 6.5-mm diameter. The blood vessels in the surrounding muscle have been infiltrated with X-ray opaque MicrofilTM contrast resin. Radial density artifacts are quite prominent, significantly worse than with titanium, and the image of the bone surrounding the pin is severely disrupted.

At the other extreme, biological scaffold materials such as PLA/PLGA and collagen need to be scanned and analyzed in their dry form in order, for example, to evaluate the porosity and pore interconnectivity parameters relating to the scaffold’s suitability for

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supporting cellular ingrowth. The resulting materials are very light and resemble cotton wool. These dry scaffolds have a low density, and require scanning by micro-CT with no X-ray filter and a low applied voltage (20–40 kV). Low X-ray energy needs to be combined with the highest possible spatial resolution to resolve fine dry structures with quite low X-ray absorption and contrast (Fig. 2). These two examples of orthopedic micro-CT applications, the metal implant in bone (contact surface) and the bioscaffold “wool” (porosity), will be examined in turn. Methods for obtaining quantitative parameters of implant osteointegration and bioscaffold porosity using purpose-built 3D image analysis software will be described in sufficient detail to allow the reader to apply these analyses.

2. Analysis of the Bone Surrounding a Metal Implant As mentioned above, metals’ high density means that the micro-CT scan should be configured for the highest sample density. There are three parameters of the micro-CT scan that can help to get the best results for an implant sample: (1) maximum applied voltage and thickest filter to get the maximum average X-ray energy; (2) scanning over 360° instead of 180°; and (3) suppression of noise by means of multiple frames averaging at each rotation step. One consequence of all these measures is a relatively long scan time. We will look at the dataset of a scan of a titanium screw implanted in a rat femur, and go through the steps involved in quantifying the amount of bone present at varying distances from the titanium implant surface.

2.1. Step 1: getting the implant dataset in the best orientation for analysis In the image shown in Fig. 1, the Ti screw comes into the bone at a diagonal angle. To standardize the analysis of the implant surface, it is convenient to resample the dataset so that the implant is close to

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Fig. 2. (a) Micro-CT cross-section and (b) maximum intensity projection (MIP) of a PLA/PLGA scaffold (SkyScan 1172; 8 micron voxels). (c) MicroCT cross-section and (d) maximum intensity projection (MIP) of a dry collagen “wool” sample (SkyScan 1172; 1.5 micron voxels).

being normal to the cross-sectional plane, i.e. it is “sticking out” of the image plane at right angles (in the z-plane). Furthermore, we are interested in analyzing the implant–bone surface, but not the inside surface of the implant if it is hollow, as in the present case. This is

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another reason for resampling the implant to a true cross-sectional orientation — the hollow center is then fully enclosed and can be easily “filled in” (as a binary image) and excluded from the analysis. There are two ways to resample a dataset into any required 3D orientation. One way is to build a rendered 3D model of the scanned object, then in the 3D software environment create a virtual plane and manipulate the plane into the desired orientation relative to the object. While this method has the advantage of direct 3D (or 3D-like) visualization and involves a single resampling step only, in practice it is time-consuming and requires many fine adjustments of the model’s and the plane’s alignment to be sure of the correct planar orientation. Note also that in 3D visual environments (such as OpenGL softwarecreated virtual environments), there is a user-selectable angle or perspective such that objects distort in relation to distance in a way simulating real-life viewing of objects or landscapes. This decreases the objectivity and precision of resampling from 3D model viewing software. The second alternative for resampling into a different 3D orientation is to make two successive resamplings in a two-dimensional (2D) plane. The resamplings are serial, so that the second recut is carried out on the images produced by the first resampling. It turns out that by making two cuts in this way, one can achieve reslicing into any 3D angle (consider that 3D angles can be defined by two “2D” angles only, the polar θ and the azimuthal π). This method requires two steps instead of one, but has the advantage of being entirely carried out in the cross-sectional analysis software without the need to perform an additional operation on a 3D model in the 3D viewing software. It also involves many fewer user operations by avoiding the repeated fine-tuning needed for model and plane recuts in 3D viewing software (in SkyScan systems, the CT-analyzerTM program for dataset cross-sectional analysis and the CT-volumeTM program for 3D viewing are separate programs that work together). However, the user can perform the resampling by either the 3D-model-based method or the serial 2D reslicing method according to individual preference. The two-step cross-sectional reslicing method is illustrated in Fig. 3. Figure 3(a) shows a cross-section from the original scan. The orientation

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Fig. 3. Two serial reslicing operations in the cross-sectional plane allow the dataset to be resampled into the plane in which the Ti implant is cut orthogonally in the cross-section, making for a more normalized and accurate measurement of the bone contact surface. In each “cut”, the dataset is resampled in the “out-of-the-page” z-direction, along the indicated red line drawn by the user. The first cut (a) creates a dataset with the pin aligned parallel to the xy-plane only. The second cut (b) creates a true cross-sectional dataset with regard to the pin (c), with its axis running exactly along the dataset z-axis.

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of a bone containing an implant during a scan is usually determined by the axial alignment of the bone along the axis of scan rotation, especially if the bone is long (note, however, that the need for dataset resampling could be obviated altogether if it is possible to align the bone sample for scanning in such a way that the metal pin, not the bone, is axially aligned to the scan rotation axis; this might be possible if the bone is cut into a small size). In the example shown here, the bone — a rat femur much longer than it is wide — is scanned vertically aligned with the scan axis as usual for a bone micro-CT scan, so that the Ti implant enters the bone at a diagonal angle which is not parallel with any of the xy-, xz-, or yz-planes. The first reslice operation [Figs. 3(a) and 3(b)] aligns the pin parallel to the xy-plane only. The second cut [Figs. 3(b) and 3(c)] creates a true cross-sectional dataset with regard to the pin, with its axis running exactly along the dataset z-axis;. this is the true cross-sectional orientation most suitable for analysis of the bone contact area near the pin, as is described below.

2.2. Step 2: creating a volume-of-interest (VOI) mask and measuring the bone “intersection” contact at and near the implant surface The large difference in density between the titanium implant and the bone makes it easy to binarize the metal implant. Figure 4 shows how, once a binary mask of the implant is created, this can first be filled to remove the central hole and then dilated; the dilated mask is then converted into the region of interest (ROI). The reason for the dilation is to allow bone contact to be measured at a selected distance away from the metal implant surface. The analysis software measures both the total surface area of the ROI and the subset of the ROI surface that is intersected by binarized bone objects. The parameter thus measured is called the “intersection surface” and corresponds to the bone contact surface. This method allows the bone contact surface to be measured at virtual ROI boundary surfaces that are at any

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Fig. 4. Measurement of the bone contact surface at a selected distance from the implant. (a) Implant and bone can be separately thresholded. (b) The binary mask of the hollow implant is filled and dilated by a selected number of pixels (here, 20), and then the dilated binary mask is converted into the region of interest (ROI). (c) The original gray-level image is reloaded into the ROI. (d) The reloaded image is thresholded to the bone, and the contact surface at the ROI periphery is measured.

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selected distance from the metal surface. This provides an extra dimension of depth to the study of bone–implant contact: the contact area can be measured as a profile with increasing distance from the implant surface. Furthermore, moving at least a small distance away from the implant is usually necessary for measuring bone contact, due to the density artifacts that affect the micro-CT cross-sectional images in the immediate vicinity (a few pixels) of the metal surface. Figure 5 shows the bone intersection surface with the successively dilated ROI around titanium implants in two rat femurs. One implant is close to the growth plate at the distal femur, while the second one has the implant further from the growth plate toward the diaphysis. Comparing the contact profiles in Fig. 5, the contact area at both sites is similarly close to the metal surface, but becomes greater at the near metaphysis than at the far metaphysis at increasing distances from the implant surface.

3. Analysis of the Porosity of Biological Scaffolds The porosity of materials such as scaffold biomaterials can be defined in a number of ways. The most straightforward one is simply the volume of space as a fraction or percentage of the total volume of space plus scaffold material. This measurement by micro-CT is done by binarizing the solid structural elements of the scaffold and then counting the solid and space voxels. The selection of a binary threshold is a complex issue: while choosing a suitable threshold by visual comparison of raw and binarized images does provide a valid basis for the comparison of different samples of the same type, some prefer the objectivity of a more automated method such as the Otsu algorithm for identifying global threshold values (Otsu 1979) or more sophisticated segmentation algorithms such as local thresholding (Waarsing et al. 2004) or the BLOB3D technique (Ketcham 2005). Twelve different segmentation techniques are evaluated by Rajagopalan et al. (2005). The spatial resolution of the scan is a critical factor with regard to the segmentation of micro-CT datasets. Higher spatial resolution

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Fig. 5. The bone intersection surface with a dilated ROI mask around a metal implant surface is measured as a profile with increasing distance from the metal implant surface. This profile is shown for two identical titanium implants in two rat femurs: one implant in the distal femur metaphysis close to the growth plate [(a), “near metaphysis”], and the other implant inserted about 7 mm away from the growth plate toward the diaphysis [(b), “far metaphysis”]. The intersection surface profiles with increasing distance from the implant surface for the two sites are shown in (c).

with smaller voxel sizes facilitates the finding of global threshold values, and makes the results of analysis less sensitive to variation in the binary threshold value. In contrast, low resolution scans are relatively more affected by the partial volume effect, as the width of structural elements in terms of image voxels decreases to close to or even below a single voxel. Therefore, the study of porosity of materials is

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benefited by high spatial resolution, especially in cases where the size of either the structural elements or the enclosed spaces is very small. It should be noted that, in scans of scaffolds at suitably high resolutions, the initial result of a global binary thresholding can be improved by image processing steps such as despeckling to remove noise dots (specifying the dot size in three dimensions) or the morphological closing procedure (dilate then erode) to “repair” broken binary images of thin structural connections. In fact, appropriate global thresholding followed by one or two such conditional binary operations (e.g. despeckle, close, smooth) can have a similar outcome as more complex adaptive thresholding or deformable surface segmentation algorithms. Moving on from the question of binarization, other parameters of porosity beyond percent porosity can be analyzed from the (binarized) image dataset from a micro-CT scan of a bioscaffold. One of these — of frequent interest to researchers in bioengineering applications — is the permeability of the scaffold, that is, the accessibility of scaffold spaces to outside space. This permeability can be qualified by a virtual object diameter; for example, a question could be asked, “How far into the scaffold can a sphere of x pixels in diameter travel?” Such a question is relevant in the case of creating scaffolds for cellular ingrowth: it is necessary to know if the holes or pores within the scaffold are wide enough to allow a cell “trying” to move into the scaffold to do so, and to what degree or depth. Using the terms “holes” or “pores” within a scaffold or any porous material raises an important point: people approaching the study of material porosity sometimes express a requirement to measure the sizes and characteristics of “pores”, as if pores are well-defined discreet entities within the material and there are a large number of them. Sometimes, this might be the case, for example, in a solid material with enclosed gas bubbles and a relatively low percent porosity. However, for the majority of porous scaffold biomaterials studied by micro-CT, the pore space is highly interconnected such that there are no discreet pores but an interconnected pore space; the number of discreet disconnected “pores” is relatively low in such materials. So, it is usually not meaningful to think in terms of properties of individual

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“pores”, but instead to measure properties of the interconnected pore space (e.g. the thickness and separation distributions measured directly in three dimensions). Furthermore, the highly interconnected nature of the pore space in fibrous or lattice-type porous materials such as bioscaffolds means that the vast majority of the porous space is accessible to the periphery of the scaffold, provided that the accessibility is defined in three dimensions. To use an analogy, the classic children’s puzzle-book task to draw a line through a maze between the rabbit and the carrot is extended into the third spatial dimension (see Fig. 6). Lin et al. (2003) defined a parameter of the “degree of interconnectivity” of pore space inside a porous material to quantify exactly this parameter — how much the inner space is connected to the outside space. However, for the reasons just outlined, this parameter is not very informative

Fig. 6. The study of the permeability of a scaffold is analogous to the classic children’s puzzle-book task to draw a line through a maze between the rabbit and the carrot; however, it is extended into the third spatial dimension.

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when applied to most biological scaffold materials, as was clearly shown when this parameter was measured accurately for polycaprolactone (PCL) biological scaffolds by Darling and Sun (2004) from high-resolution micro-CT scans. The measured values of the degree of interconnectivity ranged from 98.16% to 99.59%, while in the same samples the total percent porosity by volume ranged from 39% to 55%. This confirms that, in three dimensions, one can start from almost any voxel of pore space within such a bioscaffold and draw a convoluted line that will emerge at the periphery of the scaffold. The study of permeability or interconnectivity can be made more powerful and informative by adding the criterion of virtual sphere diameter, thus qualifying permeability as the subset of the pore space accessible from the periphery to a virtual sphere of a given diameter, in image voxels. A straightforward way to do this will be outlined below. This analysis will yield a profile of decreasing permeability with increasing diameter of the virtual spheres, which are “trying” to climb into the pore spaces of the micro-CT–generated image of the scaffold.

3.1. Step 1: delineating a volume of interest (VOI) from the scaffold scan dataset and thresholding to binary image The question of scan parameters suitable for scanning low-density bioscaffolds has been mentioned above — basically, low-energy X-rays from an unfiltered tungsten target source are appropriate. One comment on the scanning technique should be mentioned: scaffold “wool-like” materials such as PLGA/PLA or collagen itself are susceptible to deformation (“creep”) if they are compressed during handling, which at the high spatial resolutions necessary for accurate micro-CT imaging (3 micron pixels or less) can cause movement artifacts. This can be solved by careful handling, as well as by setting up a sample in the scanner sample holder and then leaving the sample to stabilize for a number of hours (or overnight) prior to scanning. From a reconstructed scan dataset, a VOI should be delineated for analysis, with dimensions of several hundred pixels in the three spatial dimensions. This can be a regular shape such as a cylinder or sphere; the latter is preferable if the measurement of anisotropy is of interest.

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In this example, binary segmentation is done by applying a global threshold, and then processing the binary images with a morphological closing procedure (employing a round kernel) followed by 3D despeckling to remove objects less than 16 voxels in size. The result is shown in Fig. 7. (a)

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Fig. 7. Binary segmentation of PLGA scaffold micro-CT scan cross-section image. (a) The original reconstructed gray-level image; (b) the “raw” binary image; and (c) the result of applying a morphological closing procedure followed by 3D noise despeckling. Some broken binary connections are restored and noise objects removed.

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3.2. Step 2: measuring the permeability from the periphery of scaffold to spheres of increasing diameter To assess the permeability from the outside of the scaffold to virtual spheres of varying diameter, a function called “shrink wrap” is used. This technique essentially wraps the ROI boundary around the solid binarized surfaces of a dataset. The ROI is “shrunk” in from the periphery, and the ROI surface can penetrate into the binarized objects in the dataset until a solid surface is encountered. This penetration can be specified to occur in either two or three dimensions, thus defining the accessibility. However, the penetration into the dataset objects can be made conditional by a subfunction called “stretch over holes”, which is based on a distance transform, so that if a pixel diameter (e.g. 8 pixels) is set, then the ROI which is being shrink-wrapped onto and into a binarized dataset cannot go through any hole or gap less than the specified 8 pixels in diameter. Therefore, this shrink-wrap function with its “stretch over holes” configuration allows the porosity of a dataset of a scaffold to be analyzed so that the subset of the internal space accessible to virtual spheres of a specified diameter can be quantified, visualized, and measured morphometrically. In this way, the subset of the internal space of a PLGA scaffold accessible to various widths of a sphere is shown in the form of 3D visual models (Fig. 8). The results of this analysis of internal spaces permeable to the periphery of virtual spheres are shown in Table 1 for a scan dataset of a PLGA scaffold [Figs. 2(a), 2(b), and 8]. We could call this analysis the “permeability to virtual spheres” or “PVS” analysis. The result takes the form of a number of parameters of the permeable space corresponding to a range of sphere diameters. These parameters include volume, surface area, thickness, spatial separation, and anisotropy. Table 1 shows the values of these parameters measured for the permeable space in the PLGA scaffold corresponding to sphere diameters of 4–24 pixels with a scan voxel size of 8.2 microns; this corresponds to 33–197 microns. The percentage of accessible space decreases sharply with increasing virtual sphere diameter, from 98% for a 4-pixel diameter to 78% for a 12-pixel diameter and 13% for a 24-pixel diameter. In contrast, the separation between accessible pore spaces

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8-pixel diameter (c)

12-pixel diameter (d)

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24-pixel diameter

Fig. 8. The “shrink-wrap” distance-transform–based function allows the internal space inside a porous material — here, a PLGA scaffold — to be analyzed in order to visualize and measure the subset of internal space accessible from the periphery by virtual spheres of varying diameter. As the sphere diameter increases from 8 to 24 pixels [(a)–(d)], the fraction of accessible internal space decreases. The pixel size of the micro-CT scan is 8 microns (SkyScan 1172).

increases: the measured values for the same 4-, 12-, and 24-pixel diameters are 52 µm, 89 µm, and 667 µm, respectively. These results show that the “shrink wrap” analysis combined with the “stretch over holes” distance transform allow a powerful analysis

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Table 1. The results of the analysis of internal spaces permeable to the periphery to virtual spheres of various diameters for a scan dataset of a PLGA scaffold (“permeability to virtual spheres” or “PVS” analysis). The result takes the form of a number of parameters of the permeable space corresponding to a range of sphere diameters. Parameter of permeable pore space Volume of scaffold analyzed Total volume of internal space Volume of accessible internal space Percent of internal space accessible Surface area Surface/volume ratio Thickness Separation Degree of anisotropy Number of discreet pore spaces

Virtual sphere diameter Unit

4 pixels 8 pixels 12 pixels 16 pixels 24 pixels (33 µm) (66 µm) (98 µm) (131 µm) (197 µm)

mm3

1.705

1.705

1.705

1.705

1.705

mm3

1.290

1.290

1.290

1.290

1.290

mm3

1.265

1.190

1.005

0.626

0.173

%

98.085

92.255

77.896

48.511

13.397

mm2 mm−1

38.497 30.431

38.297 32.184

34.486 34.323

24.584 39.289

14.177 82.051

mm mm

0.130 0.052 1.471 35

0.133 0.059 1.359 44

0.139 0.089 1.177 60

0.142 0.219 1.109 76

0.079 0.667 1.696 104

of the pore space of a scaffold material, analyzing in detail the subset of the pore space accessible to spheres of different diameters. This adds an important extra dimension to porosity analysis, which is relevant to the study of bioscaffolds that are designed for permeability to cellular ingrowth.

4. Summary In the application of nondestructive imaging techniques such as micro-CT, there is a requirement not only for the generation of 3D image datasets with sufficient contrast and resolution over

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a wide range of object densities, but also for software analytical tools to extract numerical measurements of important parameters relating to the studied sample objects. Two examples have been described here that are of importance in the orthopedic field: the analysis of implant contact (or intersection) surface, and the study of the porosity (internal pore space) of a porous bioscaffold material. Successful analysis starts with adequate imaging results. Imaging objects with high X-ray density, such as metal implants in bone, and with much lower density, such as low-molecular-weight polymers including PLGA and PLA as well as collagen, requires flexibility to alter X-ray energy in a micro-CT scan by the selection of applied voltage and filter. Once good images are obtained, the image datasets can be segmented to binary images by a range of thresholding methods. The binarized images can in turn yield a wide range of analytical endpoints by scripting software analysis that is user-friendly — not requiring any great computer programming or mathematical knowledge — and that applies a suite of logical operations to the binarized datasets, as well as by allowing flexible ROI selection. This combination of 3D imaging hardware and software will provide the orthopedic researcher with an increasingly powerful range of analytical tools for the nondestructive analysis of bone, soft tissue, and scaffold biomaterials for preclinical and clinical applications.

References Darling L, Sun W. 3D microtomographic characterization of precision extruded poly-ε-caprolactone scaffolds. J Biomed Mater Res B Appl Biomater 70B: 311–317, 2004. Ketcham RA. Computational methods for quantitative analysis of threedimensional features in geological specimens. Geosphere 1:32–41, 2005. Lin ASP, Barrows TH, Cartmell SH, Guldberg RE. Microarchitectural and mechanical characterization of oriented porous polymer scaffolds. Biomaterials 24:481–489, 2003.

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Otsu N. A threshold selection method from gray-level histograms. IEEE Trans Syst Man Cybern 9(1):62–66, 1979. Rajagopalan S, Lichun L, Yaszemski MJ, Robb RA. Optimal segmentation of microcomputed tomographic images of porous tissue-engineering scaffolds. J Biomed Mater Res 75A:877–887, 2005. Waarsing JH, Day JS, Weinans H. An improved segmentation method for in vivo microCT imaging. J Bone Miner Res 19(10):1640–1650, 2004.

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Chapter 36

Application of DXA to Assess Orthopedic Implants Tom V. Sanchez and Jing-Mei Wang

Dual-energy X-ray–based technology has developed special software to automatically identify areas overlapped by metal-containing orthopedic implants so that the surrounding bone can be isolated and monitored for follow-up over time. A body of literature has documented the scan parameters that can be used for studies involving hip arthroplasty, knee arthroplasty, and bone lengthening. The software has been demonstrated to be effective in describing patient response to different types of implants, how bone in different regions of the implant change with time, and the response of bone along the implant to treatment. High-density detection software with operator-placed regions of interest (ROIs) has demonstrated usefulness in describing changes over time, response to treatment, and the relationship between bone loss and muscle condition in knee arthroplasty. Similarly, using high-density detection software with operator-set ROIs has allowed the objective quantification and monitoring of gap mineralization in patients undergoing bone lengthening. With care taken to insure proper X-ray flux, patient positioning, and ROI positioning in analysis, this special software has proved to be an effective tool in dual-energy X-ray absorptiometry (DXA) studies of conditions involving orthopedic implants. Keywords:

DXA; hip arthroplasty; knee arthroplasty; bone lengthening.

Corresponding author: Tom V. Sanchez. Tel: +1-575-8380167; fax: +1-575-8380638; E-mail: [email protected], [email protected]

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1. Introduction The in vivo assessment of bone mineral content (BMC) is most commonly done using technology based on dual-energy X-ray absorptiometry (DXA). A DXA study involves monitoring the attenuation of two discrete X-ray energies passed across a target tissue. By evaluating the attenuation of the low and high energies via a planar scan, the system can accurately and precisely estimate bone, lean, and fat content in irregularly distributed tissue. The presence of orthopedic implants in a study presents an interesting setting in which to evaluate changes in the scan area. Orthopedic implants, in some situations, may just complicate or compromise the DXA measurement of bone mineral, lean, or fat tissue (Madsen et al. 1999; Lark et al. 2001). In other situations, it is the influence of orthopedic implants on bone mineral over time that is the phenomenon of interest for the DXA study (Soininvaara et al. 2004; Rosenthall et al. 1999). Regardless of the situation, when orthopedic implants are involved in a DXA study, the facility should be aware that patient positioning, scan speed, scan resolution, and region of interest (ROI) setting will have a critical effect on the usefulness of the results. This chapter addresses technical considerations of patient positioning, scan speed, scan resolution, and ROI setting on Norland, Lunar, and Hologic DXA-based scanners evaluating bone containing orthopedic implants.

2. Materials and Methods 2.1. Positioning of the patient 2.1.1. Norland As with all DXA studies, the positioning of a patient in a study which involves an orthopedic implant should take into consideration that the DXA scan is a planar projection of the area so that data points containing the implant will not reflect BMC. In those studies looking to follow changes over time, the subject should be positioned so that there is consistency in how the scan region is scanned. While scanners

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fitted with dynamic filtration will be able to accommodate scans which include the full beam without tissue cover (Gotfredsen et al. 1997), those scanners which do not include dynamic filtration should take steps to provide some level of tissue cover so that detectors do not experience saturation (Hagiwara et al. 1993). 2.1.2. Lunar As with all DXA studies, the positioning of a patient in a study which involves an orthopedic implant should take into consideration that the DXA scan is a planar projection of the area so that data points containing the implant will not reflect BMC. In those studies looking to follow changes over time, the subject should be positioned so that there is consistency in how the scan region is scanned. In situations where the scanner enters areas with little or no soft tissue cover, the system may encounter detector saturation; therefore, the addition of supplemental materials is advised (Li et al. 2004). 2.1.3. Hologic As with all DXA studies, the positioning of a patient in a study which involves an orthopedic implant should take into consideration that the DXA scan is a planar projection of the area so that data points containing the implant will not reflect BMC. In those studies looking to follow changes over time, the subject should be positioned so that there is consistency in how the scan region is scanned. Because the detectors in Hologic scanners operate in current mode rather than in the count mode seen in Norland or Lunar scanners, detector saturation is not a limiting factor in the study; thus, supplementary tissue cover is not needed to overcome detector saturation.

2.2. Scan speed and scan resolution 2.2.1. Norland While the scan speed and scan resolution may vary from study to study (depending on the subject being scanned), many studies

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involving orthopedic implants are carried out at between 30 mm/s and 60 mm/s (Maffulli et al. 1997; Braillon and Chotel 2003; Laursen et al. 2005; Therbo et al. 2003) and the resolution is set at 0.5 × 0.5 mm, 1.0 × 1.0 mm, or 1.5 mm × 1.5 mm (Therbo et al. 2003; Braillon and Chotel 2003; Laursen et al. 2005; Petersen 2000; Gehrchen et al. 2000). 2.2.2. Lunar While the scan speed and scan resolution may vary from study to study (depending on the subject being scanned), many studies involving orthopedic implants are carried out in fast mode with the resolution set at 0.6 mm × 1.2 mm (Li et al. 2004). 2.2.3. Hologic While the scan speed does not vary among orthopedic implant studies, most of these studies are carried out using high-resolution mode reported to be at 0.06 mm × 0.11 mm (Shetty et al. 2006).

2.3. Scan ROI setting 2.3.1. Norland When working with orthopedic materials such as implants, plates, nails, or wires, Norland equipment can activate high-density detect software, which will identify data points that contain the high density of these foreign materials. Once identified, the data from those data points will be excluded from the analysis for bone area and bone mineral, lean, or fat content. When analyzing the scan, up to seven ROIs can be placed by the operator on a scan (Fig. 1). These regions can, for example, correspond to the seven Gruen zones surrounding an implant (Gehrchen et al. 1995; Gehrchen et al. 2000), operatorplaced regions around the knee (Hernandez-Vaquero et al. 2005; Petersen 2000; Therbo et al. 2003), the acetabular region (Ang et al. 1997; Laursen et al. 2005), or operator-set regions along a gap in a

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L

609

H

Image not for diagnosis

Fig. 1. A scan of a femur with an implant. Note that high-density detect software has identified the implant and excluded that data from the scan. Note also that seven regions of interest (ROIs) corresponding to the Gruen zones have been located for analysis of bone mineral density.

bone lengthening study (Maffulli et al. 1997; Maffulli et al. 1999; Yamane et al. 1999). 2.3.2. Lunar When working with orthopedic materials such as implants, plates, nails, or wires, Lunar equipment can activate special orthopedic software, which will identify data points that contain the high density of these foreign materials. Once identified, the data from those data points will be excluded from the analysis for bone area and bone

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mineral, lean, or fat content. When analyzing the scan, ROIs can be placed by the operator on a scan. These regions can, for example, correspond to the seven Gruen zones surrounding an implant (Bloebaum et al. 2006; Kroger et al. 1997; Skoldenberg et al. 2006; Venesmaa et al. 2001a; Venesmaa et al. 2001b; Venesmaa et al. 2003), operator-placed regions around the knee (Soininvaara et al. 2004; Soininvaara et al. 2002; Li et al. 2004), or the acetabular region (Digas et al. 2006). 2.3.3. Hologic When working with orthopedic materials such as implants, plates, nails, or wires, Hologic equipment can activate metal removal software, which will identify data points that contain the high density of these foreign materials. Once identified, the data from those data points will be excluded from the analysis for bone area and bone mineral, lean, or fat content. When analyzing the scan, ROIs can be placed by the operator on a scan. These regions can, for example, correspond to the seven Gruen zones surrounding an implant (Aldinger et al. 2003; Habermann et al. 2007; Rahmy et al. 2004; Yamaguchi et al. 2003), the acetabular region (Shetty et al. 2006; Wilkinson et al. 2001), or the regions along a gap in a bone lengthening study (Eyres et al. 1993; Eyres and Kanis 1995).

3. Discussion The development of special software to identify and exclude the regions of a scan that contain high-density materials has proven to be a useful tool when a scan has unwanted artifacts such as buttons in a spine study or an implant in a whole-body scan. This software, when combined with the capability to allow operators to place ROIs wherever they desire, has proven especially useful to clinicians and researchers when pursuing studies involving hip arthroplasty, knee arthroplasty, or bone lengthening, for example. This special software has been applied to the evaluation of bone in patients with total hip arthroplasty (THA). When used in this

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application, Ang and associates (1997) were able to compare patients with a stiff collarless hip implant to patients with a flexible isoelastic hip implant; these investigators were able to show that, over 12 months, patients with the stiff collarless hip implant lost a mean of 27% of the bone along the implant, while patients with the flexible implant showed an overall increase in density. Skoldenberg and associates (2006) utilized this special software to follow 138 patients with THA for an average of 3 years, and found that bone loss in Gruen zones #1 and #7 could be related to implant stem size. To document the type and degree of change that can be expected with THA, Venesmaa and associates (2003) used this software system to follow 15 patients with THA over 5 years; over that time, the authors found that, following an initial average loss of 5%–18% in the seven Gruen zones over the first 3 months, when the patient responded well to the procedure, only minor changes in bone mineral density (BMD) were seen between 1 year and 5 years after THA. Similar findings were reported by Aldinger and associates (2003) with a larger population who were followed for 7–12 years after THA. Examining how THA reacts to treatment with a bisphosphonate, Yamaguchi and associates (2003) followed 28 patients treated with a bisphosphonate and 30 patients not treated with osteoactive drugs over 1 year; the authors found that the treated patients did not lose as much bone in the first 6 months and that they stabilized after 6 months, while at 12 months the untreated patients continued to show bone loss in the Gruen zones. Digas and associates (2006) followed acetabular bone density in five operator-set ROIs in 90 patients treated with three different methods of THA fixation; over 2 years, the authors were able to demonstrate greater regional bone loss in uncemented sockets, which they suggest may be related to stress shielding. As noted, high-density detect software with DXA has also found application in the evaluation of changes that occur with knee arthroplasty. Using operator-placed ROIs, Therbo and associates (2004) showed that BMD in 11 patients with total knee arthroplasty decreased at 3 months in a central region of the femur, but no significant losses were present at 2 years in any of the regions. In other

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studies, using operator-placed ROIs, Anchuela and associates (2001) examined the changes that occurred in 28 women in both the femur and the tibia; these authors reported that the changes found at 6 and 12 months seemed to relate to adaptive changes in femoral and tibial muscles. In another study, Soinivaara and associates (2002) reported using DXA to follow the changes over 1 year in operatorplaced regions in total knee arthroplasty patients treated with either alendronate or calcium supplements; patients treated with alendronate following arthroplasty showed no bone loss in any of the regions evaluated, while those treated with calcium supplementation showed significantly more bone loss in the distal femur, thus demonstrating the value of using DXA to follow changes with arthroplasty over time. Using high-density detect software with operator-placed ROIs enables the use of DXA to measure changes that occur in leg lengthening procedures. Maffulli and associates (1997) reported using operator-set ROIs with this enabled software to monitor progress in bone lengthening and then identify patients with fast, moderate, or slow bone accretion in the lengthening gap; the procedure was reported to be able to provide a precise methodology for monitoring the progress of bone lengthening in the individual patient. Similarly, Eyres and associates (1993) were able to assess the rate of new bone formation over 18 months of lengthening using Lunar equipment; as with the study from Hong Kong (Maffulli et al. 1999), the authors reported that this tool allowed them to follow changes over time so that they could objectively assess the progress with the lengthening. While proving useful, DXA-based measurements in these applications with orthopedic implants have conditions which should be met. Depending on the type of equipment being used, the operator would have to take care to ensure that detectors have some way to accommodate the amount of X-ray being used. In Norland equipment, this is accomplished by the dynamic filtration; in Lunar equipment, this may be accomplished by varying the scan speed and/or applying supplemental tissue cover; and in Hologic equipment, this is accomplished by using current mode operation on the

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detectors. Another condition that would have to be met is ensuring that consistent patient positioning is practiced. Because these studies are most valuable when evaluating changes over time, the operator should be aware that patient positioning is going to affect the measurement of BMD over time. Patient positioning that places the target organ perpendicular to the beam to produce a consistent scan which can be reproduced over time should be the objective. Similarly, the positioning of the analysis ROI in a reproducible fashion should also be a condition for the operator to meet. Once the positioning of the patient is managed, the operator can use physical landmarks (from the scan image and from the position of high-density materials in the scan) to consistently place the analysis ROI. With consistent positioning of the ROI, the scan will then deliver the sought-after results that can effectively reflect changes over time.

4. Summary In summary, densitometry manufacturers have developed specialized software that supports the removal of high-density-containing data points in a scan. This software has proven useful when applied to studies of patients in which there are orthopedic implants in the scan site. This specialized software has proven useful in evaluating patients with hip arthroplasty, knee arthroplasty, or bone lengthening conditions, for example. By understanding the limits of the system and by carefully positioning the patient and the analysis ROIs, the densitometrist can effectively analyze bone density in these patients so that changes in bone density can be assessed over time.

References Aldinger PR, Sabo D, Pritsch M et al. Pattern of periprosthetic bone remodeling around stable uncemented tapered hip stems: a prospective 84month follow-up study and a median 156-month cross-sectional study with DXA. Calcif Tissue Int 73:115–121, 2003.

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Anchuela J, Gomez-Pellico L, Ferrer-Blanco M et al. Muscular function and bone mass after knee arthroplasty. Int Orthop 25:253–256, 2001. Ang KC, Das De S, Goh JCH et al. Periprosthetic bone remodelling after cementless total hip replacement: a prospective comparison of two different implant designs. J Bone Joint Surg Br 79B:675–679, 1997. Bloebaum RD, Liau DW, Lester K, Rosenbaum TG. Dual-energy X-ray absorptiometry measurement and accuracy of bone mineral after unilateral total hip arthroplasty. Arthroplasty 21(4):612–622, 2006. Braillon PM, Chotel F. Bone mineral content and soft-tissue assessment in limb segments by dual-energy X-ray absorptiometry. J Clin Densitom 6(2):149–158, 2003. Digas G, Karrholm J, Thanner J. Different loss of BMD using uncemented press-fit and whole polyethylene cups fixed with cement: repeated DXA studies in 96 hips randomized to 3 types of fixation. Acta Orthop 77(2): 218–226, 2006. Eyres K, Bell MJ, Kanis JA. New bone formation during leg lengthening: evaluated by dual energy X-ray absorptiometry. J Bone Joint Surg Br 75B:96–106, 1993. Eyres KS, Kanis JA. Bone loss after tibial fracture: evaluated by dual-energy X-ray absorptiometry. J Bone Joint Surg Br 75B:473–478, 1995. Gehrchen PM, Petersen MM, Nielsen PK, Lund B. Quantification of bone remodeling in the proximal femur following uncemented total hip arthroplasty: a methodological study using dual energy X-ray absorptiometry. Eur J Exp Musculoskelet Res 4:57–61, 1995. Gehrchen PM, Petersen MM, Nielsen PK, Lund B. Influence of region size on bone mineral measurements along femoral stems in THA. Hip Int 10(4):204–208, 2000. Gotfredsen A, Baeksgaard L, Hilsted L. Body composition analysis by DEXA by using dynamically changing samarium filtration. J Appl Physiol 82(4):1200–1209, 1997. Habermann B, Eberhardt C, Feld M et al. Tartrate-resistant acid phosphatase 5b (TRAP 5b) as a marker of osteoclast activity in the early phase after cementless total hip replacement. Acta Orthop 78(2):221–225, 2007. Hagiwara S, Lane N, Engelde K et al. Precision and accuracy for rat whole body and femur bone mineral determination with dual X-ray absorptiometry. Bone Miner 22:57–68, 1993.

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Hernandez-Vaquero D, Garcia-Sandoval MA, Fernandez-Carreira JM et al. Measurement of bone mineral density is possible with standard radiographs. Acta Orthop 76(6):791–795, 2005. Kroger H, Vanninen E, Overmyer M et al. Periprosthetic bone loss and regional bone turnover in uncemented total hip arthroplasty: a prospective study using high resolution single photon emission tomography and dualenergy X-ray absorptiometry. J Bone Miner Res 12(3):487–492, 1997. Lark RK, Henderson RC, Renner JB et al. Dual X-ray absorptiometry assessment of body composition in children with altered body posture. J Clin Densitom 4(4):325–335, 2001. Laursen MB, Nielsen PT, Soballe K. DXA scanning of acetabulum in patients with cementless total hip arthroplasty. J Clin Densitom 8(4):476–483, 2005. Li MG, Nilsson KG, Nivbrant B. Decreased precision for BMD measurements in the prosthetic knee using non–knee-specific software. J Clin Densitom 7(3):319–325, 2004. Madsen OR, Egsmose C, Lorentzen JS et al. Influence of orthopaedic metal and high-density detection on body composition as assessed by dualenergy X-ray absorptiometry. Clin Physiol 19(3):238–245, 1999. Maffulli N, Cheng JCY, Sher A et al. Bone mineralization at the callotasis site after completion of lengthening. Bone 25(3):333–338, 1999. Maffulli N, Sher A, Cheng JCY, Lam TP. Dual-energy X-ray absorptiometry predicts bone formation in lower limb callotasis lengthening. Ann R Coll Surg Engl 79:250–256, 1997. Petersen MM. Bone mineral measurements at the knee using dual photon and dual X-ray absorptiometry: methodological evaluation and clinical studies focusing on adaptive bone remodeling following lower extremity fracture, total knee arthroplasty, and partial versus total meniscectomy. Acta Orthop Suppl 71(293):1–37, 2000. Rahmy AIA, Gosens T, Blake GM et al. Periprosthetic bone remodelling of two types of uncemented femoral implant with proximal hydroxyapatite coating: a 3-year follow-up study addressing the influence of prosthesis design and preoperative bone density on periprosthetic bone loss. Osteoporos Int 15:281–289, 2004. Rosenthall L, Bobyn JD, Tanzer M. Bone densitometry: influence of prosthetic design and hydroxyapatite coating on regional adaptive bone remodelling. Int Orthop 23:325–329, 1999.

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Shetty NR, Hamer AJ, Stockley I et al. Precision of periprosthetic bone mineral density measurements using Hologic Windows versus DOS-based analysis software. J Clin Densitom 9(3):363–366, 2006. Skoldenberg OG, Boden HSG, Salemyr MO et al. Periprosthetic proximal bone loss after uncemented hip arthroplasty is related to stem size: DXA measurements in 138 patients followed for 2–7 years. Acta Orthop 77(3):386–392, 2006. Soininvaara TA, Jurvelin JS, Miettinen HJA et al. Effect of alendronate on periprosthetic bone loss after total knee arthroplasty: a one-year, randomized, controlled trial of 19 patients. Calcif Tissue Int 71:472–477, 2002. Soininvaara TA, Miettinen HJA, Jurvelin JS et al. Periprosthetic tibial bone mineral density changes after total knee arthroplasty: one-year follow-up study of 69 patients. Acta Orthop Scand 75(5):600–605, 2004. Therbo M, Petersen MM, Gehrchen PM et al. Bone mineral density of the distal femur following uncemented total knee arthroplasty: a two-year follow-up of 11 knees using dual energy X-ray absorptiometry. J Orthop Traumatol 2:81–85, 2004. Therbo M, Petersen MM, Schroder HM et al. The precision and influence of rotation for measurements of bone mineral density of the distal femur following total knee arthroplasty: a methodological study using DEXA. Acta Orthop Scand 74(6):677–682, 2003. Venesmaa PK, Kroger HPJ, Jurvelin JS et al. Periprosthetic bone loss after cemented total hip arthroplasty: a prospective 5-year dual energy radiographic absorptiometry study of 15 patients. Acta Orthop Scand 74(1):31–36, 2003. Venesmaa PK, Kroger HPJ, Miettinen HJA et al. Monitoring of periprosthetic BMD after uncemented total hip arthroplasty with dual-energy X-ray absorptiometry — a 3-year follow-up study. J Bone Miner Res 16(6): 1056–1061, 2001a. Venesmaa PK, Kroger HPJ, Miettinen HJA et al. Alendronate reduces periprosthetic bone loss after uncemented primary total hip arthroplasty: a prospective randomized study. J Bone Miner Res 16(11):2126–2131, 2001b. Wilkinson JM, Peel NFA, Elson RA et al. Measuring bone mineral density of the pelvis and proximal femur after total hip arthroplasty. J Bone Joint Surg Br 83B:283–288, 2001.

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Yamaguchi K, Masuhara K, Yamasaki S et al. Cyclic therapy with etidronate has a therapeutic effect against local osteoporosis after cementless total hip arthroplasty. Bone 33:144–149, 2003. Yamane K, Okano T, Kishimoto H, Hagino H. Effect of ED-71 on modeling of bone in distraction osteogenesis. Bone 24(3):187–193, 1999.

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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

Callotasis or distraction osteogenesis is a well-established orthopedic treatment method in the correction of limb length inequality or bone defect. Plain radiography is commonly used to monitor the progress of distraction osteogenesis, with the known limitation that it can only provide qualitative measurements. Dual-energy X-ray absorptiometry (DXA) is well accepted as the standard clinical equipment for studying bone mineral density in osteoporosis quantitatively with reasonable precision. It has also been successfully used to quantify and monitor bone regeneration in callus distraction, and is able to provide valuable information for clinicians in determining the distraction rate and the timing for frame removal. This chapter summarizes the essential technical details of DXA measurement of the bone mineralization status of callus distraction osteogenesis and its clinical indications. Keywords:

Distraction; DXA; limb lengthening; bone mineralization; osteogenesis.

Corresponding author: Jack Chun-Yiu Cheng. Tel: +852-26322515; fax: +852-26377889; E-mail: [email protected]

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1. Introduction Callotasis or distraction osteogenesis is a valid and reliable technique commonly used in treating patients with leg length discrepancy, bone defect, or short stature (Aldegheri and Dall’Oca 2001; De Bastiani et al. 1987; Maffulli et al. 1997; Maffulli et al. 1999; Matsubara et al. 2006; Ng et al. 2003; Paley 1988). The rate of bone mineralization at the callus area, the alignment of the limb, and the rate of distraction are key factors that will affect the outcome of distraction osteogenesis. Radiography is the most common clinical assessment technique. It has been suggested that radiographs should be taken every week during the latent and distraction phases and biweekly during the consolidation phase in monitoring the process of distraction osteogenesis (Blane et al. 1991; Young et al. 1990). However, the accuracy of quantifying bone mineral density (BMD) by radiography is relatively unreliable. The exposure; target distance and magnification; field differences (heel effect); and orientation of the bone, soft tissue, skin folds, and other artifacts during the taking and processing will all affect the interpretation (West et al. 1987). The variation of the density could range from 5% to 10% (West et al. 1987). Thus, radiographic images can only provide a qualitative and subjective analysis of the mineralization of newly formed bone. Dual-energy X-ray absorptiometry (DXA) (Cheng et al. 2002; Maffulli et al. 1997; Maffulli et al. 1999; Reiter et al. 1997; Takata et al. 2000) and ultrasonography (Eyres et al. 1993; Shevtsov et al. 2003; Young et al. 1990) are the alternative techniques that can provide quantitative measurement with minimum radiation dose in assessing the bone quality in patients undergoing callus distraction. DXA is a noninvasive irradiation apparatus that provides quantitative analysis of BMD, and has been recommended by the World Health Organization (WHO) as the gold standard for diagnosing osteoporosis (Cummings et al. 1993; Faulkner 1998; Kanis et al. 1994; Meunier et al. 1999; World Health Organization 1994). The effective dose for a DXA scan is 1–3 µSv, which is about 1/50 of a conventional chest X-ray (Genant et al. 1996; Kalender 1992; Lewis

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et al. 1994). The precision error of different measuring sites using DXA is less than 3% (Genant et al. 1996). DXA has been applied in the prediction of fracture risk (Beck et al. 1996; Duboeuf et al. 1997; Kroger et al. 1995; McGuigan et al. 2001), diagnosis of osteoporosis (Blake and Fogelman 2001; Grampp et al. 1997; Kanis et al. 1994; Langton 1996), longitudinal monitoring of bone changes (Qin et al. 2002; Sahota et al. 2000; Szulc and Delmas 2007; Tang et al. 2001), and pediatric skeletal problems (Abe et al. 2003; Adams 1998; Cheng and Guo 1997; Cheng et al. 1999; Cheng et al. 2002; Giampietro et al. 2003; Hung et al. 2005; Ng et al. 2003). DXA has been reported to provide valuable help to clinicians in determining the distraction rate and the timing for frame removal in distraction osteogenesis (Cheng et al. 2002; Maffulli et al. 1997; Maffulli et al. 1999; Ng et al. 2003; Reiter et al. 1997). In this chapter, the authors summarize the detailed protocols and important technical considerations for quantifying bone mineral changes in the course of distraction osteogenesis using DXA.

2. Methodology The following specifications of scans are based on the authors’ experience using DXA machines (XR-36; Norland Medical Systems, Inc., WI, USA): • • •

Scanning mode: research scan Scanning resolution: 1 mm × 1 mm Scanning interval/frequency of DXA: preoperation; 1–2 days postoperation; weekly measurement during distraction period; DXA measurement performed every 3–6 months after removal of framea

a From the usual clinical practice, it has been suggested that X-rays should be taken weekly to monitor the progress of distraction osteogenesis. With the use of DXA, the frequency of X-rays taken can be reduced to a biweekly basis.

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Fig. 1. (a) DXA image of the tibia that has undergone distraction osteogenesis, clearly showing the mineralized newly formed bone at the distracted region. (b) X-ray image taken at the same time as the DXA scan on the same patient.

2.1. Limb undergoing distractionb 2.1.1. Positioning of tibia (as an example) • •

b

Patients are placed in a lying, supine position on the scanning table. The bone segment should be positioned horizontal to the scanning table.

The mineralization of newly formed bone in distraction osteogenesis can be easily seen and quantified in DXA scan [Fig. 1(a)]. However, it is difficult to measure bone regeneration in a conventional radiography [Fig. 1(b)].

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• • • •

c

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Positioning of the lengthened limb during DXA measurement.

The lower leg should be internally rotated to avoid overlapping of the tibia and the fibula.c The position is withheld with sandbags (Fig. 2). The regions of interest (ROIs) are set and the scanning is performed. The DXA image of the distracted bone should contain proximal and distal pins of the fixator that are closest to the distraction site, and clear images of the distraction site/gap and the host bone (original bone) (Fig. 3).

If an Ilizarov fixator is used, extra attention needs to be paid to the location of the rods during positioning. Since internal rotation of the lower leg is necessary to avoid overlapping of the tibia and the fibula, during the rotation, the Ilizarov fixator will also be rotated simultaneously and the position of the rod will then be changed. It is important to confirm that the rod is not positioned at the scanning region.

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Fig. 3.

DXA image of the distracted tibia.

2.2. Control limb 2.2.1. Positioning of femur (as an example) • • • • •

The nonoperative limb should be selected as a control.d The shaft of the femur should be straight and parallel to the scanning table. The position is withheld with sandbags. The ROIs of the distal femur are set and the scanning is performed. The DXA image of the distal femur is shown in Fig. 4.

3. Results and Interpretations Enable the function of high-density exclusion (if applicable). d

If one side of the tibia/femur undergoes distraction, then the contralateral limb will serve as a control; if bilateral tibias/femurs are operated on, then the humerus may be an alternative site for control.

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Fig. 4. DXA image of the control limb (distal femur). The length of the control bone is set at 2 cm (as an example).

3.1. Gap region • • •

e

The distracted region can be easily seen at around 1 week after the distraction is started. Identify the gap region (Fig. 5) The length of the gap region should be identical to the actual distracted length, which indicates that the scanning position is good.e

It is acceptable to have a 0.5–1-mm difference between the length of the gap region and the actual length of distraction.

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Fig. 5. DXA image of tibia with the actual length of distraction of 4.7 cm and original bone of 4 cm (as an example).

3.2. Original bone (host bone) • • •

f

Select a region distal to the distracted gap region as the original bone (Fig. 5).f The region of original bone should not include the pin or overlap with the fibula or rod of the frame. To ensure that the selection of the region of original bone is repeatable, set a fixed distance proximal to the distal pin (e.g. 6 cm from the distal pin; Fig. 5) and then take 4 cm (for example)

Previous studies have shown that a significant bone loss was found in the adjacent sites, particularly in the distal segment, during limb lengthening and traumatic fracture in long bone (Eyres and Kanis 1995; Kannus et al. 1994). Therefore, it is recommended to monitor the bone loss in the host bone, which is adjacent to the distraction site.

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as the length of the original bone. The selected site should be constant throughout the scanning (Fig. 5).

3.3. Control limb g • •

Select the distal region of the control bone (Fig. 4). Select a fixed distance that is proximal to the tip of the femoral condyle (e.g. 6 cm from the tip of the femoral condyle; Fig. 4), and then take 2 cm (for example) as the length of the control bone. The selected site should be constant throughout the scanning (Fig. 4).

3.4. Monitoring of the rate of bone changes for the area of distraction Bone mineral content (BMC) is used to monitor the mineralization rate of the lengthening site (Cheng et al. 2002; Maffulli et al. 1997, Maffulli et al. 1999; Ng et al. 2003). The relative percentage of BMC is calculated for three ROIs: (1) the gap region, (2) the original bone of the distracted limb, and (3) the control bone. Graphical presentation shall be provided to illustrate the mineralization status of the distraction region (Fig. 6).

4. Discussion DXA has been proven as a reliable technique for the in vivo monitoring of bone mineralization of the callus in limb lengthening (Cheng et al. 2002; Eyres et al. 1993; Hamanishi et al. 1995; Maffulli et al. 1997; Meffert et al. 2000; Ng et al. 2003; Tsumaki et al. 2004). The most challenging tasks during the course of limb lengthening are to determine the optimal rate of distraction and to select the right timing for the cease of distraction and frame removal. A distraction rate of g

The purpose of measuring the control limb is to ensure that the bone changes do not affect other skeletal sites. Therefore, the percentage change of bone mineral content in the control limb should be minimal.

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Fig. 6. Graphical presentation of the relative percentage of bone mineral content (BMC) changes of the gap region, original bone, and control limb.

0.25 mm four times a day is the standard protocol for distraction. However, it is only applicable if the rate of bone mineralization is consistent; in reality, the situation usually varies clinically case by case. Based on the change in percentage of BMC of the gap region measured by DXA, Maffulli et al. (1997) have identified three groups of bone formation: fast (0.3%–0.6% per day), moderate (0.1%–0.3% per day), and slow (<0.1% per day) formation groups in limb-lengthening patients. In addition, the rate of callus mineralization in femurs and tibias may be different (Tanaka et al. 1996). If the rate of bone formation is faster than the rate of distraction, premature fusion of callus may occur. Ng et al. (2003) reported that the premature fusion of tibial callotasis could be signified by a sharp rise in the percentage of BMC during the distraction phase. In addition to monitoring bone mineralization at the distracted gap region, DXA can also be used to measure bone loss in the host bone simultaneously. It has been suggested that when the mineralization of callotasis reaches 50%–65% of the host bone, it might be time for frame removal (Ng et al. 2003).

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Although DXA provides important information in the process of callus distraction, traditional plain radiography cannot be replaced. Radiographs provide high-resolution images for measuring the alignments of the newly formed bone, and allow early detection of corticalization or bone defects which cannot be identified by other imaging tools. With the use of DXA, the amount of X-ray exposure from plain radiographs can be reduced. A combination of DXA and radiography is recommended for monitoring distraction osteogenesis in limb-lengthening patients.

5. Summary In summary, longitudinal monitoring of the rate of bone mineralization at the callus area, the alignment of the limb, and the rate of distraction are important in patients undergoing distraction osteogenesis. In addition to plain radiography, DXA measurement may provide an objective, quantitative measurement of the bone mineralization of callotasis.

References Abe M, Sarihan H, Madenci E. Evaluation of bone mineral density with dual X-ray absorptiometry for osteoporosis in children with bladder augmentation. J Pediatr Surg 38:230–232, 2003. Adams J. Single- and dual-energy: X-ray absorptiometry. In: Genant HK, Guglielmi G, Jergas M (eds.), Bone Densitometry and Osteoporosis, Springer, Berlin, pp. 305–334, 1998. Aldegheri R, Dall’Oca C. Limb lengthening in short stature patients. J Pediatr Orthop B 10:238–247, 2001. Beck TJ, Ruff CB, Mourtada FA et al. Dual-energy X-ray absorptiometry derived structural geometry for stress fracture prediction in male U.S. Marine Corps recruits. J Bone Miner Res 11:645–653, 1996. Blake GM, Fogelman I. Bone densitometry and the diagnosis of osteoporosis. Semin Nucl Med 31:69–81, 2001. Blane CE, Herzenberg JE, DiPietro MA. Radiographic imaging for Ilizarov limb lengthening in children. Pediatr Radiol 21:117–120, 1991.

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Cheng JC, Guo X. Osteopenia in adolescent idiopathic scoliosis. A primary problem or secondary to the spinal deformity? Spine 22:1716–1721, 1997. Cheng JC, Guo X, Sher AH. Persistent osteopenia in adolescent idiopathic scoliosis. A longitudinal follow up study. Spine 24:1218–1222, 1999. Cheng JCY, Maffulli N, Sher A et al. Bone mineralization gradient at the callotasis site. J Orthop Sci 7:331–340, 2002. Cummings SR, Black DM, Nevitt MC et al. Bone density at various sites for prediction of hip fractures. The Study of Osteoporotic Fractures Research Group. Lancet 341:72–75, 1993. De Bastiani G, Aldegheri R, Renzi-Brivio L, Trivella G. Limb lengthening by callus distraction (callotasis). J Pediatr Orthop 7:129–134, 1987. Duboeuf F, Hans D, Schott AM et al. Different morphometric and densitometric parameters predict cervical and trochanteric hip fracture: the EPIDOS Study. J Bone Miner Res 12:1895–1902, 1997. Eyres KS, Bell MJ, Kanis JA. New bone formation during leg lengthening. Evaluated by dual energy X-ray absorptiometry. J Bone Joint Surg Br 75:96–106, 1993. Eyres KS, Kanis JA. Bone loss after tibial fracture. Evaluated by dual-energy X-ray absorptiometry. J Bone Joint Surg Br 77:473–478, 1995. Faulkner KG. Bone densitometry: choosing the proper skeletal site to measure. J Clin Densitom 1:279–285, 1998. Genant HK, Engelke K, Fuerst T et al. Noninvasive assessment of bone mineral and structure: state of the art. J Bone Miner Res 11:707–730, 1996. Giampietro PF, Peterson M, Schneider R et al. Assessment of bone mineral density in adults and children with Marfan syndrome. Osteoporos Int 14:559–563, 2003. Grampp S, Genant HK, Mathur A et al. Comparisons of noninvasive bone mineral measurements in assessing age-related loss, fracture discrimination, and diagnostic classification. J Bone Miner Res 12:697–711, 1997. Hamanishi C, Kawabata T, Yoshii T, Tanaka S. Bone mineral density changes in distracted callus stimulated by pulsed direct electrical current. Clin Orthop Relat Res 312:247–252, 1995.

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Hung VWY, Qin L, Cheung CSK et al. Osteopenia: a new prognostic factor of curve progression in adolescent idiopathic scoliosis. J Bone Joint Surg Am 87:2709–2716, 2005. Kalender WA. Effective dose values in bone mineral measurements by photon absorptiometry and computed tomography. Osteoporos Int 2:82–87, 1992. Kanis JA, Melton LJ 3rd, Christiansen C et al. The diagnosis of osteoporosis. J Bone Miner Res 9:1137–1141, 1994. Kannus P, Jarvinen M, Sievanen H et al. Reduced bone mineral density in men with a previous femur fracture. J Bone Miner Res 9:1729–1736, 1994. Kroger H, Huopio J, Honkanen R et al. Prediction of fracture risk using axial bone mineral density in a perimenopausal population: a prospective study. J Bone Miner Res 10:302–306, 1995. Langton CM. The clinical role of BUA for the assessment of osteoporosis: a new hypothesis. Clin Rheumatol 15:414–415, 1996. Lewis MK, Blake GM, Fogelman I. Patient dose in dual X-ray absorptiometry. Osteoporos Int 4:11–15, 1994. Maffulli N, Cheng JC, Sher A, Lam TP. Dual-energy X-ray absorptiometry predicts bone formation in lower limb callotasis lengthening. Ann R Coll Surg Engl 79:250–256, 1997. Maffulli N, Cheng JC, Sher A, Lam TP. Bone mineralization at the callotasis site after completion of lengthening. Bone 25:333–338, 1999. Matsubara H, Tsuchiya H, Sakurakichi K et al. Deformity correction and lengthening of lower legs with an external fixator. Int Orthop 30: 550–554, 2006. McGuigan FE, Armbrecht G, Smith R et al. Prediction of osteoporotic fractures by bone densitometry and COLIA1 genotyping: a prospective, population-based study in men and women. Osteoporos Int 12:91–96, 2001. Meffert RH, Inoue N, Tis JE et al. Distraction osteogenesis after acute limbshortening for segmental tibial defects. Comparison of a monofocal and a bifocal technique in rabbits. J Bone Joint Surg Am 82:799–808, 2000. Meunier PJ, Delmas PD, Eastell R et al. Diagnosis and management of osteoporosis in postmenopausal women: clinical guidelines. International Committee for Osteoporosis Clinical Guidelines. Clin Ther 21: 1025–1044, 1999.

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Ng BK, Hung VW, Cheng JC, Lam TP. Limb lengthening for short stature: a 10-year clinical experience. Hong Kong J Paediatr 8:307–317, 2003. Paley D. Current techniques of limb lengthening. J Pediatr Orthop 8:73-92, 1988. Qin L, Au S, Choy W et al. Regular Tai Chi Chuan exercise may retard bone loss in postmenopausal women: a case-control study. Arch Phys Med Rehabil 83:1355–1359, 2002. Reiter A, Sabo D, Pfeil J, Cotta H. Quantitative assessment of callus distraction using dual energy X-ray absorptiometry. Int Orthop 21:35–40, 1997. Sahota O, San P, Cawte SA et al. A comparison of the longitudinal changes in quantitative ultrasound with dual-energy X-ray absorptiometry: the four-year effects of hormone replacement therapy. Osteoporos Int 11:52–58, 2000. Shevtsov VI, Diachkova GV, Menshchikova TI, Grebenyuk LA. Radiosonographic substantiation of algorithms for examination of patients during operative lengthening of the tibia. Bull Hosp Joint Dis 61: 108–113, 2003. Szulc P, Delmas PD. Bone loss in elderly men: increased endosteal bone loss and stable periosteal apposition. The prospective MINOS study. Osteoporos Int 18:495–503, 2007. Takata S, Ikata T, Yonezu H, Inoue A. Effects of lower-leg lengthening on bone mineral density and soft tissue composition of legs in a patient with achondroplasia. J Bone Miner Metab 18:339–341, 2000. Tanaka K, Kurokawa T, Nakamura K et al. Callus formation in femur and tibia during leg lengthening: 7 patients examined with DXA. Acta Orthop Scand 67(2):158–160, 1996. Tang GW, Yip PS, Li BY. The profile of bone mineral density in Chinese women: its changes and significance in a longitudinal study. Osteoporos Int 12:647–653, 2001. Tsumaki N, Kakiuchi M, Sasaki J et al. Low-intensity pulsed ultrasound accelerates maturation of callus in patients treated with opening-wedge high tibial osteotomy by hemicallotasis. J Bone Joint Surg Am 86: 2399–2405, 2004. West JD, Mayor MB, Collier JP. Potential errors inherent in quantitative densitometric analysis of orthopaedic radiographs. A study after total hip arthroplasty. J Bone Joint Surg Am 69:58–64, 1987.

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World Health Organization. Assessment of Fracture Risk and Its Application to Screening for Postmenopausal Osteoporosis. WHO Technical Report Series. WHO, Geneva, 1994. Young JW, Kostrubiak IS, Resnik CS, Paley D. Sonographic evaluation of bone production at the distraction site in Ilizarov limb-lengthening procedures. Am J Roentgenol 154:125–128, 1990.

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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

XtremeCT is a three-dimensional (3D) high-resolution peripheral quantitative computed tomography (HRpQCT) device that enables the acquisition of both volumetric bone mineral density (BMD) and bone structure. This chapter provides general information on the specific features of XtremeCT, and a guide to perform XtremeCT measurements using standard measurement protocols for in vivo human application. Its potential for studying large animals in vivo and ex vivo is also mentioned. Keywords:

High-resolution pQCT; XtremeCT; volumetric bone mineral density; bone microarchitecture; humans; animals; in vivo; in vitro.

1. Introduction XtremeCT is a high-resolution peripheral quantitative computed tomography (HRpQCT) device that is developed mainly for the representation and quantitative measurement of three-dimensional (3D) Correspondence author: Maximilian A. Dambacher. E-mail: [email protected]

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Fig. 1. XtremeCT with the patient in place on a comfortable and adjustable seat during CT scanning of the (a) distal forearm and (b) lower leg.

bone structures in vivo in humans (Dambacher et al. 2007) (Fig. 1). Given the specific features of XtremeCT, however, the device can also be extended to large animals for both in vivo and ex vivo or in vitro applications, as well as for evaluations of appreciated materials. This chapter provides general information on the specific features of XtremeCT, and a step-by-step guide to perform XtremeCT measurements using standard measurement protocols for in vivo applications in humans. Specific userdefined measurement protocols can be developed for various applications.

2. Specific Features of XtremeCT (Dambacher et al. 2007; SCANCO Medical) •

• •

Type: high-resolution pQCT for human (Fig. 1) and large animal in vivo measurements of both volumetric bone mineral density (BMD) and bone microarchitecture X-ray: microfocus X-ray source, 70 µm spot size, 60 kVp/40 keV (<1 mA), no shielding required Detector: 3072 × 255 elements, fiber optic taper, 41 µm pitch

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• •

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Resolution (standard): 4.2 lp/mm (10% MTF on the projections) Image matrix: 1536 × 1536 (standard measurement for tibia and radius with 900 mA); possibly up to 3072 × 3072 Specimen size: field of view, 126 mm; maximum scan length, >150 mm Scan time: <3 minutes for one stack of a total of 110 slices (1536 × 1536, continue mode) Stack height: 9 mm Effective dose: 3 µSv (for a standard measurement with 95 mA) Operator safety: outside a range of 2 m from the scanner, the machine does not show detectable stray radiation; thus, no radiation at the normal operator position Power input: 208–230 VAC, 50–60 Hz, 10 A Scanner weight and dimensions (W × D × H): 475 kg and 1380 mm × 930 mm × 1460 mm

3. Software for Complete Imaging and Evaluation Solution • • • • • • • • • • • •

Data acquisition Online and offline reconstruction Sophisticated two-dimensional (2D) and 3D evaluation 3D visualization and animation Volumetric density measurements of trabecular and cortical regions Structural measurements Baseline and follow-up measurements based on automatic matching of common region (see Sec. 4.4.2) Archiving Finite element (FE) analysis 64-bit software (only 64-bit software allows the handling of very big datasets) Database Browser access (Web-based access)

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4. Operation Using the Standard Scanning and Measurement Program (XtremeCT manual book) 4.1. Preparation before scanning • • • •

•

•

Turn on the key of the scanner (30 minutes before the first measurement of the day). Turn on the computer and the monitor. It takes approximately 5 minutes before the computer is ready to start. Log in by typing in the username, and then click OK. Press the button Precalibrate to conduct a precalibration (when the control file is selected, the system needs to be calibrated; a precalibration must be done every hour). Edit operator data and edit patient data. By the first measurement of the patient, the patient data need to be registered. It is very important that the registration is consequently done. Follow the instructions in the section Edit Patient to fill in the fields. It is recommended to have a special tape for phantom measurements labeled “PHANT1”. Further recommendations for backups of the phantom measurements are as follows:





•

•

Daily phantom per week: It is very important to run daily phantom measurements prior to the first measurement of a patient every day, and to compare the results with the reference phantom measurement. The whole procedure of daily phantom measurement will take approximately 45 minutes (30 minutes for the warming up of the X-ray tube and about 15 minutes for the measurement and evaluation). Weekly phantom per month: The phantom has to be measured to control the functionality and stability of the system.

Old measurements can be deleted, but make sure to always keep all daily phantom measurements from the last week and all weekly measurements from the last month on the computer. Raw data have higher priority for backup than image data, since the image data can always be reconstructed from the raw data. Instruct the patient for positioning and stable scanning. It is very important to instruct the patient not to move or talk during the scanning. Apart from scanning in site for measuring the distal

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Fig. 2. Position of the patient in bed for CT scanning of the (a) distal forearm and (b) lower leg.

radius and tibia (Fig. 1), the application can also be extended to patients who are not able to sit during scanning, i.e. to position the patient in bed for scanning both the forearm and the leg (Fig. 2).















Perform the calibration (if necessary). Select and use a cast fixator. Select an adequate cast fixator to fit the patient for either forearm or leg scanning (Fig. 3), and then fix the cast into the cast slider of the machine. Test the size of the arm pad. The arm must lie stable in the hand cast. Position the hand and arm in the hand cast. Try to get the hand as far in as possible. Fixate the cast in the cast slider. Move the hand cast completely into position and snap in the cast at the gantry. Check that the cast is positioned correctly. Measure the scout view. After closing the bore cover and positioning the patient correctly, the scout view measurement has to be conducted. The scout view is used to determine the measurement area where a reference line has to be set carefully on the hump in the joint space (Fig. 4). For the follow-up measurements, the reference line has to be set exactly at the same location. Only in this way can changes in bone density and architecture be reliably determined. In the Scout View window, adjust the start and end positions by clicking on the Reference

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(a)

(b)

Fig. 3. Carbon fiber cast for fixation of the (a) arm and (b) leg before CT scanning.

Line button; the first reference scout view will be shown for every follow-up measurement of the same patient.

4.2. Starting of the measurement program To start the measurement, click on the SCAN button. During the measurement, the patient must not move or talk at all. It is very important that the patient is informed about and understands this. During the measurement, the operator should not talk or move so as not to distract the patient.

4.3. Validity of the measurement Control that the measurement is valid by looking at the reconstructed slice of the measurement (Figs. 5 and 6). If no artifacts can be found

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Fig. 4. After the patient is correctly positioned, the scout view measurement is conducted to determine the measurement area where a reference line has to be set carefully on the hump in the joint space of the (a) distal radius or (b) distal tibia.

Fig. 5. XtremeCTR — a third-generation pQCT machine — is developed mainly for the visualization and quantitative measurement of 3D skeleton structures of humans in vivo, including the distal radius and distal tibia. (a) Cross-sectional and (b) sagittal images of the distal radius of a normal subject; (c) cross-sectional and (d) sagittal images of the distal radius of an osteoporotic patient.

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Fig. 6. 3D reconstructed skeleton structures of humans in vivo, including the distal forearm and distal tibia. (a) Cross-sectional and (b) sagittal images of the distal radius of a normal subject; (c) cross-sectional and (d) sagittal images of the distal radius of an osteoporotic patient.

in the image, the measurement is valid. If movement artifacts are found in the image, the measurement is not valid and a new measurement needs to be made.

4.4. Starting of the evaluation program Start the evaluation program by clicking the button with the evaluation symbol. After starting the evaluation program, the Select Sample and Measurement window appears. On the left-hand side of the window, there is a list of patients whose measurements have not yet been archived. Click on the desired patient, and then all of the measurements of this patient will appear on the right-hand side. Click on the

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desired measurement and confirm by clicking the OK button. All images that belong to this measurement are now loaded and will appear on the screen.

4.4.1. Define the volume of interest •

•

•

Draw a contour on the first image. The first image is automatically chosen; the images can also be chosen manually by entering the desired number of slices in the box under the image. Click the Draw Contour button and draw the contour in the image as close to the bone edge as possible. Press the left mouse button as long as you are drawing. It is important to draw in a counterclockwise direction around the bone. If correction of the contour is needed, click on the correct button and then draw over the contour you want to modify. Here, you must also draw in a counterclockwise direction. The original contour has to be crossed at least twice. The part between the first and the last points of intersection will be replaced by the new drawing.

4.4.2. Start the automatic contour detection by clicking on the contouring button With a standard resolution of 82 µm, XtremeCTR enables the measurement and monitoring of changes to the 3D bone structure in vivo in humans (Fig. 7). This opens a new horizon to evaluate the efficacy of drugs developed for prevention and treatment of osteoporosis and potentially also for osteoporotic fracture repair. Automatic standard evaluation includes the following (Fig. 8): • • • •

Trabecular and cortical evaluation of the radius or tibia Density in different regions, including Ct, Tb, etc. Structural indices such as Tb.N, Tb.Th, Tb.Sp, and Ct.Th Matching of the 3D region of interest for follow-up scans that is calculated from the common region (Rüegsegger 1994)

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Fig. 7. Monitoring of trabecular bone loss in the distal radius of a postmenopausal women using XtremeCT. (a) 3D distal radius at baseline; (b) 3D distal radius of the same region made 5 months after baseline measurement. The arrows demonstrate trabecular bone structural deterioration and loss.

5. Accuracy and Precision (Reproducibility) of XtremeCT Long-term reproducibility is a relative measure of the precision of repeated measurements over a more or less prolonged period of time. Reproducibility depends on the method used and on the value of the mineral density of intact bone at baseline. This device features a reproducibility in vivo of 0.7%–1.5% for total trabecular and cortical BMD and 2.5%–4.4% for trabecular architecture,

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Fig. 8. Datasheet of a standard XtremeCT report of a distal forearm that contains all of the density and structural data for diagnosis of osteoporosis and/or for comparison of research studies.

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with a resolution of 82 µm (Dambacher et al. 2007). However, it is recommended to define the precision for each ethnic, age, and gender-specific group (Boutroy et al. 2005; Dambacher et al. 2007).

6. Applications of XtremeCT The recent use of this latest development for human applications suggests that XtremeCTR has the following characteristics: •

• • • • •

Ιt has an ability to discriminate between osteopenic women with and without a prior history of fracture for distal extremities (Figs. 5 and 6). It can identify fast and slow bone losers. It is suitable for studying large skeletons in vitro, such as the hip (Fig. 9) and spine. Its quantitative assessment of the trabecular microarchitecture allows for an improved assessment of fracture risk. It can better monitor the treatment effects of both injectable biomaterials and drugs. It allows for prospective studies to assess osteopenic patients at increased risk of fracture.

Fig. 9. An in vitro 2D image front view of a human femoral head scanned at a resolution of 80 µm.

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Fig. 10. Rabbit vertebra scanned in vitro at 40 µm. (a) 2D transversal slice; (b, c) 3D reconstruction from two different views.

•

It is suitable for preclinical studies using large animals in vivo (Fig. 10) (Boutroy et al. 2005).

7. Summary The following are the key features of the XtremeCT for skeleton applications: •

• • • •

It provides valuable information about the complex trabecular network in the extremities in vivo for humans and about all skeletons of the human cadaver in vitro; it can also be used for in vivo large animal applications. It has good reproducibility and accuracy. It allows a low dose (3 mSv) to be given to the patient. It has an automated standard evaluation procedure, but is fully flexible for other applications. It can also be used for animals and in vitro scanning.

References Boutroy S, Bouxsein ML, Munoz F, Delmas P. In vivo assessment of trabecular bone microarchitecture by high-resolution peripheral quantitative computed tomography. J Clin Endocrinol Metab 90:6508–6515, 2005.

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Rüegsegger P. The use of peripheral QCT in the evaluation of bone remodelling. Endocrinologist 4(3):167–176, 1994. SCANCO Medical AG. Avaliable at http://www.scanco.ch/ XtremeCT manual book. Scanco Medical AG, Switzerland.

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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

This chapter describes quantitative computed tomography (QCT) image processing techniques for bone mineral density (BMD) and structure assessments of the proximal femur and vertebrae, as well as their applications in clinical trials for assessing osteoporosis therapy efficacy, in studying bone loss due to weightlessness, in understanding age-related changes in bone, and in examining how BMD and geometric characteristics correlate to fracture risk. With intrasubject rigid registration, we observe improved longitudinal measurement precision by reducing operator errors, and directly visualize bone loss of astronauts associated with long-duration spaceflight. To develop a general framework for three-dimensional (3D) bone modeling and population comparison, we adapt intersubject registration techniques to transform groups of hip QCT scans into a common reference space. We apply this technique to study the spatial distribution of microgravityinduced bone loss in the proximal femur. Keywords:

Quantitative computed tomography (QCT); proximal femur; spine; image registration; bone mineral density (BMD); osteoporosis; spaceflight.

Corresponding author: Wen-Jun Li. Tel: +1-415-3534932; fax: +1-415-3539423; E-mail: [email protected]

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1. Introduction Osteoporotic skeletal fracture is an epidemic problem in the elderly, and is primarily caused by the age-related loss of bone mass and deterioration of bone structure. Bone mineral density (BMD) is a surrogate measure of osteoporosis. In clinical settings, BMD is generally measured by dual-energy X-ray absorptiometry (DXA) (Stein et al. 1987; Mazess et al. 1989); more recently, three-dimensional (3D) quantitative computed tomography (QCT) imaging has become feasible with the advent of fast helical and multidetector CT scanners and the cost reduction in computer processing power. Compared to DXA, which produces projectional two-dimensional (2D) images, QCT imaging permits compartment-specific 3D BMD and geometry assessment. Densitometric and structural assessment techniques based on volume reconstructions of the proximal femur and vertebrae from QCT scans have been developed, such as those at the University of California, San Francisco, USA (Lang et al. 1997; Lang et al. 1999; Lang et al. 2004) and the University of Erlangen, Germany (Kang et al. 2003). Computer algorithms used to analyze QCT images generally involve manual interaction, which introduces intraoperator or interoperator precision errors and can affect the ability of this technique to resolve small bone changes. To reduce manual interaction, we have developed an automatic image registration algorithm for 3D hip and spine images (Li et al. 2006a). After registration of longitudinal images, the follow-up scan can be automatically segmented and analyzed. Characterizing the impact of a disease process or a drug therapy on the integrity of bone depends on understanding how the disease or treatment effect is distributed throughout the bone of interest. Recently, we have developed an approach using intersubject image registration and statistical atlas construction to model the proximal femur three-dimensionally (Li et al. 2007). We transformed the hip QCT scans of 16 astronauts to a common reference hip space, and observed subregions inside the proximal femur that experienced the most significant bone loss due to long-duration spaceflight. In this chapter, we describe techniques for analyzing hip and spine QCT images and their applications. We focus on the techniques

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recently developed in our lab, including quantification of bone parameters (BMD, geometry, bone strength indices), longitudinal measurement based on image registration, and 3D bone modeling and population comparison.

2. Materials The image acquisition of two QCT image datasets is described below.

2.1. Hip and spine QCT scans of postmenopausal women Twenty pairs of repeat QCT scans were acquired, including 10 hip pairs and 10 spine pairs. The subjects were a group of postmenopausal women. For the hip group, the mean age was 63 years ± 2 years, and the T-score was −1.05 ± 1.30; for the spine group, the mean age was 64 years ± 3 years, and the T-score was −0.76 ± 1.69. Each patient was scanned twice with repositioning after a time interval of 15 minutes between scans. The scanner was a Philips Mx8000 16-slice detector system (Philips Medical Systems, Eindhoven, The Netherlands). QCT images were calibrated to BMD using a solid hydroxyapatite calibration phantom (Image Analysis, Columbia, KY, USA). The scanning parameter settings were 90 kVp and 280 mAs for the hip scans, and 90 kVp and 140 mAs for the spine scans. Scans used 3-mm-thick sections with a 3-mm/s table speed.

2.2. QCT scans of astronauts before and after spaceflight QCT scans from 16 crew members (44.6 years ± 4.0 years) of the second through eighth International Space Station (ISS) missions were acquired. Preflight scans were performed 30–60 days prior to launch, while postflight scans were performed within 7–10 days of landing and 1 year after landing. QCT images were acquired using a helical CT scan protocol (GE HiSpeed Advantage; GE Medical Systems, Milwaukee, WI, USA) at scanning settings of 80 kVp, 280 mAs, 3-mm slice thickness, and a pitch of 1. The in-plane pixel size was 0.9375 mm.

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3. Methods 3.1. Bone measurements of individual QCT scans Densitometric and structural assessments of the proximal femur based on CT scans — such as the approaches developed by Lang et al. (1997 and 2004) and Kang et al. (2003) — generally involve segmentation of the entire proximal femoral bone envelope, with combinations of mathematical morphology and thresholding, and edge detection approaches to derive the regions of interest (ROIs) for quantification. Lang et al. (1999) also developed a volumetric spinal QCT approach in which anatomic landmarks such as the vertebral endplates and the spinous process are used to fix the 3D orientation of the vertebral body. Some of the segmented femur and vertebra ROIs are shown in Fig. 1. Below, we describe the QCT scan analysis techniques developed by Lang et al. (1997 and 1999) in more detail. We will mainly use the hip scan analysis (Lang et al. 1997) for illustration. The basic methods for vertebral analysis are similar and are described in Lang et al. (1999). 3.1.1. Bone segmentation and bone-fixed anatomic coordinate systems •

•

•

•

Calibration: Volumetric data are converted from Hounsfield units (HU) to BMD (mg/cm3 calcium hydroxyapatite) using a calibration phantom scanned simultaneously with the patient. Proximal femur contouring: The external contours of the proximal femur are determined from each axial slice using a regiongrowing technique, and are used to generate a 3D binary mask of the hip. Definition of bone-fixed anatomic coordinate systems: The CT and binary data are resampled along the direction of the femoral neck axis, which is determined manually from the CT images in the coronal and sagittal views. Determination of ROIs: The femoral neck ROI is defined as a portion of the binary image bounded in the femoral neck axis by fixed percentages of the distance between the intertrochanteric

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Fig. 1. Regions of interest (ROIs) in (a) proximal femur, (b) femoral neck, and (c) vertebra. For the femur and femoral neck, cortical, integral, and trabecular ROIs are shown in the coronal view. For the vertebra, both lateral and axial views are shown, including the integral, cortical, and trabecular ROIs of the vertebral centrum as well as the integral ROI of the total vertebral integral bone.

•

and femoral head cross-sectional area (CSA) maxima (the medial limit is 75% of this distance, and the lateral limit is 25%). The integral trochanteric ROI is bounded by the lateral limit of the femoral neck region and the lateral limit of the CSA curve. Finally, the femoral neck and trochanteric subregions are combined to form the total femur ROI. Trabecular and cortical bone extraction: Trabecular ROIs are extracted from the integral ROIs by a 4-mm erosion of the external contours. Residual cortical bone is removed from the trabecular ROIs by use of a 350-mg/cm3 global threshold. The cortex

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is determined by including those voxels above the 350-mg/cm3 threshold in the total femur ROI. 3.1.2. Bone parameter quantification •

•

•

Volumetric BMD: The scan analysis program calculates the average BMD in the femoral neck, the trochanter, and the total femur, where the ROIs are derived as described above. The calculations are performed for the cortical, trabecular, and integral regions, respectively. Bone geometry: For the ROIs derived above, we can determine the integral, cortical, and trabecular bone volumes discretely. Geometric measurements also include the CSA along the femoral neck axis, including the smallest CSA of the femoral neck and the largest CSA through the trochanteric region. The femoral neck axis length is determined as the length of the femoral neck axis between the medial limit of the femoral head and the base of the trochanter. Bone strength indices: For the hip, the moments of inertia are calculated in the body-fixed principal axes (Corcoran et al. 1994) at the location of the femoral neck slice of minimum CSA (CSAmin). These are used to derive the femoral neck bending strength index (fnBSI) (Lang et al. 2004). For vertebra analyses, a 10-mm-thick section through the midvertebra is also determined and is used to calculate the vertebral compressive strength index (Lang et al. 2004).

3.2. Intrasubject rigid image registration Intrasubject registration aligns scans with global translations and rotations, i.e. rigid transformations, where the femur or a vertebra is considered as a rigid body during the transformations. 3.2.1. Rigid registration algorithm The QCT image dataset used to develop our rigid registration was described in Sec. 2. The registration algorithm is based on mutual

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Diagram of the main steps of rigid registration.

information. It works as follows, and is illustrated schematically in Fig. 2. •

• •

Image cropping: Prior to registration, the baseline and follow-up CT images are cropped to include only the bone structure to be registered (either the left or right hip, or either the L1 or L2 vertebra). The images are also resampled to the isotropic voxel size and blurred to reduce noise. Bone segmentation: The baseline scan is segmented to create a binary bone mask using a region-growing algorithm. Resampling and voxel selection: The images are resampled to a selected resolution. The voxels from the segmented binary mask are used to create a list of voxels for calculating image similarity during the registration process.

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Image similarity calculation: To calculate the mutual information, we use an adaptive bin size method similar to that described in Zhu and Cochoff (2002). At each registration step, based on the current configuration of the baseline and transformed follow-up images, a normalized mutual information (Studholme et al. 1999) is calculated. Optimization: The program then iteratively searches for the optimal transformations in the six-dimensional (6D) parameter space (three translations and three rotations) using simplex optimization (Press et al. 1999). To speed up the registration and to avoid local maxima at the startup, we apply a multiresolution scheme (Studholme et al. 1997; Hill et al. 2001).

3.2.2. Longitudinal measurement based on rigid registration After registration, the transformed follow-up image is automatically processed for segmentation and definition of the ROIs based on the morphometric features identified in the baseline analysis. Note that instead of simply superposing the ROIs defined from the baseline onto the registered follow-up image, we allow the registered follow-up image to be resegmented and the ROIs redefined. This allows the program to take into account possible periosteal apposition effects thought to be associated with aging and anabolic drug therapies (Oxlund et al. 1993; Riggs et al. 2004). When the ROIs are defined, quantification of femoral and spinal parameters for the registered image is subsequently performed. 3.2.3. Evaluation of measurement precision using repeat scans To calculate the precision error for each BMD, geometry, and bone strength index measurement, we use the precision definition given by Glüer et al. (1995), i.e. the root mean square of the coefficients of variation (CVs) calculated from the baseline and registered follow-up scan pairs.

3.3. Intersubject nonrigid registration and population-based statistical atlas In order to compare different bones three-dimensionally, the geometric correspondences among homologous locations need to be

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established. This is achieved by nonrigid registration. Once such correspondences are established, the scans can be resampled in a common reference space to build a statistical atlas. 3.3.1. Nonrigid registration Figure 3 illustrates the use of registration to compare homologous locations in scans from different subjects. In Fig. 3 (3D rendering), after the source scan [Fig. 3(b)] is registered toward the target scan [Fig. 3(a)], it is resampled to create a new image [Fig. 3(c)] that has the same dimensions as the target scan. To determine the grayscale value at each voxel of the resampled image, for instance (i, j, k), the transformation derived from the registration is applied to calculate its homologous location, (x, y, z), in the original source scan. The BMD

Fig. 3. Nonrigid registration establishes the geometric correspondence between homologous locations in (a) the target scan and (b) the source scan. (c) The registered image is a resampled image of the original source image (b). For each voxel in (c), the grayscale bone mineral density (BMD) value is taken from the corresponding location in (b).

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value at (x, y, z) will then be used as the BMD value at (i, j, k) in the resampled image [Fig. 3(c)], and can be compared with the BMD value at (i, j, k) in the target scan [Fig. 3(a)]. When (x, y, z) has noninteger values, interpolation will be applied (we use trilinear interpolation) to obtain the BMD value around that location. The resampled image [Fig. 3(c)], i.e. the registered image, looks like a “warped” image from the original scan [Fig. 3(b)]. We have adapted a nonrigid registration algorithm originally developed for brain magnetic resonance images by Collins and colleagues (1995 and 1997). It is an automatic method for volumetric image data. This nonrigid registration algorithm estimates local rigid transformations to achieve global nonrigid registration. The main steps are illustrated in Fig. 4. The original registration algorithm is described in detail by Collins and Evans (1997) and Collins et al. (1995).

Fig. 4.

Diagram of the main steps of nonrigid registration.

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3.3.2. Atlas construction and statistics We have constructed a femoral atlas using the spaceflight QCT image datasets described in Sec. 2.2 (Li et al. 2007). The postflight scans to be described here are those scanned shortly after the landing (not those scanned after 1 year). •

• •

•

•

Preprocessing: Similar to rigid registration, image preprocessing includes image cropping, resampling to isotropic voxels, and bone segmentation. Rigid registration: We apply rigid registration to automatically align hip QCT scans, as described earlier in Sec. 3.2.1. Nonrigid registration: A typical scan is selected to serve as the reference/target scan for registration. The main steps of nonrigid registration are illustrated in Fig. 4. Statistical atlas: After the scans of the 16 subjects are registered, they are resampled in the common reference space to form a femur statistical atlas. We subtract the preflight and postflight average images to visualize the distribution of bone loss between these two groups. Voxel-by-voxel statistics: Each voxel location in the common atlas space corresponds to 16 elements from the registered preflight scans and 16 elements from the registered postflight scans. A paired t-test is performed to obtain localized t-statistics, and such voxel-by-voxel t-tests are performed through the total proximal femur. Due to the large amount of tests performed, we use false discovery rate (FDR) (Genovese et al. 2002) to provide correction for multiple comparisons.

4. Applications 4.1. Bone quantification based on individual QCT scans 4.1.1. Clinical trials of osteoporosis drugs A recent study (Black et al. 2003) applied the hip and spine QCT analysis techniques described in Sec. 3.1 to investigate whether the

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use of antiresorptive drugs, such as alendronate and risedronate, and parathyroid hormone, which stimulates bone formation, would provide a therapeutic advantage by combining different mechanisms for the reduction of the risk of fracture. There was no evidence of synergy between parathyroid hormone and alendronate. Changes in the volumetric density of trabecular bone and in the cortical tissue volume at the hip suggested that the concurrent use of alendronate might reduce the anabolic effects of parathyroid hormone. The continuation of the above trial (Black et al. 2005) observed that, over 2 years, alendronate therapy after 1-year parathyroid hormone therapy led to significant increases in BMD in comparison with the results for placebo after 1-year parathyroid hormone therapy — a difference particularly evident for BMD in trabecular bone at the spine on quantitative CT. These results have clinical implications for therapeutic choices after the discontinuation of parathyroid hormone. 4.1.2. In vivo fracture correlation A recent cross-sectional study comparing women imaged within 48 hours of a hip fracture to age-matched and body-size–matched controls showed that hip fracture was significantly associated with reduced BMD in the cortical, integral, and trabecular compartments, as well as reduced measures of cortical volume and thickness (Cheng et al. 2007). Fracture status was also associated with increased femoral neck CSA. In addition, the study found that measures of cortical geometry and trabecular BMD were independently associated with hip fracture. QCT was also employed to characterize the association of femoral neck density and geometry parameters with incident hip fracture in men in Orwoll et al.’s (2006) prospectively designed study. 4.1.3. Age-related bone changes Meta et al. (2006) reported QCT differences in proximal femoral compartmental BMD, bone mineral content, bone geometry, and bone strength indices between healthy young women and healthy elderly women. Cross-sectional changes in femoral neck BMD and

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geometry were consistent with the presence of periosteal apposition (Riggs et al. 2004). With age, there was greater loss of trabecular and cortical bone in the femoral neck region than in the trochanteric region, and a greater increase in bone size at the trochanteric region compared with the femoral neck region. 4.1.4. Bone loss due to long-duration spaceflight Bone loss due to weightlessness is considered one of the most intractable problems of long-duration spaceflight. Recently, Lang et al. (2004) conducted the first systematic investigation using QCT imaging to study the effects of spaceflight in the vertebrae and hip. The subjects and image acquisition were described in Sec. 2.2. The results indicated that, despite extensive exercise programs designed to maintain mechanical loading of the skeleton, crew members on the ISS (International Space Station) showed as much bone loss as did their counterparts one decade ago on the Soviet space station Mir (Vico et al. 2000). In addition, Lang et al. (2006) studied the effect of re-exposure to Earth’s gravity on the proximal femoral BMD and structure of astronauts 1 year after the missions. It was observed that the readaptation of the proximal femur to Earth’s gravity entailed an increase in bone size and an incomplete recovery of volumetric BMD. Proximal femoral bone mass was substantially recovered in the year after spaceflight, but measures of BMD and estimated bone strength showed only partial recovery. The data indicate that recovery of skeletal density after long-duration space missions may exceed 1 year.

4.2. Longitudinal measurement using intrasubject rigid registration An example of registered images is displayed in Fig. 5, which compares the matched proximal femoral cross-section from the baseline and the aligned follow-up image as well as their difference image. The boundaries are drawn on the baseline image and directly superimposed on the follow-up image, showing good qualitative agreement with the bone edge on the registered follow-up image.

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Fig. 5. Coronal cross-section from the baseline (left), the aligned followup (middle), and the difference image (right) of a repeat scan pair. The femur boundaries outlined from the baseline image are superimposed onto the follow-up image to check their alignment.

4.2.1. Improving measurement precision In the study of Li et al. (2006a), the precision errors of bone parameters measured in the proximal femur and spine using rigid registration were compared with the manual results. For the proximal femur, such parameters included femoral neck, trochanter and total femur BMDs (cortical, trabecular, and integral) and volumes (cortical and integral), CSAs (minimum and maximum), and femoral neck bending strength index. For vertebrae L1 and L2 (treated separately), measurements included midvertebral and total vertebral BMDs (cortical, trabecular, and integral), simulated DXA BMD, vertebral lengths (anteroposterior and lateral), CSA, and compressive strength index. Some of these parameters are shown in Table 1. By using automated registration, most of the BMD, geometry, and strength index precision errors were smaller than those obtained by manual analysis of individual scans. 4.2.2. 3D visualization of bone loss due to long-duration spaceflight We applied our hip registration technique to align the preflight and postflight QCT scans so that the subregional bone changes could be directly visualized side by side (Li et al. 2006b). Figure 6 shows images (in axial view) obtained from registering the preflight and

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Table 1. Comparison of hip and spine measurements obtained from 10 repeat scan pairs using manual analysis and automated registration. The first numerical column shows the average values of bone parameters, along with their standard deviations. This is followed by the root mean square (RMS) errors between the baseline and follow-up scans in physical units. Precision is calculated as the RMS of the coefficients of variation (CVs) of the 10 pairs, with the CV deviations shown on the far right. Measurement in physical units

Hip measurements Neck integral BMD (g/cm3) Neck trabecular BMD (g/cm3) Neck cortical BMD (g/cm3) Total femur integral BMD (g/cm3) Total femur trabecular BMD (g/cm3) Total femur cortical BMD (g/cm3) CSA_min (cm2) fnBSI (cm3)

Spine measurements 3D trabecular BMD (g/cm3) 3D integral BMD (g/cm3) 3D cortical BMD (g/cm3) CSA (cm2) Compressive strength index (g2/cm4)

CV (%)

Average value ± standard deviation

Errorregistration (Errormanual)

Precisionregistration (Precisionmanual)

Deviationregistration (Deviationmanual)

0.273 ± 0.039

0.005 (0.005) 0.005 (0.006) 0.012 (0.022) 0.004 (0.004) 0.001 (0.001) 0.009 (0.010) 0.233 (0.226) 0.025 (0.037)

1.31 (1.56) 4.53 (5.85) 1.61 (2.89) 0.87 (1.14) 0.82 (0.72) 1.24 (1.36) 1.51 (1.44) 3.66 (5.09)

0.80 (1.17) 2.74 (3.21) 1.06 (2.30) 0.67 (0.59) 0.63 (0.30) 0.86 (0.69) 0.93 (0.87) 2.08 (3.07)

0.004 (0.007) 0.003 (0.004) 0.006 (0.006) 0.217 (0.383) 0.012 (0.017)

1.92 (3.56) 0.90 (1.19) 2.11 (1.89) 1.61 (2.41) 3.31 (5.33)

0.94 (2.65) 0.51 (0.85) 1.00 (1.26) 0.87 (1.79) 2.29 (3.36)

0.089 ± 0.032 0.521 ± 0.030 0.262 ± 0.036 0.109 ± 0.033 0.511 ± 0.030 10.715 ± 0.815 0.530 ± 0.082

0.143 ± 0.037 0.224 ± 0.033 0.190 ± 0.022 9.774 ± 1.603 0.253 ± 0.113

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Fig. 6. Visualization of bone loss in the proximal femur after rigid image registration. Images were scanned from an astronaut (a) before and (b) after a half-year spaceflight. Thinning of cortical bone and loss of trabecular bone can be observed.

postflight scans of an astronaut. In order to better illustrate the bone changes, false colors were applied to the CT values. After registration, bone changes at the corresponding regions of the preflight and postflight scans could be directly visualized. For example, we can clearly see the thinning of cortical bone and loss of trabecular bone after the spaceflight.

4.3. 3D bone modeling and population comparison using statistical atlas 4.3.1. Intersubject registration An example of the registered images is shown in Fig. 7 (in coronal view). In the top row, one slice is shown from each of two QCT scans, i.e. the target and the source, from two different subjects (before registration). From their superimposed image on the far right, we can see the body positioning difference, such as from the different shaft orientations. After registering the source scan toward the target scan using rigid registration, they were well aligned, as can be seen by comparing the superposition images in the first and second rows.

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Fig. 7. Intersubject registration of hip images (in coronal view). The source scan is rigidly and nonrigidly transformed toward the target scan. Arrows indicate the location where anatomical differences between the target and the source, and the warping effect, can be easily seen.

However, anatomic variability of these two femora can be seen from the rigidly registered images (middle row), for example, at the location pointed by the arrows. Notice that registration was performed only for the femur, not including the acetabulum; therefore, the acetabular parts remained unaligned. After nonrigid registration, as shown in the bottom row of Fig. 7, the nonrigidly registered (warped) source image appeared like the target. The warping effect from rigid to nonrigid registration can be seen, for example, by comparing the transformed source images in the middle and bottom rows at the location pointed by the arrow. 4.3.2. 3D bone modeling and population comparison We performed intersubject registration (rigid and nonrigid) for the spaceflight QCT scans (Li et al. 2007). After registration, all of the

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Fig. 8. Comparison of preflight and postflight hip models (integrated from scans of 16 subjects). (a) Subtraction of the preflight and postflight average images. Regions I and II, shown as highlighted regions in the inset, experienced greater bone loss. (b) Voxels where bone loss shows statistical significance in false discovery rate (FDR) analysis (marked in white).

scans were transformed to a common reference space, and the average preflight and postflight images were calculated. Subtraction between the two average images illustrated regional variation of bone loss through the proximal femur [Fig. 8(a)]; we see that regions marked by I and II show greater magnitude in bone loss compared to the rest. Based on voxel-by-voxel t-tests and FDR analysis (with the q-value set at 0.05), in Fig. 8(b), regions I and II also show statistical significance in bone loss while most other regions do not.

5. Discussion Compared to DXA, which measures bone properties based on 2D projectional images, QCT imaging makes it possible to measure the differential response of trabecular and cortical bones to aging, drug treatment, and mechanical unloading, as well as the correlation between bone density and structural characteristics and fracture risk. In QCT image analysis, errors due to manual operations may hinder this technique in resolving small bone differences. Longitudinal bone measurements of the hip and spine, as demonstrated by our registration algorithm, can be automated to reduce user interaction and to

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improve measurement precision. Intrasubject registration also makes it possible to directly visualize subregional bone changes. By applying intersubject registration, we integrated hip scans into a common reference hip space. By comparing the preflight and postflight models for 16 astronauts experiencing 4–6 months of spaceflight, we observed regions inside the femur that showed the most significant bone loss in the proximal femur. Focusing bone measurement on such most responsive regions can potentially improve our understanding of bone response to mechanical unloading as well as to disease and treatment. The fundamental limitation of registration-based image processing is that registration inevitably introduces errors due to data interpolation during image transformation (partial volume effect) and misregistration. Registration errors make this technique unsuitable for quantifying thin structures such as the femoral and vertebral cortices. With higher image resolution and improved registration techniques, registration-induced errors may be reduced. In addition, in population-based statistical atlas analyses, we generally assume that the anatomic topology of femoral and vertebral structures is the same among populations; however, this assumption may not hold precisely. The QCT image processing software packages developed at our laboratory are mainly written in C and currently run in a UNIX environment (Linux). When using the scan analysis program (Sec. 3.1), users are required to identify typical bone anatomic features such as the femoral neck axis and the vertebra axis. The registration programs (Secs. 3.2 and 3.3) are automatic (no anatomic landmarks need to be identified); however, before registration, the contours of the proximal femur or the specific vertebra need to be provided so that the bone structure to be registered is separated from the soft tissue or adjacent bones (such as the acetabulum or a neighboring vertebra). We generally use the contours generated by the scan analysis programs, but they can be created using other image editing applications as well. The hip and spine QCT scan analysis programs have been used routinely in several institutes in the U.S. and in Iceland, and they are currently being integrated with the registration programs.

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6. Summary QCT imaging provides a noninvasive assessment of compartmental bone properties including BMD and bone geometry. Bone strength indices can also be derived. Automatic intrasubject registration of the hip and spine can improve longitudinal bone measurements by reducing errors due to user interaction. With intrasubject registration, longitudinal bone changes can also be directly visualized. By constructing a population-based statistical atlas using intersubject registration, groups of QCT images can be integrated and compared.

Acknowledgments This study was supported by NASA Grant NNJ04HF78G and NIH grant NIH-R42-AR45713.

References Black DM, Bilezikian JP, Ensrud KE et al. One year of alendronate after one year of parathyroid hormone (1–84) for osteoporosis. N Engl J Med 353(6):555–565, 2005. Black DM, Greenspan SL, Ensrud KE et al. The effects of parathyroid hormone and alendronate alone or in combination in postmenopausal osteoporosis. N Engl J Med 349(13):1207–1215, 2003. Cheng X, Li J, Lu Y et al. Proximal femoral density and geometry measurements by quantitative computed tomography: association with hip fracture. Bone 40(1):169–174, 2007. Collins DL, Evans AC. Animal: validation and applications of nonlinear registration-based segmentation. Int J Pattern Recognit Artif Intell 11(8):1271–1294, 1997. Collins DL, Holmes CJ, Peters TM, Evans AC. Automatic 3-D model-based neuroanatomical segmentation. Hum Brain Mapp 3(3):190–208, 1995. Corcoran TA, Sandler RB, Myers ER et al. Calculation of cross-sectional geometry from CT images with application in postmenopausal women. J Comput Assist Tomogr 18(4):626–633, 1994. Genovese CR, Lazar NA, Nichols T. Thresholding of statistical maps in functional neuroimaging using the false discovery rate. Neuroimage 15(4):870–878, 2002.

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Glüer CC, Blake G, Blunt BA et al. Accurate assessment of precision errors: how to measure the reproducibility of bone densitometry techniques. Osteoporos Int 5(5):262–270, 1995. Hill DL, Batchelor PG, Holden M, Hawkes DJ. Medical image registration. Phys Med Biol 46(3):R1–R45, 2001. Kang Y, Engelke K, Kalender WA. A new accurate and precise 3-D segmentation method for skeletal structures in volumetric CT data. IEEE Trans Med Imaging 22(5):586–598, 2003. Lang T, LeBlanc A, Evans H et al. Cortical and trabecular bone mineral loss from the spine and hip in long-duration spaceflight. J Bone Miner Res 19(6):1006–1012, 2004. Lang TF, Keyak JH, Heitz MW et al. Volumetric quantitative computed tomography of the proximal femur: precision and relation to bone strength. Bone 21(1):101–108, 1997. Lang TF, Leblanc AD, Evans HJ, Lu Y. Adaptation of the proximal femur to skeletal reloading after long-duration spaceflight. J Bone Miner Res 21(8):1224–1230, 2006. Lang TF, Li J, Harris ST, Genant HK. Assessment of vertebral bone mineral density using volumetric quantitative CT. J Comput Assist Tomogr 23(1): 130–137, 1999. Li W, Kezele I, Collins L et al. Voxel based modeling and quantification of the proximal femur using inter-subject registration of quantitative CT images. Bone 41(5):888–895, 2007. Li WJ, Sode M, Saeed I, Lang T. Automated registration of hip and spine for longitudinal QCT studies: integration with 3D densitometric and structural analysis. Bone 38(2):273–279, 2006a. Li WJ, Sode M, Saeed I, Lang T. Image registration of proximal femur with substantial bone changes: application in 3D visualization of bone loss of astronauts after long duration spaceflight. Prog Biomed Opt Imaging 7(30):61443B.1–61443B.5, 2006b. Mazess RB, Collick B, Trempe J et al. Performance evaluation of a dual energy X-ray bone densitometer. Calcif Tissue Int 44:228–232, 1989. Meta M, Lu Y, Keyak JH, Lang T. Young–elderly differences in bone density, geometry and strength indices depend on proximal femur subregion: a cross sectional study in Caucasian-American women. Bone 39(1):152–158, 2006.

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Orwoll ES, Marshall LM, Chan BK et al. Measures of hip structure are important determinants of hip fracture risk independent of BMD. J Bone Miner Res 21(Suppl 1):S35, 2006. Oxlund H, Ejersted C, Andreassen TT et al. Parathyroid hormone (1–34) and (1–84) stimulate cortical bone formation both from periosteum and endosteum. Calcif Tissue Int 53(6):394–399, 1993. Press WH, Teukolsky SA, Vetterling WT, Flannery BP. Numerical Recipes in C. Cambridge University Press, Cambridge, UK, 1999. Riggs BL, Melton LJ, Robb RA et al. Population-based study of age and sex differences in bone volumetric density, size, geometry, and structure at different skeletal sites. J Bone Miner Res 19(12):1945–1954, 2004. Stein JA, Lazewatsky JL, Hochberg AM. Dual energy X-ray bone densitometer incorporating an internal reference system. Radiology 165(Suppl): 313, 1987. Studholme C, Hill DL, Hawkes DJ. Automated three-dimensional registration of magnetic resonance and positron emission tomography brain images by multiresolution optimization of voxel similarity measures. Med Phys 24(1):25–35, 1997. Studholme C, Hill DLG, Hawkes DJ. An overlap invariant entropy measure of 3D medical image alignment. Pattern Recognit 32(1):71–86, 1999. Vico L, Collet P, Guignandon A et al. Effects of long-term microgravity exposure on cancellous and cortical weight-bearing bones of cosmonauts. Lancet 355(9215):1607–1611, 2000. Zhu YM, Cochoff SM. Influence of implementation parameters on registration of MR and SPECT brain images by maximization of mutual information. J Nucl Med 43(2):160–166, 2002.

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Chapter 40

Micro-Finite Element Analysis of Bone He Gong, Ming Zhang and Ling Qin

Micro-finite element (micro-FE) analysis is a numerical technique to obtain the mechanical properties of bone or artificial bone-like structures as they relate to their microstructures. The micro-FE approach was developed for use in cooperation with images obtained from microcomputed tomography (CT), peripheral quantitative computed tomography (pQCT), or magnetic resonance imaging (MRI). This chapter describes the establishment of the micro-FE model and the factors that may influence the numerical results. Our study on the microstructural and mechanical parameters of a trabecular bone from a vertebral body is used as an example to illustrate the process of building the micro-FE model and the analysis of results. Some recent developments and applications of this technique are also introduced. The computational data provided by the micro-FE technique may help us to better understand the bone mechanical properties as well as the failure mechanisms associated with osteoporosis, osteoarthritis, loosening of implants, and cellmediated adaptive bone remodeling processes. Keywords:

Micro-FE; failure behavior; apparent anisotropic elastic properties; loading conditions; in vivo; bone remodeling process; linear elastic analysis; nonlinear analysis; degree of mineralization; voxel conversion.

Corresponding author: Ming Zhang. Tel: +852-27664939; fax: +852-23624365; E-mail: [email protected]

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1. Introduction Micro-finite element (micro-FE) analysis is a numerical technique to obtain the mechanical properties of bone or artificial bone-like structures as they relate to their microstructures. The micro-FE approach was developed for use in cooperation with images obtained from in vitro imaging techniques such as micro-computed tomography (micro-CT), in which case a resolution of up to 10 µm can be reached. Recent studies showed that the micro-FE approach can also provide an adequate evaluation of bone mechanical properties for in vivo conditions, based on the existing imaging techniques that can be applied to bone in vivo, with a resolution adequate to visualize individual trabeculae. In this sense, peripheral quantitative computed tomography (pQCT) can be used with an isotropic spatial resolution of 165 µm (Laib et al. 1997; Ulrich et al. 1999), a whole-body magnetic resonance imaging (MRI) scanner can provide a resolution of approximately 150–200 µm in plane at a plane thickness of 300–350 µm (Lang et al. 1998; Laib et al. 1998; Wehrli et al. 1998), and in vivo micro-CT can provide a resolution of 16 µm for in vivo animal study (Dambacher et al. 2007; Gasser and Ingold 2007). With the above imaging techniques, the morphologies of bone or artificial bone-like structures can be obtained by a large number of sequential cross-sectional images. Three-dimensional (3D) trabecular structures are reconstructed from these digitized images as voxel grids, with voxels representing bone tissue or marrow. By converting voxels representing bone tissue to equally shaped eight-node brick elements, a micro-FE model is generated that can represent the trabecular structure in great detail (van Rietbergen et al. 1996). The micro-FE technique can be used to obtain accurate and complete characterization of bone mechanical behavior, such as a complete evaluation of apparent anisotropic elastic properties (Kabel et al. 1999; van Rietbergen et al. 2002). The tissue Young’s modulus can be obtained by comparison between experimental tests and micro-FE results (Homminga et al. 2002; Morgan et al. 2003). The bone failure load can also be predicted (Pistoia et al. 2002; Pistoia et al. 2004); the results predicted from micro-FE analyses agreed well with those

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measured experimentally. Furthermore, micro-FE analysis can be used to quantify the loading conditions of bone at the tissue level (Gong et al. 2007a; Gong et al. 2007b; Homminga et al. 2004; van Rietbergen et al. 1999), as well as those of bone tissue-engineering scaffolds for the load transfer from the biomaterial structure to the biological entities (Lacroix et al. 2006). Recently, this technique was implemented to identify the location of bone tissue at the highest risk of initial failure (Eswaran et al. 2007). Another application of the micro-FE technique is to analyze bone mechanical properties in vivo for the improvement of the prediction of bone fracture risk or for the evaluation of the efficacy of drugs used to treat bone diseases based on pQCT or MRI measurements (Pistoia et al. 2004; van Rietbergen 2001; van Rietbergen et al. 2002). In addition, the validation of micro-FE results with mechanical testing is possible for in vitro study. In general, the computational data provided by the micro-FE technique can help us to better understand bone mechanical properties as well as the failure mechanisms associated with osteoporosis, osteoarthritis, loosening of implants, and cell-mediated adaptive bone remodeling processes. Accordingly, this chapter describes the establishment of the micro-FE model and the factors that may influence the numerical results. Some recent developments and applications of this technique are also introduced. Such a numerical simulation technique provides a useful platform for the evaluation of bone mechanical properties.

2. Methods The technical steps to run micro-FE analysis are as follows: (1) Preparations • •

Selection of a volume of interest Image processing




Filtering using the IPL gauss_ip command Segmentation using the IPL threshold command Removal of unconnected parts using the IPL cl_rank_exact command

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(2) Preprocessing and solving • • •

Commencement of the program using the IPLFE FEM aim_file_name command Inputting of parameters or files Selection of output parameters

(3) Postprocessing • •

Commencement of the program by the IPLFE FE_post command Selection of output files

The majority of these functions are illustrated below.

2.1. Imaging A micro-CT, pQCT, axial CT, or MRI system is used to reconstruct the microarchitecture of bone or artificial bone-like structures. 2.1.1. Selection of a volume of interest (VOI) The volume of interest (VOI) is the basis for the micro-FE model. For trabecular bone, a representative volume should be at least five intertrabecular lengths in size (approximately 3–5 mm). It is not possible to obtain meaningful mechanical properties for specimens smaller than this minimal size (Scanco Medical FE software manual, 2004). To obtain a complete characterization of the apparent anisotropic elastic properties, a cubic specimen that can be tested in its three orthogonal directions is necessary; otherwise, only a limited number of elastic parameters can be obtained. For example, meaningful elastic properties can only be obtained for the longitudinal direction in the case of cylindrical specimens. 2.1.2. Voxel size and accuracy The micro-FE models are created by the voxel conversion technique from the voxel data. With this technique, voxels in the model are

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converted to equally shaped brick elements such that the resulting micro-FE model has the exact same geometry as the reconstruction it is derived from. Accordingly, it is necessary to consider the voxel size and accuracy first. In general, a smaller element size will increase the accuracy of the results; however, the central processing unit (CPU) time needed to solve the problems is mainly determined by the number of elements. The choice of the resolution will thus be a trade-off between accuracy and CPU time. There are three criteria for evaluating voxel size and accuracy: (1) As a rule of thumb, it has been stated that a micro-FE model should have at least four elements through a cross-section of the microstructural components that form the structure (Guldberg et al. 1998). (2) Homminga et al. (2002) used the criteria of
f =

Ê 8 ¥ number of elements ˆ Ln Á ˜ Ë number of nodes ¯

.

Ln 2 The recommended minimal mesh fractal number is 1.83 for a rod-like architecture and 2.42 for a plate-like architecture (the maximal mesh fractal number is 3.0 for a hexahedron model). 2.1.3. Image processing After choosing an appropriate voxel size, the usual Gaussian filtering procedure can be applied to reduce noise artifacts. Special care should be taken when choosing the filter parameter to make sure that the data do not become too blurry. An initial micro-FE model is then obtained by segmentation of the data. The threshold value has an important effect on the volume

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fraction of the generated model and, accordingly, a large effect on the results of the micro-FE analyses. The threshold value can be chosen by comparing the volume fraction of the final model with that measured by another calibrated technique (e.g. Achimedes principle or QCT measurement). Ouyang et al. (1997) developed a global threshold criterion for high-resolution MRI. The segmentation threshold for each image was determined from the average gray-level values within six calibration regions of interest (ROIs): three ROIs in the air, tendon, and cortical bone, respectively, yielding low graylevel values; and one in bone marrow and two in subcutaneous fat, yielding high gray-level values. For each subject, the six calibration ROIs were placed on all five contiguous middle slices and the mean gray-level value in each ROI was measured. Averaging over all five slices, the following gray-scale values were defined: (1) ATC, the mean gray-level value of all the ROIs in air, tendon, and cortical bone; and (2) FAT, the mean gray-level value of the ROIs in marrow and subcutaneous fat. The segmentation threshold must separate image pixels representing bone marrow from those representing bone trabeculae. The segmentation threshold therefore lies somewhere between the high (bright) graylevel values of FAT and the low (dark) gray-level values of ATC. A single threshold value, TH, was calculated for all five image slices as follows: ATC ˆ Ê TH = ATC + d Á 1 ˜, Ë FAT ¯ where δ is an empirical constant. The threshold determined by the equation is the mean ATC value over the five slices plus a modification term that takes into account the dynamic range of the grayscale in each image. This is an entirely empirical approach, and other approaches could be derived based on the same rationale and study goal; however, this approach can be used as a means of standardizing the analysis of images obtained at different times and in different subjects. Following this, parts that are poorly connected to the main structure are removed. This is necessary to avoid numerical problems during the FE analyses, and will not affect the results of these analyses since

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the poorly connected parts do not contribute to the stiffness of the specimens and do not carry loads.

2.2. Loading conditions There are two kinds of loading conditions for micro-FE analysis. The first one is to simulate experimental tests for the calculation of overall (apparent) properties of the structure. For example, the model can be used to simulate a compression test on a bone specimen in order to calculate the apparent stiffness of the specimen (Gong et al. 2007; Kabel et al. 1999; van Rietbergen et al. 2002). It is also possible to specify user-defined test conditions. The second one is to calculate tissue-level mechanical loading conditions for external loads working on the structure (Eswaran et al. 2007; Homminga et al. 2004; van Rietbergen et al. 1999). For a femoral head, it is useful to artificially add a cup or cartilage layer that intersects a face of the VOI in order to simplify the application of loading condition (van Rietbergen et al. 1999).

2.3. Boundary conditions Scanco Medical FE software (Scanco Medical AG, Bassersdorf, Switzerland) has a library from which the boundary conditions for standard experimental tests can be chosen. It is also possible to specify user-defined testing conditions. The micro-FE model generally consists of a very large number of brick elements. Special-purpose iterative (conjugate gradient) solvers in combination with an element-by-element matrix–vector multiplication scheme have been developed to ensure the solution of such large problems within reasonable CPU time limits (van Rietbergen et al. 1996). In the case of small-strain linear elastic analysis, the results on tissue-level stress and apparent modulus can be scaled from the tissue modulus.

3. Example 3.1. Micro-CT scanning A micro-CT system (µCT40; Scanco Medical AG, Bassersdorf, Switzerland) was used to reconstruct the microarchitecture of the

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trabecular cubes from the L4 vertebral body of a 63-year-old male cadaver. The spatial resolution for specimen scanning was set to 20 µm. During each scanning, the vertebral body was scanned continuously with increments of 20-µm thickness for 250 slices. The region of interest (ROI) was chosen as a 5 mm3 × 5 mm3 × 5 mm3 cube in the center of the vertebral body. The image was segmented with a threshold to obtain the same volume fraction as that measured by Archimedes-based calibration. Table 1 lists the architectural parameters evaluated by a direct method in the system for a three-dimensional (3D) model. A 3D reconstruction of the trabecular cube was generated using a built-in program of the micro-CT system [Fig. 1(a)].

3.2. Micro-FE modeling Scanco Medical Finite Element Software 1.02 (Scanco Medical AG, Bassersdorf, Switzerland) was used to perform micro-FE analysis in order to simulate the axial compression tests in three orthogonal directions: longitudinal, medial-lateral, and anterior-posterior. The micro-CT reconstruction model was used to construct the micro-FE model by directly converting image voxels representing hard tissue (20 µm in size) to eight-node brick finite elements. A fixeddisplacement boundary condition was chosen for micro-FE analysis: Table 1.

3D microstructural parameters of the trabecular cube.

Microstructural parameter BV/TV SMI H1 (mm) H2 (mm) H3 (mm)

Value

Microstructural parameter

Value

0.0913 1.5543 1.4403 1.0963 0.8806

Conn.D. (1/mm3) Tb.N (1/mm) Tb.Th (mm) DA Tb.Sp (mm)

2.4637 1.1179 0.1321 1.6356 0.8668

BV/TV: bone volume fraction; SMI: structure model index; H1, H2, H3: the three radii of the mean intercept length (MIL) ellipsoid; Conn.D.: connectivity density; Tb.N: trabecular number; Tb.Th: trabecular thickness; DA: degree of structural anisotropy; Tb.Sp: trabecular separation.

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(b)

Fig. 1. (a) 3D reconstruction of the microstructure of a trabecular cube from the L4 vertebral body of a 63-year-old male cadaver. (b) The loading and boundary conditions when compressed in the longitudinal direction.

all nodes at the bone–plate interface were constrained in the plane of the plate, with all other surfaces unconstrained, to simulate the boundary conditions used in other mechanical testing procedures (Judex et al. 2003; Morgan et al. 2003). A displacement of 1% compressive strain was applied to the trabecular cube in the longitudinal direction, anterior-posterior direction, and medial-lateral direction, respectively. Figure 1(b) shows the loading and boundary conditions when compressed in the longitudinal direction. Bone material properties at the tissue level were assumed to be homogeneous and isotropic with an elastic modulus of 15 GPa (Rho et al. 1997). The isotropic Poisson’s ratio of bone material was set to be 0.3 (Homminga et al. 2002).

3.3. Results Table 1 shows the 3D microstructural parameters of the trabecular cube. The number of bone elements in the micro-FE model was 657 731 and the number of nodes was 870 977. The computational

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Table 2.

Mechanical parameters calculated from the simulation of compression tests.

Direction of compression test

Apparent Young’s modulus (MPa)

Tissue von Mises stresses (MPa)

Longitudinal Anterior-posterior Medial-lateral

401.720 37.449 31.920

61.836 ± 48.691 19.794 ± 17.424 14.741 ± 10.022

time for each micro-FE analysis was about 8 hours. Table 2 lists the mechanical parameters calculated from the simulation of compression tests. It was shown that the vertebral trabecular specimen displayed a near transversely isotropic mechanical behavior. Figure 2 shows the von Mises stress distributions of the trabecular cube compressed in the three orthogonal directions. Figure 3 consists of histograms showing the distributions of tissue von Mises stresses in the compressions of the three orthogonal directions. When compressed in the longitudinal direction, there was 71% of tissue volume with von Mises stresses lying within the mean ± SD, 95.48% below 155 MPa, and 98.69% below 200 MPa. When it was compressed in the medial-lateral direction, there was 90.75% of tissue volume with von Mises stresses within the range of the mean ± SD, 95.41% below 36 MPa, and 99.27% below 80 MPa. When it was compressed in the anterior-posterior direction, there was 90.32% of tissue volume with von Mises stresses lying within the mean ± SD, 93.01% below 44 MPa, and 98.13% below 80 MPa.

4. Discussion There are some factors that may have an effect on the precision and/or accuracy of the micro-FE result.

4.1. Jagged surfaces caused by unique cubic elements — a precision-related factor The modeling of trabecular bone with unique cubic elements produces jagged surfaces, which can lead to errors in the stress/strain

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(b)

(c)

Fig. 2. Von Mises distributions of the trabecular cube compressed in (a) the longitudinal direction, (b) the anterior-posterior direction, and (c) the medial-lateral direction.

calculations, especially at the bone surfaces (Guldberg and Hollister 1994). However, researchers have found that, although local oscillations and errors in the calculated stresses and strains might exist, their effect on the distributions and the calculated average and standard deviations is small (Guldberg and Hollister 1994; van Rietbergen et al. 1999). Hence, this modeling method is sufficiently converged, and only a minor part of the wide variation in tissue stresses and strains can be due to numerical artifacts related to these jagged surfaces.

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(b)

(c)

Fig. 3. Histograms of the distributions of tissue von Mises stresses compressed in (a) the longitudinal direction, (b) the anterior-posterior direction, (c) the medial-lateral direction.

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4.2. High-friction vs. low-friction boundary condition — an accuracy-related factor Micro-FE analysis can be used to simulate mechanical tests that are performed on the specimens, whereby laterally fixed boundary conditions at the loaded surfaces are called high-friction boundary conditions. Ladd and Kinney (1998) determined that the difference between a laterally fixed and a laterally free compression test can be as much as 25% for apparent stiffness. Whether to choose a high-friction or low-friction boundary condition depends on the experimental conditions in the mechanical test. For example, Homminga et al. (2002) chose the laterally fixed boundary conditions because the plates in the mechanical test were not purposefully lubricated, although the marrow from the specimens may have acted as a mild lubricant. However, if there is movement at the loaded surfaces, this would influence all of the specimens to the same extent.

4.3. Linear vs. nonlinear analysis — a precisionand accuracy-related factor Micro-FE analysis for the calculation of the elastic behavior of trabecular bone has been the subject of many studies. There have also been some studies that used linear FE analysis to predict the failure behavior of bone. For example, Pistoia et al. (2002 and 2004) used linear elastic micro-FE models to predict the distal radius strength. They assumed that bone failure would be initiated as soon as a significant part of the bone tissue was strained beyond a critical limit, which was taken as the tissue yield strain. The application of this technique can lead to a better prediction of the bone failure load for bone in vivo than is possible from dual-energy X-ray absorptiometry (DXA) measurements, structural parameters, or a combination thereof, although the failure loads predicted differed in absolute value. As another example, Eswaran et al. (2007) predicted the locations of bone tissue at high risk of initial failure during compressive loading of the human vertebral body. They assumed that all bone tissues within each vertebra having either the maximum or minimum principal strain beyond

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its 90th percentile was the tissue at highest risk of initial failure within the vertebral body. This study provided a new insight into the micromechanics of failure of the trabecular and cortical bone within the human vertebra, and the consistency of their results with those from a single fully nonlinear analysis supported the validity of their approach to identify those regions in which the tissue was most likely to fail initially. Of course, the application of linear FE analysis to investigate failure behavior may have some limitations. For the simulation of bone failure behavior, where strain-dependent changes in material properties and large deformation can play an important role (Bevill et al. 2006), a nonlinear micro-FE approach is needed. Such analyses are much more complex and computationally expensive than linear analyses. With the development of computer technology, however, such nonlinear micro-FE analyses could be more feasible. At present, whether to use linear or nonlinear analysis depends on the objective of the study.

4.4. Degree of mineralization of bone material — a precision- and accuracy-related factor The best tool to analyze stresses and strains in bone during in vivo loading conditions is large-scale FE analysis (van Rietbergen et al. 1996). However, in most of the simulations in the literature, Young’s modulus was assumed to be homogeneous. Recently, van Ruijven et al. (2007) investigated the effect of an inhomogeneous distribution of mineralization on stress and strain distributions in the human mandibular condyle during static clenching. They found that the trabecular bone of the condyle was subjected to a larger range of stresses and strains than the cortical bone and that an inhomogeneous mineral distribution reduced the trabecular stiffness substantially, which in turn led to an increase of the condyle deformation, cortical strains, trabecular strains, and mediolateral trabecular displacements. Hence, it was concluded that neglect of the inhomogeniety of the mineral distribution could result in a large underestimation of the stresses and strains. The renewal process of bone results in an inhomogeneous

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distribution of mineralization (Mulder et al. 2005); therefore, when doing micro-FE analysis for bone tissue in which the bone remodeling process is active, the inhomogeniety of mineral distribution should be taken into consideration. As for Poisson’s ratio, Ladd and Kinney (1998) analyzed the sensitivity of the calculated elastic moduli to the variations in Poisson’s ratio of the tissue material. They found that all apparent elastic moduli varied by less than 2% over a range of tissue-level Poisson’s ratios from 0.15 to 0.35. Hence, the effect of variations in Poisson’s ratio on bone tissue is negligible.

4.5. Small bone samples vs. larger pieces of bones — a precision-related factor Studies applying the micro-FE technique to small bone samples have produced valuable information about load transfer in trabecular architecture (Gong et al. 2007a; Gong et al. 2007b; Judex et al. 2003). However, this cannot be transferred to the situation of bone in situ, as the boundary forces for an excised specimen are not representative of the intact situation. Nevertheless, this does not affect comparative study. Recently, micro-FE analyses were applied to obtain the trabecular tissue mechanical properties throughout larger pieces of bones or whole joints (Homminga et al. 2004; Pistoia et al. 2002; Pistoia et al. 2004; van Rietbergen et al. 1999). With this procedure, assumptions about boundary conditions can seriously influence the results as well (Pistoia et al. 2004). Moreover, computational power needs to be further developed to enable the solving of such large problems within a reasonable CPU time.

5. Summary The micro-FE technique can be used to obtain accurate and complete characterization of bone mechanical behavior as well as the in vitro and in vivo loading conditions of bone at the tissue level, and can also be used to predict bone failure behavior. It can provide important information needed for the diagnosis of bone quality and bone failure risk.

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As soon as research on devices is further developed, the remaining uncertainties in the micro-FE results will be eliminated to provide sufficient resolution for resolving the trabecular network in vivo; then, the micro-FE technique can be extensively used for in vitro clinical investigations.

Acknowledgments The related work was supported by The Hong Kong Polytechnic University Research Grant (No. G-YX64, 1-BB81) and a grant from the National Natural Science Foundation of China (10502021).

References Bevill G, Eswaran SK, Gupta A et al. Influence of bone volume fraction and architecture on computed large-deformation failure mechanisms in human trabecular bone. Bone 39(6):1218–1225, 2006. Dambacher MA, Neff M, Radspieler HT et al. In vivo bone mineral density and structures in humans: from Instom over Densiscan to XtremeCT. In: Qin L, Genant HK, Griffith J, Leung KS (eds.), Advanced Bioimaging Technologies in Assessment of the Quality of Bone and Scaffold Materials, Springer Verlag, Berlin, pp. 65–78, 2007. Eswaran SK, Gupta A, Keaveny TM. Locations of bone tissue at high risk of initial failure during compressive loading of the human vertebral body. Bone 41(4):733–739, 2007. Gasser JA, Ingold P. Osteoporosis research with the vivaCT40. In: Qin L, Genant HK, Griffith J, Leung KS (eds.), Advanced Bioimaging Technologies in Assessment of the Quality of Bone and Scaffold Materials, Springer Verlag, Berlin, pp. 451–462, 2007. Gong H, Zhang M, Qin L. Mechanical properties of vertebral trabeculae with aging evaluated with microCT. In: Qin L, Genant HK, Griffith J, Leung KS (eds.), Advanced Bioimaging Technologies in Assessment of the Quality of Bone and Scaffold Materials, Springer Verlag, Berlin, pp. 463–474, 2007a.

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Gong H, Zhang M, Qin L, Hou Y. Regional variations in the apparent and tissue-level mechanical parameters of vertebral trabecular bone with aging using micro-finite element analysis. Ann Biomed Eng 35(9): 1622–1631, 2007b. Guldberg RE, Hollister SJ. Finite element solution errors associated with digital image-based mesh generation. 1994 Proceedings of the ASME Bioengineering Conference, pp. 147–148, 1994. Guldberg RE, Hollister SJ, Charras GT. The accuracy of digital image-based finite element models. J Biomech Eng 120:289–295, 1998. Homminga J, McCreadie BR, Ciarelli TE et al. Cancellous bone mechanical properties from normals and patients with hip fractures differ on the structure level, not on the bone hard tissue level. Bone 30(5):759–764, 2002. Homminga J, van Rietbergen B, Lochmuller EM et al. The osteoporotic vertebral structure is well adapted to the loads of daily life, but not to the infrequent ‘error’ loads. Bone 34(3):510–516, 2004. Judex S, Boyd S, Qin YX et al. Adaptations of trabecular bone to low magnitude vibrations result in more uniform stress and strain under load. Ann Biomed Eng 31:12–20, 2003. Kabel J, van Rietbergen B, Odgaard A, Huiskes R. Constitutive relationships of fabric, density, and elastic properties in cancellous bone architecture. Bone 25(4):481–486, 1999. Lacroix D, Chateau A, Ginebra MP, Planell JA. Micro-finite element models of bone tissue-engineering scaffolds. Biomaterials 27:5326–5334, 2006. Ladd AJC, Kinney JH. Numerical errors and uncertainties in finite-element modeling of trabecular bone. J Biomech 31:941–945, 1998. Laib A, Hauselmann HJ, Ruegsegger P. In vivo high resolution 3D-QCT of the human forearm. Technol Health Care 6:329–337, 1998. Laib A, Hildebrand T, Hauselmann HJ, Ruegsegger P. Ridge number density: a new parameter for in vivo bone structure analysis. Bone 21:541–546, 1997. Lang T, Augat P, Majumdar S et al. Noninvasive assessment of bone density and structure using computed tomography and magnetic resonance. Bone 22:149s–153s, 1998. Morgan EF, Bayraktar HH, Keaveny TM. Trabecular bone modulus–density relationships depend on anatomic site. J Biomech 36:897–904, 2003.

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Mulder L, Koolstra JH, Weijs WA, van Eijden TMGJ. Architecture and mineralization of developing trabecular bone in the pig mandibular condyle. Anat Rec 285A:659–666, 2005. Ouyang X, Selby K, Lang P et al. High resolution magnetic resonance imaging of the calcaneus: age-related changes in trabecular structure and comparison with dual X-ray absorptiometry measurements. Calcif Tissue Int 60:139–147, 1997. Pistoia W, van Rietbergen B, Lochmuller EM et al. Estimation of distal radius failure load with micro-finite element analysis models based on three-dimensional peripheral quantitative computed tomography images. Bone 30(6):842–848, 2002. Pistoia W, van Rietbergen B, Lochmuller EM et al. Image-based microfinite-element modeling for improved distal radius strength diagnosis — moving from “bench” to “bedside”. J Clin Densitom 7(2):153–160, 2004. Rho JY, Tsui TY, Pharr GM. Elastic properties of human cortical and trabecular lamellar bone measured by nanoindentation. Biomaterials 18:1325–1330, 1997. Scanco Medical FE software manual, version 1.03. Scanco Medical AG, Bassersdorf, Switzerland, 2004. Ulrich D, van Rietbergen B, Laib A, Ruegsegger P. Load transfer analysis of the distal radius from in vivo high-resolution CT imaging. J Biomech 32:821–828, 1999. van Rietbergen B. Micro-FE analyses of bone: state of the art. Adv Exp Med Biol 496:21–30, 2001. van Rietbergen B, Majumdar S, Newitt D, MacDonald B. High-resolution MRI and micro-FE for the evaluation of changes in bone mechanical properties during longitudinal clinical trials: application to calcaneal bone in postmenopausal women after one year of idoxifene treatment. Clin Biomech 17:81–88, 2002. van Rietbergen B, Muller R, Ulrich D et al. Tissue stresses and strain in trabeculae of a canine proximal femur can be quantified from computer reconstructions. J Biomech 32:443–451, 1999. van Rietbergen B, Weinans H, Polman BJW, Huiskes R. Computational strategies for iterative solutions of large FEM applications employing voxel data. Int J Numer Methods Eng 39:2743–2767, 1996.

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van Ruijven LJ, Mulder L, van Eijden TMGJ. Variations in mineralization affect the stress and strain distributions in cortical and trabecular bone. J Biomech 40:1211–1218, 2007. Wehrli FW, Hwang SN, Ma J et al. Cancellous bone volume and structure in the forearm: noninvasive assessment with MR microimaging and image processing. Radiology 206(2):347–357, 1998.

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Chapter 41

The Characterization of Cortical Bone Water 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

A nuclear magnetic resonance (NMR) spin-spin (T2) relaxation technique is described for determining water distribution changes in cortical bone tissue. The advantages of using the NMR T2 relaxation technique for bone water distribution are illustrated. The Carr-Purcell-Meiboom-Gill (CPMG) sequence-based T2 relaxation data can be used to determine the porosity, and its inversion T2 relaxation spectrum can be transformed to a pore-size distribution with the longer relaxation times corresponding to larger pores. The free induction decay (FID)-based T2 relaxation data can be inverted to T2-FID relaxation distribution, and this distribution can then be transformed to bound and mobile water distribution with the longest relaxation time corresponding to mobile water and the middle relaxation time corresponding to bound water. The technique is applied to quantify apparent changes in porosity as well as bound and mobile water in cortical bone age, microdamage, and disuse. Overall bone porosity is determined using the calibrated NMR fluid volume from the proton relaxation data divided by overall bone volume. The NMR bound and mobile water changes are determined and found from cortical bone specimens obtained from different-aged donors Corresponding author: Qing-Wen Ni. E-mail: [email protected], [email protected]

691

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A Practical Manual for Musculoskeletal Research and functional disused turkey. It is also demonstrated that the NMR T2 relaxation data is sensitive to changes resulting from the creation of microdamage in cortical bone, which can be interpreted as an effective increase in bone porosity; the result indicates that the detection of cortical bone microdamage is possible by this technique. The obtained information may be used as a measure of bone quality describing porosity and water content, both of which may be important determinants of bone strength and fracture resistance. Keywords:

NMR; spin-spin relaxation; NMR system; bone; cortical porosity; pore size; water distribution; microdamage; disuse.

1. Introduction Quantification of measures of bone quality such as porosity, water distribution (bound and mobile), microdamage accumulation, and other microstructural characteristics may lead to a more accurate measure of bone strength and therefore fracture risk. It has been shown that agerelated increases in bone porosity without significant changes in bone mineral density (BMD) result in a decrease in bone strength (McCalden et al. 1993). Bone fracture toughness is also significantly correlated to changes in porosity, water distribution (bound and mobile water) (Yeni et al. 1997; Yeni et al. 1998), microarchitecture, osteonal morphology, collagen integrity, and microdamage (Currey et al. 1996; Yeni et al. 1998), all of which are measures of bone quality. Bone is a two-component composite material in which the mineral phase (mainly hydroxyapatite) confers the strength (Zioupos 2001) and stiffness (Currey 1988), while the organic matrix (mainly type I collagen) primarily influences the toughness of bone (Wang et al. 2001; Zioupos et al. 1999; Zioupos 2001). While mineral and collagen each contribute to bone’s competency, as do microarchitecture (e.g. porosity and osteonal morphology), macrostructure (e.g. curvature of diaphysis and thickness of cortical shell), and in vivo microdamage (e.g. microcracks and diffuse cracks), their interaction with water is equally important to the mechanical behavior of bone. Early studies demonstrated that bone stiffness, tensile strength, and hardness increase, whereas strain at fracture and energy to fracture decrease, following the dehydration of bone tissue (Dempster and Liddicoat

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1952; Evans 1973; Evans and Lebow 1951; Sedlin and Hirsch 1966; Smith and Walmsely 1959; Yamada and Evans 1970). In a comprehensive review on the porosity of bone, Martin (1984) described that small changes in porosity lead to significant changes in the stiffness and strength of both compact and spongy bone. In another study, McCalden et al. (1993) reported that the capability of bone to absorb energy during fracture strongly correlates with the porosity of human bone. The total fluid contained within bone tissue includes fluid explicitly within the intrinsic microstructural porosity (Fantazzini et al. 2003; Fantazzini et al. 2004; Ni et al. 2004; Wang and Ni 2003), fluid contained within microcracks and diffuse damage (Ni and Nicolella 2005), and fluid within the extracellular matrix. The distribution of water within bone tissue appears to change throughout life. It has been reported that water in bone tissue changes with skeletal growth (Jonsson et al. 1985) and with progressive mineralization (Robinson 1975; Robinson 1979). The observation that mineral content increases with age, tapering at elder years (Mueller et al. 1966; Timmins and Wall 1977), implies that the amount of water in the tissue would likely be reduced in the elderly skeleton. The influence of water removal on the strength and toughness of cortical bone has been studied by Nyman et al. (2006). Their results showed that loss of water in the collagen phase decreases the toughness of bone, whereas loss of water associated with the mineral phase decreases both bone strength and toughness; however, in that paper, the loss of water was achieved by the dehydration method for such mechanism studies. Among the current techniques, quantitative computed tomography (QCT) and dual-energy X-ray absorptiometry (DXA) can determine the volumetric density of compact bone (Genant et al. 1997). However, they cannot provide detailed information on compact bone microstructure, such as porosity and pore size changes. Conventionally, histomorphometry of biopsy bone samples can provide a direct assessment of bone microstructural characterization including pore size distribution, but it does not observe actual three-dimensional structures and requires an invasive surgical procedure.

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Recently, magnetic resonance imaging (MRI) has been applied for characterization of bone marrow (Mulkern et al. 1994; Traber et al. 1996). High-resolution MR images that resolve trabecular bone structure have been obtained in vitro at high field strength (Chung et al. 1993; Hipp et al. 1996) and in vivo using clinical scanners at 1.5 tesla (Jara et al. 1993; Majumdar et al. 1993; Selby et al. 1996). In addition, solid state MRI microscopy has been developed to demonstrate human dental anatomy using a proton 300 MHz (7.1 tesla) spectrometer to reach a resolution of 195 µm (Appel and Baumann 2002). An extremely high-resolution MRI system has been developed to study microscopic features of normal and Leber’s hereditary optic neuropathy (LHON) human optic nerves with a proton 500 MHz (12 tesla) microimaging system to yield a resolution of 30 µm (Sadun et al. 2002). However, no current MRI technology can obtain usable imaging from human cortical bone or bone microdamage. Synchrotron-radiation-based computer microtomography has been applied to biomineralized tissues by Prymark et al. (2005); their results showed that the obtained resolution can be on a micrometer scale (1–2 µm) that is very powerful, but it gives no information on porosity or water distribution. On the other hand, nuclear magnetic resonance (NMR) proton spin-spin (T2) or spin-lattice (T1) relaxation time measurements and analytical processing techniques have been used to determine microstructural characteristics including the porosity, pore size distribution, and permeability of various types of fluid-filled porous media with characteristic pore sizes ranging from submicron to submillimeter (Appel and Baumann 2002; Chui et al. 1995; Gallegos et al. 1987; Glaves and Smith 1989; Howard et al. 1993; Kenyon 1997; Prymark et al. 2005; Sadun et al. 2002). Using this NMR relaxation technique, a wide range of pore sizes can be probed with the requirement of less pore-shape assumptions (Liaw et al. 1996). This is because the observed proton NMR relaxation signals are a convolution of the relaxation of fluid in the various pores throughout the observed system. Since this method is noninvasive and nondestructive, the use of this technique has great potential in the biomedical field.

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Similar to the other porous media, the amplitude of the proton NMR Carr-Purcell-Meiboom-Gill (CPMG) (Carr and Purcell 1954; Meiboom and Gill 1958; Strange 1994) sequence-based spin-spin relaxation data may be used to determine the porosity of bone if water-based fluids fill up the major portion of compact bone natural pores. Moreover, its inversion T2 relaxation distribution can be transformed to a pore size distribution after the surface relaxivity is known. The NMR proton T2 relaxation rate (1/T2) is known to depend on the surface-to-volume ratio with the proportionality of the surface relaxivity constant. By testing human compact bone samples from the elderly and younger populations, we determine whether or not the low-field NMR spin-spin relaxation (T2) measurement and its inversion T2 spectrum technique are suitable for detecting age-related changes in the porosity and pore size distribution of human compact bones. In addition, we investigate the use of the NMR inversion T2-FID spectrum derived from NMR free induction decay (FID) measurements for estimating the bound and free water distribution in vitro in bone specimens from the different age groups. This new information on water distribution within bone tissue will be used for further bone mechanical property studies and a more accurate assessment of bone quality. In this paper, the focus is to demonstrate that bone porosity, pore size distribution, water distribution changes, and damage including microporosity of cortical bone can be detected by NMR relaxation techniques. Here, we hypothesize that the NMR relaxation technique can nondestructively and noninvasively assess bound water (water bound to collagen and mineral phases) and mobile water (water within the pores) as well as characterize bone microdamage and bone disuse. The results of these measurements can be directly used for further determination of cortical bone mechanical properties.

2. Background 2.1. Porosity determination The essential point for our experiment is that signal decay is fast for solids (the characteristic decay time T2 is normally <100 µs), but

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longer for liquids. It is known that the total amplitude of T2 relaxation envelopes, measured by NMR CPMG spin-echo train, is a representation of the liquid phase inside the pores (Sadatmanesh and Ehsani 1997; Strange 1994). The NMR CPMG magnetization amplitude of liquid inside the pores can be transferred to the volume of water. For example, if the water volume Vw, the corresponding measured NMR CPMG magnetization amplitude Mw (which is obtained from the NMR signal at relaxation time back to zero of the total amplitude envelope), and the obtained bone NMR signal amplitude Ml are known, then the volume of the liquid (water) inside the bone is Vl = (Vw Ml)/Mw. The volume of bone is determined by Archimedes’ water displacement principle (i.e. a body immersed in a fluid will be buoyed up by a force equal to the weight of the fluid that it displaced). Thus, the calibrated bone volume is VB = (Wair – Wwater)/dwater, where VB is the volume of bone, Wair is the weight of bone in the air, Wwater is the weight of bone in the water, and dwater is the density of water. After the volume of the bone sample (VB) is known, the porosity of bone can be estimated as Vl/ VB.

2.2. Relationship between NMR data and pore sizes Based on the low-field NMR principle with Hτ ≈ 0.1 gauss/s maintained (Straley et al. 1997), the diffusion effect may be negligible. Here, we accept Brownstein and Tarr’s (1979) assumption that the relaxation rate 1/T2 is proportional to the surface-to-volume (S/V) ratio of the pore: 1 ÊSˆ = rÁ ˜ , Ë V ¯ pore T2

(1)

where ρ is the surface relaxivity, i.e. a measure of the pore surface’s ability to enhance the relaxation rate. Equation (1) indicates that the NMR relaxation time is proportional to the pore size. For a porous compact bone, the observed NMR magnetization will depend upon the broad distribution of T2 values (i.e. all pore sizes).

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This implies that NMR transverse relaxation (T2) data can be expressed as a sum of exponential functions: m

M (t i ) =

Ê -t i ˆ ˜, Ë 2, j ¯

 f (T2, j ) exp Á T j =1

(2)

where f (T2, j ) is proportional to the number of spins which relax with a time constant T2, j , and M(t i ) is the NMR magnetization decay from fluid-saturated compact bone. Equation (2) can be inverted into a T2 relaxation time distribution. Thus, instead of estimating a single relaxation time from a magnetization decay, it is possible to estimate a spectrum or distribution of relaxation times f (T2, j ). Since T2 depends linearly upon pore size, the T2 distribution corresponds to pore-size distribution with the longer relaxation times having the larger pores.

2.3. Determination of bound and mobile water distribution In this paper, we demonstrate that the NMR instrument with a larger magnet and gap devised by the Southwest Research Institute (SwRI) (San Antonio, TX, USA) can detect three components — protons in solid, bound, and liquid-like or mobile phases — from the equilibrium nuclear magnetization of bone, i.e. Ms+b+l, by simple NMR FID measurement. The three components are due to the different T2-FID relaxation times of the components.

3. Method 3.1. NMR measurements A SwRI-built 0.5–40 MHz broad-line NMR system with an electromagnet 19 inches in diameter and a 4-inch gap was set up at proton frequencies of 2 MHz and 27 MHz for these measurements. Laboratory-built 1.5-inch-diameter and 1-inch-diameter radiofrequency (RF) coils were used in the experiment. 1H spin-spin (T2)

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relaxation profiles were obtained by using the NMR CPMG — 90° [− τ − 180° – τ (echo)]n – TR — spin-echo method with a 9.5-µs-wide 90° RF pulse, τ of 1000 µs, and TR (sequence repetition rate) of 15 s. Each T2 profile, which included 1000 echoes (one scan with n = 1000), was acquired and sixth-four scans were used for the bone porosity measurements. The FID signal was sampled and recorded at 2-µs intervals using a 9.5-µs-wide 90° RF pulse for the bone mobile water and bound water measurements. For each FID profile, 1000–2000 data points were acquired in one scan. An inversion relaxation technique was used to invert both CPMG and FID data to a T2 relaxation distribution spectrum (Fantazzini et al. 2003; Fantazzini et al. 2004; Labadie et al. 1994; Wang and Ni 2003; Ni et al. 2004).

3.2. Histomorphometric analysis To verify the efficacy of the NMR technique, histomorphometry of bone specimens was performed to measure the porosity and pore size distribution induced by Haversian canals and osteocytic lacunae. After the NMR measurements, the specimens were reused for this purpose. A slice of cross-section was cut from each bone specimen on a Buehler slicing machine. These slices were embedded in polymethylmethacrylate for subsequent polishing and lapping on a Buehler grinding/lapping machine. Using a custom-built image acquisition and processing system, the porosity and pore size distribution of each bone sample were measured under a magnification of 50× for large pores (e.g. Haversian canals including resorption bays) and 200× for small pores (e.g. osteocytic lacunae) using a code written by an image processing software (NIH Image). The histomorphometric measurements were repeated at four to eight random locations within each slice of cross-section to ensure statistical validity.

4. Results 4.1. Determination of porosity and pore size distribution 4.1.1. Samples Eight middiaphyses of human cadaver femurs from donors aged 21–89 years old were collected through a local tissue bank and the

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Musculoskeletal Transplant Foundation (Edison, NJ, USA) for this study. All samples were cleaned of soft tissues and bone marrow. These femurs were divided into two age groups, i.e. younger than 45 years old and older than 63 years old, with four femurs in each of the groups. Prior to testing, the bone samples were fresh wrapped in gauze and stored at –20°C (only the gauze was saturated with phosphate buffered saline [PBS], pH 7.4, 99+% H2O solution). 4.1.2. NMR porosity and validation by histomorphometry measurement Figure 1 shows an example of NMR CPMG relaxation decay data for normalized signals (assuming they are from the same volume) for compact bone samples aged 21 years and 89 years, plotted in a semilog scale. These decay signals demonstrate multiexponential relaxation behavior. The higher-amplitude signal is for the 89-year-old bone sample, while the lower-amplitude signal is for the 21-year-old bone sample. The intensity differences between them are proportional to the porosity differences. After baseline correction, the NMR

Fig. 1. NMR CPMG spin relaxation decay signals from 89-year-old (higher amplitude) and 21-year-old (lower amplitude) human cortical bone samples.

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porosities of these eight compact bone samples were estimated as listed in Table 1. Figure 2 shows the microphotographs of bone microstructures observed on the samples of a 21-year-old, an 89-year-old, and a 78year-old donor. These microphotographs demonstrate similar porosity changes as seen in the porosity values determined by NMR. Table 2 lists the bone porosities measured directly from the microphotographs by areal proportion (histomorphometric method) visually. These porosities were calculated from the average of four different Table 1.

Bone porosity measured by the NMR method.

Sample number

Age

Sex

Sample volume VB (cm3)

Porosity Vl/VB (%)

1 2 3 4

21 29 34 45

F M M M

6.8 8.96 11.6 6.4

6.2 7.6 7.1 12.9

Mean ± standard deviation 5 6 7 8

63 73 78 89

Mean ± standard deviation

8.5 ± 3.5 F F F M

6.6 13.0 9.0 17.5

14.3 15.1 15.9 20.8 16.5 ± 2.9

Fig. 2. Microphotograph spectra for human compact bone samples (a) 21 years old, (b) 89 years old, and (c) 78 years old.

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Bone porosity measured by the histomorphometric method.

Sample number

Age

Sex

Average porosity area

Total area

Porosity (%)

1 2 3 4

21 29 34 45

F M M M

5299 5854 5274 7264

76 800 76 800 76 800 76 800

6.9 7.6 6.9 9.5

Mean ± standard deviation 5 6 7 8

63 73 78 89

F F F M

7.7 ± 1.2 11 159 10 476 12 242 14 454

76 800 76 800 76 800 76 800

Mean ± standard deviation

14.5 13.6 15.9 18.8 15.7 ± 2.3

areas on a cross-section in each sample. Figure 3 shows a good correlation between the NMR porosity and the microphotograph porosity (histomorphometric method), with r 2 = 0.93 from a linear regression fit. The trend of porosity increasing with age is consistent in both the NMR results and the histomorphometric results, as seen in Fig. 3. For the histomorphometric method, it is difficult to measure small pore areas in the microphotograph visually. Because the pore sizes can range from submicron to hundreds of microns, the histomorphometric results may be underestimated. Therefore, the NMR method may be more sensitive and complete than the histomorphometric method, since the histomorphometric method depends on the selected area while the NMR method measures the whole or bulk of the sample. 4.1.3. NMR pore size distribution and validation by histomorphometry measurement As a reasonable assumption, we can define the relaxation time distribution as a measure of an effective pore size distribution in bone, which may involve not only actual pores (e.g. Haversian canals, lacunae, and

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Fig. 3. Correlation between the NMR porosity and the histomorphometric porosity.

canaliculi) as described earlier. For convenience in the later discussion, the term “pore size” is referred to as “effective pore size”. Using the T2 relaxation data shown in Fig. 1, after baseline correction, the obtained inversion T2 relaxation distribution patterns for the 21- and 89-year-old compact bones are shown in Fig. 4. Figure 5 shows the typical spectra of the T2 distribution and pore size distribution obtained by the histomorphometric method; the histograms show the relationship of each peak to the corresponding anatomic cavity [Fig. 5(a)] and the pore size distributions of Haversian canals and osteocytic lacunae [Fig. 5(b)] as determined by bone histomorphometry. By comparing Fig. 5 and Fig. 4, it can be assumed that the size distributions of lacunae and Haversian canals are closely related to the P1 and P2 peaks in the NMR inversion T2 spectrum, respectively. This in turn suggests that smaller pore sizes are contributed at T2 < 10 ms, while medium and larger pore

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Fig. 4. Inversion T2 relaxation time spectra from human cortical bone samples (a) 21 years old and (b) 89 years old.

Fig. 5. Typical spectra of (a) the T2 distribution of NMR measurements and (b) the pore size distribution obtained by histomorphometry.

sizes are contributed at T2 > 10 ms. These smaller pore sizes may be from lacuna cavities (they may also include canaliculi). Since the pore sizes of canaliculi are in the submicron range, their values of porosity could be in the error range (standard deviation) of the porosity of lacunae.

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4.2. Determination of water distribution (bound and mobile water) 4.2.1. Samples Bone samples were collected from 10 female donors in the middiaphysis section, and were divided into two age groups. The middleaged group included bone obtained from donors aged 42, 49, 53, 54, and 58 years old; while the old-aged group included bone obtained from donors aged 72, 75, 78, 81, and 87 years old. An axial slice of cortical bone with a thickness of 2 mm was taken from the medial side of each cross-section using a diamond saw under constant irrigation. The final dimensions of the specimens were approximately 25 mm × 5 mm × 2 mm. For male water distribution studies, similar cross-sections and sample sizes were cut from 10 male donors and divided into two age groups. The middle-aged group included bone obtained from male donors aged 51, 52, 55, 57, and 59 years old; while the old-aged group included bone obtained from male donors aged 69, 76, 76, 77, and 87 years old. All of the specimens were stored in PBS-soaked gauze at −20°C; and prior to NMR measurements, the samples were completely thawed at room temperature. 4.2.2. Bound and mobile water Figure 6 shows an example of NMR CPMG relaxation decay data for normalized signals (assuming they are from the same volume) for female cortical bone samples aged 49 and 87 years old, plotted in a semilog scale, indicating multiexponential relaxation behavior. The higher-amplitude signal is for the 87-year-old bone, while the loweramplitude signal is for the 49-year-old bone. Using the T2 relaxation decay data shown in Fig. 6 after baseline correction, the inversion T2 relaxation distribution patterns for these two specimens are shown in Fig. 7. The T2 relaxation times are distributed in a wide range from submillisecond to second, indicating a wide range of pore size distributions, since longer T2 relaxation times correspond to larger pore sizes (Fantazzini et al. 2003; Ni et al.

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Fig. 6. NMR CPMG spin relaxation decay signals from 49-year-old (lower amplitude) and 87-year-old (higher amplitude) human cortical bone samples.

(a)

(b)

Fig. 7. Inversion T2 relaxation time spectra from human cortical bone samples (a) 49 years old and (b) 87 years old.

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Fig. 8. NMR FID spin relaxation decay signals from 49-year-old (lower amplitude) and 87-year-old (higher amplitude) human cortical bone samples.

2004). Comparing the elder (87-year-old) bone to middle-aged (49-year-old) bone, the volume fraction of the larger pores is significantly increased for the elder bone, since the intensity of its T2 spectrum at longer relaxation times is much larger compared to the spectrum from the middle-aged bone. These data reaffirm that microstructural changes in cortical bone can be detected and quantified by this low-field NMR technique. Figure 8 shows an example of an NMR FID signal from the same cortical bone as in Fig. 7, plotted in a semilog scale. These decay signals also demonstrate multiexponential relaxation behavior. Using the T2-FID relaxation data shown in Fig. 8, the inversion T2-FID relaxation distribution patterns for these two samples are shown in Fig. 9. The three peaks in the spectra, from left to right, result from protons in the solid component (peak near 11 µs), protons in bound water (peak near 210 µs), and protons in mobile water (peak near 2.7 ms), respectively. After obtaining the NMR intensity of a known volume of a reference sample of water for calibration, the intensity of the second and third peaks can be converted into the volume of water bound to the

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(a)

(b)

Fig. 9. Inversion T2-FID relaxation time spectra from human cortical bone samples (a) 49 years old and (b) 87 years old.

collagen and mineral and the volume of water inside the pores, respectively. Thus, the water distribution within cortical bone can be determined. 4.2.3. Difference between CPMG T2 and T2-FID Figure 7 shows the CPMG T2-derived relaxation times from the mobile water (protons) inside the pores; while Fig. 9 shows the FIDderived T2-FID relaxation times from protons in all material phases including solid, bound, and mobile (or liquid-like) phases. The T2 relaxation distributions for liquid-like phase are from milliseconds to seconds (Fig. 7); while the T2-FID relaxation time distributions are only from microseconds to milliseconds, even for the three different phase components (Fig. 9).

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The major difference between the T2 relaxation times obtained using the NMR FID and CPMG methods is that the T2 relaxation time constant is significantly shorter in the FID technique than in the CPMG technique. This is because in the FID measurement, using a simple 90° RF pulse, the free induction signal decays at a time T2-FID that is often determined primarily by field inhomogeneity, since nuclei in different parts of the field precess at slightly different frequencies and hence quickly get out of phase with each other (Farrar and Becker 1971). Our ingenious CPMG measurement is a practical method for overcoming the inhomogeneity problem. The method consists of the application of a 90°-τ-180° sequence (see Sec. 3.1) and the observation at time 2τ of free induction “echo” (rephrase nuclear magnetization) to obtain a natural spin-spin relaxation time T2 (Farrar and Becker 1971; Rubin and Lanyon 1985; Rubin et al. 1995). The contribution of inhomogeneity in magnetic field to the FID precludes the use of the decay time T2-FID as a measure of T2. From our FID result, it is suitable to separate the solid, bound, and mobile water phases within the bone and obtain the intensities of the different phase components. This result cannot provide detailed information such as water distribution in the different phases; however, with CPMG T2 data, it can provide the mobile water distribution within bone.

4.3. Microdamage 4.3.1. Samples A human femur (male, age 58 years) was sectioned into three components: distal, middle, and proximal. The proximal section was further divided radially into five blanks that were machined to final dimensions, resulting in five specimens used for this study. The geometry of each specimen was a rectangular shape that was nominally 3 mm in width, 4 mm in height, and 35 mm in length. The use of geometrically well-defined, machined cortical bone specimens allows greater control over the stress and strain applied and measured on each bone specimen; consequently, the measured damage and NMR spectra can be confidently compared in the context of a known

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mechanical damage state. We have chosen to generate microdamage through cyclic fatigue loading of specimens, since this will allow us to achieve a certain level of control (albeit incomplete) over the amount of microdamage in a bone sample. Thus, by acquiring an NMR relaxation signal prior to damage, we will be able to quantify particular changes in the NMR signal specifically due to microdamage. The use of four-point bending allows each specimen to be used for both fatigue-loading-induced predamage and postdamage. The portion of the specimen that is outside of the four-point bending support pins remains unstressed during bending (the bending moment is zero in this section); this portion of the specimen is used for predamage histological characterization, while the gauge region of the specimen (the portion of the specimen between the inner loading points) is used for postdamage histological characterization. The bone specimens were then subjected to cyclic four-point bending under load control (Gibson et al. 1995). The initial load was determined by applying a small diagnostic bending load to each specimen in order to determine the specimen stiffness. From this stiffness measurement, the bone elastic modulus and the load required to produce 5000 microstrain in the outer specimen tensile fiber was determined using beam bending theory. Using a custom-developed LabView data acquisition and control program, each specimen was cyclically loaded until a 20% reduction in secant stiffness (the ratio of maximum applied load to maximum beam deflection) was achieved in order to ensure the presence of bone microdamage (Burr et al. 1998), at which point the test was automatically stopped. All specimens were tested at 37°C in an environmentally controlled chamber under constant irrigation of PBS solution. After cyclic loading, a second NMR measurement was performed on the specimen (postdamage). 4.3.2. Predamage cortical bone Table 3 shows the porosity results using NMR and histomorphometry measurements from five bone samples. These five samples were cut from the proximal portion of the same block of cortical bone at different radial locations. The NMR porosity was calculated from the

dHaversian (µm)

Porosity (%)

T2P 1 (ms)

5.74 5.85 5.99 5.91 5.94 5.89 ± 0.10

58.5 60.1 54.9 59.1 66.9 59.9 ± 4.4

10.5 10.7 9.9 8.0 11.3 10.1 ± 1.3

1.98 2.00 2.54 2.19 2.44 2.23 ± 0.25

T2P 2 (ms)

ρlacunae (µm/ms)

ρHaversian (µm/ms)

67.5 0.452 0.217 60.7 0.456 0.248 50.8 0.368 0.270 59.6 0.421 0.248 71.4 0.380 0.234 62.0 ± 7.9 0.416 ± 0.041 0.243 ± 0.020

Note: T2P1, median T2 time of peak P1 (lacunae); T2P2, median T2 time of peak P2 (Haversian canals); dlacunae, median nominal diameter of osteocyte lacunae; dHaversian, median nominal diameter of Haversian canals; ρ lacunae, surface relaxivity of lacunae; ρ Haversian, surface relaxivity of Haversian canals.

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Determination of the porosity and surface relaxivity of predamage bone samples.

Table 3.

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maximum amplitude of the CPMG signal obtained from the liquid inside the pores. The histomorphometric porosity was calculated from the ratio of the sum of the estimated lacuna and Haversian areas to the area of the sample cross-section. The surface relaxivity was determined empirically in this study based on the results of pore sizes from histomorphometry and the corresponding T2 times (by NMR measurement) (Wang and Ni 2003). Two constants (lacunae and Haversian canal) of ρ were calculated by using Eq. (1), where the median pore size diameter was obtained from the histomorphometry results and the median T2 relaxation time constant was obtained from the inversion T2 relaxation spectrum. Then, the obtained relaxivity ρ was substituted back into Eq. (1) to convert the T2 relaxation time spectrum into a pore-diameter distribution. In Table 3, peaks 1 (P 1) and 2 (P 2) are the peaks in the inversion T2 relaxation spectrum (see Fig. 4) and are assumed to be the size distributions of lacunae and Haversian canals, respectively (Ni et al. 2004; Wang and Ni 2003). 4.3.3. Postdamage cortical bone Figures 10 and 11 show the results of the NMR CPMG relaxation decay data for samples 1 and 3 (in Table 1 and Table 3) at predamage and postdamage conditions, respectively. The signal with the higher amplitude and slower decay is the postdamage signal, while the predamage signal has a lower amplitude and decays faster. The intensity differences between them are proportional to the porosity differences. Figures 10 and 11 demonstrate that the NMR CPMG relaxation method is sensitive to detecting differences between predamage and postdamage bone samples. Figure 13 shows that the porosity is increased by about 5.3% (sample 1 in Table 3) after damage as compared with before damage, while Fig. 14 shows only a 1.6% increase (sample 3 in Table 3) after damage. It is known that the decay signal corresponds to the protons contributed from all selected regions of pooled fluid within the bone (i.e. intrinsic bone pores plus newly created microcracks). Thus, changes between the predamage decay signal and the postdamage decay signal in this range of the

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Fig. 10. NMR CPMG spin relaxation decay signals from the same proximal section (sample 1) of predamage and postdamage cortical bones.

Fig. 11. NMR CPMG spin relaxation decay signals from the same proximal section (sample 3) of predamage and postdamage cortical bones.

decay component are due to the creation of bone microdamage and the subsequent fluid that is pooled within the microcracks, since the intrinsic in vitro specimen porosity attributable to lacunae, canaliculi, or Haversian canals has not changed.

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Fig. 12. Specimen 1 postdamage microstructure. The large pores represent the Haversian canals, while the smaller pores are the osteocyte lacunae. The microdamage (cracks) evident in this micrograph results in an increase in NMR-measured porosity.

Figure 12 shows the microphotographs of postdamage bone microstructures observed on sample 1. Using the T2 relaxation data in Figs. 10 and 11, after baseline correction, the obtained inversion T2 relaxation distribution patterns for predamage and postdamage bone samples of specimens 1 and 3 are shown in Figs. 13 and 14, respectively. Comparing the predamage and postdamage bone samples, it is found that the total volume of the small pore sizes (lacuna range) indicates a smaller overall change; however, the average of the larger pore sizes (Haversian range) is increased. In these inversion T2 relaxation spectra (Figs. 13 and 14), it is apparent that the T2 relaxation shifts into a longer time constant domain after damage. These changes could be related to the size or length of cracks, where the volume of pooled fluid within a crack would be much larger than the fluid pooled within a lacuna. This result is consistent with the observation in Fig. 12, where the area of a crack is much larger than the area of a lacuna. Crack widths varied in measurement from <1 micron to >5 microns. Table 4 lists a comparison of the NMR and histomorphometric porosities as well as T2 media of osteocyte lacunae and Haversian

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Fig. 13. Inversion T2 relaxation time spectra from the predamage and postdamage cortical bones of sample 1.

Fig. 14. Inversion T2 relaxation time spectra from the predamage and postdamage cortical bones of sample 3.

canals between predamage and postdamage samples. The results show that before damage, NMR-determined porosity is in good agreement with histomorphometry-determined porosity. Using linear regression, the estimate of the correlation coefficient between NMR and histology was 0.92 for the predamage porosity measurement with a standard error of 0.4, while the correlation coefficient was 0.83 for postdamage porosity measurement with a standard error of 0.6. Due to the inherent differences in bone tissue, the level of the damage could be sample- or location-dependent. The average porosity increase for NMR measurement was about 4.0%, compared to an average increase in histologically determined porosity of 8.8% and a decrease in bone elastic modulus of 20% after damage induced by fatigue loading in the same specimens.

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T2P1 (ms)

T2P2 (ms)

Porosity (%)

NMR

Porosity (%)

Porosity increase (%)

Histology

T2P1 (ms)

T2P2 (ms)

Porosity (%)

Porosity increase (%)

1 10.5 2.0 67.5 9.0 11.1 5.3 2.2 94.1 9.5 5.6 2 10.7 2.0 60.7 11.5 11.2 4.4 2.2 86.3 12.2 5.5 3 9.9 2.5 50.8 8.9 10.1 1.6 2.6 85.6 9.6 7.1 4 8.0 2.2 59.6 5.2 8.2 2.8 2.1 85.0 6.8 23.6 5 11.3 2.4 71.4 11.7 12.0 5.9 2.5 89.7 12.0 2.1 Average ± 10.1 ± 1.3 2.23 ± 0.25 62.0 ± 7.9 9.3 ± 2.6 10.5 ± 1.5 4.0 ± 1.8 2.33 ± 0.23 88.1 ± 3.8 10.0 ± 2.2 8.8 ± 8.4 standard deviation

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The major differences between NMR-determined porosity and histomorphometrically measured porosity could be attributed to the following factors. Since four-point bending is used, the portion of the rectangular specimen outside of the loading pins is not stressed and remains undamaged. The NMR result is based on the whole sample volume (including the undamaged portion), while the histomorphometric result is based on a small area from a thin cross-section of the specimen around the damaged area in this test. The occurrence of damage or cracks in bone is nonhomogeneous and may not be adequately captured on each thin cross-section, while the NMR technique accounts for all of the damage within the specimen regardless of where it occurs. In addition, histomorphometric measurements cannot be on the same section of predamage or postdamage bone sample, while NMR measurements can be on the same block of the bone sample. Furthermore, the assumption of a penny-shaped crack in computing the contribution of the induced microdamage to the increase in measured porosity may contribute to discrepancies in porosity, since the actual shape of the microcracks is not known. The shape of the cracks may vary from penny-shaped to elliptical, thus contributing to errors in the porosity calculation. It is reasonable to assume that the NMR result is more reliable than the histomorphometry result in this microdamage test. An interesting finding is that the NMR T2 median is sensitive to changes in the sample from predamage to postdamage, particularly in the size range of Haversian canals. In Table 4, the average T2 median for Haversian canals is 62 ms ± 8 ms for predamage and 88 ms ± 4 ms for postdamage. This implies that the range of the size or length of cracks in the Haversian canals could be related to the NMR measurement results, thus suggesting that changes or percentage shifts in the T2 median could be used to estimate the degree of damage.

4.4. Bone disuse 4.4.1. Samples The cortical bone of the middiaphyses of 30 male turkeys was dissected from freshly slaughtered animals. These samples were categorized as

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normal or 4-week disuse treated by functionally isolated osteotomies. The bone samples were cut 2 cm long using a diamond saw with saline solution. All samples were cleaned of soft tissues and bone marrow. With this cortical bone sample, the fat signal could be neglected in the NMR relaxation and free induction measurements (Ni et al. 2007). As previously described, the left ulna of each turkey was functionally isolated via transverse epiphyseal osteotomies (Rubin and Lanyon 1985; Rubin et al. 1995). Using a template as a guide, each of the distal metaphyseal ends of the ulna was isolated and covered with a stainless steel cap filled with 5 mL of polymethylmethacrylate. The detailed procedure is described in Qin et al. (1998). The animals with left ulnae functionally isolated were left unloaded, with ventral and distal clamps fixed to Steinman pins to minimize mechanical loading during the 4-week experimental period. The turkeys were permitted normal activity, and were housed with others. Daily cleaning procedures (i.e. wound care and changing of clamps) were similar among the turkeys, which were subjected to loading. There were 15 control and 15 disuse samples (half-year-old male turkeys) for porosity and water distribution studies. All animal samples were collected from the Department of Biomedical Engineering and the Department of Orthopedics, State University of New York at Stony Brook, USA. The samples were fresh wrapped in gauze and stored at –20°C (only the gauze was saturated with PBS, pH 7.4, deionized water solution). 4.4.2. Determination of porosity and water distribution changes between normal and disuse groups Figure 15 shows an example of NMR CPMG relaxation decay data for normalized signals (assuming they are from the same volume) for control (sample C14) and disuse (sample D14) cortical bone samples from halfyear-old turkeys, plotted in a semilog scale. These decay signals demonstrate multiexponential relaxation behavior. The higher-amplitude signal is for disuse bone samples, while the lower-amplitude signal is for normal bone samples. The intensity differences between them are proportional to the porosity differences. Table 5 lists the NMR

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Fig. 15. NMR CPMG spin relaxation decay signals from disuse (higher amplitude) and normal (lower amplitude) turkey cortical bone samples. Both signals show multiexponential relaxation behavior plotted in a semilog scale.

porosities and bound-to-mobile water distribution ratios from the turkey bone samples of the 4-week disuse group. After baseline correction, Fig. 16 shows the inversion T2 relaxation distribution patterns for control sample C14 and disuse sample D14. The T2 relaxation times are distributed in a wide range from submillisecond to second, indicating a wide range of pore size distributions, since longer T2 relaxation times correspond to larger pore sizes. Comparing the disuse bone to normal bone, the volume fraction of the larger pores is significantly increased for the disuse bone, since the intensity of its T2 spectrum at longer relaxation times is much larger compared to the spectrum from the normal bone. These data reaffirm that microstructural changes in cortical bone can be detected and quantified by this low-field NMR technique. Figure 17 shows an example of NMR FID (T2-FID) signals from the bone of control sample C14 and disuse sample D14, plotted in a semilog scale. These decay signals also demonstrate multiexponential relaxation behavior. Using the T2-FID relaxation data in Fig. 17, the inversion T2-FID relaxation distribution patterns for these two samples are shown in Fig. 18. There are three separated peaks obtained in the spectra that demonstrate, from left to right, protons in the solid

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Table 5. Porosities and bound-to-mobile water ratios obtained from NMR measurements. Normal

Disuse

Sample number

Porosity

Bound water/ free water

Porosity

Bound water/ free water

4 5 7 10 11 13 14 15 17 18 23 27 28 30 31 Mean ± standard deviation

4.88 5.92 8.05 5.93 7.48 10.87 8.01 5.18 10.95 8.29 7.72 14.08 12.84 7.19 10.32 8.31 ± 2.68

2.58 1.83 0.93 2.09 1.15 1.16 1.04 1.13 0.71 1.32 1.84 0.71 0.63 0.67 0.52 1.22 ± 0.61

16.94 10.62 21.04 9.61 27.47 15.91 15.07 14.4 16.02 15.5 12.58 15.28 29.57 22.11 17.97 17.34 ± 5.62

0.62 0.97 0.61 0.53 0.29 0.50 0.51 0.86 0.76 0.49 0.66 0.56 0.17 0.26 0.81 0.57 ± 0.22

Fig. 16. Distributions of T2 relaxation times that reflect individual pore sizes for bone samples C14 (lower intensity) and D14 (higher intensity).

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Fig. 17. NMR FID spin relaxation decay signals from disuse (higher amplitude with slower decay) and normal (lower amplitude with faster decay) turkey cortical bone samples. Both signals show multiexponential relaxation behavior plotted in a semilog scale. (a)

(b)

Fig. 18. Inversion T2-FID relaxation time spectra for bone tissue of (a) control sample C14 with a bound-to-mobile water ratio of about 1.0 and (b) disuse sample D14 with a bound-to-mobile water ratio of about 0.5.

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component (peak near 9–10 µs), protons in bound water (peak near 150–160 µs), and protons in mobile water (peak near 1.2–1.3 ms), respectively. Presumably, the peak located at the shortest relaxation time (near 10 µs) represents the protons in the solid phase, while the peak located at the longest relaxation time (near 1.3 ms) represents the protons in the mobile phase. Therefore, it is reasonable to assign the peak located at the middle relaxation time (near 150 µs) as representing the protons in the bound phase. For better normalization and comparison, the changes in water distribution are characterized as the ratio of bound water intensity to mobile water intensity in Table 5. In Table 5, the significant differences between the values of normal and disuse porosity were found by paired t-test (p < 0.001); similarly, the significant differences between the ratios of bound water to free water in the normal and disuse samples were found (p < 0.001).

5. Summary A new low-field NMR methodology has been described for estimating the age, microdamage, functional disuse porosity and water distribution, pore size, and microstructural changes in cortical bone from NMR spin-spin relaxation data. The porosity is determined by the calibrated NMR fluid volume divided by overall bone volume, and the (effective) pore size distribution is represented by the relaxation time distribution based upon the fact that larger pores have longer relaxation times. The FID T2 relaxation data can be inverted to T2-FID relaxation distribution, and this distribution can then be transformed to bound and mobile water distribution with the longest relaxation time corresponding to mobile water and the middle relaxation time corresponding to bound water. In these studies, the NMR porosities are in good correlation with the results obtained from histomorphometric measurements, and the T2 relaxation (or pore size) distributions are similar to the distributions obtained from histomorphometric measurements. The results of these studies suggest that the age, microdamage, functional disuse porosity and water distribution, and pore size

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changes in cortical bone can be detected using this technique. By comparing the NMR technique with the histomorphometric method, the NMR method may have more advantages than other traditional methods, such as being rapid, nondestructive, and noninvasive as well as providing three-dimensional (bulk-of-sample) results. In contrast, the histomorphometry method is destructive; determines pore sizes from the sample cross-sectional area, which varies in orientation with respect to the cross-section; and does not give actual three-dimensional results. NMR porosity is independent of the sample orientation and provides results from the fluid in all of the pores. More recently, solid state MRI microscopy has been developed to demonstrate human dental anatomy using a 300 MHz (7.1 tesla) proton spectrometer to reach a resolution of 195 µm (Appel and Baumann 2002). An extremely high-resolution MRI system has been developed to study microscopic features of normal and LHON human optic nerves with a 500 MHz (12 tesla) proton microimaging system to yield a resolution of 30 µm (Sadun et al. 2002). It is believed that, in the near future, extremely high-resolution MRI can be developed to determine the microstructure of compact bone. However, our low-field NMR methodology is an effective tool to characterize microstructural changes rapidly in cortical bone with an inexpensive instrument and test. We feel that additional in vitro experiments must be conducted first, whereby the actual amount of microstructural changes is quantified using established histological methods to establish the relationship between bone microstructural change and NMR T2 relaxation as well as the relationship between damage, porosity, and changes in the NMR relaxation signal. With these relationships determined, it may be possible to predict the degradation of bone mechanical properties nondestructively using this method. After this in vitro methodology is fully developed, further studies will test the feasibility of localized human cortical measurements for in vivo applications. It may be possible to use an NMR singlesided-access sensor approach for volume-selective measurement of cortical bone in vivo, in which the use of specially shaped magnets and

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RF coils are used to selectively probe a discrete volume (e.g. a thin slice or a cylindrical volume) of bone without interference from surrounding soft tissues. This type of NMR approach may be sensitive to a selected section of bone tissue and insensitive to the surrounding soft tissue. Therefore, an NMR sensor development needs to be pursued once the above in vitro technology has been established.

References Appel TR, Baumann MA. Solid-state nuclear magnetic resonance microscopy demonstrating human dental anatomy. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 94:256–261, 2002. Brownstein KR, Tarr CE. Importance of classical diffusion in NMR studies of water in biological cells. Phys Rev A 19:2446, 1979. Burr DB, Turner CH, Naick P et al. Does microdamage accumulation affect the mechanical properties of bone? J Biomech 31:337–345, 1998. Carr HY, Purcell EM. Effects of diffusion on free precession in nuclear magnetic resonance experiments. Phys Rev 904(3):630, 1954. Chui MM, Philips RJ, McCarthy M. Measurement of the porous microstructure of hydrogels by nuclear magnetic resonance. J Colloid Interface Sci 174:336, 1995. Chung H, Wehrli FW, Williams JL, Kugelmass SD. Relationship between NMR transverse relaxation, trabecular bone architecture and strength. Proc Natl Acad Sci USA 90:10250, 1993. Currey JD. The effect of porosity and mineral content on the Young’s modulus of elasticity of compact bone. J Biomech 21:131–139, 1988. Currey JD, Brear K, Zioupos P. The effects of ageing and changes in mineral content in degrading the toughness of human femora. J Biomech 29:257–260, 1996. Dempster WT, Liddicoat RT. Compact bone as a non-isotropic material. Am J Anat 91:331–362, 1952. Evans FG. Mechanical Properties of Bone. Thomas, Springfield, IL, 1973. Evans FG, Lebow M. Regional differences in some of the physical properties of the human femur. J Appl Physiol 3:561–572, 1951. Fantazzini P, Bortolotti V, Brown RJS et al. Two 1H-nuclear magnetic resonance methods to measure internal porosity of bone trabeculae: by

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solid-liquid signal separation and by longitudinal relaxation. J Appl Phys 95:339–343, 2004. Fantazzini P, Brown RJS, Borgia GC. Bone tissue and porous media: common features and differences studied by NMR relaxation. Magn Reson Imaging 21:227–234, 2003. Farrar TC, Becker ED. Pulse and Fourier Transform NMR. Academic Press, New York, 1971. Gallegos DP, Munn K, Smith DM, Stermer DL. A NMR technique for the analysis of pore structure: application to materials with well-defined pore structure. J Colloid Interface Sci 119:127, 1987. Genant HK, Cann CE, Ettinger B, Cordan GS. Qualitative computed tomography of vertebral spongiosa: a sensitive method for detecting early bone loss after oophorectomy. Ann Intern Med 97:699, 1997. Gibson VA, Stover SM, Martin RB et al. Fatigue behavior of the equine third metacarpus: mechanical property analysis. J Orthop Res 13:861–868, 1995. Glaves CL, Smith DM. Membrane pore structure analysis via NMR spinlattice relaxation experiments. J Memb Sci 46:167, 1989. Hipp JA, Jansujwicz A, Simmons CA, Snyder B. Trabecular bone morphology using micro-magnetic resonance imaging. J Bone Miner Res 11:286, 1996. Howard JJ, Kenyon WE, Staley C. Proton magnetic resonance and pore size variation in reservoir sandstones. SPE Formation Eval 8:194, 1993. Jara H, Wehrli FW, Chung H, Ford JC. High-resolution variable flip angle 3D MR imaging of trabecular microstructure in vivo. Magn Reson Med 29:528, 1993. Jonsson U, Ranta H, Stromberg L. Growth changes of collagen crosslinking, calcium, and water content in bone. Arch Orthop Trauma Surg 104:89–93, 1985. Kenyon WE. Petrophysical principles of applications of NMR logging. Log Anal 38:21, 1997. Labadie C, Lee J, Vetek G, Springer JR. Relaxographic imaging. J Magn Reson 105:99–112, 1994. Liaw HK, Kulkarni R, Chen S, Watson AT. Characterization of fluid distributions in porous media by NMR techniques. AIChE J 42(2):538, 1996.

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Majumdar S, Gies A, Jergas M et al. Quantitative measurement of trabecular bone structure using high resolution gradient echo imaging of the distal radius. Proc Soc Magn Reson Med: 455, 1993. Martin RB. Porosity and specific surface of bone. Crit Rev Biomed Eng 10(3):179, 1984. McCalden RW, McGeough JA, Barker MB, Court-Brown CM. Age-related changes in the tensile properties of cortical bone: the relative importance of changes in porosity, mineralization, and microstructure. J Bone Joint Surg Am 75(8):1193, 1993. Meiboom S, Gill D. Modified spin-echo method for measuring nuclear relaxation times. Rev Sci Instrum 29:688, 1958. Mueller KH, Trias A, Ray RD. Bone density and composition: age-related and pathological changes in water and mineral content. J Bone Joint Surg Am 48:140–148, 1966. Mulkern R, Meng J, Oshio K et al. Bone marrow characterization in the lumbar spine with inner volume spectroscopic CPMG imaging studies. J Magn Reson Imaging 4:585–589, 1994. Ni Q, King JD, Wang X. Characterization of human bone structure changes by low field NMR. Meas Sci Technol 15:58–66, 2004. Ni Q, Nicolella DP. The characterization of human cortical bone microdamage by nuclear magnetic resonance. Meas Sci Technol 16:659–668, 2005. Ni Q, Nyman J, Wang X et al. Assessment of water distribution changes in human cortical bones by nuclear magnetic resonance. Meas Sci Technol 18:715–723, 2007. Nyman JS, Roy A, Shen XM et al. The influence of water removal on the strength and toughness of cortical bone. J Biomech 39:931–938, 2006. Prymark O, Tiemann H, Sotje I et al. Application of synchrotron-radiationbased computer microtomography to selected biominerals: embryonic snails, statoliths of medusae, and human teeth. J Biol Inorg Chem 10:688–695, 2005. Qin Y, Rubin CT, McLeod KJ. Non-linear dependence of loading intensity and cycle number in the maintenance of bone mass and morphology. J Orthop Res 16:482–489, 1998. Robinson RA. Physicochemical structure of bone. Clin Orthop 208:263–315, 1975.

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Robinson RA. Bone tissue: composition and function. Johns Hopkins Med J 145:10–24, 1979. Rubin CT, Gross TS, McLeod KJ, Bain SD. Morphologic stages in lamellar bone formation stimulated by potent mechanical stimulus. J Bone Miner Res 10:488–495, 1995. Rubin CT, Lanyon LE. Regulation of bone mass by mechanical strain magnitude. Calcif Tissue Int 37:411–417, 1985. Sadatmanesh H, Ehsani R. Non-destructive evaluation of concrete and wood properties using NMR. Insight 39(2):75, 1997. Sadun AA, Carelli V, Bose S et al. First application of extremely high-resolution magnetic resonance imaging to study microscopic features of normal and LHON human optic nerve. Ophthalmology 109:1085–1091, 2002. Sedlin ED, Hirsch C. Factors affecting the determination of the physical properties of femoral cortical bone. Acta Orthop Scand 37:29–48, 1966. Selby K, Majumdar S, Newitt DC, Genant HK. Investigation of MR decay rates in microphantom models of trabecular bone. J Magn Reson Imaging 6:549–559, 1996. Smith JW, Walmsley R. Factors affecting the elasticity of bone. J Anat 93:503–523, 1959. Straley C, Rossinl D, Vinega H, Tutunjian P. Core analysis by low-field NMR. Log Anal 38:84, 1997. Strange JH. Cryoporometry: a new NMR method for characterising porous media. Nondestr Test Eval 11:261, 1994. Timmins PA, Wall JC. Bone water. Calcif Tissue Res 23:1–5, 1977. Traber F, Block W, Layer G et al. Determination of 1H relaxation times of water in human bone marrow by fat-suppressed turbo spin echo in comparison to MR spectroscopic methods. J Magn Reson Imaging 6:541–548, 1996. Wang X, Bank RA, TeKoppele JM, Agrawal CM. The role of collagen in determining bone mechanical properties. J Orthop Res 19:1021–1026, 2001. Wang X, Ni Q. Determination of cortical bone porosity and pore size distribution using a field NMR approach. J Orthop Res 21(2):312–319, 2003. Yamada H, Evans FG. Strength of Biological Materials. Williams & Wilkins, Baltimore, MD, 1970. Yeni YN, Brown CU, Norman TL. Influence of bone composition and apparent density on fracture toughness of the human femur and tibia. Bone 22:79–84, 1998.

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Yeni YN, Brown CU, Wang Z, Norman TL. The influence of bone morphology on fracture toughness of the human femur and tibia. Bone 21:453–459, 1997. Zioupos P. Ageing human bone: factors affecting its biomechanical properties and the role of collagen. J Biomater Appl 15:187–229, 2001. Zioupos P, Currey JD, Hamer AJ. The role of collagen in the declining mechanical properties of aging human cortical bone. J Biomed Mater Res 45:108–116, 1999.

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Chapter 42

Dynamic Contrast-Enhanced Magnetic Resonance Imaging of the Musculoskeletal System: Basic Principles and Clinical Applications in Bone Sarcomas and Rheumatoid Arthritis Yi-Xiang Wang

Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) is the rapid acquisition of serial MRI images before, during, and after the administration of an MR contrast agent. Unlike conventional enhanced MRI, which simply provides a snapshot of enhancement at one point in time, DCE-MRI permits a fuller depiction of the wash-in and wash-out contrast kinetics, and thus provides insight into the microcirculation of the studied tissues or lesions. With the dynamic signal intensity change postcontrast agent injection, empirical measures such as maximal signal intensity enhancement and initial enhancement slope can be easily obtained. Such data are also amenable to two-compartment pharmacokinetic modeling, from which parameters based on the rates of exchange between the compartments can be generated. DCE-MRI can be used to characterize masses, stage tumors, and noninvasively monitor therapy. Measures of contrast uptake by dynamic MRI have demonstrated a convincing ability to aid in diagnosing the presence of viable tumors and to measure the response for a

Corresponding author: Yi-Xiang Wang. E-mail: [email protected]

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A Practical Manual for Musculoskeletal Research range of human tumors. While questions remain about how to best extract noninvasive pharmacokinetic measures of drug access from these novel dynamic imaging methods, scientists and clinicians are optimistic that these methods can provide important new clinical measures which reflect the range of biological variations within and between naturally occurring solid tumors. Efforts to standardize DCE-MRI acquisition, analysis, and reporting methods will allow wider dissemination of this useful functional imaging technique. Keywords:

DCE-MRI; osteosarcoma; rheumatoid arthritis; pharmacokinetic model; microcirculation.

1. Introduction Dynamic contrast-enhanced magnetic resonance imaging (DCEMRI) acquires serial MR images before, during, and after the administration of an intravenous contrast agent. Unlike conventional MRI, which simply provides one or more snapshots of the scanned target, DCE-MRI provides richer, more complete information on the regional microcirculation of the target tissue. DCE-MRI has been used to characterize masses, stage tumors, and noninvasively monitor therapy. In recent years, DCE-MRI has grown with the development of antiangiogenic strategies for tumor therapy, which reduce both the number of vessels and their permeability. While there is currently strong interest in new angiogenic inhibitors, more conventional therapies can also be monitored with DCE-MRI because vessel loss is a final common pathway for many therapies. Cytotoxic chemotherapy and vaccine immunotherapy can result in changes in MRI enhancement kinetics within a tumor. Therapeutic radiation can also be monitored with DCE-MRI because decreased vascularity is anticipated with successful treatment early after therapy. In addition, local therapies, such as cryotherapy and radiofrequency ablation, can be monitored with this technique (Choyke et al. 2003; Knopp et al. 2001). In the musculoskeletal system, DCE-MRI can be used to evaluate the pathophysiological status of many diseases where the microcirculation is disturbed, such as tumors, inflammations, and osteonecrosis (Taylor et al. 2000). This chapter describes DCE-MRI image acquisition

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and data analysis, and discusses DCE-MRI applications in the evaluation of osteosarcoma and rheumatoid arthritis.

2. DCE-MRI Data Acquisition Techniques Before DCE-MRI sequences, routine T1- and T2-weighted (T1W/ T2W) images are obtained to provide anatomic information for the lesion or tissue in question. This also helps to select the disease areas where DCE-MRI is performed. Quantification of contrast agent concentration in tissue necessitates the acquisition of a T1 value of the target tissue before DCE-MRI (i.e. the native T1 value of each pixel within imaging areas), or the T1 value within a region of interest (ROI), if the signal-to-noise ratio (SNR) does not allow pixel-by-pixel analysis. For gradient-echo–based sequences, this usually involves a number of images with different flip angles while keeping other parameters consistent. Low-molecular-weight gadolinium chelate contrast agents (such as Gd-DTPA) are used for DCE-MRI studies. In most tissues except the brain, testes, and retina, these small molecular agents pass rapidly into the extravascular-extracellular space (EES) — also called the leakage space — at a rate determined by the permeability of the microvessels, their surface area, and blood flow. Gadolinium exerts a paramagnetic effect on nearby water protons, causing them to relax more rapidly on T1-weighted sequences. Signal intensity increases proportionate to the concentration of gadolinium chelate. Pharmacokinetic modeling (see below) requires a nearly linear relationship between gadolinium chelate concentration and signal intensity. In fact, exact linearity is never perfectly achieved in MRI, but can only be approximated with low doses of gadolinium chelates (Donahue et al. 1994). Linearity, while desirable, is not an absolute necessity, as many clinically important phenomena can be observed with slightly nonlinear gadolinium concentration–signal intensity relationships. For DCE-MRI studies, the dosage of contrast agents, the method of contrast administration, the appropriate speed and length of image acquisition, the optimal spatial resolution and coverage, and the

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appropriate method of analyzing the data all depend on what is being studied. Spatial sampling (single or multiple slices, in-plane spatial resolution, and slice thickness) and temporal sampling (the rate at which the time-varying signal intensity curve is sampled) are interrelated variables that are limited by the SNR and defined by the target tissue. The purpose of DCE-MRI is not to provide the ultimate in spatial resolution and tumor conspicuity — this can be accomplished by anatomic MR procedures. Greater spatial resolution, as in the case of detecting residual foci of tumor, requires longer time periods for the collection of images and, therefore, slower temporal sampling rates. Faster temporal sampling, as in the case of blood volume mapping, is usually restricted to one or two slices. Faster imaging rates and smaller voxel sizes decrease the SNR, and may be detrimental to the reliability and sensitivity of the calculations of variables. When low-molecular-weight contrast agents are used to image bone sarcoma, maximum uptake of the contrast agent into the tumor EES will usually occur within the first 3 minutes after contrast agent injection. In some of the best perfused regions of an osteosarcoma, uptake of the contrast agent can equilibrate in as little as 25–30 seconds. The sampling rate should adequately sample the fastest accumulation rate. To improve the scan speed, gradient echo sequences with short repetition time/echo time (TR/TE) and low flip angle are used. With modern MRI scanners, it is possible to achieve a data sampling rate of every 3 seconds with multiple slices. After injection, scanning is repeated until approximately 8–10 minutes of data have been accumulated. Before DCE-MRI, a shortened version of the DCE sequence can be run to ensure adequate SNR and exact section positioning. Gadolinium chelate is usually given after five or more images have been acquired, so that the steady status is achieved and the baseline tissue signal ahead of contrast agent arrival is obtained. Nowadays, automatic injection (Medrad, Indianola, PA, USA) has replaced manual contrast agent injection, using a standard injection rate followed by a 20-mL saline flush. Dyke et al. (2003) detailed their techniques for the DCE-MRI evaluation of bone sarcoma. Image acquisition was performed with a

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1.5-T MR system. Anatomical MRIs included T1W imaging and fatsaturated T2W imaging in the transverse, sagittal, and coronal planes. The sagittal plane was prescribed for DCE-MRI. Gd-DTPA was administered at a flow rate of 2 mL/s (2 mL = 1 mmol), with a standard dosage of 0.1 mmol/kg. DCE-MRI was acquired with a multislice fast spoiled gradient-echo sequence. The entire tumor was imaged contiguously with 10–12-mm-thick sections. Acquisition parameters included 9/2 ms TR/TE, 30° flip angle, 20–24 cm field of view, and 256 × 128 matrix (yielding a voxel resolution of 12–20 mm3). These parameters provided a temporal resolution between 4.75 and 9.45 seconds per image, and approximately five points on the initial slope of the blood pool time–intensity curve. Data were acquired at a total of 20–40 time points in imaging times of less than 5 minutes. To study vertebral body blood perfusion, Griffith et al. (2006) reported a single-slice fast data acquisition approach. MRI was performed using a 1.5-T MR scanner. DCE-MRI was acquired in the transverse plane through the mid-L3 vertebral body. The DCE sequence was performed with a fast T1W gradient-echo sequence (TR/TE, 2.7/0.95 ms; prepulse inversion time, 400 ms; flip angle, 15°; section thickness, 10 mm; field of view, 250 mm; acquisition matrix, 256 × 256; one signal averaging). A total of 160 dynamic images were obtained with a temporal resolution of 543 ms per image. A bolus of gadolinium chelate at a dose of 0.15 mmol/kg was injected at a rate of 2.5 mL/s through a 20-gauge intravenous catheter inserted in an antecubital vein. DCE-MRI differs significantly from most conventional MRI exams, and greater care must be exercised in the contrast agent injection rate as well as the dose and image timing. Therefore, DCE-MRI should be performed mainly in well-controlled environments. Failure to obtain DCE-MRI sequences in a uniform manner will confound the results of serial studies.

3. DCE-MRI Empirical Measures Empirical (semiquantitative) measures describe tissue enhancement using a number of descriptors derived from the time–signal intensity

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enhancement curve postcontrast agent injection (Fig. 1). These measures include onset time (time from the contrast agent injection to the first increase in tissue signal enhancement), initial and mean gradients of the upsweep of enhancement curves, maximum signal intensity, wash-out gradient, etc. Oftentimes, the signal intensity measures are normalized to baseline intensity or compared with other normal tissues in the imaging plane. The uptake integral or initial area under the (time–signal) curve (AUC) has also been studied. Enhancement slope (ES), or initial rate of enhancement (IRE), can be defined as the rate of enhancement between 10% and 90% of the maximum signal intensity difference between maximum signal intensity postenhancement (Imax) and signal intensity prior to enhancement (Ibase). The maximal signal intensity enhancement (ME) and enhancement slope (ES) can be calculated according to Eqs. (1) and (2), repectively: È (I - I base ) ˘ ME = Í max ˙ ¥ 100 I base Î ˚

(1)

Ï [(I - I base )] ¥ 0.8 ¸ ES = Ì max ˝ ¥ 100, Ó[I base ¥ (t 90% - t 10% )] ˛

(2)

where t10% and t90% are the time intervals when the rise in signal intensity reaches 10% and 90% of the maximum signal intensity difference between Ibase and Imax, respectively. In a more simple way, the enhancement slope can also be calculated as follows: Ï ¸ (I max - I base ) ES = Ì ˝ ¥ 100, Ó[I base ¥ (t max - t base )] ˛

(3)

where tbase denotes the starting time corresponding to the image acquisition time at which the contrast agent reaches the tissue and tmax

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Fig. 1. A characteristic signal-enhancement-over-time curve showing an increase in tissue signal following the injection of gadolinium contrast agent. MR signal intensity is a relative value without a unit. The maximal signal intensity enhancement (ME) corresponds to the plateau region of the plot after equilibrium of contrast enhancement is established. The dashed line corresponds to the enhancement slope (ES), or the initial rate of enhancement (IRE). Reproduced from Reece et al. (2002) with permission, © John Wiley & Sons Ltd.

denotes the time at which the contrast agent concentration in tissue EES reaches its maximum. ME and ES are derived from the first-pass phase of signal intensity enhancement, and are considered to represent the arrival of contrast material into the arteries and capillaries and its diffusion into the extracellular space. ME and ES time–signal intensity curve indices are dependent on many factors, including the concentration of the contrast material bolus, blood volume, tissue permeability, and available leakage space. Elevated interstitial fluid pressure in some tumor types may also be an important physiological barrier to contrast agent exchanges. Therefore, although these indices are often predictive of perfusion, they are not a direct measure of perfusion. Additionally, both ME and ES values will be affected by variations in the precontrast T1 value of tissues, T1(0), which is one factor in MR signal. Empirical measures have the advantage of being relatively straightforward to calculate, but they have a number of limitations. These limitations include the fact that they do not accurately reflect

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contrast agent concentration in the tissue of interest, and can be influenced by scanner settings (including gain and scaling factors). These factors limit the usefulness of empirical measures, and make betweenpatient and between-system comparisons difficult. Furthermore, it is often unclear what these parameters reflect physiologically and how robust they are to variations in cardiac output. Evelhoch (1999) has suggested that the AUC parameter, when normalized to muscle tissue, parallels K trans estimates over a wide range of tissue input functions using mathematical simulations. Nevertheless, it is clear that empirical measures have a close, albeit complex and undefined, link to underlying tissue physiology and contrast agent kinetics. In spite of a lack of consistency in the empirical measures extracted from DCE-MRI data, clinical investigators who studied patients with breast carcinoma, cervical carcinoma, or bone sarcoma and who correlated their DCE-MRI measures with histology have consistently reported that regions which enhanced brightly and/or reached near-maximum enhancement rapidly (within the first 60– 90 seconds after contrast injection) were correlated with blood vessels and viable tumor regions. These studies predict that empirical measures derived from DCE-MRI will allow the identification of malignant regions with sufficient accuracy to measure angiogenesis or response at least as good as measures such as microvessel density and histopathological evaluation of necrosis (Choyke et al. 2003; Knopp et al. 2001).

4. DCE-MRI Pharmacokinetic Model Measures Pharmacokinetic model measures apply established compartmental methods to model the rates at which low-molecular-weight solutes (gadolinium chelates) transfer between plasma and EES of tissues, and the extent to which these agents accumulate in the EES. An imaging protocol for pharmacokinetic model analysis includes a means of measuring the native T1 value, a reproducible delivery of the contrast agent, and a T1W image acquisition whose signal change over time can be reliably converted to concentration change over time for the contrast agent. Knowledge of the physics of the MR signal and

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the relaxation properties of water protons allow for the conversion of the signal change in tissue due to the local contrast agent into the corresponding concentration of contrast agent, which is present in the local volume and contributes to MR signal increase. The equations relating the signal of the DCE-MRI image to the contrast agent concentration depend on the nature of the MRI sequence (Workie et al. 2004). The rates at which the contrast agent enters the tissue EES from the vascular space are determined by the concentrations of contrast agent in plasma and EES, and by the size and permeability of the capillary–EES interface. Pharmacokinetic model measures include the transfer constant of the contrast agent (K trans, formally called the permeability–surface area product per unit volume of tissue), the leakage space as a percentage of unit volume of tissue (ve), and the rate constant (kep). These standard parameters are related mathematically (kep = K trans/ve). As low-molecular-weight contrast media do not cross cell membranes, their volume of distribution is effectively the interstitial space. Over a period typically lasting several minutes to hours, the contrast agent diffuses back into the vasculature (described by the rate constant kep) from which it was excreted (usually by the kidneys). For modeling the relation of the concentration in the tissue to the concentration–time curve for the central compartment (plasma or arterial input function, AIF), a linear two-compartment model is commonly chosen (Figs. 2 and 3). The first compartment is defined as the plasma or central compartment. This compartment is connected to the second compartment, the EES or the leakage space of the tissue (designated as the peripheral compartment), by linear exchange processes in both directions and by an irreversible elimination of tracer from both or one of the compartments. The essential features are covered by the generalized kinetic model in Eq. (4) (Tofts et al. 1999), where the MR signal contribution of the intravascular contrast agent to the signal in target tissue is assumed to be negligible: dC lesion = K trans C p - K epC lesion , dt

(4)

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Fig. 2. A two-compartment tissue model describing the kinetics of exchange of gadolinium chelate between the plasma and the interstitial or leakage space, with an irreversible elimination from the plasma compartment characterized by a rate constant kel min−1 (Tofts 1997).

Fig. 3. DCE-MRI study showing the arterial input function (signal change over time in the abdominal aorta; curve in green) and the signal change over time in a lumbar vertebral body which is a peripheral compartment (line in blue). The x-axis is time in seconds. A recirculation of contrast agent in the central compartment is also seen (indicated by the black vertical line).

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where Clesion and Cp are the concentrations of the agent in the EES and plasma space, respectively. Thus, the concentration of contrast agent within the tissue is determined by the blood plasma concentration curve and two parameters: the transfer constant, Ktrans, and the EES fractional volume index, ve. In relating the shape of the enhancement curve obtained from dynamic MRI studies, Ktrans controls the height of the Clesion curve and kep = Ktrans/ve controls the shape of the curve; the smaller the value of kep, the more delayed the enhancement. Ktrans is a function of flow (perfusion) and permeability; the greater the flow and permeability, the greater the Ktrans value. The concept of “extraction fraction” developed by Renkin (1959) provides a precise understanding of the relationship: Ktrans = F × EF, where F is flow and EF is the extraction fraction or the fraction of contrast agent that diffuses into the EES during initial passage of the bolus of contrast agent, assuming negligible initial concentration in EES; and EF = 1 − e(−PS/F ), where PS is the permeability–surface area product and F is flow. If permeability is high, then EF approximates 1, Ktrans = F, and the lesion contrast agent concentration is “flow limited” (Kety 1951). On the other hand, if permeability is low compared to F, then EF approximates PS/F, Ktrans = PS, and the lesion contrast agent concentration is “permeability limited” (Kety 1951). Pharmacokinetic model analysis approaches have two advantages over empirical measures: (1) pharmacokinetic model analysis allows comparison between groups of investigators and patients, and (2) pharmacokinetic model analysis yields insight into the pathophysiology of target tissues. Modeling the effect of altering physiological parameters in the two-compartment model showed that, for a given leakage space (EES), improving the permeability increased the initial contrast accumulation rate and decreased the time to maximum enhancement. For a fixed permeability, increasing the EES had no effect on the initial accumulation rate, but increased the magnitude and time of maximum enhancement.

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For both empirical measures and pharmacokinetic modeling, summary measures over extended ROIs can work well for homogeneous tissues. However, many lesions such as tumors are spatially heterogeneous prior to any treatment, and treated tumors show even more heterogeneity. Ideally, heterogeneous lesions should be evaluated on a pixel-by-pixel basis to produce a parametric map of the lesion and to facilitate the detection of small foci of residual tumor. This map can be used for qualitative inspection to identify small foci of viable tumor. In addition, it is easy to display a parameter/volume histogram for the entire tumor and to provide a statistically valid summary measure of this distribution that is correlated to the percentage of necrosis. However, pixel-by-pixel basis demands a satisfactory SNR. For image analysis, color-encoded images based on the pharmacokinetic model can be displayed; from the color-encoded images, specific ROI enhancement curves can be generated (Fig. 4). Another useful display technique is that of the observer interactively moving a cursor within the tumor while generating time–signal curves to probe for enhancement characteristics within the tumor. For tumors, it should be kept in mind that there is a significant difference between the core of a tumor mass, which tends to be necrotic, and the rim of the tumor, which can be highly proliferatic. Histograms or cluster plots of the parameters from pharmacokinetic models can be used as a global depiction of lesion heterogeneity and therapeutic response. A DCE-MRI study can thus be analyzed using both empirical measures and pharmacokinetic model measures.

5. Clinical Applications of DCE-MRI in Bone Sarcomas Osteosarcoma is the most common malignant bone tumor of childhood (Gherlinzoni et al. 1992). Fortunately, the use of preoperative chemotherapy has produced a dramatic improvement in the outlook for children with this disease (Link et al. 1986). Recent studies show that at least 70% of children with osteosarcoma now survive. The degree of necrosis in osteogenic and Ewing sarcomas after a course of induction chemotherapy before surgery is prognostic for event-free

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Fig. 4. Osteosarcoma of the thigh. (a) Projection color-coded MRI demonstrates the vascular perfusion of the tumor with heterogeneity. Time–signal curves obtained through avascular core (b) and vascular portions of the tumor (c) indicate the extremes of vascular heterogeneity within a tumor. Reproduced from Choyke et al. (2003) with permission, © John Wiley & Sons Ltd.

survival (Meyers et al. 1998). For example, a combination of good necrosis and wide surgical margins was associated with only one recurrence in 93 patients; in contrast, patients who had a poor necrosis response developed a local recurrence in 4 of 14 cases, despite wide surgical margins (Link et al. 1993). These data suggest that it is important to tailor surgery on the basis of local response to chemotherapy. Noninvasive testing of tumor viability can be performed early in the course of therapy and serially throughout therapy. This may allow earlier identification of inferior responders, giving clinicians the opportunity to identify a population of patients who may benefit from a change in therapy type or intensity. No satisfactory clinical methods exist for determining the response to cytotoxic drugs before resection. Without imaging, assessing the

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effects of chemotherapy relies on histologic examination of a representative section of the resected tumor. A complete response is defined as disappearance of the soft tissue mass and absence of malignant cells detectable by microscopic examination of the marrow. For osteosarcomas, the intramedullary tumor volume typically does not decrease because of the extended rigid matrix structure; the percentage necrosis of this tumor is strongly correlated with the outcome, whereas volume changes are not. The effectiveness of conventional static MRI has been limited because signal intensities of viable and necrotic tumor, edema, and hemorrhage overlap on T2-weighted images (Lawrence et al. 1993). In addition, the use of short-time inversion recovery (STIR) and contrast-enhanced T1-weighted MRI overestimates the extent of viable tumor because of the enhancement of nonmalignant reactive tissues. It should be noted that the occurrence of spontaneous necrosis within osteogenic sarcomas rarely accounts for more than 25% of the total necrotic fraction (Springfield et al. 1991); thus, any finding of substantially greater necrotic fraction in patients undergoing chemotherapy may be attributed to the effects of the therapy. Compared with information gained via single-slice pathologic examination, MRI allows for the assessment of the degree of necrosis throughout the entire tumor. DCE-MRI has demonstrated a convincing ability to detect the presence of viable tumor and measure response. Erlemann et al. (1990) reported that viable bone sarcoma reaches peak enhancement rapidly (less than 90 seconds postinjection) and that DCEMRI–based parameters can distinguish between malignant tumor and tumor invasion of muscle from peritumoral edema and other confounding pathologies. Lang et al. (1995) showed that viable tumor and extraosseous tumor infiltrating muscle had the highest initial slopes, significantly differentiating viable tumor in bone from nonneoplastic bone lesions, edematous or normal muscle, or marrow. This is because the maximum enhancement is influenced by the EES fractional volume, which is increased enormously for many types of tumors (Jakobsen et al. 1995). Fletcher et al. (1992) published results in 20 patients with bone and soft tissue sarcoma. The DCE-MRI images were analyzed for one or more ROIs of various shapes and

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sizes; these ROIs were selected to allow sampling of as much of the enhancing portion of the tumor as possible. The percent slope values were evaluated with the examination immediately before resection at the completion of chemotherapy and were compared with histologic response. A critical percent slope value of 44% per minute was used to differentiate responsive tumors from unresponsive tumors with a sensitivity of 89%, a specificity of 100%, and an accuracy of 95%. Islands of persisting enhancement suggesting hyperpermeability implied the presence of tumor that had escaped the effects of therapy. These regions usually correlate directly with foci of chemoresistant tumor, and often represent the more aggressive clones within a tumor. DCE-MRI also provides a noninvasive measure of drug access. Using contrast agent uptake and/or elimination to predict which tumors will be unresponsive to cytotoxic therapy relies on the premise that the delivery of a contrast agent can provide a measure of perfusion, blood volume, or capillary permeability. This assessment of microcirculation can be crucial for the delivery of chemotherapy and the efficacy of radiotherapy and hypothermic therapy. Reddick et al. (1999) reported a DCE-MRI study at the time of presentation and upon the completion of preoperative chemotherapy in 34 patients with primary osteosarcoma. They found that greater initial Kep was associated with higher disease-free survival estimates, whereas lower Kep after preoperative chemotherapy was predictive of improved outcome. Consequently, larger values for regional access at the time of diagnosis would be expected to correspond to a greater decrease in Kep during therapy. The relationship between regional access (Kep) and improved disease-free survival estimates is consistent with the hypothesis that the transfer rate of low-molecular-weight MR contrast agents between the vasculature and the extracellular fluid acts as a surrogate measure of drug delivery. A small value for the effective regional access (Kep) after preoperative chemotherapy may indicate that a large proportion of the tumor tissue is necrotic and that its microvasculature is reduced. It has been reported that both the empirical model and the pharmacokinetic model have good correlation, and that both predict

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tumor necrosis. Dyke et al. (2003) reported that the histogram analysis of initial enhancement slope from the tumor correlated well with percentage necrosis as determined at pathologic examination, as did a two-compartment pharmacokinetic model. Both methods predicted tumors with clinically important degrees of necrosis (i.e. ≥90%) in a large majority of their cases. However, like most imaging techniques, DCE-MRI cannot substitute for histopathology. Microscopic disease may still be present even when a DCE-MRI shows no evidence of tumor.

6. Clinical Applications of DCE-MRI in Rheumatoid Arthritis Rheumatoid arthritis (RA) is a chronic debilitating disease associated with early joint damage. Poor disease control may result in long-term irreversible disability. Improving the management of arthritis demands a reliable, noninvasive method for monitoring the degree of inflammation and therapeutic response during the early phase of the disease. MRI techniques have shown remarkable sensitivity for detecting changes in synovial inflammation. Synovitis is a characteristic feature of RA associated with the continued synovial inflammation that leads to structural damage, including erosion and joint space narrowing. DCE-MRI has been used to evaluate the disease activity of RA. After intravenous injection of a gadolinium chelate, the MR signal of highly vascular inflamed synovium enhances significantly (Fig. 5). Clinical studies have shown that the initial enhancement rate over the synovium is correlated with clinical measures such as joint swelling (Cimmino et al. 2003). Ostergaard et al. (1996) demonstrated that the rate of early synovial enhancement reflects synovial inflammatory activity, and that subclinical changes may be revealed by DCE-MRI. Kalden-Nemeth et al. (1997) reported a study using DCE-MRI to monitor synovitis of several joints in a group of patients receiving therapy with antitumor necrosis factor-alpha monoclonal antibody. They found that those given a high dose of the antibody demonstrated a marked improvement in clinical parameters as well as a

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Fig. 5. Five characteristic DCE-MRI sagittal scans across synovial space following gadolinium enhancement. The colour-coded spectrum demonstrates minimal to maximal enhancement (red to yellow). Reproduced from Reece et al. (2002) with permission, © John Wiley & Sons Ltd.

highly significant reduction in gadolinium uptake, and that changes in signal intensity of the synovium on MRI correlated with clinical indicators of inflammation. Reece et al. (2002) used DCE-MRI as an efficacy assessment tool for differentiating treatment effect in a randomized, active-controlled trial comparing leflunomide and methotrexate therapy for 4 months. DCE-MRI scans were obtained at baseline and at 4 months, and the initial rate of enhancement and the maximal enhancement were calculated from the signal intensity curves. The results showed that leflunomide treatment was associated with a significantly greater improvement in the initial rate of enhancement compared with methotrexate treatment, while average values of maximal enhancement indicated reduction of inflammation with both leflunomide and methotrexate.

7. Summary Clinical application of DCE-MRI in the musculoskeletal system is expanding. Efforts to standardize DCE-MRI acquisition, analysis, and reporting methods will allow wider dissemination of this valuable functional imaging technique. In clinical trials, DCE-MRI is likely to assume increasing importance, given the variety of new biological and pharmacologic agents currently being developed for use in oncology and RA treatment.

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References Choyke PL, Dwyer AJ, Knopp MV. Functional tumor imaging with dynamic contrast-enhanced magnetic resonance imaging. J Magn Reson Imaging 17:509–520, 2003. Cimmino MA, Innocenti S, Livrone F et al. Dynamic gadolinium-enhanced magnetic resonance imaging of the wrist in patients with rheumatoid arthritis can discriminate active from inactive disease. Arthritis Rheum 48:1207–1213, 2003. Donahue KM, Burstein D, Manning WJ, Gray ML. Studies of Gd-DTPA relaxivity and proton exchange rates in tissue. Magn Reson Med 32:66–76, 1994. Dyke JP, Panicek DM, Healey JH et al. Osteogenic and Ewing sarcomas: estimation of necrotic fraction during induction chemotherapy with dynamic contrast-enhanced MR imaging. Radiology 228:271–278, 2003. Erlemann R, Sciuk J, Bosse A et al. Response of osteosarcoma and Ewing sarcoma to preoperative chemotherapy: assessment with dynamic and static MR imaging and skeletal scintigraphy. Radiology 175:791–796, 1990. Evelhoch JL. Key factors in the acquisition of contrast kinetic data for oncology. J Magn Reson Imaging 10:254–259, 1999. Fletcher BD, Hanna SL, Fairclough DL, Gronemeyer SA. Pediatric musculoskeletal tumors: use of dynamic contrast-enhanced MR imaging to monitor response to chemotherapy. Radiology 184:243–248, 1992. Gherlinzoni F, Picci P, Bacci G, Campanacci D. Limb sparing versus amputation in osteogenic sarcoma. Correlation between local control, surgical margins and tumor necrosis: Istituto Rizzoli experience. Ann Oncol 3(Suppl 2):S23–S27, 1992. Griffith JF, Yeung DK, Antonio GE et al. Vertebral marrow fat content and diffusion and perfusion indexes in women with varying bone density: MR evaluation. Radiology 241:831–838, 2006. Jakobsen I, Lyng H, Kaalhus O, Rofstad EK. MRI of human tumor xenografts in vivo: proton relaxation times and extracellular tumor volume. Magn Reson Imaging 13:693–700, 1995. Kalden-Nemeth D, Grebmeier J, Antoni C et al. NMR monitoring of rheumatoid arthritis patients receiving anti-TNFα monoclonal antibody therapy. Rheumatol Int 16:249–255, 1997. Kety SS. The theory and applications of the exchange of inert gas at the lungs and tissues. Pharmacol Rev 3:1–41, 1951.

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Knopp MV, Giesel FL, Marcos H et al. Dynamic contrast-enhanced magnetic resonance imaging in oncology. Top Magn Reson Imaging 12:301–308, 2001. Lang P, Honda G, Roberts T et al. Musculoskeletal neoplasm: perineoplastic edema versus tumor on dynamic postcontrast MR images with spatial mapping of instantaneous enhancement rates. Radiology 197:831–839, 1995. Lawrence JA, Babyn PS, Chan HS et al. Extremity osteosarcoma in childhood: prognostic value of radiologic imaging. Radiology 189:43–47, 1993. Link MP, Eilber F. Osteosarcoma. In: Pizzo PA, Poplack DG (eds.), Principles and Practice of Pediatric Oncology, Vol. 2, JB Lippincott, Philadelphia, PA, p. 841, 1993. Link MP, Goorin AM, Miser AW et al. The effect of adjuvant chemotherapy on relapse-free survival in patients with osteosarcoma of the extremity. N Engl J Med 314:1600–1606, 1986. Meyers PA, Gorlick R, Heller G et al. Intensification of preoperative chemotherapy for osteogenic sarcoma: results of the Memorial Sloan–Kettering T12 protocol. J Clin Oncol 16:2452–2458, 1998. Ostergaard M, Stoltenberg M, Henriksen O, Lorenzen I. Quantitative assessment of synovial inflammation by dynamic gadolinium-enhanced magnetic resonance imaging. A study of the effect of intra-articular methylprednisolone on the rate of early synovial enhancement. Br J Rheumatol 35:50–59, 1996. Reddick WE, June S, Taylor JS, Fletcher BD. Dynamic MR imaging (DEMRI) of microcirculation in bone sarcoma. J Magn Reson Imaging 10:277–285, 1999. Reece RJ, Kraan MC, Radjenovic A et al. Comparative assessment of leflunomide and methotrexate for the treatment of rheumatoid arthritis, by dynamic enhanced magnetic resonance imaging. Arthritis Rheum 46:366–372, 2002. Renkin EM. Transport of potassium-42 from blood to tissue in isolated mammalian skeletal muscles. Am J Physiol 197:1025–1210, 1959. Springfield DS, Schakel ME, Spanier SS. Spontaneous necrosis in osteogenic sarcoma. Clin Orthop 263:233–237, 1991. Taylor JS, Wilburn E, Reddick WE. Evolution from empirical dynamic contrast-enhanced magnetic resonance imaging to pharmacokinetic MRI. Adv Drug Deliv Rev 41:91–110, 2000.

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Tofts PS. Modeling tracer kinetics in dynamic Gd-DTPA MR imaging. J Magn Reson Imaging 7:91–101, 1997. Tofts PS, Brix G, Buckley DL et al. Weisskoff RM. Estimating kinetic parameters from dynamic contrast-enhanced T(1)-weighted MRI of a diffusable tracer: standardized quantities and symbols. J Magn Reson Imaging 10:223–232, 1999. Workie DW, Dardzinski BJ, Graham TB et al. Quantification of dynamic contrast-enhanced MR imaging of the knee in children with juvenile rheumatoid arthritis based on pharmacokinetic modelling. Magn Reson Imaging 22:1201–1210, 2004.

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Chapter 43

Noninvasive Evaluation of Knee Cartilage Morphology by Magnetic Resonance Imaging Yi-Xiang Wang

Attrition and eventual loss of articular cartilage are crucial elements in the pathophysiology of osteoarthritis. Preventing the breakdown of cartilage is believed to be critical in order to preserve the functional integrity of a joint. Magnetic resonance imaging (MRI) and advanced digital postprocessing techniques have opened novel possibilities for in vivo quantitative analysis of cartilage morphology, structure, and function in health and disease. Techniques of semiquantitative scoring of human knee cartilage pathology and quantitative assessment of human cartilage have recently been developed. Though cartilage represents a thin layer of material relative to the size of voxels typically used for MRI, cartilage thickness and volume have been quantified in human and in small animals. MRI-detected cartilage loss has been shown to be more sensitive than radiography-detected joint space narrowing. Progress made in MRI technology in the last few years allows longitudinal studies of knee cartilage with an accuracy good enough to follow disease-caused changes and to evaluate the therapeutic effects of chondroprotective drugs. Keywords:

Cartilage; knee; osteoarthritis; joint space narrowing; animal model; MRI; radiographs.

Corresponding author: Yi-Xiang Wang. E-mail: [email protected]

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1. Introduction Osteoarthritis (OA) occurs in more than 60% of people over the age of 65 years. Currently, there exists no well-accepted medical treatment for OA with structure or disease modification efficacy. Attrition and eventual loss of articular cartilage are crucial elements in the pathophysiology of OA. Because of the avascular nature and small chondrocyte population in adults, the capacity of injured or degenerated cartilage to synthesize and secrete its extracellular matrix is poor. The healing response to cartilage injury and degeneration also decreases with age. Though articular cartilage lacks nerves, recent studies showed that cartilage pathology is associated with clinical symptoms (Link et al. 2003; Sowers et al. 2003). Preventing the breakdown of cartilage is believed to be critical for preserving the functional integrity of a joint. A number of promising therapeutic agents and surgical procedures are currently under development in this regard. Besides to investigate the pathophysiology of cartilage generation, there is a significant need for a noninvasive method of monitoring OA to judge the success of potential chondroregenerative and surgical treatments. Magnetic resonance imaging (MRI) offers a unique opportunity to characterize various pathologies of articular cartilage in vivo. MRIdetected cartilage loss has been shown to be more sensitive than radiography-detected joint space narrowing. Amin et al. (2005) reported that cartilage loss was significantly associated with semiquantitatively graded joint space narrowing of weight-bearing radiographs in the femoral–tibial joint; whereas there was a substantial proportion of knees in which cartilage loss was detected with MRI, but no radiographic joint space narrowing was observed. Raynauld et al. (2004) described no significant change in the medial femoral-tibial compartment of weight-bearing semiflexed radiographs positioned with fluoroscopy in 32 patients with OA over 2 years, but reported a highly significant change of cartilage volume from MRI in both the medial and lateral femoral-tibial compartments. The knee is the largest weight-bearing joint in the body, and is therefore most commonly affected by OA. This chapter discusses

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the in vivo MRI methodology of morphological assessment of knee cartilage.

2. MRI Acquisition Techniques for Human Knee Given that knee cartilage is only 1.3–2.5 mm thick in healthy human subjects, and also because of its complex morphology, knee cartilage presents a challenge for MRI (Eckstein et al. 2001; Hudelmaier et al. 2001; Wang 2007). The challenge is even greater in OA patients, as decrease in signal and thinning make delineation of the articular cartilage more difficult. MR images capable of resolving various structures of knee joint require a good signal-to-noise ratio (SNR), good tissue contrast, and good spatial resolution. The SNR is the ratio between the intensities of the signals from tissue and background. Tissue contrast is the difference between the signal intensities of a target tissue (or a lesion) and the surrounding or adjacent tissues. Spatial resolution refers to the smallest size of details that is visible on images; it is determined by the thickness of the slice and the size of the smallest element of the image (i.e. the pixel). MR instruments help to determine the quality of images. As the magnetic field strength increases, the SNR increases. The quality of the radiofrequency (RF) coil receiving the signal also significantly affects the SNR. The SNR, image acquisition time, and spatial resolution are interdependent, and optimizing one of these parameters at a given magnetic field strength necessitates sacrifices in the others. Therefore, a delicate balance has to be achieved among these parameters for optimal delineation of a knee joint. Most studies on human knee articular cartilage MRI have so far been performed with a threedimensional (3D) data acquisition mode using 1.5-tesla whole-body scanners and dedicated RF extremity coils. For human studies, MR images should be acquired within a reasonable examination time (<20 minutes per pulse sequence) in order to avoid movement artifacts, maintain patient comfort, and contain costs. The pattern of joint structures as seen on MR images can be modified in various ways by the choice of MR pulse sequences. Numerous

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studies have evaluated different MR pulse sequences, including T1weighted gradient echo (GE) sequence with fat suppression and fatsuppressed T2- or intermediate-weighted fast spin echo sequence, in order to achieve a high SNR and tissue contrast of the joint. The MR sequences that have been most commonly used for cartilage assessment are fat-suppressed T1-weighted spoiled gradient echo sequences. Different MRI vendors name these sequences differently (e.g. FLASH, fast low-angle shot; SPGR, spoiled gradient recalled acquisition at steady state). Fat suppression (FS) is important for increasing the dynamic range between cartilage and adjacent structures, and for eliminating chemical shift artifacts at the cartilage– bone interface. FS enhances the contrast for the cartilage, and it has been reported that it can lead to better reproducibility of the volumetric measurements (Sittek et al. 1996). FS is accomplished either by spectral fat saturation using a prepulse tuned to the resonant frequency of fat (chemical-shift-selective fat suppression), by frequencyselective water excitation, or by the short-tau inversion-recovery (STIR) technique. The GE sequence allows a very short time of echo (TE), which improves signal sensitivity when small structures are imaged. FS GE sequences with short TEs and relatively large flip angles provide T1weighted images where the intra-articular fluid is less intense than the cartilage and fat is suppressed, therefore maximizing the contrast between cartilage, fluid, and marrow, with cartilage showing a bright signal. Cartilage demonstrates intermediate signal in intermediateand T2-weighted sequences; whereas synovial fluid is bright and the internal structure of the cartilage displays a more heterogeneous signal, and internal pathological changes may be more readily displayed (Fig. 1). Because of the relatively short T2 relaxation times in articular cartilage, especially in deep cartilage adjacent to the bone interface, MR sequences for quantitative volume measurements should be used with a TE as short as possible, preferably below 10 ms. A longer TE leads to cartilage signal decay; Eckstein et al. (2000) reported a significant underestimation of tibial cartilage thickness compared to computed tomography (CT) arthrography when using a GE sequence with a TE of 11 ms. It is particularly important to avoid this

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Fig. 1. (a) Sagittal fat-saturated SPGR image of the knee in a patient with early OA and a cartilage fissure (arrow). (b) Sagittal FS intermediateweighted image of the same patient. Note the difference in contrast, with bright cartilage signal in (a) and intermediate cartilage signal in (b). Joint effusion in (b) with bright signal improves visualization of the cartilage fissure. Reproduced from Eckstein et al. (2006), with permission of John Wiley & Sons Ltd.

confounding effect of T2 relaxation on volume measurement in longitudinal studies on OA progression. Most studies have used the sagittal scan plane for cartilage evaluation. Although there is no current consensus on the optimal resolution for imaging knees in OA, sections 1.5 mm thick and an in-plane resolution of 0.3 mm have commonly been used, as these allow total coverage of the knee in imaging times of 10–12 minutes. In a systematic comparison of images with different in-plane resolutions, Hardya et al. (2000) reported significantly larger precision errors for cartilage volume measurements derived from lower-resolution (0.55 mm × 0.55 mm) images than for those from higher-resolution (0.28 mm × 0.28 mm) images in the femur and tibia. Cicuttini and coworkers (2004) measured the rate of progression in tibial cartilage using sagittal images and reformatted coronal images. They reported a higher rate of progression of tibial cartilage loss in the reformatted coronal vs. original sagittal images (Cicuttini et al. 2004; Wluka et al. 2002). These findings indicate that changes may be more readily

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detected in coronal views of the knee. Which protocol (sagittal or a combination of axial and coronal scans) is preferable remains to be confirmed by future studies. A higher magnetic field could potentially increase the quality of cartilage images at 3.0 T. It was found that the SNR and contrast-tonoise ratio (CNR) efficiency for cartilage increased by a factor of 1.8 when compared to 1.5 T for spoiled gradient echo sequences. Cartilage volume and thickness measurements at 3.0 T showed only small (nonsignificant) differences as compared with measurements at 1.5 T (Eckstein et al. 2005; Kornaat et al. 2005b). Studies have shown that knee bends and squatting can cause a reduction of approximately 5% in patellar cartilage volume and thickness, and this effect can last for approximately 90 minutes (Eckstein et al. 2001). To avoid differences in subject conditions due to differences in the level of physical activity prior to imaging, for cartilage volume and thickness measurements, study subjects need to rest for 1 hour prior to the image acquisition.

3. MRI Acquisition Techniques for Animal Knee As tissue samples from early-stage human OA tissue cannot be reliably obtained, a number of animal models have been devised to investigate the pathogenesis of OA, whereby the same joint can be studied at the earliest and at much later stages of disease within a predictable time span. OA animal models can be divided into two main categories: spontaneous joint degeneration models and experimentally induced OA models. Spontaneous OA in the knee joints of mice and guinea pigs has been described (Bendele 2001; Bendele et al. 1999). Experimentally induced OA models are further subdivided into biochemically and biomechanically induced OA. Biochemical induction is achieved by the intra-articular injection of chemicals that cause lysis of specific cartilage constituents, such as collagen, proteoglycans, and/or chondrocytes. Biomechanical induction involves the initiation of a biomechanical instability through surgical transaction of a ligament such as an anterior cruciate ligament, or partial removal of the meniscus (meniscectomy), or a combination of both. OA animal

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models are also used to evaluate potential new treatments. It is a great advantage for research that the time course of OA in animal models is much more rapid than the development of OA pathology in humans. Knee is the most commonly used joint for OA induction; compared with other joints, it is easily assessable to intervention and assessment. MRI has already been applied to investigate a variety of OA animal models, including mouse, rat, guinea pig, rabbit, monkey, goat, and dog (Wang 2007). OA models of small animals are preferred due to economic and ethical reasons. The high-resolution requirement for small animal knee MRI demands high performance of MR instruments. A higher magnetic field offers a higher SNR; this can be traded with better spatial resolution. It is technically difficult to construct a very high-field MR scanner with a large horizontal bore to hold large animals. Due to this limitation and also due to cost constraints, high-field MR research scanners are usually equipped with small bores which can hold up to the size of rabbits or rats. For imaging of large animals like dog and goat, clinical human scanners are commonly used, with a magnetic field of 1.5–3 tesla, mostly together with an RF coil designed for human knee. For signal optimization, suitable RF coils for animal knee can be custom-made and interfaced to clinical human scanners. For small animals, small bore research scanners are preferred, usually with a magnetic field of 4.7–7 tesla. The RF coils are usually home-made, or made by some small specialist companies; the most commonly used one is the single-turn solenoid RF coil (Fan et al. 1987), which is designed to open at the top. Animals can be placed on a Perspex platform with one hind leg extending through the RF coil, with the knee centered in it. While designed to completely cover the knee joint of the animal species imaged, the length and diameter of the coil are optimized to minimize the image field of view so as to get a good filling factor. To prevent motion, the animal’s paw of the scanned leg can be secured to a secondary lower platform. Similar MR sequences for human studies are used for animal studies. With high-field MR scanners, it is feasible to obtain 3D data sets of around 100 µm resolution with a scanning duration of less than 1 hour (Faure et al. 2003; Wang et al. 2006). In Tessier et al.’s (2003) paper, the MR image acquisition protocol for guinea pig knee cartilage evaluation

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was detailed. A 4.7-tesla magnet and a FS 3D GE sequence were used. The length and diameter of the solenoid RF coil used were optimized to minimize the image field of view (30 mm × 30 mm × 30 mm). Prior to the acquisition of the 3D data set, the overall positioning of the guinea pig was checked using a fast multislice sagittal GE image to ensure that the leg was placed adequately within the RF coil. Then, a transverse image of the knee was used to select the orientation of the sagittal view of the 3D images such that they were parallel to the medial condyle. The image matrix was zero-filled to 512 × 256 × 128 after 3D Fourier transform, and an apparent image resolution of 59 µm × 117 µm × 234 µm was achieved. The highest resolution (59 µm) was chosen to be across the cartilage thickness (approximately 330 µm). For assessment of the rat knee joint, Wang et al. (2006) used a 4.7-T magnet; the RF coil was an inhouse–built double-balanced matched 3-cm-diameter copper sheet solenoid, with 1 cm in length. A 3D data set at the sagittal plane was acquired using a spoiled multiecho FS 3D GE sequence (TR = 75 ms; flip angle = 30°; five echoes of TE1 = 2.8 ms, TE2 = 6.0 ms, TE3 = 9.2 ms, TE4 = 12.5 ms, and TE5 = 15.7 ms). Echo summation provided a means of enhancing the SNR, and enabled the acquisition of high-resolution 3D images of the rat knee in approximately 50 minutes. The images covered the entire knee joint with a resolution of 59 µm × 117 µm × 234 µm. During the early stages of OA, articular cartilage constituents degenerate before any substantial morphological changes occur. During disease progression, changes in the tissue MR relaxation values (T1, T2, and T1ρ [or T1 in the rotating frame]), magnetic transfer, and delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) (Fig. 2) reflect early alteration in the tissue architecture and biochemical composition (Wang 2007). However, the clinical relevance and reproducibility of these techniques still remain to be validated.

4. Morphological Evaluation of Human Knee Cartilage Knee cartilage morphological evaluation includes qualitative assessment of articular cartilage pathology, semiquantitative scoring of articular cartilage pathology, and quantitative assessment of articular

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Fig. 2. T1-weighted coronal MR image series of a normal rabbit knee post-intravenous injection [Gd(DTPA)2−]. Gd(DTPA)2− uptake in the articular cartilage region is more apparent from difference images (calculated pixel by pixel as the difference between postcontrast and precontrast signal intensity). A bright signal gradually built up in the cartilage, and reached the maximum within 45 minutes after Gd(DTPA)2− injection. Reproduced from Laurent et al. (2003), with permission, © John Wiley & Sons Ltd.

cartilage volume and thickness. In this chapter, semiquantitative scoring of articular cartilage pathology and quantitative assessment of articular cartilage volume are discussed.

4.1. Semiquantitative scoring of human knee cartilage pathology A number of semiquantitative scoring methods have been developed for the evaluation of articular cartilage on MR images (Disler et al. 1996; Drape et al. 1998). Most of these methods grade the severity of cartilage thinning from 0 to 3 or 4, based on subjective evaluations by one or more experienced readers. These systems commonly differentiate between cartilage lesions of <50% depth, >50% depth, and full-thickness cartilage lesions. Peterfy and colleagues (2004) described a scoring system in which cartilage lesions are graded according to both depth and extent along the joint surface using an eight-point scale (Fig. 3). This score is part of a more comprehensive scoring system (i.e. Whole-Organ MRI Score) in which multiple features are graded within the knee, such as articular cartilage integrity, subarticular bone marrow abnormality,

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Fig. 3. Eight-point scale for semiquantitative scoring of articular cartilage signal and morphology. Grade 0 = normal thickness and signal; grade 1 = normal thickness, but increased signal on T2-weighted MR images; grade 2.0 = partial-thickness focal defect <1 cm in greatest width; grade 2.5 = fullthickness focal defect <1 cm in greatest width; grade 3 = multiple areas of partial-thickness (grade 2.0) defects intermixed with areas of normal thickness, or a grade 2.0 defect wider than 1 cm but <75% of the region; grade 4 = diffuse (≥75% of the region) partial-thickness loss; grade 5 = multiple areas of full-thickness loss (grade 2.5), or a grade 2.5 lesion wider than 1 cm but <75% of the region; and grade 6 = diffuse (≥75% of the region) full-thickness loss. Reproduced from Peterfy et al. (2004), with permission of Osteoarthritis Research Society International.

subarticular cysts, subarticular bone attrition, and marginal osteophytes. The surface areas of the knee joint are subdivided into 15 different regions or anatomical landmarks in the extended knee (Fig. 4). Cartilage signal and morphology are scored in each of the 14 articular surface regions (excluding region S of tibia) using the fat-suppressed T2-weighted fast spin echo images and the fat-suppressed 3D spoiled gradient echo images. Peterfy et al. (2004) reported that, despite the complexity of the system and the expanded scale (eight-point), interobserver agreement among two trained readers was high. Another compartment-based scoring system termed the Knee OA Scoring System has also been published recently (Kornaat et al. 2005a).

4.2. Quantitative assessment of human cartilage Due to the relatively low contrast in some areas of the joint surface, fully automated segmentation of cartilage from MR images has not

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Fig. 4. Regional subdivision of the articular surfaces. The patella (left image) is divided into medial (M) and lateral (L) regions, with the ridge considered part of the M region. The femur and tibia are also divided into M and L regions (right image), with the trochlear groove of the femur considered part of the M region, and the boundary between MF and LF is defined by a plane aligned with the lateral wall of the femoral notch. Region S represents the portion of the tibia beneath the tibial spines. The femoral and tibial surfaces are further subdivided into anterior (A), central (C), and posterior (P) regions (middle image). Region A of the femur corresponds to the patellofemoral articulation; region C, the weight-bearing surface; and region P, the posterior convexity which articulates only in extreme flexion. Region C of the tibial surface corresponds to the uncovered portion between the anterior and posterior horns of the meniscus centrally and the portion covered by the body of the meniscus peripherally. Reproduced from Peterfy et al. (2004), with permission of Osteoarthritis Research Society International.

yet been achieved. Computer-generated measurements based on signal intensity or predefined shape are not always reliable. Editing of automatically generated segmentations, or complete manual segmentation by experienced readers, is frequently necessary; therefore, cartilage segmentation remains time-consuming (Fig. 5). After segmentation, computation of the cartilage volume is achieved by simply summing the voxels attributed to the segmented cartilage (Fig. 6). Osteophytes are excluded from segmentation. In the absence of OA, Hudelmaier et al. (2001 and 2003) reported a 0.3%–0.5% reduction in cartilage thickness per annum due to cartilage thinning during normal aging. Other authors have reported a faster rate of cartilage loss. For example, Hanna et al.

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Fig. 5. 3D gradient echo fat-suppressed MRI at the sagittal plane. Bone cortex, bone marrow, and fat tissue appear as dark signals. Muscle appears as gray signal, and cartilage appears as bright signal. The femur cartilage has been manually segmented at this slice.

(2005) found a significant (−2.8% annual) reduction in total tibial cartilage volume over a 2-year period. With data summarized from studies on OA patients in the published literature, Eckstein et al. (2006) reported that the annual loss of cartilage volume was −136 µL (−4.1%) in the patella, −90 µL (−5.6%) in the medial tibia, and −107 µL (−6.0%) in the lateral tibia. However, results varied between published studies, with the annual rate of change ranging from −0.3% to −7.4% in the medial tibia (Eckstein et al. 2006). Because attrition in cartilage volume occurs at a very slow rate, measurement precision is of critical importance. Precision errors can be expressed as the standard deviation (SD) or coefficient of variation [CV (%); SD divided by the mean value] of repeated measurements. Use of the CV is most appropriate when the SD is proportional to volume, for instance, when comparing precision in different joint surfaces of the knee (i.e. medial tibia vs. total femur). If the SD is

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Fig. 6. 3D rendering of femur cartilage. This is achieved by segmentation of the slices containing femur cartilage in a 3D knee MRI data set, after which a computer program is used to generate the 3D volume of the segmented region of interest (ROI). Reproduced from Ding et al. (2005a), with kind permission of Springer Science + Business Media.

independent of volume, it is more appropriate to compare SD values directly. It should be noted that when examining patients with severe OA, the CV will be larger than that in healthy volunteers, even if the absolute error (SD) is similar; this is because patients with severe OA have less cartilage volume. The CV of cartilage volume in medial tibia of healthy volunteers has been reported to be in the range of 2%–3.5%. Precision errors for cartilage volume across studies at 1.5 T for each cartilage component have recently been summarized (Eckstein et al. 2006). Several studies examined the interobserver and intraobserver precision of repeated analyses. It has been confirmed that segmenting all images in a subject’s series by the same skilled reader is more accurate than segmenting by different readers. To alleviate reader bias, the results of several readers can be averaged. Resegmentation of the same data set over a period of 1 year by the same user created larger errors than the segmentation of the data set twice during one session

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(Eckstein et al. 2002). Therefore, to ensure the smallest CV in longitudinal studies, comparative analyses should be performed in one postprocessing session. Blinding the user to the order of the exams is necessary in order to avoid bias. Cartilage volume provides a first step in the analysis of cartilage morphology. More comprehensive information can be derived by separating the cartilage volume into its two factors, namely, the cartilage thickness and the size of joint surface area. Specific algorithms are required to differentiate between these factors. Other variables such as percent cartilaginous (or denuded) joint surface area, cartilage surface curvature, and lesion size and depth can also be investigated. Additionally, overall volume and mean thickness for an entire cartilage plate may be relatively insensitive to regional/focal changes; thus, several investigators have developed techniques for displaying regional thickness patterns. Kshirsagar et al. (1998) suggested that analyzing subvolumes within the joint surface by such techniques can reduce precision errors relative to those from analyses of the entire cartilage plate; this was recently confirmed by a study by Koo et al. (2005) for the central weight-bearing regions of the femoral condyles. These types of “local” approaches to measuring changes in cartilage morphology are technically challenging, but they can be important because the overall cartilage volume can remain constant even if there are focal changes in cartilage thickness and denuded bone. In order to track local/regional thickness changes over time, the bone interfaces from two data sets are “matched” so that the thickness distribution can be compared on a point-by-point basis. Koo et al. (2005) suggested a “trimmed” region defined to avoid errors arising at the edges of articulating surfaces, which are difficult to segment yet may be involved to only a minor degree in the disease process.

5. Morphological Evaluation of Animal Knee Cartilage Although cartilage represents a thin layer of material relative to the size of voxels typically used for MRI, cartilage thickness and volume

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have been quantified in animals as small as guinea pigs and rabbits. Cartilage of OA involves the initial swelling phase, and the later attrition and loss as well as defect phases. These phases have been demonstrated in experimental animal models by sequential MRI (Tessier et al. 2003; Calvo et al. 2001). Tessier et al. (2003) studied 19 male Dunkin–Hartley guinea pigs with MRI at 3, 6, 9, 10.5, and 12 months of age. They found that at 6 months, swollen cartilage was observed in all animals; at 9 months, marked fragmentation of the medial tibial cartilage was seen in areas not covered by the meniscus; and at 12 months, focal thinning of the cartilage was apparent with occasional full cartilage loss. Tessier et al. (2003) detailed their segmentation methodology for guinea pig tibia cartilage. Segmentation of the cartilage of the medial plateau was performed on sagittal slices covering the medial side only; these slices were selected on the basis that they covered the “flattest” region of the medial tibial surface. The slices covering the inner side where the cruciate ligament is attached were excluded because the images were subject to significant partial volume averaging. Slices at one timepoint were matched as close as possible to those obtained at subsequent timepoints based on anatomical references. Thus, for any animal, the number of slices analyzed was equal for all timepoints. However, the number of segmented slices was different between animals to account for the difference in the overall size of the tibial plateau. Their data showed that maximal cartilage loss (36%) occurs from the medial side of the tibial plateau from 9 to 12 months of age. In small animals like rats, due to the small size of their knee joint, assessment of cartilage thickness is challenging but has recently proved to be feasible. With a 7-tesla MR scanner, Faure et al. (2003) reported that MRI could be used to detect arthritis and joint changes at a very early stage in living rats with rheumatoid arthritis. With a rat meniscus transection OA model 44 days postsurgery, MRI was able to demonstrate qualitatively the decrease of cartilage thickness and loss of cartilage in some areas, and focal neocartilage proliferation at the joint margin (Wang et al. 2006).

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6. Clinical Relevance of Cartilage Pathologies Demonstrated by MRI Satisfactory specificity and sensitivity for detecting chondral lesions by MRI have been demonstrated in cadaveric knees and in vivo with arthroscopic verification (Disler et al. 1996; Kawahara et al. 1998; Yoshioka et al. 2004). Knee cartilage defect severity, as measured from sagittal T1-weighted FS SPGR sequences, has been shown to be significantly associated with urinary levels of C-terminal cross-linking telopeptide of type II collagen (Ding et al. 2005b). An association was also reported between cartilage defects and body mass index (Ding et al. 2005a). Link et al. (2003) showed that, in patients with OA, the degree of cartilage pathology seen in MR images is associated with clinical symptoms. In a cross-sectional study, Hunter et al. (2003) observed a significant negative association of patellar cartilage volume with the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score in a population of 133 postmenopausal women. Wluka et al. (2004) reported a weak association between tibial cartilage volume and symptoms (WOMAC score) at baseline in a sample of 132 patients with symptomatic early knee OA, and worsening of symptoms over a 2-year period was associated with tibial cartilage loss. These data suggest that treatment targeted at reducing the rate of knee cartilage loss in subjects with symptomatic OA may relate to clinical outcomes and delay knee replacement. MRI can be used to carry out in vivo longitudinal follow-up in the same animal and track the disease, monitor its progress, and see how it responds to potential treatments. In a recent study of monoiodoacetate-induced arthritis model in rats (Wang et al. 2005), MRI demonstrated that intra-articular soft tissue inflammatory changes peaked at day 3 and started regression afterwards; bony damages appeared at day 14 and peaked at day 21, with hallmarks of repair visible by day 35. A similar biphase pain response was observed clinically with the 1-mg monoiodoacetate dose group, peaking at days 3 and 21. Therefore, MRI was able to characterize the pathological course of the arthritis model, which enables the

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establishment of a link between the structural changes and joint clinical discomfort. There are several disease- or structure-modifying OA drugs under clinical development. Although joint space narrowing on weightbearing radiographs is still the accepted surrogate marker for demonstrating structural change by regulatory agencies, this is expected to change in the near future given the limitations of radiography (Amin et al. 2005; Raynauld et al. 2004), and MRI will very likely replace radiography in this context. By measuring the volume of medial tibia cartilage in a guinea pig spontaneous OA model, MRI demonstrated that doxycycline treatment halved the cartilage loss as compared to the vehicle treatment (Tessier et al. 2006). Clinical efficacy data on human subjects are expected to be published soon.

7. Summary Progress made in MRI technology in the last few years allows longitudinal studies of human knee cartilage with an accuracy good enough to follow disease-caused changes and to evaluate the therapeutic effects of chondroprotective drugs. In animal experimental settings, high-field MRI can noninvasively provide detailed images of joints and be used to carry out in vivo longitudinal follow-up in the same animal as well as track the disease, monitor its progress, and see how it responds to potential treatments.

References Amin S, LaValley MP, Guermazi A et al. The relationship between cartilage loss on magnetic resonance imaging and radiographic progression in men and women with knee osteoarthritis. Arthritis Rheum 52:3152–3159, 2005. Bendele A, McComb J, Gould T et al. Animal models of arthritis: relevance to human disease. Toxicol Pathol 27:134–142, 1999. Bendele AM. Animal models of osteoarthritis. J Musculoskelet Neuronal Interact 1:363–376, 2001.

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Calvo E, Palacios I, Delgado E et al. High-resolution MRI detects cartilage swelling at the early stages of experimental osteoarthritis. Osteoarthritis Cartilage 9:463–472, 2001. Cicuttini FM, Wluka AE, Wang Y, Stuckey SL. Longitudinal study of changes in tibial and femoral cartilage in knee osteoarthritis. Arthritis Rheum 50:94–97, 2004. Ding C, Cicuttini F, Scott F et al. Knee structural alteration and BMI: a cross-sectional study. Obes Res 13:350–361, 2005a. Ding C, Garnero P, Cicuttini F et al. Knee cartilage defects: association with early radiographic osteoarthritis, decreased cartilage volume, increased joint surface area and type II collagen breakdown. Osteoarthritis Cartilage 13:198–205, 2005b. Disler DG, McCauley TR, Kelman CG et al. Fat-suppressed threedimensional spoiled gradient-echo MR imaging of hyaline cartilage defects in the knee: comparison with standard MR imaging and arthroscopy. Am J Roentgenol 167:127–132, 1996. Drape JL, Pessis E, Auleley G et al. Quantitative MR imaging of chondropathy in osteoarthritic knees. Radiology 208:49–55, 1998. Eckstein F, Burstein D, Link TM. Quantitative MRI of cartilage and bone: degenerative changes in osteoarthritis. NMR Biomed 19:822–854, 2006. Eckstein F, Charles HC, Buck RJ et al. Accuracy and precision of quantitative assessment of cartilage morphology by magnetic resonance imaging at 3.0T. Arthritis Rheum 52:3132–3136, 2005. Eckstein F, Heudorfer L, Faber SC et al. Long-term and resegmentation precision of quantitative cartilage MR imaging (qMRI). Osteoarthritis Cartilage 10:922–928, 2002. Eckstein F, Reiser M, Englmeier KH, Putz R. In vivo morphometry and functional analysis of human articular cartilage with quantitative magnetic resonance imaging — from image to data, from data to theory. Anat Embryol (Berl) 203:147–173, 2001. Eckstein F, Stammberger T, Priebsch J et al. Effect of gradient and section orientation on quantitative analysis of knee joint cartilage. J Magn Reson Imaging 11:161–167, 2000. Fan M, Gonord P, Kan S, Taquin J. A UHF probe for NMR micro-imaging experiments. Magn Reson Med 4:591–596, 1987. Faure P, Doan BT, Beloeil JC. In-vivo high resolution three-dimensional MRI studies of rat joints at 7 T. NMR Biomed 16:484–493, 2003.

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Hanna F, Ebeling PR, Wang Y et al. Factors influencing longitudinal change in knee cartilage volume measured from magnetic resonance imaging in healthy men. Ann Rheum Dis 64:1038–1042, 2005. Hardya PA, Newmark R, Liu YM et al. The influence of the resolution and contrast on measuring the articular cartilage volume in magnetic resonance images. Magn Reson Imaging 18:965–972, 2000. Hudelmaier M, Glaser C, Englmeier KH et al. Correlation of knee-joint cartilage morphology with muscle cross-sectional areas vs. anthropometric variables. Anat Rec 270A:175–184, 2003. Hudelmaier M, Glaser C, Hohe J et al. Age-related changes in the morphology and deformational behavior of knee joint cartilage. Arthritis Rheum 44:2556–2561, 2001. Hunter DJ, March L, Sambrook PN. The association of cartilage volume with knee pain. Osteoarthritis Cartilage 11:725–729, 2003. Kawahara Y, Uetani M, Nakahara N et al. Fast spin-echo MR of the articular cartilage in the osteoarthrotic knee. Correlation of MR and arthroscopic findings. Acta Radiol 39:120–125, 1998. Koo S, Gold GE, Andriacchi TP. Considerations in measuring cartilage thickness using MRI: factors influencing reproducibility and accuracy. Osteoarthritis Cartilage 13:782–789, 2005. Kornaat PR, Ceulemans RY, Kroon HM et al. MRI assessment of knee osteoarthritis: Knee Osteoarthritis Scoring System (KOSS) — interobserver and intra-observer reproducibility of a compartment-based scoring system. Skeletal Radiol 34:95–102, 2005a. Kornaat PR, Reeder SB, Koo S et al. MR imaging of articular cartilage at 1.5T and 3.0T: comparison of SPGR and SSFP sequences. Osteoarthritis Cartilage 13:338–344, 2005b. Kshirsagar AA, Watson PJ, Tyler JA, Hall LD. Measurement of localized cartilage volume and thickness of human knee joints by computer analysis of three-dimensional magnetic resonance images. Invest Radiol 33: 289–299, 1998. Laurent D, Wasvary J, O’Byrne E, Rudin M. In vivo qualitative assessments of articular cartilage in the rabbit knee with high-resolution MRI at 3 T. Magn Reson Med 50:541–549, 2003. Link TM, Steinbach LS, Ghosh S et al. Osteoarthritis: MR imaging findings in different stages of disease and correlation with clinical findings. Radiology 226:373–381, 2003.

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Peterfy CG, Guermazi A, Zaim S et al. Whole-Organ Magnetic Resonance Imaging Score (WORMS) of the knee in osteoarthritis. Osteoarthritis Cartilage 12:177–190, 2004. Raynauld JP, Martel-Pelletier J, Berthiaume MJ et al. Quantitative magnetic resonance imaging evaluation of knee osteoarthritis progression over two years and correlation with clinical symptoms and radiologic changes. Arthritis Rheum 50:476–487, 2004. Sittek H, Eckstein F, Gavazzeni A et al. Assessment of normal patellar cartilage volume and thickness using MRI: an analysis of currently available pulse sequences. Skeletal Radiol 25:55–62, 1996. Sowers MF, Hayes C, Jamadar D et al. Magnetic resonance-detected subchondral bone marrow and cartilage defect characteristics associated with pain and X-ray–defined knee osteoarthritis. Osteoarthritis Cartilage 11:387–393, 2003. Tessier J, Bowyer J, Heapy C et al. Doxycycline slows MRI-assessed cartilage volume loss in the guinea pig model of osteoarthritis. Proc 14th Int Soc Magn Reson Med, Seattle, WA, 2006. Tessier JJ, Bowyer J, Brownrigg NJ et al. Characterisation of the guinea pig model of osteoarthritis by in vivo three-dimensional magnetic resonance imaging. Osteoarthritis Cartilage 11:845–853, 2003. Wang YX. In vivo magnetic resonance imaging of animal models of knee osteoarthritis. Lab Anim (in press), 2008. Wang YX, Heapy C, Pickford R et al. MRI structural changes and joint discomfort: an investigation in the mono-iodoacetate induced arthritis model in rats. Proc 22nd Eur Soc Magn Reson Med Biol, Basel, Switzerland, 2005. Wang YX, Westwood FR, Moores SM et al. In vivo high-resolution threedimensional magnetic resonance imaging of a rat knee osteoarthritis model induced by meniscal transection. Proc 14th Int Soc Magn Reson Med, Seattle, WA, 2006. Wluka AE, Stuckey S, Snaddon J, Cicuttini FM. The determinants of change in tibial cartilage volume in osteoarthritic knees. Arthritis Rheum 46:2065–2072, 2002. Wluka AE, Wolfe R, Stuckey S, Cicuttini FM. How does tibial cartilage volume relate to symptoms in subjects with knee osteoarthritis? Ann Rheum Dis 63:264–268, 2004.

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Yoshioka H, Stevens K, Hargreaves BA et al. Magnetic resonance imaging of articular cartilage of the knee: comparison between fat-suppressed threedimensional SPGR imaging, fat-suppressed FSE imaging and fatsuppressed three-dimensional DEFT imaging and correlation with arthroscopy. J Magn Reson Imaging 20:857–864, 2004.

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Chapter 44

Cell Traction Force Microscopy for Musculoskeletal Research James Hui-Cong Wang, Bin Li and Jeen-Shang Lin

Cell traction force (CTF) is essential for controlling cell shape, enabling cell motility, and maintaining cellular homeostasis. As such, CTF plays a critical role in wound healing and angiogenesis of musculoskeletal tissues. Cell traction force microscopy (CTFM) — a modern technology to determine CTF — is briefly presented in this chapter, followed by a detailed description of the materials and methods necessary for its implementation. Finally, examples are given to illustrate many potential applications of CTFM technology in musculoskeletal investigation. Keywords:

Cell traction force (CTF); cell traction force microscopy (CTFM); tendon fibroblasts; myofibroblasts.

1. Introduction Cells develop intracellular tension, apply tensile forces on other cells, and exert traction forces on the extracellular matrix (ECM) (Burridge and Chrzanowska-Wodnicka 1996; Harris et al. 1981). In particular, cell traction force (CTF) is essential in controlling cell shape, permitting cell movement (Beningo et al. 2001; Ingber 2003; Pourati et al.

Corresponding author: James Hui-Cong Wang. Tel: +1-412-6489102; fax: +1-412-6488548; E-mail: [email protected]

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1998), and maintaining cellular tensional homeostasis (Brown et al. 1998; Eckes and Krieg 2004). CTFs are generated by the interaction of actin filaments with motor protein myosin II as well as by actin polymerization (Wang and Lin 2007). These forces are transmitted to the ECM via focal adhesions (FAs), which form a physical link between the actin cytoskeleton and the ECM (Burton et al. 1999). FAs consist of diverse structural and signaling proteins, including vinculin, integrin family, kinases, and phosphatases. Therefore, biological, biochemical, or biomechanical stimuli acting on individual cells through the ECM will likely cause changes in the assembly and activity of FA proteins, the actin cytoskeleton, and the interaction between actin filaments and myosin II. Thus, CTF may serve as a useful biophysical marker to characterize cell phenotypic changes. Examples of applications of this cellular biophysical marker may include those in response to the alteration in mechanical loading conditions on musculoskeletal tissues, musculoskeletal tissue injuries, and wound healing. As such, knowledge of CTFs will aid in a better understanding of musculoskeletal tissue physiology and pathology. To determine CTFs, various techniques have been devised. These include thin silicone membranes (Harris et al. 1980; Oliver et al. 1995), microfabricated cantilevers (Galbraith and Sheetz 1997), micropost force sensor arrays (du Roure et al. 2005; Li et al. 2007; Tan et al. 2003), and cell traction force microscopy (CTFM) (Butler et al. 2002; Dembo and Wang 1999; Yang et al. 2006). Among these methods, CTFM is considered to be the most efficient and reliable for determining traction forces of individual cells or a group of cells (Wang and Lin 2007). This chapter will give a brief review of CTFM and of the materials and methods needed for its implementation. Examples of CTFM applications are also given to demonstrate the significant potential of this technology in musculoskeletal investigation.

2. Theoretical Background of CTFM Current CTFM methods involve four steps: (1) fabricating fluorescent microbead-embedded elastic polyacrylamide gel (PG) as the cell

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General scheme of CTFM and its implementation.

growth substrate; (2) obtaining a pair of substrate microscopy images first when cells are attached and then after the cells are removed, denoted as force-loaded and null-force images, respectively; (3) determining the PG substrate displacement field from the image pair; and (4) computing CTFs based upon the extracted substrate displacements. We begin by discussing the last two steps first, as steps (1) and (2) will be described in detail in Sec. 4. The general scheme of CTFM and its implementation are illustrated in Fig. 1.

2.1. Determining the substrate displacement field Determining the substrate displacement field requires the matching of a pixel from the null-force image to one in the force-loaded image. This is known as the image registration problem. The fluorescent microbeads embedded in the substrate serve as markers for tracking the movement of the substrate under CTFs. Because of mixing during the preparation of the substrate, microbeads distribute randomly within the PG substrate. This introduces intensity variation throughout an image that, in turn, gives spatially distinguishable

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characteristics. PG substrate, as described below, can be readily made in laboratories. Image registration is carried out under the consideration that image intensity change is a result of substrate surface movement caused by CTFs. This consideration is called optical flow (Sonka et al. 1993). Matching pixels between these two images is equivalent to finding a proper mapping function for the optical flow at each pixel of the null-force image. Each pixel on an image has a gray value often characterized by an eight-bit integer; the brighter the pixel, the higher its gray value. Two approaches are currently used in CTF image registration. Dembo and Wang (1999) and Butler et al. (2002) based their methods on finding matching areas with similar contrast between a pair of images; similarity is defined by cross-correlation. Even though their approaches differ in details, the basic frameworks of these two methods are quite similar. Both Dembo and Wang (1999) as well as Butler et al. (2002) employed the technique of a grid drawn on the null-force image. At each grid node, a fixed-size window centered at the node defines a region. The cross-correlations of this region with regions of the same size within a given range in the force-loaded image are computed using fast Fourier transform. A match is defined as the region in the force-loaded image that gives the highest correlation. However, this type of image registration can result in two issues. First, the matching does not reflect the fact that displacement is generally not uniform, and there currently is no provision for rotational movement. Second, there is no assurance that correct correspondence is always attained. As an alternative, Yang et al. (2006) adopted a feature-based registration method. The registration first extracts microbeads as clusters of high-intensity spots. This is followed by matching the features between the null-force and force-loaded pair of images based upon the perceived levels of strains. The procedure offers the advantage that the results are verifiable through manual visual inspection. Two complicating issues, however, must also be considered. First, microbeads may be mismatched, and it is likely that the rate of successful matching is uneven across the substrate surface,

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which makes error quantification difficult. Second, when there is only sparse matching between microbeads in locations of interest, the displacement field will have gaps and hence becomes ill-defined. Both the Dembo and Wang (1999)/Butler et al. (2002) and the Yang et al. (2006) methods of registration are in need of further improvement to give higher resolution results, and this is the current focus of research in CTFM. Once the matching between images is completed, the displacement field can be readily obtained through coordinate subtraction between identified pairs.

2.2. Determining cell traction forces After the substrate displacement field is established, CTFs can be determined. This, however, requires a mechanical model. The PG substrate has been shown to exhibit a linear elastic behavior. Two mechanical models have been used. Both Dembo and Wang (1999) and Butler et al. (2002) assume that the substrate is a semi-infinite half-space, i.e. the substrate extends infinitely in both horizontal and vertical directions. The half-space assumption allows the use of the Boussinesq equation, a simple analytical force–displacement relationship. The application of the Boussinesq equation means that only the substrate surface enters analysis, and thus the degrees of freedom of a problem are substantially reduced. On the other hand, Yang et al. (2006) modeled the true dimensions of the PG substrate and applied a three-dimensional (3D) finite element method (FEM) for the determination of CTFs. Computationally, the determination of CTF from the displacement field constitutes an inverse problem. This inverse problem is often ill-posed, that is, a slight perturbation in the displacement data can cause a large fluctuation in the estimated CTFs. Such oversensitivity is undesirable as, for an elastic system, the force fluctuation should be proportional to the noise perturbation. To address this potential problem, Dembo and Wang (1999) applied a regularization algorithm in which they dictated CTFs to be smooth; this constraint dampens out the high-fluctuation part of noise.

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Butler et al. (2002) estimated forces on a grid and, in the process, also removed the effects of high-frequency components in the noise to stabilize the solution. Yang et al. (2006) used brick elements formed with a regular lattice, and thus achieved similarly stable results. The 3D FEM analysis is computationally efficient, even though a much larger number of variables is involved. The PG substrate used in CTFM is generally 70–100 µm thick, and can be modeled with five to seven layers of elements. By using a regular lattice, not much overhead is introduced for generating FE meshes. A typical force computation including meshing using five layers of elements along the depth with a mesh size of 4 µm × 4 µm on each layer surface, or an equivalent of ~9000 nodes, takes less than 1 minute with a 1.2 GHz Pentium PC using the commercial software ANSYS (Yang et al. 2006).

3. Materials • • • • • • • • • • • • • •

NaOH (Sigma, St. Louis, MO, USA), 0.1 N Petri dish with uncoated glass bottom (MatTek, Ashland, MA, USA), Φ 35 mm 3-aminopropyltrimethoxysilane (Sigma, St. Louis, MO, USA) Phospate buffered saline (PBS) buffer (Sigma, St. Louis, MO, USA) Glutaraldehyde solution (Sigma, St. Louis, MO, USA), 0.5% Acrylamide (Sigma, St. Louis, MO, USA), 5% N,N ′-methylenebisacrylamide (bisacrylamide; Sigma, St. Louis, MO, USA), 0.1% Fluorescent beads (Molecular Probes, Eugene, OR, USA), Φ 0.2 µm Cover glass (Fisher Scientific, Pittsburgh, PA, USA), Φ 12 mm N-sulfosuccinimidyl-6-[4′-azido-2′-nitrophenylamino] hexanoate (sulfo-SANPAH; Pierce, Rockland, IL, USA), 5 mM HEPES buffer (Sigma, St. Louis, MO, USA), 200 mM Collagen type I (Angiotech Biomaterials, Palo Alto, CA, USA), 100 µg/mL Steel ball (Microball Co., Peterborough, NH, USA), Φ 0.64 mm, 7.2 g/cm3 Trypsin-EDTA (Invitrogen, Carlsbad, CA, USA), 0.5% (10×)

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4. Methods 4.1. Activating the glass surface Five drops of 0.1 M NaOH are added to a glass in the center of a 35-mm dish and air-dried. Next, 3-aminopropyltrimethoxysilane is smeared onto the dried cover glass. After 5 minutes, the dish is washed and soaked with distilled water. The dish is then immersed in 0.5% glutaraldehyde in PBS for 30 minutes, followed by extensive washing with distilled water for 30 minutes and then air-drying.

4.2. Fabricating the PG substrate An acrylamide/bisacrylamide mixture (110 µL), containing 5% acrylamide and 0.1% bisacrylamide, is added to the pretreated glass of the dish after being mixed with 0.2-µm-diameter fluorescent beads (1/125 volume of acrylamide mixture). The mixture is covered with a small, circular piece of cover glass (Φ 12 mm) and turned upside down to facilitate the movement of microbeads to the surface of the cover glass by gravity. After 45 minutes, the mixture is cured and forms a small gel disk with a Young’s modulus of ~3 kPa. The circular cover glass is then carefully removed, followed by extensive washing with distilled water.

4.3. Conjugating type I collagen to the PG surface To activate the free surface of the PG substrate to which matrix protein can be conjugated, 100 µL of 5 mM sulfo-SANPAH in 200 mM HEPES is pipetted on the gel surface. After exposing the dish to ultraviolet light for 5 minutes, the sulfo-SANPAH solution is removed and the process is repeated once more, followed by washing the dish with PBS twice. The PG is then incubated with 130 µL of collagen type I (100 µg/mL) and stored overnight at 4°C.

4.4. Measuring the PG thickness First, the microscope is focused on the bottom of the gel as determined by the embedded fluorescent microbeads, and the z-position

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of the microscope is recorded. Second, the microscope is refocused on the top of the gel, and the z-position is recorded again. The difference between these two z-positions is the thickness of the PG substrate. Alternatively, gel thickness can be estimated by the following formula: total volume of gel/gel area.

4.5. Determining the Poisson ratio and Young’s modulus of the PG substrate To determine the Poisson ratio of the PG substrate, a published method (Li et al. 1993) is used. Briefly, a steel ball (Φ 0.64 mm) is placed on the surface of the gel embedded with the fluorescent microbeads. The indentation caused by the weight of the steel ball is measured with a microscope focusing mechanism by following the vertical position of the fluorescent microbeads under the center of the ball as described above. Young’s modulus is determined according to the method previously described (Lo et al. 2000). In brief, Young’s modulus is calculated as Y = 3(1 − ν 2)f/4d3/2r1/2, where f is the force exerted on the gel; d is the indentation; r is the radius of the steel ball; and ν is the Poisson ratio, which can be assumed to be 0.3.

4.6. Culturing cells on the PG substrate After trypsinization, cells are dispersed in medium and a 130-µL cell suspension is added to the collagen-coated PG substrate in a dish. The cell density is chosen such that there are ~3000 cells on each gel. The cells are allowed to spread on the gel for 6 hours before cell images are taken. The exact spreading time, however, may need to be adjusted for different types of cells.

4.7. Image acquisition First, the phase contrast image of individual cells and the fluorescence image of the embedded fluorescent microbeads at the same region are recorded through a CCD camera attached to a fluorescence microscope. This fluorescence image is termed a force-loaded image. Next,

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the medium in dish is extracted and 2 mL of trypsin (0.5%) is added to detach cells from the PG substrate. Finally, after cells are detached, the fluorescence image of fluorescent microbeads in the same view and z-plane is taken. This image is termed a null-force image.

4.8. CTF computation CTFs are determined by computation on a desktop computer using ANSYS software, based on the method previously described (Yang et al. 2006).

5. Remarks Although the fabrication process for PG substrates is straightforward, care must be taken to obtain consistent stiffness and uniform coating of the collagen layer on the gel surfaces. Failure to make uniform PG substrates and achieve even coating of collagen type I on the PG surface will affect cell morphology and alter cell behavior, resulting in large variations and inconsistent CTF results from one experiment to another. In addition, isolated individual cells with a similar shape and spreading area should be sampled to reduce variations in CTF values, and thus increase statistical power for detecting differences in CTF between treatment and control groups.

6. Examples of CTFM Application 6.1. Determining traction forces of individual human tendon fibroblasts To illustrate that CTFM is an effective technology which can determine traction forces of various types of cells, including those from musculoskeletal tissues, we applied CTFM to determine the traction forces of human patellar tendon fibroblasts (HPTFs). Briefly, a PG substrate was made and conjugated with collagen type I according to the method described here. The HPTFs were plated to the PG substrate and allowed to spread for 6 hours. Cells with an elongated shape on each

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gel were chosen for imaging. The force-loaded and null-force images were used to determine the substrate displacement field and CTFs, based on the published method (Yang et al. 2006). The cell image, displacement field, and cell traction stress distribution are given in Fig. 2. (a)

(b)

(c)

Fig. 2. A CTFM application to determine CTFs. (a) Human patellar tendon fibroblast grown on PG substrate embedded with fluorescent microbeads (not shown on the phase contrast image). (b) Substrate displacement field. (c) CTF field. The displacements and CTFs were determined by a matching algorithm and 3D FEM analysis, respectively. Adopted with permission from Fig. 6 in Yang et al. (2006).

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The maximum displacement obtained was ~1.2 µm, and the maximum traction stress was ~250 Pa. The CTFs were concentrated in the front and rear of the cell, which is consistent with the general understanding of fibroblast traction mechanisms.

6.2. Detecting differentiated cells by CTFM To show that CTFM can be used to detect differentiated cells, we used rabbit corneal stroma cells as an example because there are wellestablished culture protocols to induce these cells to differentiate into fibroblasts or myofibroblasts. According to the published protocols (Anderson et al. 2004), rabbit corneal stroma cells were obtained and differentiated into fibroblasts in DMEM (GIBCO-BRL, Grand Inland, NY, USA) solution containing 10% fetal bovine serum (FBS), 20 ng/mL of bFGF, 5 µg/mL of heparin, 100 U/mL of penicillin, and 100 µg/mL of streptomycin. These differentiated fibroblasts expressed minimum α-smooth muscle actin (α-SMA) [Fig. 3(a)], which is a specific marker of myofibroblasts (Hinz et al. 2001; Chen et al. 2007).

Fig. 3. Determining CTFs of fibroblasts and differentiated myofibroblasts by CTFM. (a) Fibroblasts expressed minimum α-SMA protein. When they were differentiated into myofibroblasts by TGF-β1 (2 ng/mL) treatment, they expressed a high level of α-SMA. (b) There was a significant difference in CTFs generated by fibroblasts and myofibroblasts. Each bar represents the mean ± SD of >30 cells from three independent experiments. Adopted with permission from Fig. 2 in Chen et al. (2007).

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Fig. 4. Detecting myofibroblast differentiation by CTFM. After fibroblasts were differentiated into myofibroblasts, two populations of cells were clearly separated by their large differences in CTF distributions.

To obtain myofibroblasts, the fibroblasts were treated with 2 ng/mL of TGF-β1 (R&D Systems, Minneapolis, MN, USA) for 24 hours. These cells expressed a high level of α-SMA [Fig. 3(a)]. Then, CTFM was used to determine the CTFs of individual fibroblasts and myofibroblasts. It was found that the fibroblasts generated a mean CTF of ~100 Pa [Fig. 3(b)]. In contrast, once these fibroblasts were differentiated into myofibroblasts, their mean CTF was three times that of the fibroblasts. On the CTF histogram in Fig. 4, the two cell populations — myofibroblasts and fibroblasts — are clearly distinguishable. The myofibroblast population shifted toward the right, meaning that the overall CTF of the myofibroblast population was larger than that of the fibroblast population. Thus, CTFM may be used to detect cell differentiation such as myofibroblast differentiation in this example.

6.3. Determining traction forces of a group of cells To demonstrate that CTFM can also be used to determine the traction forces of a group of cells, we first applied microcontact printing technology (Whitesides et al. 2001) to make fibronectin-coated, square microislands on PG substrates. Then, NIH 3T3 cells were plated on the

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Fig. 5. The application of CTFM to a group of micropatterned cells. (a) Phase contrast image of a group of cells on a square microisland. (b) The corresponding CTF distribution of the cell island.

microisland and a monolayer of cells (or a square cell island) was formed on it after 1 day in culture [Fig. 5(a)]. Similar to the application of CTFM to single cells, CTFM was applied to determine the CTF distribution of the square cell island. Large CTFs were found to locate at the corners and around the edges of the cell island [Fig. 5(b)]. The results suggest that the cells at the boundary generated much larger traction forces than those in the middle region of the cell island.

Acknowledgments We thank Mr Jianxin Chen for his assistance in the application of CTFM to determine traction forces of fibroblasts and myofibroblasts. We also thank Michael Lin for his assistance in preparing this review. Finally, we gratefully acknowledge the funding support of NIH grants AR049921 and AR113046 and the Arthritis Investigator Award (J. H. C. Wang).

References Anderson S, DiCesare L, Tan I et al. Rho-mediated assembly of stress fibers is differentially regulated in corneal fibroblasts and myofibroblasts. Exp Cell Res 298:574–583, 2004.

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Beningo KA, Dembo M, Kaverina I et al. Nascent focal adhesions are responsible for the generation of strong propulsive forces in migrating fibroblasts. J Cell Biol 153:881–888, 2001. Brown RA, Prajapati R, McGrouther DA et al. Tensional homeostasis in dermal fibroblasts: mechanical responses to mechanical loading in three-dimensional substrates. J Cell Physiol 175:323–332, 1998. Burridge K, Chrzanowska-Wodnicka M. Focal adhesions, contractility, and signaling. Annu Rev Cell Dev Biol 12:463–518, 1996. Burton K, Park JH, Taylor DL. Keratocytes generate traction forces in two phases. Mol Biol Cell 10:3745–3769, 1999. Butler JP, Tolic-Norrelykke IM, Fabry B, Fredberg JJ. Traction fields, moments, and strain energy that cells exert on their surroundings. Am J Physiol Cell Physiol 282:C595–C605, 2002. Chen J, Li H, Sundarraj N, Wang JH. Alpha-smooth muscle actin expression enhances cell traction force. Cell Motil Cytoskeleton 64:248–257, 2007. Dembo M, Wang YL. Stresses at the cell-to-substrate interface during locomotion of fibroblasts. Biophys J 76:2307–2316, 1999. du Roure O, Saez A, Buguin A et al. Force mapping in epithelial cell migration. Proc Natl Acad Sci USA 102:2390–2395, 2005. Eckes B, Krieg T. Regulation of connective tissue homeostasis in the skin by mechanical forces. Clin Exp Rheumatol 22:S73–S76, 2004. Galbraith CG, Sheetz MP. A micromachined device provides a new bend on fibroblast traction forces. Proc Natl Acad Sci USA 94:9114–9118, 1997. Harris AK, Stopak D, Wild P. Fibroblast traction as a mechanism for collagen morphogenesis. Nature 290:249–251, 1981. Harris AK, Wild P, Stopak D. Silicone rubber substrata: a new wrinkle in the study of cell locomotion. Science 208:177–179, 1980. Hinz B, Celetta G, Tomasek JJ et al. Alpha-smooth muscle actin expression upregulates fibroblast contractile activity. Mol Biol Cell 12:2730–2741, 2001. Ingber DE. Mechanosensation through integrins: cells act locally but think globally. Proc Natl Acad Sci USA 100:1472–1474, 2003. Li B, Xie L, Starr ZC et al. Development of micropost force sensor array with culture experiments for determination of cell traction forces. Cell Motil Cytoskeleton 64:509–518, 2007. Li Y, Hu Z, Li C. New method for measuring Poisson’s ratio in polymer gels. J Appl Polym Sci 50:1107–1111, 1993.

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Lo CM, Wang HB, Dembo M, Wang YL. Cell movement is guided by the rigidity of the substrate. Biophys J 79:144–152, 2000. Oliver T, Dembo M, Jacobson K. Traction forces in locomoting cells. Cell Motil Cytoskeleton 31:225–240, 1995. Pourati J, Maniotis A, Spiegel D et al. Is cytoskeletal tension a major determinant of cell deformability in adherent endothelial cells? Am J Physiol 274:C1283–C1289, 1998. Sonka M, Hlavac V, Boyle R. Image Processing, Analysis and Machine Vision, 1st ed. Chapman & Hall, London, 1993. Tan JL, Tien J, Pirone DM et al. Cells lying on a bed of microneedles: an approach to isolate mechanical force. Proc Natl Acad Sci USA 100:1484–1489, 2003. Wang JH, Lin JS. Cell traction force and measurement methods. Biomech Model Mechanobiol 6:361–371, 2007. Whitesides GM, Ostuni E, Takayama S et al. Soft lithography in biology and biochemistry. Annu Rev Biomed Eng 3:335–373, 2001. Yang Z, Lin JS, Chen J, Wang JH. Determining substrate displacement and cell traction fields — a new approach. J Theor Biol 242:607–616, 2006.

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Chapter 45

Nanoindentation: Techniques and Technical Considerations for Musculoskeletal Research Suzanne Ferreri, Stefan Judex and Yi-Xian Qin

Nanoindentation is a relatively new technique that can achieve high-resolution characterization of material properties in musculoskeletal tissues. It has been shown to be an effective tool in describing changes in bone mineralization that result from disease and aging by quantifying the material’s direct response to mechanical loading. Several studies have shown that the results of nanoindentation are highly sensitive to parameters such as sample preparation, loading protocol, indent location, and sample storage. This chapter describes the techniques for a successful nanoindentation protocol and addresses key technical considerations. Keywords:

Nanoindentation; material property; mineralization; loading; mapping; high resolution.

1. Introduction Nanoindentation is an emerging technology that has recently found applications in musculoskeletal research. The principles behind nanoindentation evolved from traditional microindentation tests Corresponding author: Yi-Xian Qin. Tel: +1-631-6321481; fax: +1-631-6328577; E-mail: [email protected]

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used to measure hardness, such as the Vickers and Knoop tests. Subsequently, the development of this technique was driven by the demands of the microelectronics industry to investigate the performance of thin films and microelectromechanical systems (MEMS) devices. Although still a relatively new technique to the field, it possesses several characteristics that make it particularly attractive for answering research questions related to musculoskeletal biomechanics. First, nanoindentation uses a sharp indenter tip and highly sophisticated load-sensing technology to probe mechanical properties at extremely small length scales. Depending on the type of material and indenter tip, mapping of material properties can be achieved at a resolution on the order of 10 nm. This is often useful in studies addressing the spatial heterogeneity found within native and diseased tissues such as bone and cartilage (Hengsberger et al. 2002; Hoffler et al. 2000). A second advantage of nanoindentation’s high spatial resolution is that it allows for the characterization of material properties within various organizational structures such as lamellar and interstitial bone (Rho et al. 1999a; Rho et al. 1999b; Rho et al. 2001a) as well as superficial and deep zones in cartilage (Ferguson et al. 2003). This can be useful when addressing the effects of disease or treatment, as it provides a means of keying in on the mechanical response of tissues undergoing specific stages of the modeling or remodeling cycle. A third advantage of nanoindentation’s high spatial resolution is that it can be used in concert with complementary imaging modalities. For example, several researchers have combined nanoindentation studies with high-resolution atomic force microscopy (AFM) imaging to characterize the local topology of a specimen’s surface before and after indentation (Hengsberger et al. 2001; Ho et al. 2004; Pelled et al. 2007; Xu et al. 2003; Zhang et al. 2002); this allows for a greater understanding of the local geometric effects present throughout the indentation process. Other researchers have used infrared microscopy to complement the findings of nanoindentation studies in order to better understand the underlying chemical composition associated with a sample’s response to mechanical loading (Busa et al. 2005; Hofmann et al. 2006).

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Finally, the information obtained from nanoindentation studies can be used as inputs for powerful modeling and simulation tools, which address bone’s macro-scale response to loading. One such example, known as “virtual biopsy”, uses finite element (FE) analysis to approximate the response of cancellous bone to applied loads, and has been established as an effective tool in predicting fatigue (Muller et al. 1994; Van Rietbergen et al. 1999). Nanoindentation, while a powerful technique, has only recently been used as a tool in the field of musculoskeletal research. As a result, standards have yet to be defined for the instrumented indentation testing of biological materials such as bone and cartilage. In light of this fact, nanoindentation studies are highly sensitive to several key parameters such as sample preparation, loading protocol, indent location, and sample storage (Mittra et al. 2006). Therefore, future studies should carefully consider these factors in the design of experiments and the reporting of results. The focus of this chapter is to outline the key materials and methods required for a successful nanoindentation protocol, and to highlight technical considerations relevant to the design of experiments and interpretation of results.

2. Materials 2.1. Sample extraction •

Cutting instrument capable of clamping and accurately positioning the sample relative to the cutting plane





The tool should be capable of cutting under constant water irrigation to lubricate and prevent overheating of the sample surface. Examples include a variable-speed diamond wheel saw (e.g. Model 650, South Bay Technology, Inc., San Clemente, CA, USA; or Diamond Wafering Blade, Buehler Ltd, Lake Bluff, IL, USA) or a diamond band saw (Exakt Instruments, Oklahoma City, OK, USA).

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Pulsatile water irrigation system to remove marrow, particularly in samples containing trabecular bone
Examples include Waterpik (Water Pik Technologies, Inc., Fort Collins, CO, USA).

2.2. Fixation •

A series of increasing concentrations of ethanol (70%, 80%, 90%, and 100% EtOH) — for fixing and dehydrating the bone sample

2.3. Embedding Some examples of embedding materials used in nanoindentation studies of bone include epoxy resin (e.g. EpoThin; Buehler, Lake Bluff, IL, USA) (Mittra et al. 2006; Rho et al. 1997; Xu et al. 2003), photoelastic epoxy (Photoelastic Division, Measurements Group, Raleigh, NC, USA) (Hoffler et al. 2000; Mittra et al. 2006), PMMA (Hengsberger et al. 2001; Hengsberger et al. 2002; Turner et al. 1999), Spurr’s resin (Donnelly et al. 2006a), and MAS Epoxy (Mittra et al. 2006). •

•

Disposable embedding molds (e.g. Peel-Away Mold or Flat Bottom Embedding Capsule, both from Electron Microscopy Sciences, Inc., Hatfield, PA, USA) Vacuum chamber and pump (Fisher Standard Maxima C Plus Vacuum Pumps, Fisher Scientific)

2.4. Polishing materials • • • •

Abrasive silicon carbide papers with particle sizes of 220 µm, 330 µm, 600 µm, and 1000 µm (Buehler, Lake Bluff, IL, USA) Diamond suspension with particle sizes of 3 µm, 1 µm, 0.25 µm, and 0.05 µm (Buehler, Lake Bluff, IL, USA) Manual or automatic polishing wheel (PowerPro; Buehler, Lake Bluff, IL, USA) Ultrasonic cleaner (Model 2510; Branson, Inc., Danbury, CT, USA)

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2.5. Indentation hardware •

Commercially available nanoindentation systems — for measuring the loads and displacements required by nanoindentation
Currently available systems include the Nano Indenter® XP (MTS Nano Instruments, Oak Ridge, TN, USA), the NanoTest (Micro Materials Ltd, Wrexham, UK), the Ultra-MicroIndentation System (UMIS) (Fischer-Cripps Laboratories, Sydney, Australia), the Ultra Nano Hardness Tester (CSM Instruments, Needham, MA, USA), and the TriboScope (Hysitron, Inc., Minneapolis, MN, USA). Hysitron’s TriboScope can also be mounted onto an existing AFM machine to more precisely determine indent locations. Dedicated machine optics are also used to precisely place indents within a sample.

•

An appropriate indenter tip





•

Examples include Berkovich, cube corner, and conospherical tips. Indenter tips are usually purchased from the same company as the commercial nanoindenter to ensure compliance with the transducer.

Calibration grade samples of fused quartz and single-crystal aluminum — for calibration of the tip area function and machine optics



These samples should be very smooth and relatively flat. They may also be purchased from the same company as the commercial nanoindenter.

3. Methods 3.1. Sample preparation 3.1.1. Technique Samples should be cut in the plane of interest, cleaned of soft tissue using the water jet, and dehydrated in a series of ethanol solutions (70%, 80%, 90%, and 100% EtOH), allowing the sample to remain in each solution for a minimum of 48 hours. Samples should then be

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embedded in a low-viscosity epoxy resin such as EpoThin (Buehler, Lake Bluff, IL, USA), and be allowed to cure under negative pressure in a vacuum chamber for at least 24 hours. Once cured, the samples should be polished on an automatic polishing wheel using first abrasive papers (particle sizes of 220, 330, 600, and 1000), followed by a polishing cloth with diamond suspensions (3 µm, 1 µm, 0.25 µm, and 0.05 µm). Polishing at each step should be performed for at least 5 minutes under moderate pressure to ensure a uniformly smooth surface. Upon completion of each step, the mean surface roughness of the sample should be equivalent to that of the grinding surface. Care should be taken to maintain parallel ends of the embedded sample. Finally, since the polishing process generates a large amount of loose particulate, the sample should be ultrasonically cleaned prior to storage and testing. Once the samples are clean, contact with the polished surface should be minimized to prevent accumulation of oils and dust. Immediately before testing, compressed air may be used to clear the surface of debris due to storage and handling. While this protocol for sample preparation works well for most bones, variations may be necessary depending on the particular study. The following subsection discusses technical considerations pertaining to sample preparation that should be acknowledged when developing a protocol or interpreting results. 3.1.2. Technical considerations for sample preparation (1) Dehydration vs. rehydration It is often necessary to dehydrate bone prior to embedding, particularly in cases where the bone is highly porous, as is the case in trabecular bone. Macroscopic testing of bone has shown that material properties are affected by bone’s hydration level (Townsend et al. 1975). Similarly, nanoindentation studies have shown that the elastic modulus and hardness vary depending on the level of hydration: elastic modulus increased by as much as 50%, while hardness increased by as much as 100% (Bushby et al. 2004; Hengsberger et al. 2001; Hengsberger et al. 2002; Ho et al. 2004; Hoffler et al. 2005; Rho et al. 1999a). Furthermore, dehydration was shown to have a slightly greater effect on osteons compared with interstitial bone (Rho and Pharr 1999).

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It is thought that water plays a role in the stability of collagen fibrils (Melacini et al. 2000), and that excessive drying can lead to tissue shrinkage and cracking. However, for some types of bone, sample dehydration greatly improves the quality of the sample surface after polishing. Thus, it has been suggested that samples may undergo a period of rehydration in physiologic solution after embedding/ polishing to restore bone’s native mechanical characteristics (Akhtar et al. 2006; Hoffler et al. 2005). (2) Embedding material Embedding materials are often used to provide support for porous tissues such as trabecular bone and to improve the effectiveness of the polishing process (Rho et al. 1997). An embedding material should have material properties similar to those of the sample to be tested, and should demonstrate good adhesion to the bone surface. Depending on the particular application, some important features include (i) high or low viscosity to control infiltration into the submicron porosities, (ii) slow or rapid cure times to affect the sample storage, and (iii) opacity/ color to affect the ability of certain features to be seen under the system’s optics. However, the effect of embedding material on measured parameters is not fully understood. One study in trabecular bone found that increasing the modulus of the embedding material resulted in a 36% increase in bone tissue hardness, but not modulus (Mittra et al. 2006); this may be due to the large porosities found in trabecular bone and the potential for indentation depth to approach trabecular thickness. It is also unclear whether or not embedding samples of cortical bone affects measured material properties. One study found that embedding in resin decreased microhardness by 4% (Evans et al. 1990), while another determined that there were no differences in modulus or hardness for samples embedded in PMMA and those that were not (Hoffler et al. 2005). (3) Surface preparation Surface preparation plays an important role in the reproducibility of data obtained using nanoindentation. Because nanoindentation relies heavily on analytic solutions during data analysis, a sample’s surface characteristics should be close to those considered in the analytic

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solution. In particular, the sample surface should be flat and continuous such that it makes full contact with the penetrating tip, and the surface should be perpendicular to the loading axis. Several studies have highlighted the effects of surface roughness on the measurement of bone mechanical properties. For samples with a surface roughness of >3 µm, shallow indents (<250 nm) produced highly variable data (Donnelly et al. 2006a; Hoc et al. 2006), suggesting nanoindentation’s limitations as the mean surface roughness approaches the maximum indentation depth. Thus, the mean surface roughness should be substantially less than that of the maximum indentation depth, and a larger number of indents must be made for situations requiring indentation depths <250 nm. It was also shown that polishing may result in uneven surfaces, as thin lamellae are more susceptible to wear compared with thick lamellae (Xu et al. 2003). Interestingly, differences between thin and thick lamellae were identified for surfaces prepared using a microtome, but not for those that had been polished (Xu et al. 2003). (4) Selection of indent location Bone’s inherent heterogeneity at the length scales tested using nanoindentation requires careful selection of the indent location. Several studies have noted the distinct difference in material properties between osteonal and interstitial bone (Hoffler et al. 2000; Hoffler et al. 2005; Rho et al. 1999b), where elastic modulus can vary by as much as 10% (Hoffler et al. 2005). Differences have also been observed in material properties between young and old osteons (Huja et al. 2006). Therefore, all indents should be placed within one structure to minimize variability in the data and to improve the statistical power of the experiment. For cases where it is not possible to constrain indents to either osteonal or interstitial bone, a larger number of indents should be made, particularly for cases of high heterogeneity such as Haversian bone. It has also been shown that modulus and hardness vary within substructures, such as thin and thick lamellae, due to the alternating orientation of collagen fibers (Xu et al. 2003). However, it is often difficult to delineate such structures using a nanoindenter’s optics. Subsequently, an

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increasing number of studies have combined nanoindentation with atomic force microscopy technology to image bone’s surface topography in order to better select the desired indent location (Balooch et al. 2004). 3.1.3. Selection of indenter tip An appropriate indenter tip should be selected for each study. The Berkovich tip is characterized by an included angle of 142.3°, a halfangle of 65.35°, and a radius of curvature between 100 nm and 200 nm. It is the most commonly used tip for the characterization of bone matrix because it resists wear and allows probing of the sample at greater depths; it is also less expensive than other tips. The cube corner tip has an included angle of 90° and a much smaller radius of curvature (40–100 nm), and can be used to probe at very shallow depths. The cube corner tip produces a smaller damage field and can therefore be used to achieve a higher resolution for spatial mapping of material properties. The conospherical tip has a much larger tip radius (~3 µm), and is used to indent softer materials including cartilage. Each of the aforementioned tips is also available with a longer shaft, and can be used to test submerged samples.

3.2. Calibrations 3.2.1. Calibration of the tip area function The calculation of material hardness requires an understanding of the tip’s projected cross-sectional area at the point of maximum load. Due to inherent deviations in tip geometry, particularly near the tip, the projected cross-sectional area over a range of depths is best described by fitting a mathematical function to experimental data. This should be done by making at least 100 indents in a sample of fused quartz, and then increasing the peak load with each indent uniformly over the range of the transducer. Fused quartz is used to calibrate the tip area function because of its low modulus-to-hardness ratio. Each indent should use a triangular load function with a constant

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loading/unloading rate. The computed contact area, A, is then calculated for each indent using the following equation: 2

A=

pÊ S ˆ , 4 ÁË E r ˜¯

where S is the contact stiffness determined from the initial portion of the unloading curve (typically between 95% and 20% of the maximum applied load), and Er is the reduced modulus of the fused quartz and the diamond indenter tip (Er = 69.6 GPa). The following sixth-order polynomial is then fit to a plot of computed contact area, A(hc), versus contact depth, hc, where C0 is a constant equal to 24.5 for a Berkovich tip (Oliver and Pharr 1992; Oliver and Pharr 2004): A (hc ) = C 0hc2 + C1hc + C 2hc1 2 + C3hc1 4 + C 4hc1 8 + C5hc1 16 . This approach works well for describing the contact area for a variety of indenter geometries, including Berkovich, conical, and spherical tips. The higher-order terms are useful for describing variations in tip geometry, particularly near the tip, improving the quality of the tip area function. However, for cases where the tip geometry is far from ideal, or in situations where greater accuracy is required, it may be useful to tailor the tip area function to specifically address the range of indentation depths used in the study. Finally, with increased use and subsequent wear, the tip geometry will deviate from the ideal, requiring that a new set of load displacement data be obtained and a new area function calculated. The tip-blunting rate depends on the type and frequency of indents as well as the nature of the substance being indented. 3.2.2. Calibration of machine optics It has been shown that material properties vary in bone, including within an osteon and between osteonal and interstitial bone. Thus, careful placement of indent locations is critical for minimizing unwanted

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variability and interpreting results. The location of the optics’ focal point must be calibrated relative to the tip of the indenter. This is often done by generating a recognizable pattern of indents on a sample of single-crystal aluminum. These points are then located using the system’s optics, and a coordinate map is defined to relate the position of the indenter tip to the focal point of the optical microscope. Under normal use, the position of the focal point relative to the indenter tip should remain stable; however, for cases that require high precision, optical calibration should be performed more frequently.

3.3. Quasi-static elastic indentation Quasi-static indentation refers to the process by which an indenter is driven into the surface of a sample, held for a certain period of time at a constant load or displacement, and then withdrawn. Load displacement data are recorded and used to calculate mechanical properties such as elastic modulus and hardness. Examples of two load–displacement curves can be seen in Fig. 1. As the tip is driven into the sample, the material undergoes both elastic (recoverable) and plastic (permanent) deformation; and upon withdrawal from the sample, the force exerted on the tip by the material reflects only the material’s elastic characteristics. Thus, the unloading portion of the curve is used to calculate elastic modulus and hardness during quasi-static data analysis. Bone, however, has been shown to exhibit viscoelastic characteristics at both the macro-scale and micro-scale. The maximum load is held constant for a period of time prior to unloading (at least 10 seconds) to dissipate any remaining viscoelastic effects; this helps to ensure that the unloading portion of the curve solely reflects the elastic response of the material. It is important to note that data analysis is based on a series of analytic solutions describing the indentation of an infinite elastic half-space (Oliver and Pharr 2004); this requires the assumptions that the penetrating tip makes full contact with the opposing surface, and that the material experiences no pile-up surrounding the indent. Once experimental load displacement data have been obtained, a curve is fit to the initial portion of the unloading curve, typically

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Fig. 1. Force–displacement curves for two indents made in a sample of cortical bone taken from the middiaphysis of a mouse tibia. The sample was dehydrated, embedded in epoxy resin, and polished to a mean surface roughness of 0.05 µm. Indents were made using a Berkovich tip under displacement control at a constant loading rate of 150 nm/s. The two curves represent unique indents (A and B, depicted in the SEM image of Fig. 2) with maximal loads of 2760 µN and 5970 µN, which correspond to maximum penetration depths of 317 and 468, respectively. Stiffness is calculated as the slope of the initial portion of the unloading curve, as depicted by the dashed lines.

between 95% and 20% of the maximum applied load. The curve is described using a power-law relation of the form P = a(h - h f )m , where P, h, and hf represent the measured force, depth, and maximum depth, respectively (Oliver and Pharr 1992; Oliver and Pharr 2004), and the parameters α and m are power-law fitting constants. The stiffness, S, is then calculated by taking the first derivative of the power-law relation, P, with respect to depth (∂P/∂h) and evaluating at the maximum load, Pmax. As the indenter penetrates the sample, it is assumed that the sample surface “sinks in”, resulting in a contact

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Fig. 2. Scanning electron microscopy (SEM) image of indents A and B described in Fig. 1. Indents were made using a three-sided Berkovich tip with an approximate radius of curvature of 100 nm.

depth that is less than the maximum penetration depth, hmax. The maximum contact depth, hc, is defined by the following equation: hc = hmax - 0.75

Pmax . S

Material hardness, H, is then calculated using the formula

H =

Pmax , A (hc )

where A(hc) is determined by evaluating the tip area function at the maximum contact depth (Oliver and Pharr 1992; Oliver and Pharr 2004). The reduced modulus, Er, which reflects the material properties of both the sample and the indenter, is determined using the formula Er =

S p , 2 Ac

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where Ac is the contact area evaluated at the contact depth, hc (Oliver and Pharr 1992; Oliver and Pharr 2004). The elastic modulus for the sample, Es, is then calculated using the following equation:

Es =

E i ¥ E r ¥ (1 - n s 2 ) E i + E r ¥ (n i 2 - 1)

,

where Ei and νi are the elastic modulus and Poisson’s ratio of the indenter, respectively, and νs is the Poisson’s ratio of the sample (Oliver et al. 1992; Oliver et al. 2004). 3.3.1. Technical considerations for quasi-static indentation (1) Indentation depth A variety of maximum indentation depths have been used in previous nanoindentation studies of bone, yet it remains unclear whether or not indentation depth affects measured modulus and hardness. One study found that the modulus was unaffected by indentation at depths up to 500 nm (Mittra et al. 2006); while others have shown that for depths less than 200 nm, Young’s modulus increased with increasing depth (Hoc et al. 2006). It has also been shown that measured material properties are highly variable when obtained using indents made at depths less than 250 nm (Hoffler et al. 2005; Vanleene et al. 2006); this may be because as the indentation depth approaches the mean surface roughness, it becomes difficult to measure applied force and predict the contact area. In addition, it is important to note that for cancellous bone, the maximum indentation depth may approach the thickness of the individual trabeculae, resulting in a measurement that reflects not only the material properties of bone but also those of the embedding material. (2) Loading rate Numerous studies have used a variety of loading/unloading rates, including low (50 µN/s [Donnelly et al. 2006b; Hofmann et al.

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2006] and 100 µN/s [Hoffler et al. 2000]), medium (300– 400 µN/s [Fan et al. 2002; Hengsberger et al. 2001; Rho et al. 1997; Roy et al. 1999]), and high (750 µN/s [Turner et al. 1999]). However, the effect of the loading rate on measured material properties remains unclear. One study found that material properties were dependent on the loading rate and that the modulus was proportional to the strain rate raised to the power of 0.06 (Fan et al. 2002), similar to another study at the macro-scale (Carter and Hayes 1976). Other studies also observed that elastic modulus increased with increasing loading rate (Fan and Rho 2003; Hoffler et al. 2005; Vanleene et al. 2006). However, one conflicting study found no differences in material properties obtained using different loading rates (Mittra et al. 2006). Inconsistencies among the available data may be the result of differences in species, sample preparation, and loading protocol.

3.4. Dynamic indentation For many biological tissues, quasi-static indentation is not sufficient to fully describe the material’s response to mechanical loading. For example, bone has been shown to exhibit strain rate dependency at both the macro-scale (Carter and Hayes 1976) and micro-scale (Fan and Rho 2003; Hoffler et al. 2005; Vanleene et al. 2006), suggesting that it may be better characterized using viscoelastic material properties. The complex modulus, which is defined as the sum of the storage and loss moduli, characterizes a material in terms of both its static and dynamic responses to loading. At the macro-scale, dynamic mechanical analysis (DMA) (Bushby et al. 2004) is used to determine storage and loss moduli. This principle was recently adapted for nanoindentation applications (Loubet 1996) and was used to determine the complex modulus of bone by superimposing small-amplitude (~3–5 nm) oscillations on a quasi-static indentation test at a frequency between 10 Hz and 200 Hz (Muller et al. 1994). Dynamic indentation is performed by superimposing a smallamplitude, sinusoidal load onto a constantly applied load. The displacement amplitude X0, displacement force F0, and phase response

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are then used to calculate the storage modulus E ′ and loss modulus E ′′, using the following equations:

X0 = tan(f) =

F0 (K s + K i - M w 2 )2 + [(Ci + C s )w ]2 (C i + C s )w Ks + Ki - Mw2

E ¢¢ ÈÎwC s (1 + n) p ˘˚ = E¢ ÈK s (1 + n) p ˘ Î ˚

A (hc ) A (hc )

,

where Ki and Ks represent the stiffness of the indenter shaft springs and tip-sample contact, respectively; M is the mass of the indenter; and ω is the excitation frequency of the tip. Ci and Cs are the damping coefficients of the displacement-sensing capacitor and sample, respectively; ϕ is the phase shift; and A(hc) is the contact area evaluated at contact depth hc as described previously. In the case of bone, the storage modulus reflects mineral content; while the loss modulus reflects damping characteristics, which result from collagen and water content.

4. Experimental Design Considerations Nanoindentation is a powerful tool that is highly sensitive to differences in bone’s intrinsic material properties. As such, it is necessary to consider several issues related to the design of experiments and interpretation of data.

4.1. Species It is widely accepted that material properties at the macro-scale vary among species, yet it remains unclear whether this difference is due to the tissue’s intrinsic material properties or differences in

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morphology. Nanoindentation has been used to determine the mechanical properties of bone at the nanoscale for a variety of species including mice (Akhter et al. 2004; Silva et al. 2004), fish (Rho et al. 2001b), horses (Rho et al. 2001a), cows (Tai et al. 2005), and humans (Roy et al. 1999). While it is often difficult to compare studies due to compounding effects of differences in sample preparation and testing, it does appear that material properties vary among species.

4.2. Age Changes in bone mineralization and organization have been observed in a variety of species with increasing age; however, it is unclear whether or not these changes result in alterations to the tissue’s mechanical characteristics. One study showed that while elastic modulus varied, it did not significantly depend on age (Zysset et al. 1998). Conversely, other studies have noted a significant loss in tissue stiffness with increasing age (Diab et al. 2005; Nalla et al. 2006), and this change may be due in part to a loss of collagen organization (Diab et al. 2005).

4.3. Indent location 4.3.1. Skeletal site It is widely accepted that bone is sensitive to mechanical loading and exhibits a morphology adapted to the local loading environment. Not surprisingly, bone’s material properties at the nanoscale have also been shown to depend on the skeletal site. Differences have been detected between the femoral neck and diaphysis (Hoffler et al. 2000; Zysset et al. 1999), as well as in the distal radius and lumbar vertebra (Hoffler et al. 2000). Nanoindentation has also shown sensitivity to differences in and around the growth plate (Lee et al. 1998) and between the articular cartilage and subchondral bone (Ferguson et al. 2003).

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4.3.2. Trabecular bone vs. cortical bone There is conflicting information with regards to whether or not material properties differ between trabecular and cortical bone (Turner et al. 1999; Zysset et al. 1999). It is important to note that the discrepancy observed within such studies may result from trabecular bone’s high sensitivivity to parameters such as embedding and orientation.

4.4. Anatomic orientation It has been widely established that material properties are anisotropic at the whole-tissue level. Therefore, it is not surprising that material properties measured by nanoindentation have been shown to depend on anatomic orientation. In general, previous studies have shown that small changes in sample orientation do not affect material properties measured by nanoindentation (Fan et al. 2002; Hofmann et al. 2006); however, large changes in sample orientation do significantly affect the elastic modulus and hardness (Fan et al. 2002; Hofmann et al. 2006; Rho et al. 2001a, Rho et al. 2001b; Roy et al. 1999; Swadener et al. 2001). This suggests that bone behaves anisotropically at the submicron length scale. The effects of anisotropy can also be seen in trabecular bone, where it was shown that material properties vary when measured perpendicular and transverse to the medial axis of the trabeculae (Roy et al. 1999).

4.5. Storage One may also want to consider the manner and length in which specimens are stored. It has been shown that after 6 months of storage, tissue modulus but not hardness was affected (Mittra et al. 2006). Therefore, samples should be stored frozen until they are ready to be tested; and once embedded, samples should be tested as soon as possible.

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4.6. Pathology Nanoindentation probes mechanical properties at the nanometer range, and therefore is sensitive to changes in mineralization and collagen organization. For these reasons, it has been used to investigate the effects of various pathologies on bone’s intrinsic material properties. Nanoindentation has been used to study the effects of ovariectomy (OVX) on bone in both rats (Guo and Goldstein 2000; Hengsberger et al. 2005) and monkeys (Li et al. 2005). It was also used to study conditions such as osteogenesis imperfecta (Fan et al. 2006; Weber et al. 2006) and osteopetrosis (Jamsa et al. 2002).

References Akhtar R, Eichhorn SJ, Mummery PM. Microstructure-based finite element modelling and characterisation of bovine trabecular bone. J Bionic Eng 3:3–9, 2006. Akhter MP, Fan Z, Rho JY. Bone intrinsic material properties in three inbred mouse strains. Calcif Tissue Int 75:416–420, 2004. Balooch G, Marshall GW, Marshall SJ et al. Evaluation of a new modulus mapping technique to investigate microstructural features of human teeth. J Biomech 37:1223–1232, 2004. Busa B, Miller LM, Rubin CT et al. Rapid establishment of chemical and mechanical properties during lamellar bone formation. Calcif Tissue Int 77:386–394, 2005. Bushby AJ, Ferguson VL, Boyde A. Nanoindentation of bone: comparison of specimens tested in liquid and embedded in polymethylmethacrylate. J Mater Res 19:249–259, 2004. Carter DR, Hayes WC. Bone compressive strength: the influence of density and strain rate. Science 194:1174–1176, 1976. Diab T, Sit S, Kim D et al. Age-dependent fatigue behaviour of human cortical bone. Eur J Morphol 42:53–59, 2005. Donnelly E, Baker SP, Boskey AL, van der Meulen MC. Effects of surface roughness and maximum load on the mechanical properties of cancellous bone measured by nanoindentation. J Biomed Mater Res A 77:426–435, 2006a.

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Donnelly E, Williams RM, Downs SA et al. Quasistatic and dynamic nanomechanical properties of cancellous bone tissue relate to collagen content and organization. J Mater Res 21:2106–2117, 2006b. Evans GP, Behiri JC, Currey JD, Bonfield W. Microhardness and Young’s modulus in cortical bone exhibiting a wide range of mineral volume fractions, and in a bone analogue. J Mater Sci Mater Med 1:38–43, 1990. Fan Z, Rho JY. Effects of viscoelasticity and time-dependent plasticity on nanoindentation measurements of human cortical bone. J Biomed Mater Res A 67:208–214, 2003. Fan Z, Smith PA, Eckstein EC, Harris GF. Mechanical properties of OI type III bone tissue measured by nanoindentation. J Biomed Mater Res A 79:71–77, 2006. Fan Z, Swadener JG, Rho JY et al. Anisotropic properties of human tibial cortical bone as measured by nanoindentation. J Orthop Res 20: 806–810, 2002. Ferguson VL, Bushby AJ, Boyde A. Nanomechanical properties and mineral concentration in articular calcified cartilage and subchondral bone. J Anat 203:191–202, 2003. Guo XE, Goldstein SA. Vertebral trabecular bone microscopic tissue elastic modulus and hardness do not change in ovariectomized rats. J Orthop Res 18:333–336, 2000. Hengsberger S, Ammann P, Legros B et al. Intrinsic bone tissue properties in adult rat vertebrae: modulation by dietary protein. Bone 36:134–141, 2005. Hengsberger S, Kulik A, Zysset P. A combined atomic force microscopy and nanoindentation technique to investigate the elastic properties of bone structural units. Eur Cell Mater 1:12–17, 2001. Hengsberger S, Kulik A, Zysset P. Nanoindentation discriminates the elastic properties of individual human bone lamellae under dry and physiological conditions. Bone 30:178–184, 2002. Ho SP, Goodis H, Balooch M et al. The effect of sample preparation technique on determination of structure and nanomechanical properties of human cementum hard tissue. Biomaterials 25:4847–4857, 2004. Hoc T, Henry L, Verdier M et al. Effect of microstructure on the mechanical properties of Haversian cortical bone. Bone 38:466–474, 2006. Hoffler CE, Guo XE, Zysset PK, Goldstein SA. An application of nanoindentation technique to measure bone tissue lamellae properties. J Biomech Eng 127:1046–1053, 2005.

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Hoffler CE, Moore KE, Kozloff K et al. Heterogeneity of bone lamellar-level elastic moduli. Bone 26:603–609, 2000. Hofmann T, Heyroth F, Meinhard H et al. Assessment of composition and anisotropic elastic properties of secondary osteon lamellae. J Biomech 39:2282–2294, 2006. Huja SS, Beck FM, Thurman DT. Indentation properties of young and old osteons. Calcif Tissue Int 78:392–397, 2006. Jamsa T, Rho JY, Fan Z et al. Mechanical properties in long bones of rat osteopetrotic mutations. J Biomech 35:161–165, 2002. Lee FY, Rho JY, Harten RJ et al. Micromechanical properties of epiphyseal trabecular bone and primary spongiosa around the physis: an in situ nanoindentation study. J Pediatr Orthop 18:582–585, 1998. Li J, Sato M, Jerome C et al. Microdamage accumulation in the monkey vertebra does not occur when bone turnover is suppressed by 50% or less with estrogen or raloxifene. J Bone Miner Metab 23(Suppl):48–54, 2005. Loubet JL. Some measurements of viscoelastic properties with the help of nanoindentation. NIST Special Publication 896, pp. 31–34, 1996. Melacini G, Bonvin AM, Goodman M et al. Hydration dynamics of the collagen triple helix by NMR. J Mol Biol 300:1041–1049, 2000. Mittra E, Akella S, Qin YX. The effects of embedding material, loading rate and magnitude, and penetration depth in nanoindentation of trabecular bone. J Biomed Mater Res A 79:86–93, 2006. Muller R, Hildebrand T, Ruegsegger P. Non-invasive bone biopsy: a new method to analyse and display the three-dimensional structure of trabecular bone. Phys Med Biol 39:145–164, 1994. Nalla RK, Kruzic JJ, Kinney JH et al. Role of microstructure in the agingrelated deterioration of the toughness of human cortical bone. Proceedings of the First TMS Symposium on Biological Materials Science, Vol. 26, pp. 1251–1260, 2006. Oliver WC, Pharr GM. An improved technique for determining hardness and elastic modulus using load and displacement sensing indentation experiments. J Mater Res 7:1564–1583, 1992. Oliver WC, Pharr GM. Measurement of hardness and elastic modulus by instrumented indentation: advances in understanding and refinements to methodology. J Mater Res 19:3–20, 2004. Pelled G, Tai K, Sheyn D et al. Structural and nanoindentation studies of stem cell-based tissue-engineered bone. J Biomech 40:399–411, 2007.

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Rho JY, Currey JD, Zioupos P, Pharr GM. The anisotropic Young’s modulus of equine secondary osteons and interstitial bone determined by nanoindentation. J Exp Biol 204:1775–1781, 2001a. Rho JY, Mishra SR, Chung K et al. Relationship between ultrastructure and the nanoindentation properties of intramuscular herring bones. Ann Biomed Eng 29:1082–1088, 2001b. Rho JY, Pharr GM. Effects of drying on the mechanical properties of bovine femur measured by nanoindentation. J Mater Sci Mater Med 10: 485–488, 1999. Rho JY, Roy ME, Tsui TY, Pharr GM. Elastic properties of microstructural components of human bone tissue as measured by nanoindentation. J Biomed Mater Res 45:48–54, 1999a. Rho JY, Tsui TY, Pharr GM. Elastic properties of human cortical and trabecular lamellar bone measured by nanoindentation. Biomaterials 18: 1325–1330, 1997. Rho JY, Zioupos P, Currey JD, Pharr GM. Variations in the individual thick lamellar properties within osteons by nanoindentation. Bone 25: 295–300, 1999b. Roy ME, Rho JY, Tsui TY et al. Mechanical and morphological variation of the human lumbar vertebral cortical and trabecular bone. J Biomed Mater Res 44:191–197, 1999. Silva MJ, Brodt MD, Fan Z, Rho JY. Nanoindentation and whole-bone bending estimates of material properties in bones from the senescence accelerated mouse SAMP6. J Biomech 37:1639–1646, 2004. Swadener JG, Rho JY, Pharr GM. Effects of anisotropy on elastic moduli measured by nanoindentation in human tibial cortical bone. J Biomed Mater Res 57:108–112, 2001. Tai K, Qi HJ, Ortiz C. Effect of mineral content on the nanoindentation properties and nanoscale deformation mechanisms of bovine tibial cortical bone. J Mater Sci Mater Med 16:947–959, 2005. Townsend PR, Rose RM, Radin EL. Buckling studies of single human trabeculae. J Biomech 8:199–201, 1975. Turner CH, Rho J, Takano Y et al. The elastic properties of trabecular and cortical bone tissues are similar: results from two microscopic measurement techniques. J Biomech 32:437–441, 1999. Van Rietbergen B, Muller R, Ulrich D et al. Tissue stresses and strain in trabeculae of a canine proximal femur can be quantified from computer reconstructions. J Biomech 32:165–173, 1999.

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Vanleene M, Mazeran P, Tho M. Influence of strain rate on the mechanical behavior of cortical bone interstitial lamellae at the micrometer scale. J Mater Res Soc 21:2093–2097, 2006. Weber M, Roschger P, Fratzl-Zelman N et al. Pamidronate does not adversely affect bone intrinsic material properties in children with osteogenesis imperfecta. Bone 39:616–622, 2006. Xu J, Rho JY, Mishra SR, Fan Z. Atomic force microscopy and nanoindentation characterization of human lamellar bone prepared by microtome sectioning and mechanical polishing technique. J Biomed Mater Res A 67:719–726, 2003. Zhang Y, Cui FZ, Wang XM et al. Mechanical properties of skeletal bone in gene-mutated stöpsel dtl28d and wild-type zebrafish (Danio rerio) measured by atomic force microscopy-based nanoindentation. Bone 30:541–546, 2002. Zysset PK, Guo XE, Hoffler CE et al. Mechanical properties of human trabecular bone lamellae quantified by nanoindentation. Technol Health Care 6:429–432, 1998. Zysset PK, Guo XE, Hoffler CE et al. Elastic modulus and hardness of cortical and trabecular bone lamellae measured by nanoindentation in the human femur. J Biomech 32:1005–1012, 1999.

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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

The protocols presented in this chapter focus on the micromechanical tensile testing of osteonal/interstitial bone, single trabeculae, and small animal bone specimens. Micromechanical testing of these small samples is technically challenging and often time-consuming. To alleviate the possible difficulties, the protocols for specimen preparation, apparatus and fixture design, data acquisition/interpretation, and associated techniques are provided for researchers in order to adapt these testing methodologies to their research needs. Keywords:

Micromechanical test; tension; osteonal bone; interstitial bone; trabecular bone.

1. Introduction The high morbidity, mortality, and treatment costs associated with age- and disease-related bone fragility pose significant problems in the health care of our populations. Bone is a hierarchical composite material that exhibits different structures, from the microstructural to the

Corresponding author: Xiao-Du Wang. Tel: +1-210-4585565; fax: +1-210-4586504; E-mail: [email protected]

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ultrastructural level. For instance, the microstructural features of cortical bone consist of osteons, interstitial tissue, Haversian canals, lamellae, cement lines, canaliculi, and lacunae; whereas trabecular bone has a porous structure (75%∼95% porosity) filled with bone marrow, is characterized by interconnected rods and plate-like structures, and contains only lamellae and lacunae. The complex structure of bone makes it difficult to elucidate the underlying mechanisms of age- and disease-related bone fractures. Furthermore, bone is a living tissue that adapts its structure over time to changes in its mechanical environment (i.e. the bone remodeling process). Thus, bone is a mixture of structurally and chronologically different tissues in which changes at local regions (e.g. microstructural and ultrastructural changes) can adversely impact the overall quality of bone. For these reasons, assessments of local tissue properties at the microscale have become necessary in bone research. Many previous studies have attempted different micromechanical testing methodologies for assessing the local tissue properties of bone (e.g. tensile, bending, torsion, and buckling tests). Among these micromechanical testing protocols, tensile tests may be preferred over other testing modes. For example, the bending test is at best a structural test; in such tests, it is difficult to determine the stress–strain curve from the load-deflection data because the assumption of linear elasticity from simple beam theory is no longer valid for any measurement of the postyield behavior of bone (Lucchinetti 2000). Similarly, buckling and cantilever beam deflection are also structural tests, which raise the same concerns as bending tests (Mente and Lewis 1989; Townsend et al. 1975). On the other hand, there are only limited uses of torsional tests in testing bone tissues because of the limitations in test apparatus and associated techniques. The tensile test is a straightforward technique that allows for the assessment of preyield-to-postyield and failure behaviors of a material. Thus, it has been widely used to investigate bone mechanical properties such as Young’s modulus, yield strain and strength, ultimate strength, and other postyield behaviors (Bowman et al. 1996; Currey 1959; Kaplan et al. 1985; Lindahl and Lindgren 1967; McCalden et al. 1993; Melick and Miller 1966; Nyman et al. 2005; Pattin et al.

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1990). Tensile testing at the microscopic scale is challenging because it requires custom-designed devices and methods to prepare small specimens and to carry out the testing. In the literature, microtensile testing has been performed on osteonal (Ascenzi and Bonucci 1964; Ascenzi and Bonucci 1967; Reyes et al. 2007) and interstitial bone tissue. In addition, the elastic properties of rod-like single trabeculae have been measured using tensile testing protocols (Bini et al. 2002; McNamara et al. 2006; Rho et al. 1993; Ryan and Williams 1989). In some other cases, microbeams or bars of cortical bone were prepared and tested in tension for small animal models (Choi et al. 1990; Ramasamy and Akkus 2007; Rho et al. 1993). This chapter focuses on the protocols for the preparation and micromechanical tensile testing of specimens from (1) osteons/ interstitial bone, (2) trabecular bone samples, and (3) small animal bone. Techniques will be described for obtaining, selecting, and preparing bone samples for micromechanical testing. Next, the methods for collecting the experimental data will be addressed. Results will then be presented to illustrate how to interpret the experimental results for determination of the material properties of the specimens. Finally, potential problems that may be encountered in specimen preparation and testing will be highlighted, along with tips and solutions that will assist the researcher in obtaining good results. When relevant, alternative methods for preparing and testing will be presented, and the pros and cons of each method discussed.

2. Micromechanical Tensile Testing of Osteonal/Interstitial Tissues Micromechanical testing of osteonal and interstitial bone tissue involves challenges in sample preparation and testing. The ultimate tensile strength of osteonal tissue was first obtained by Ascenzi and Bonucci (1964) using direct tensile loading of a gripped specimen; however, this method provided limited information regarding the other material properties of the tissue. Subsequently, more complete characterizations of the tensile properties of osteons were obtained using a microwave extensometer by Ascenzi’s group (Ascenzi and

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Bonucci 1967; Ascenzi and Bonucci 1968; Ascenzi et al. 1982), and an optical extensometer was developed in our laboratory. We (Reyes et al. 2007) have extended the techniques for acquiring and testing osteonal tissue (secondary osteons) to the acquisition and testing of interstitial tissue. From these tests, stress–strain diagrams can be obtained to determine the material properties of the tissue (e.g. stiffness, strength, and toughness). Comparisons can then be made between the mechanical properties of anatomic regions (osteonal vs. interstitial bone), genders, and age groups, as well as correlations between mechanical and compositional (organic and inorganic phases) properties for each tissue type. This information can then be compared to the bulk properties of cortical bone. Through the application of these methods, the heterogeneous nature of bone can be addressed and a more complete characterization of the tissue can be obtained.

2.1. Materials •

• • • • • • •

Tissue: osteonal bone from human donors or animals (e.g. bovine and canine)
Unless the goal of the research is to study a specific disease condition, the donors or animals should be free of any known bone diseases. Band saw: Ryobi BS902 band saw — for removing a longitudinal section of bone Diamond saw: Buehler IsoMet — for obtaining thin (3 mm) cross-sections from the rough cut of the longitudinal section Sandpaper (grits #600, #1200, and #4000) — for lapping bone specimens Silicon carbide and alumina suspension (0.05 µm) — for polishing bone surface Phosphate-buffered saline (PBS) — for irrigating bone cross-section during microcore acquisition and for storage of tissue Microvials: Fisher Flat Top Microcentrifuge Tubes (Cat. No. 05408-129) — for pretest and posttest storage of specimens Distilled water — for hydrating specimens during specimen preparation and tensile testing

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• •

Forceps — for manipulating microcores Microscopes — for imaging of specimens






•

•

•

•

• • •

Nikon Measurescope UM-2 — for use with the computer numerically controlled (CNC) machine to target and image the tissue of interest Meiji ML 8500 light microscope — for classifying microcores Trinocular boom microscope — for use with a digital camera to obtain photographic strain measurements

Computer numerically controlled (CNC) milling machine: Light Machines proLIGHT — for obtaining microcores (position repeatability, <5 µm; displacement resolution, ∼1.0 µm) Eccentric coring bit — for use with the CNC machine in obtaining microcores from bone cross-sections
The eccentric coring bit is made from a carbide blank with a cutting edge ~3 mm long and 0.125 mm offset from the bit centerline. Stage micrometer: Klarman Rulings, Inc. KR-814 (1 mm × 3 mm), with finest scale of 0.2 mm — for measuring dimensions of osteonal and interstitial microcores Specimen holders: made of Lexan — for use in mounting the microcores in the fabrication of the dog-bone–shaped specimens which will be mechanically tested Specimen preparation fixture — for use in positioning and holding the Lexan holders, while the microcore is being fixed and glued Cyanoacrylate glue: BSI Adhesives gap-filling glue — for attaching a microcore to the Lexan holders Mechanical testing system:






•

817

EnduraTEC Elf 3300 or equivalent with a displacement resolution <2.0 µm — for conducting mechanical test Omega LCF A-10 10-lb load cell Upper and lower specimen fixtures with slots, into which the Lexan holders of the dog bone specimen are inserted

Fiber optic lighting: Fiber-Lite MI-150 — for illumination of the specimen during testing

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Tissue paper — for removing water from the specimen prior to testing Digital cameras:
Sony DSC-H2 digital camera — for photographically documenting specimens
Motic 3000 CCD camera — for obtaining test images during mechanical testing Image analysis software: ImageJ software (http://rsb.info.nih. gov/ij/) — for calculation of specimen dimensions

2.2. Methods 2.2.1. Tissue preparation • • • •

Obtain a section (~50 mm long) from the middiaphysis of a femur using a band saw. Obtain a cross-section (~3 mm thick) from the middiaphysis section using a diamond saw. Lap the distal surface of the cross-section using successive grits of sandpaper (#600, #1200, and #4000). Polish the distal surface of the cross-section using a 0.05-µm alumina suspension.

2.2.2. Specimen acquisition (Fig. 2) • •

•

Secure the cross-section to the stage of the CNC machine. Center the tissue of interest (Fig. 1) using the microscope attached to the milling head of the CNC machine (see Sec. 2.4, note 1). The region of tissue should be positioned to fit within the second ring of a reticule as viewed through the eyepiece of the microscope at 100× [Fig. 2(b)]. Move the eccentric coring bit to the area of interest, and core out the target tissue at a rotational speed of 3600 RPM and a feed rate of 5 mm/min.



Irrigate the specimen using PBS to prevent heat-induced damage. The bit should descend into the tissue to a depth of ~2.8 mm [Fig. 2(c)], producing a microcore ~2.8 mm long and 0.25 mm in diameter [Fig. 2(d)].

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Fig. 1. A pictorial representation to identify secondary osteons and interstitial bone regions in human cortical bone. The secondary osteons (Onew, dashed outline) are newly formed bone tissues exhibiting an intact cement line, whereas the interstitial bone regions (Int, solid outline) and older osteons (Oold, dotted outline) represent tissues formed previously. Adapted from Nyman et al. (2006), with permission of Elsevier.

• •

Pluck the microcore from the cross-section carefully using forceps. Specimens can also be acquired using alternative methods (see Sec. 2.4, note 2).

2.2.3. Specimen classification •

Classify the microcore using a transmitted light microscope as an acceptable osteonal or interstitial sample (Fig. 3).
For osteonal tissue, the microcore should have a Haversian canal spanning through the testable region (central 1.5 mm; grades 1 and 2).
For interstitial tissue, the microcore should not have a Haversian canal spanning through the testable region (central 1.5 mm; grades 4 and 5).

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Fig. 2. (a) The specimen acquisition system consists of a CNC machine and a microscope with video camera for targeting and photographing tissue samples. (b) The tissue is targeted for the selection of a secondary osteon. (c) The eccentric coring bit descends into the tissue to produce a microcore. (d) The resulting microcore is 2.8 mm long and 0.25 mm in diameter. Adapted from Nyman et al. (2006), with permission of Elsevier.

•

•

•

Reject microcores that possess flaws which may lead to a premature failure of the specimen during testing. Examples of flaws include Haversian canals that “run out” from the gauge region of the specimen. Rotate and photograph the specimen about its long axis at 90° intervals against the stage micrometer to document dimensions of the microcore and Haversian canal. Place the microcore in a PBS-filled microvial and store in a freezer at −20°C until the time of specimen mounting.

2.2.4. Specimen mounting •

Place the Lexan holders in the specimen preparation fixture and align them ~1.5 mm apart.

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Fig. 3. Osteonal and interstitial tissues are viewed using transmitted light microscopy. For osteons, the Haversian canal should extend through the test region (grades 1 and 2). For interstitial specimens, there should be no Haversian canal in the test region (grades 4 and 5). Adapted from Nyman et al. (2006), with permission of Elsevier.

• •

•

Drop the microcore into the slots of the Lexan holders. Glue each end of the microcore to the Lexan holders using a small drop of cyanoacrylate glue, and allow it to cure completely (see Sec. 2.4, note 3). The completed dog-bone–shaped specimens have an overall length of 8 mm with a gauge region of ~1.5 mm (Fig. 4; see Sec. 2.4, note 4). Obtain scaled images of the completed specimen to be used in stress analysis.

2.2.5. Specimen testing • • • • • •

Slide the specimen in place within the slots of the grips secured on the bench-top mechanical testing system (Fig. 5). Hydrate the specimen with a bead of water. Preload the specimen in tension to a load of 0.01 N. Illuminate the specimen using fiber optic lighting. Remove the bead of water by wicking it away using tissue paper. Focus the camera to the specimen through the microscope.

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Fig. 4. A completed specimen ready for mechanical testing. The Lexan holders at each end are mounted to the fixtures of the micromechanical testing system [see Fig. 5(b)].

Fig. 5. (a) The mechanical testing system consists of a bench-top system for gathering force data and a microscopy system with attached camera for gathering optical strain data. (b) Close-up of specimen within the test fixtures.

• •

Begin image acquisition simultaneously with loading (see Sec. 2.4, note 5). Begin monotonic loading of the specimen while the specimen is still hydrated. Rehydrate and repeat from step 5 if necessary. Load

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to failure in tension at a loading rate of 0.005 mm/s and a sampling rate of 5–10 Hz (see Sec. 2.4, note 6). Specimens can also be tested using an alternative method (see Sec. 2.4, note 7).

2.2.6. Data preparation •

•

•

•

Synchronize the force data to the photographs of the specimen that have been taken throughout the loading process. Find the test image that shows the beginning of specimen failure, and match it to the failure point of the force–displacement data. Then, match subsequent prefailure images to corresponding force– displacement data points backwards as a function of time. Calculate strain from the test images using custom-written image analysis software. Measure in pixels the original gauge length between designated points on opposite sides of the failure plane using the first image of the series, and measure subsequent changes in length between the two points on an image-by-image basis (Fig. 6). Calculate stress by dividing the applied force by the original crosssectional area of the specimen, which is determined using the images of both the microcores acquired during specimen classification and the pretest dog-bone–shaped specimen. By using two images taken perpendicular to each other, the cross-sectional area of the Haversian canal can be determined and subtracted from the cross-sectional area of the microcore (Fig. 7). The scaled images of the dog-bone–shaped specimen can be used to determine where the specimen failed and where the cross-sectional area needs to be determined. Match the stress and strain data on a time basis to create a stress vs. strain plot for the test (Fig. 8).

2.3. Results From the stress vs. strain plot, material properties such as the stiffness (slope of the 0.2% offset line), yield stress (σy), failure strength (σsc),

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Fig. 6. Optical strain measurements are based upon identifying features (black circled areas) on the first image of the test series (a) and tracking the relative displacement of the points on subsequent images (b).

Fig. 7. The cross-sectional area of the microcore is calculated using two scaled images of the specimen taken from perpendicular views. The measurements of the Haversian canal from each view represent the major and minor axes of an ellipse (grey circle), which is subtracted from the total crosssectional area of the specimen.

fracture strain (εf), and toughness (area under the stress–strain curve) can be determined as shown in Fig. 8.

2.4. Notes •

The objective of the microscope should be positioned a known distance from the cutting head of the CNC machine. This allows

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Fig. 8. Stress–strain diagram produced from mechanical testing and optical strain systems. From this diagram, material properties such as stiffness (slope of the 0.2% offset line), yield stress (σy), strength (σsc), failure strain (εf), and work of fracture (shaded area) can be determined.

•

the coordinates of the targeted tissue to be programmed into the CNC machine so as to control the positioning of the coring tool. Ascenzi and Bonucci (1964, 1967, and 1968) present alternative methods for obtaining osteonal tissue. The first method (Ascenzi and Bonucci 1964; Ascenzi and Bonucci 1967) consists of preparing a longitudinal section of femoral shaft that has been ground to a thickness of 20–50 µm. Under polarized light (at 80–100 magnification), the section is dissected with a sharp needle. The resulting specimen consists of a thin section of osteonal tissue with a square coupon of bone at each end. The second method (Ascenzi and Bonucci 1968) is similar in principle to our approach. In this method, a sharpened steel needle is eccentrically inserted on a dentist’s drill and used to core out a region of osteonal tissue from a 500-µm-thick cross-section of femoral bone. The specimens are then tested in compression. The benefit of the coring approach utilized by our laboratory and by Ascenzi and Bonucci (1968) is the ability to easily identify secondary osteons and regions of interstitial tissue. The drawback

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of this approach is the possibility that the microcore may have flaws or a Haversian canal running out of the core (for osteonal specimens) or into the core (for interstitial specimens) that cannot be determined a priori. One technique that may circumvent this problem is imaging the femoral cross-sections using highresolution micro-computed tomography (CT). The predominant orientation of the Haversian canals can thereby be determined, allowing the bone specimen to be lapped at an angle that would align the Haversian canals of the secondary osteon with the axis of the coring tool. This technique would improve the likelihood of obtaining a usable specimen, and may also allow for the identification of candidate tissue from a three-dimensional (3D) model of the cross-section. It is helpful to transfer the glue drop from the glue dispenser to the specimen using the tip of a hypodermic needle. Each specimen should be rotated and examined about its long axis to determine if glue has run out of the slots and into the test region, thereby compromising the specimen. If possible, the images should encompass the entire test region at the highest possible magnification. This will allow the researcher to determine the manner in which the specimen failed (e.g. a break in the gauge region or at the glue interface, or a pull-out from the Lexan holder), and whether or not the results of the test should be accepted. The loading rate is dependent on the image acquisition rate of the camera system (e.g. Motic 3000 acquires one image per second) and the dehydration of the specimen. A compromise must be reached between loading at a rate so rapid that it produces motion blur in the images and loading at a rate so slow that the specimen dries out prior to completing the test. Ascenzi and Bonucci (1964 and 1967) present an alternative method for tensile testing osteonal tissue. Using the specimen dissection technique described earlier (see Sec. 2.4, note 2), the specimens are tensile tested with a microwave extensometer. Osteonic lamellae obtained using the coring technique (Ascenzi and Bonucci 1968) have also been tested using a microwave

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extensometer (Ascenzi et al. 1982). The results of our studies of human femoral osteonal and interstitial tissues reveal average values for a failure strength of 119 MPa and 147 MPa, a modulus of elasticity of 11.4 GPa and 13.8 GPa, and an elongation at failure of 0.06 and 0.05, respectively (pooled male and female specimens for each tissue type).

3. Micromechanical Tensile Testing of Trabecular Tissues The mechanical properties of trabecular bone at the macroscopic level have been determined by mechanical testing and ultrasound techniques (Ashman and van Buskirk 1987; Carter and Hayes 1977; Keaveny and Hayes 1993; Keaveny et al. 1994; Linde 1994). However, the mechanical properties determined from the macroscopic level only reflect the mechanical properties of trabecular bone at the structural level, not at the material level. The material properties of trabecular bone tissue can be determined by micromechanical tests of single trabeculae, which have a diameter of ~0.15 mm and a length of ~2 mm (Hernandez et al. 2005; Rho et al. 1993).

3.1. Materials • • • • • • • • •

Bone tissue: trabecular bone Water jet — for removing bone marrow Low-speed diamond saw — for cutting trabecular bone sections PBS — for storing tissues prior to use Scalpels — for removing single trabeculae from trabecular bone sections Forceps — for harvesting individual trabeculae Grip assembly — for holding specimens during micromechanical testing (Fig. 9; see Sec. 3.4 for additional examples of grip assembly) Cyanoacrylate glue — for attaching specimens to grip assembly Micromechanical testing machine: commercial or custom-made testing system that is suitable for testing sample specimens (see Sec. 3.4 for an example of a custom-made device)

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Fig. 9. Schematic diagram of a microtensile testing apparatus. Adapted from Rho et al. (1993), with permission of Elsevier.

•

Microscopes





• •

Stereomicroscope — for dissecting single trabeculae from trabecular bone sections Transmitted light microscope with video camera — for capturing images of specimens during deformation

Staged micrometer — for measuring specimen dimensions ImageJ software — for calculation of specimen geometry measurements from captured images

3.2. Methods 3.2.1. Specimen preparation •

Cut a thin section of trabecular bone to a thickness of 0.15 mm from a specific anatomical location using a low-speed diamond saw.

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Fig. 10. Trabecular bone slices (a), from which single trabecula tensile test specimens (b) are harvested and fabricated. Adapted from Bini et al. (2002), with permission of Elsevier.

• •

•

Remove the bone marrow from trabecular sections using a water jet. Under the examination of a dissection microscope or a stereotype microscope, isolate single trabeculae from the trabecular section using a scalpel blade and forceps (Fig. 10). Store specimens of single trabeculae in PBS, and place them in a −20°C freezer before mechanical testing.

3.2.2. Specimen geometry • • •

Place a staged micrometer under a transmitted light microscope with a video camera. Take an image of the staged micrometer, and use it to calibrate the measurement system of the ImageJ software. Take images of the single trabeculae using a video camera connected to the transmitted light microscope, and then transfer them to the ImageJ software and process them using the calibrated measuring system.

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Approximate the diameter and length of the single trabeculae from the acquired images as the average values of three measurements for each dimension.

3.2.3. Micromechanical testing • • • • • • •

Place two grip rods in a custom-designed fixture and align them ~2 mm apart to minimize the bending under a stereomicroscope. Attach each end of single trabeculae into the hole of the grip rod using cyanoacrylate glue. Allow the glue to cure completely. Move and fix the specimen with the grip rods to a micromechanical testing apparatus. Attach a clip-on extensometer to the grip rods to measure displacement. Apply tensile load to the specimen with a strain rate of 0.001 per second. Record the signals from load cells and extensometers.

3.3. Results The load–displacement curve can be obtained from the microtensile test of single trabeculae. Stress is calculated by dividing the applied force by the average cross-sectional area of the trabeculae, whereas strain is estimated as the ratio between the displacement detected by the extensometer and the initial gauge length. The elastic modulus can be calculated from the linear region of the stress–strain curve. According to Ryan and Willlams (1989), single trabeculae from bovine femur had an average elastic modulus of 1 GPa. In addition, Rho et al. (1993) conducted tensile tests on single trabeculae from human tibiae; the average Young’s modulus of trabecular bone was reported as 10.4 GPa. Recently, Bini et al. (2002) used single trabeculae from human femur to perform microtensile testing; Young’s modulus of single trabeculae ranged from 1.41 GPa to 1.89 GPa.

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3.4. Notes •

•

•

•

A specially designed apparatus for testing small bone samples is shown in Fig. 9 (Rho et al. 1993). It includes a force transducer for measuring load, an extensometer for measuring strain, and a mechanism for applying tensile load to the specimen. In addition, a commercial testing system can be used for micromechanical testing. For example, a motorized tensile substage (Electroscan E-3; Ernest F. Fullam, Inc., Latham, NY, USA) has been employed to conduct a tensile test of single trabeculae (Hernandez et al. 2005). The error sources in a tensile test lie in the grip of single trabeculae. It is a challenge to grip such a small sample. When glues are used in the grip assembly to secure individual trabeculae, the glue deformation also contributes to the measurement of the displacement for the bone sample. Therefore, the determination of Young’s modulus may be hindered because the difference between the bone deformation and the glue deformation cannot be distinguished. In addition, misalignment of the sample may introduce uncontrolled shear stress in the bone; the shear stress will have an influence on the calculation of Young’s modulus of bone tissue. Great care should be taken to excise single trabeculae from trabecular sections. The specimens can be taken by extracting the portion located between two adjacent mutual supports (Bini et al. 2002). Only the straight specimens can be kept for testing (Ryan and Williams 1989). Grips are essential for a tensile test because the major concern in a tensile test is slippage in the grips. One grip assembly has been developed for the tensile testing of single trabeculae (Ryan and Williams 1989). It contains a grip body and a grip slide. The grip body is pin-connected to the base of the stationary frame of a testing machine. The grip slider is attached to the moving cross-head of the test frame. The grip slider can slide on the grip body through two guide rods, which prevent any rotation or bending of the specimen during testing. The specimen can be held in place by clamp plates and tightened with thumbscrews.

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Fig. 11. Digital photographs of single trabeculae within the holders. Adapted from Hernandez et al. (2005), with permission of Elsevier.

•

•

•

As shown in Fig. 11, another grip assembly has been designed to hold samples of single trabeculae during a tensile test (Hernandez et al. 2005). A pair of rectangular brass holders contains tapped holes with a diameter of 1.2 mm. Single trabeculae are placed within the tapped holes and potted in cyanoacrylate glue. The rectangular brass holders are then connected with the cross-heads of the micromechanical testing system. Force is usually measured by a load cell connected to one of the two grip rods (Rho et al. 1993) or a guide block (Ryan and Williams 1989). The load cell could be (1) a trapezoidal cantilever beam holding a four-arm strain-gauge bridge which measures the applied tensile load (Ryan and Williams 1989), (2) an aluminum channel with two biaxial strain gauges connected in a full-bridge configuration (Rho et al. 1993), or (3) a commercially available low-force transducer (Bini et al. 2002; Hernandez et al. 2005). Strain can be measured using extensometers. Strain-gauge extensometers can be mounted on the grip body and the grip slide to measure the specimen elongation (Ryan and Williams 1989). Strain can also be measured using a custom laser Michelson interferometer (Bini et al. 2002).

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4. Micromechanical Tensile Testing of Mouse Bone Samples Mechanical properties of mouse bone have been studied by mechanical testing of the whole bone (Kunnel et al. 2002; Margolis et al. 2004; McBride 1998; LaMothe et al. 2005; Silva et al. 2006), which gives the mechanical properties of bone at the structural level. In order to obtain mechanical properties at the tissue level, micromechanical testing approaches are required. Here, we present a microtensile test of microbeams from mouse bone samples (Ramasamy and Akkus 2007).

4.1. Materials • • • • • • • • •

Bone tissue: mouse femur Scalpels — for removing soft tissues from bone surface PBS — for storing tissues prior to use Calcium-supplemented solution — for maintaining wetness and calcium content of bone specimens during testing Micromachining system — for machining specimens into the desired shape and size Microscope — for measuring the dimensions of prepared specimens Micromechanical test system — for performing microtensile tests Load cell — for measuring the applied force Displacement gauge — for measuring the displacement

4.2. Methods 4.2.1. Specimen preparation • •

•

Remove soft tissues from the femur surface using a scalpel. Pot the proximal and distal ends of femur into two brass potting blocks using a cold curing dental cement (Henry Schein Nelville, NY, USA) [Fig. 12(a)]. Secure the potted specimen in a supporting brass channel [Fig. 12(b)], i.e. a femur assembly.

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Fig. 12. A schematic illustration for specimen preparation. (a) Put the femur into brass blocks. (b) Secure the brass blocks in a supporting brass channel. (c–g) Completely remove the other three quadrants by machining to make the anterior quadrant a tensile specimen. Adapted from Ramasamy and Akkus (2007), with permission of Elsevier.

• •

•

Place and secure the femur assembly in a water bath attached to a micromilling machine. Machine the region of interest of the femur into a coupon shape using the micromilling machine [Figs. 12(c)–12(g)] with a 1/8′′-diameter carbide cutting tool. The micromilling machine consists of three translation stages (1 µm resolution), which can be moved manually in x-y-z direction by rotating the attached micrometers. Measure the dimensions of prepared specimens using a calibrated microscopy system.

4.2.2. Micromechanical testing •

Testing apparatus: Immediately after specimen preparation, attach the femur assembly into the grips of the testing machine (ELF 3200;

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•

•

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EnduraTEC, MN, USA) and then remove the supporting brass channel from the femur assembly. Custom-designed fixtures may be needed to eliminate misalignment in the axes of the two grips. Testing protocols: Perform microtensile under displacement control, e.g. 10 µm/s (Ramasamy et al. 2007). Spray calcium-supplemented solution on the specimens to keep the specimens wet. Force measurement: Measure the force applied to the specimen by a load cell attached to one of the two loading fixtures, and record it into the computer connected to the control box of the test system. Displace measurement: Measure displacement with a displacement gauge with a 0.5-mm deflection range (Model 3540; Epsilon Technologies, WY, USA) attached to the upper grip.

4.3. Results The monotonic microtensile test provides a load–displacement curve of bone samples at the microscopic level, which can be easily converted to a strain–stress relationship with known specimen size. In addition, the study by Ramasamy and Akkus (2007) on tensile testing of mouse femur microbeams demonstrated that the average Young’s modulus of the anterior quadrant (1.9 GPa) differed from that of the posterior quadrant (1.3 GPa), which means that the anterior quadrant is more capable of sustaining tensile loads than the posterior quadrant in mouse femurs.

4.4. Notes • •

•

The method described above can be applied to test bone samples from other animals as well. For human cortical bone, a specially designed microtensile apparatus is needed, as has been mentioned in the previous section (Rho et al. 1993). Bone samples should be stored at ≤ −20°C and wrapped with PBS-soaked gauze before specimen preparation. During specimen

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preparation and micromechanical testing, keep the specimens wet using PBS or calcium-supplemented solution. Harvest bone sections from the middiaphysis of the femur or tibia, and keep the anatomical location choice consistent for the same study. Mechanical test fixture is needed to ensure alignment in the axes of the upper and lower grips. Choose a load cell with appropriate capacity (e.g. 44 N) and resolution (e.g. 0.1 N). Collect data at an appropriate rate to get smooth results, e.g. 50 Hz. Other than measuring displacement, an extensometer with appropriate resolution (e.g. 1 µm) attached to both grips may be used to measure strain (Rho et al. 1993). Moreover, surface displacement can be measured by optical determination, as shown in Sec. 2 and in other studies (Bini et al. 2002; Reyes et al. 2007; Shahar et al. 2007).

References Ascenzi A, Benvenuti A, Bonucci E. The tensile properties of single osteonic lamellae: technical problems and preliminary results. J Biomech 15:29–37, 1982. Ascenzi A, Bonucci E. The ultimate tensile strength of single osteons. Acta Anat (Basel) 58:160–193, 1964. Ascenzi A, Bonucci E. The tensile properties of single osteons. Anat Rec 158(4):375–386, 1967. Ascenzi A, Bonucci E. The compressive properties of single osteons. Anat Rec 161(3):377–391, 1968. Ashman RB, van Buskirk WC. The elastic properties of a human mandible. Adv Dent Res 1(1):64–66, 1987. Bini F, Marinozzi A, Marinozzi F, Patanè F. Microtensile measurements of single trabeculae stiffness in human femur. J Biomech 35:1515–1519, 2002. Bowman SM, Zeind J, Gibson LJ et al. The tensile behavior of demineralized bovine cortical bone. J Biomech 29(11):1497–1501, 1996. Carter DR, Hayes WC. The compressive behavior of bone as a two-phase porous structure. J Bone Joint Surg Am 59(7):954–962, 1977.

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Choi K, Kuhn JL, Ciarelli MJ, Goldstein SA. The elastic moduli of human subchondral, trabecular, and cortical bone tissue and the size-dependency of cortical bone modulus. J Biomech 23(11):1103–1113, 1990. Currey JD. Differences in the tensile strength of bone of different histological types. J Anat 93:87–95, 1959. Hernandez CJ, Tang SY, Baumbach BM et al. Trabecular microfracture and the influence of pyridinium and non-enzymatic glycation-mediated collagen cross-links. Bone 37(6):825–832, 2005. Kaplan SJ, Hayes WC, Stone JL, Beaupre GS. Tensile strength of bovine trabecular bone. J Biomech 18(9):723–727, 1985. Keaveny TM, Guo XE, Wachtel EF et al. Trabecular bone exhibits fully linear elastic behavior and yields at low strains. J Biomech 27(9): 1127–1136, 1994. Keaveny TM, Hayes WC. A 20-year perspective on the mechanical properties of trabecular bone. J Biomech Eng 115(4B):534–542, 1993. Kunnel JG, Gilbert JL, Stern PH. In vitro mechanical and cellular responses of neonatal mouse bones to loading using a novel micromechanicaltesting device. Calcif Tissue Int 71(6):499–507, 2002. LaMothe JM, Hamilton NH, Zernicke RF. Strain rate influences periosteal adaptation in mature bone. Med Eng Phys 27(4):277–284, 2005. Lindahl O, Lindgren AG. Cortical bone in man. II. Variation in tensile strength with age and sex. Acta Orthop Scand 38(2):141–147, 1967. Linde F. Elastic and viscoelastic properties of trabecular bone by a compression testing approach. Dan Med Bull 41(2):119–138, 1994. Lucchinetti E. Micromechanical testing of bone trabeculae — potentials and limitations. J Mater Sci 35(24):6057–6064, 2000. Margolis DS, Lien YHH, Lai LW, Szivek JA. Bilateral symmetry of biomechanical properties in mouse femora. Med Eng Phys 26(4):349–353, 2004. McBride D. Bone geometry and strength measurements in aging mice with the oim mutation. Calcif Tissue Int 62(2):172–176, 1998. McCalden RW, McGeough JA, Barker MB, Court-Brown CM. Age-related changes in the tensile properties of cortical bone. The relative importance of changes in porosity, mineralization, and microstructure. J Bone Joint Surg Am 75(8):1193–1205, 1993. McNamara LM, Ederveen AG, Lyons CG et al. Strength of cancellous bone trabecular tissue from normal, ovariectomized and drug-treated rats over the course of ageing. Bone 39(2):392–400, 2006.

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Melick RA, Miller DR. Variations of tensile strength of human cortical bone with age. Clin Sci 30(2):243–248, 1966. Mente PL, Lewis JL. Experimental method for the measurement of the elastic modulus of trabecular bone tissue. J Orthop Res 7(3):456–461, 1989. Nyman JS, Reyes M, Wang X. Effect of ultrastructural changes on the toughness of bone. Micron 36(7–8):566–582, 2005. Nyman JS, Roy A, Acuna RL et al. Age-related effect on the concentration of collagen crosslinks in human osteonal and interstitial bone tissue. Bone 39(6):1210–1217, 2006. Pattin CA, Carter DR, Caler WE. Cortical bone modulus reduction in tensile and compressive fatigue. Trans 36th Orthop Res Sot 15:50, 1990. Ramasamy JG, Akkus O. Local variations in the micromechanical properties of mouse femur: the involvement of collagen fiber orientation and mineralization. J Biomech 40(4):910–918, 2007. Reyes M, Acuna R, Wang X. Micromechanical tensile testing of cortical bone tissue. Trans Orthop Res Soc 32: poster no. 1354, 2007. Rho JY, Ashman RB, Turner CH. Young’s modulus of trabecular and cortical bone material: ultrasonic and microtensile measurements. J Biomech 26(2):111–119, 1993. Ryan SD, Williams JL. Tensile testing of rodlike trabeculae excised from bovine femoral bone. J Biomech 22(4):351–355, 1989. Shahar R, Zaslansky P, Barak M et al. Anisotropic Poisson’s ratio and compression modulus of cortical bone determined by speckle interferometry. J Biomech 40(2):252–264, 2007. Silva MJ, Brodt MD, Wopenka B et al. Decreased collagen organization and content are associated with reduced strength of demineralized and intact bone in the SAMP6 mouse. J Bone Miner Res 21(1):78–88, 2006. Townsend PR, Rose RM, Radin EL. Buckling studies of single human trabeculae. J Biomech 8(3–4):199–201, 1975.

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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

Standard mechanical testing methodologies are typically conducted in line with ASTM (American Society for Testing and Materials) or BS (British Standards) guidelines. However, the nature of musculoskeletal tissues — which are generally inhomogeneous, anisotropic, porous, viscoelastic composite materials with widely varying mechanical properties — means that standard testing methodologies are often inappropriate. Depending on the nature of the specimen and the type of testing to be conducted, the equipment and methodology used in mechanical testing of musculoskeletal tissues vary widely. It is practically impossible to dictate an optimum approach that should be used in the mechanical testing of musculoskeletal tissues. Several approaches may be equally valid, and the strategy adopted will generally depend on the particular requirements of individual studies. As such, a “how to” guide to biomechanical testing is almost impossible to compile and of limited practical use. This chapter is therefore written from the opposite, cautionary perspective, indicating common approaches to the biomechanical testing of musculoskeletal tissues as well as considerations that need to be taken into account during each stage of testing. Once the aim of a biomechanical test or series of tests has been established, it is hoped that this chapter will help in the choice of an appropriate strategy and indicate areas where particular care should be taken. Keywords:

Musculoskeletal tissues; mechanical testing; in vitro study.

Corresponding author: Daniel Hung-Kay Chow. Tel: +852-27667674; fax: +852-23342429; E-mail: [email protected]

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1. Introduction Biomechanical testing is important in many experimental studies as an endpoint measure to evaluate and confirm the effects of various treatments on musculoskeletal tissues prior to their clinical trials or applications (Turner and Burr 1993; Burstein and Wright 1994). Standard mechanical testing methods are typically conducted in line with ASTM (American Society for Testing and Materials) or BS (British Standards) guidelines. However, due to the nature of musculoskeletal tissues, which are generally inhomogeneous, anisotropic, porous, viscoelastic composite materials with widely varying mechanical properties (Burstein and Wright 1994; Turner and Burr 1993), standard testing methodologies are often inappropriate and the equipment and methodology used in biomechanical testing of musculoskeletal tissues vary widely. It is practically impossible to dictate an optimum approach that should be used in the biomechanical testing of musculoskeletal tissues, as the strategies adopted generally depend on the particular requirements of individual studies. Nevertheless, the mechanical testing of musculoskeletal tissues can be divided into three major stages: (1) Tissue storage, handling, preparation, and mounting; (2) Testing methodologies; and (3) Data analysis. These are dealt with separately below, but a general guiding principle in the mechanical testing of musculoskeletal tissues is to always consider how the in vitro testing environment relates to the in vivo physiological environment of the specimen.

2. Materials and Methods 2.1. Tissue storage, handling, preparation, and mounting Prior to biomechanical testing on musculoskeletal tissue, proper tissue storage, handling, preparation, and mounting are extremely important for assuring the quality of results.

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2.1.1. Storage •

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Tissue autolysis begins within hours following the removal of bone from the body, and storage by deep freezing (below −15°C) is commonly required (Burstein et al. 1972).a Refrigeration (4°C) is not appropriate for storing collagenous tissues for extended periods of longer than approximately 12–24 hours. If the specimen cannot be tested within 24 hours of excision from the donor, deep freezing is preferable. Bone specimen kept at −20°C with saline has been suggested to be the best condition for long-term preservation (Ashman 1982; Pelker et al. 1984).b Freezing will tend to cause drying, and the specimens should be thoroughly moistened and bagged (preferably airtight) before freezing to minimize this effect. Most tissues will retain moisture during excision; but to prevent any drying of the tissues, physiological saline or similar (such as Ringer’s solution) should be used to keep the tissue moist during tissue excision prior to storage. Deep freezing has a limited effect on the mechanical properties of tissues (Smeathers and Joanes 1988). This has been the subject of many papers, but should be considered with respect to the nature of the testing to be undertaken. For specimens of small size, the effect of freezing (cell necrosis and tissue breakdown) on the mechanical properties should be taken into account. Rapid freezing by immersing the specimen in liquid nitrogen for a few seconds may help to limit cellular damage in smaller samples such as meniscus, ligament, tendon, etc. Smaller specimens may require more rapid defrosting (immersion in warm saline or similar), but larger specimens can be thawed out at room temperature. Packing material should only be removed once the specimen has thawed.

If possible, tissues should be tested fresh. Prolonged freezing (longer than a month or so), however, should be avoided, as should repeated thawing and freezing. A specimen should be frozen only once, and kept in a frozen state for less than 1 month if at all possible. b

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2.1.2. Handling •

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In handling specimens, a documented history of the donor should be kept. Note should be taken of the donor’s age, level of activity (for laboratory animals), and any illness that could confound the results of testing. For cadaveric specimens, the body weight, body height, gender, and cause of death should be recorded. A careful visual inspection of all specimens should be carried out to check for abnormalities, structural damage, etc.c Appropriate tissue handling facilities should be available, and usual precautions (gloves, clothing, mask, sharps disposal, sterilization of equipment) should be taken. Indirect contact with or transfer of body fluids must be avoided. All instruments used in the preparation of cadaveric specimens should be sterilized after use, and preferably restricted for use in cadaveric preparation. Specimens should always be kept moistened with physiological saline (0.96 g/L of NaCl) or similar (Ringer’s solution) and never be allowed to dry out, unless this is specifically intended; otherwise, their mechanical properties will be affected (Evans 1973). If ligaments, tendons, etc. are to be excised, dimensions should be recorded in situ before excision, as the strain that exists in the specimen in situ may need to be considered. Specimens have a limited lifetime in vitro before irreversible biological degradation occurs. The effect of this degradation on the mechanical properties depends on factors including the tissue type, specimen size, and testing conditions, and is therefore difficult to account for accurately. The rate of tissue degradation depends on the temperature and other environmental conditions. If the specimen has to be tested at higher temperatures (e.g. body temperature), special consideration should be paid to the testing time. Testing at room temperature

Larger osteoligamentous structures (e.g. joints and sections of long bone) should be radiographed to check for fracture, tumor, or other morphological abnormalities that may not be found under visual inspection.

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(23°C) increases Young’s modulus of bone by about 2%–4% compared to a test at 37°C (Ashman 1982; Bonfield and Tully 1982). Specimens should be thawed completely before testing; but once thawed, the testing should be completed as soon as possible.d

2.1.3. Preparation and mounting •

Careful mounting is essential to the accuracy of testing, as insecure or misaligned specimens will skew the results (Fig. 1). While

Fig. 1. A specially designed jig is used for mounting a spine specimen. The jig allows three-dimensional linear and rotational adjustment (indicated by arrows), so that the specimen can be properly aligned with the jig for load application. Radiograph(s) can be taken to ensure that the alignment of the specimen resembles the in vivo situation. d If it is not possible to complete testing within 6 hours or so following thawing, the mechanical effects of changes in the composition of soft tissues may need to be investigated.

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the time available for testing is often limited due to the biological nature of the specimen, mounting should never be rushed.e Custom-made mounting jigs and frames (such as clamps) are commonly used to attach specimens to the test equipment (Fig. 2), and are often very convenient for use with certain specimen types in specific tests.f A well-designed jig can save time, while providing a consistent physiological alignment and loading pattern. The mechanical properties (rigidity and viscoelastic properties) of the jig should be considered, as a jig that is 10 times stiffer than the specimen will still give a 10% error in the strain recorded by the testing machine. All mounting materials, frames, jigs, etc. that are likely to come into contact with specimens or be otherwise contaminated should be either for single use (disposed of appropriately immediately following testing) or sterilized following testing.g Dissection to obtain smaller specimens such as cartilage, ligament, tendon, etc. should be carried out with great care. Fiber bundles (fascicles) are normally parallel, but are often branched and intertwined. Dissection to produce uniform strips should be carried out under a microscope.

(1) Mounting of soft tissues •

Ligaments, tendons, etc. to be tested in tension should be mounted securely so that slipping cannot occur, but at the same disruption of the tissue ends should be minimized.h

e If there is any problem with the mounting, it is better to reject the specimen than to carry on with the testing. Due to the limited in vitro lifetime of specimens, preparation and mounting are often performed during thawing, so that once the specimen has thoroughly thawed it is ready for testing. Storage overnight in a refrigerator to thaw partially and then warming to testing temperature during preparation and mounting is often a convenient option. f Specimen preparation depends on the nature of the testing. In general, however, great care is needed during mounting to avoid damaging, overstraining, or misalignment of the specimen. g Mounting jigs and frames are commonly made from stainless steel, aluminum, nonporous plastics (Perspex, PVC), etc. h Clamping of soft tissues may result in stress concentration at the site of clamping, and this should be considered.

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Fig. 2. Custom-made mounting jigs. (a) Indenters with different diameters and adjustable stage for indentation test. (b) Clamps and holders for screw pull-out test. (c) Jigs for 3-point or 4-point bending test. (d) Angleadjustable holder and clamps for tendon/ligament tensile test.

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Clamp design may help minimize stress concentration (Fig. 3), but in any case the clamped ends should be observed carefully during testing for any sign of slippage or fiber damage. Excessive clamping force at ligament or tendon ends will tend to induce premature tissue failure at the clamping site. Clamping augmented by cyanoacrylate cement (Super Glue) fixation is a less traumatic

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Fig. 3. An example of a clamp design for ligaments and tendons. The clamp is cylindrical in shape and acts as a roller, with the specimen looped over the top of the roller. The roller can be rotated using the knurled knob, and clamped securely at either end by the screws. This design means that the clamped ends of the specimen are shielded from direct loading, resulting in less stress concentration and less chance of the specimen slipping through the clamps.

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method to secure the specimen for testing (Fig. 4) (Hukins et al. 1990).i Testing ligaments and tendons in situ (complete with attachments) will help to ensure that the in vitro testing environment resembles the in vivo physiological environment, and may be necessary to establish failure mechanisms. For instance, the ligamentum flavum will fail at the bony attachment with the laminae (Hukins et al. 1990), but this will not be found if the excised ligament is tested. The ligament and bony attachments need to be tested as a whole unit.

(2) Mounting of hard tissue and/or joints •

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Larger specimens such as joints often require stripping of the muscles and adjacent fibrous tissues to allow secure mounting.

Cyanoacrylate is not recommended for thicker fibrous specimens, as the inner fibers will be shielded and thus not properly secured. More reliance on clamping is required in these cases, and suturing the ligament ends will often help to provide a secure fixation.

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Fig. 4. Photos showing the embedding process for an irregular specimen for screw pull-out testing. (a) Thawed bone specimen wrapped by gauze soaked with 0.9% saline is fixed into a custom-made jig. (b) The specimen is wrapped with aluminum foil. An adhesive stick is used to fill the gap between the specimen and the jig in order to make a circular barrier with standardized diameter, so that resin in liquid form cannot flux in. The radius (distance between the screw and the resin) is thus standardized, reducing the error during the pull-out test. (c) Resin is poured into a mold with standard size. (d) The bone specimen is embedded in the resin block with standard size. (e) The pull-out test is performed by a standard material testing machine after the block has been attached to the custom-made apparatus.

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This procedure should be consistent between specimens and performed carefully. Larger stiffer specimens (joints, bones, etc.) often need to be mounted or potted. Bone cement, dental cement, or fast-drying epoxy resin adhesives (Fig. 5) are all suitable, but curing times and temperatures may need to be considered; augmentation with bone screws, K-wires, etc. is recommended. Potting material such as epoxy, bone cement, or dental cement is often difficult to remove, and should not be used to fix specimens directly to testing machines. Reusable or disposable potting cups that can be screwed or clamped securely into the testing machine should be used (Figs. 5 and 6).j

If the alignment of joints is important, then if possible this should be checked by radiography immediately prior to testing with the specimen mounted in the test apparatus.

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Fig. 5. An example of a fixation of a bony specimen using a potting cup. The L2 vertebral body is embedded in epoxy resin using the metal cup shown, which is fixed into the testing machine. The L1 vertebra is prepared for embedding by augmenting with screws, as shown. Note: the specimen is kept moist with physiological saline.

(3) Mounting of external transducers •

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Any external transducer (strain gauges, etc.) should be mounted carefully in order to try and keep the specimen as close to the physiological condition as possible (Fig. 7). The region where strain gauges are mounted will often have to be dry and cleared of all soft tissues, but the rest of the specimen should be kept moist.k

Any internal transducer (e.g. to measure the pressure in joint capsule) should be as minimally invasive as possible (miniaturized) and introduced carefully. This may require the aid of or supervision by a surgeon. The effects of introducing internal (invasive) transducers need to be considered very carefully, and statistical comparison of the results against a control group without placement of invasive transducers is preferable. Although in some situations the effect of external transducers is not so critical, the mechanical impedance of the transducer still needs to be considered (Fig. 7). For example, if a displacement transducer is stiffer than the specimen, it will reinforce the specimen and make it appear stiffer than it really is.

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Fig. 6. Potting cups of different sizes that are used to fix bone specimens (femoral neck) with epoxy resin before compression test. The ones with a white plastic head on the right are the compression rods connected to the material testing machine.

Fig. 7. An extensometer can be used to measure the deformation of specimen with small strain.

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(4) Testing conditions •

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The experimental conditions need careful consideration. In most cases, testing is done at room temperature with the specimen kept moistened with saline. However, viscoelastic properties are temperature- and moisture-dependent (Chae et al. 2003; Helvatjoglu-Antoniades et al. 2007; Pietrucha 2005). It may be necessary to control the temperature and maintain 100% humidity (Fig. 8), depending on the nature of the specimen and testing. Replication of the strains found in vivo may be required by straining the specimen to dimensions found in vivo. For testing specimens where the in vivo prestrain is along a single longitudinal axis (such as ligaments and tendons), this can be done simply by measuring the contraction on excision and then distracting the specimen to an equivalent strain prior to testing. For anisotropic materials such as skin, where the contraction on excision is in two or more dimensions, replication of in vivo prestrains may be more complex. Better results may be found by using a comparatively stiff template to reinforce the tissue prior to excision. The template and

Fig. 8. It is necessary to control the temperature and maintain 100% relative humidity for testing tissues.

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the tissue can then be excised and mounted en bloc before the template is carefully removed, leaving the tissue mounted in its in vivo configuration (Choi et al. 1990). The configuration and dimensions of tissues during the testing procedure (as well as just mounting) should also be taken into account. While tissues such as ligaments and tendons have an obvious longitudinal axis that indicates the direction of major loading, connective tissue “sheets” such as cartilage and skin typically experience loads along several axes simultaneously. Compression of a cartilage specimen will cause lateral expansion along a perpendicular axis, in accordance with Poisson’s ratio (Black 1976; Aspden 1990); while restriction of the expansion along the perpendicular axis will increase the stiffness (Aspden 1990). The boundary conditions of the tissue should therefore be considered, and the use of oversized specimens may help to replicate physiological conditions (Fig. 9).l

2.2. Testing methodologies 2.2.1. Testing apparatus •

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Much of biomechanical testing involves stress or strain application using some form of hydraulic material or universal testing machine, with common brands including Instron (www.instron. com; Instron Corp., 825 University Ave., Norwood, MA 020622643, USA) and MTS (www.mts.com; MTS Systems Corp., 14000 Technology Drive, Eden Prairie, MN 55344, USA). These are very convenient in that several parameters (e.g. load, displacement, torque, etc.) can all be recorded while the specimen is loaded under stress or strain control.m

Anatomical measurements (such as cross-sectional area) to determine stresses in the tissue should be made prior to testing if possible, as deformation of soft tissues can be quite high; and some permanent deformation is generally found, even under low loading levels. m Testing machines come in a wide range of degrees of sophistication. The simplest ones are limited to axial tension and compression loading, while the more complex ones will allow bending and axial torque to be applied and recorded either separately or simultaneously. However, it should be noted that there are some important considerations when using these machines that should not be overlooked.

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rigid constraint specimen

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Fig. 9. Boundary effects in nondiscrete tissues. This experiment to determine the tensile modulus of a skin specimen will give different values if the specimen is (a) unconstrained, (b) constrained, or (c) partially constrained by oversizing. An equal stress applied at the ends of the specimen will result in different strains, as illustrated. Similar effects can be seen in other nondiscrete musculoskeletal tissues such as cartilage, fascia, and cancellous bone.

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It is quite possible to input parameters into the machine which it is not capable of matching, so care should be taken on this account. If a specimen is to be strained over a period of less than half a second, it is unlikely that the hydraulics of the testing machine will be able to respond sufficiently quickly to achieve this strain rate. The machine will respond as fast as it can, but it is better to check (rather than just assume) that the machine is performing as required when using large or rapidly applied loads. Loading using universal testing machines should preferably be under strain or displacement control to avoid large changes in the strain rate because the observed stiffness of viscoelastic tissues is strain-rate–dependent. A common feature of universal testing machines is that the motion of the specimen is constrained to a few simple directions and a single axis. For example, axial torque will always be applied and

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measured around the axis of the testing machine; whereas the in vivo rotational axis of the specimen may not be aligned with the axis of the testing machine, and indeed may not occupy a unique position (the axis of rotation may change with load). However, adaptations such as a self-aligning mounting may in some cases be used to avoid such problems (Fig. 10). It should also be noted that testing machines constrain the motion of the specimen by applying a unique strain pattern and measuring the stress response. When larger strains or displacements are produced (such as flexion or extension of a joint or a series of joints), the strain patterns found in vivo should also be considered.n

Fig. 10. A self-aligned mounting system for use in conjunction with testing equipment that has a single unique axis of rotation (dotted line). Two sliding rails perpendicular to each other will allow free translation in the horizontal plane without rotation, and the application of a torsional load via this arrangement will therefore allow the axis of rotation of the specimen to align with that of the testing machine. n

In cases such as those indicated in the above point, constrained testing machines may not be appropriate; instead, testing by the application of an approximate physiological force to the unconstrained specimen may be a better approach.

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Examination of the motion and strain response of the specimen as well as comparison with in vivo results (from radiography, etc.) will indicate whether a physiological response is seen, and thus whether the testing is physiologically relevant.

2.2.2. Loading or testing jigs and associated monitoring devices •

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The alternatives to testing machines are often simple loading jigs, which apply a force to the unconstrained end of the specimen via deadweight, spring, etc. The resultant motion or strain of the specimen is therefore dependent on the force vector applied rather than on the motion constraints of the testing machine. In general, the force applied in vivo may be fairly complex, and the motion and/or strains produced in vitro should be compared to those in vivo so as to establish whether the loading regime represents a reasonable physiological loading pattern. While loading by deadweight, spring, etc. in a simple jig allows the force to be recorded, the displacement/strain also generally needs to be recorded. This can be done optically (by radiography or CCTV monitoring of calibrated markers, as used in gait analysis), mechanically (by displacement transducer), or electronically (by strain transducers or position sensors). Advances in robotics have allowed specimens to be tested without motion constraint, while at the same time automatically recording the force and displacement data.o

2.2.3. Specimens for testing: intact vs. operated specimens •

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The testing of intact specimens is normally aimed at finding the mechanical properties of normal tissue, but can also be used to

While this sort of testing requires highly sophisticated testing equipment that is not typically available, the advantages of universal testing equipment are retained without constraints on the motion of the specimen. However, it should be noted that the working load of many robotic arms is low, and a higher working load is often achieved at the cost of positional accuracy of the robotic arm.

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examine the mechanical effect of pathological conditions, if specimens can be obtained. The operated condition refers to some alteration (e.g. sectioning of tissues) that is used to mimic a pathological situation in a systematic way, such as sectioning of the knee ligaments to mimic an injured knee following anterior cruciate ligament (ACL) rupture, etc. Operated conditions frequently involve mimicking an injury and then repairing it with instrumentation in order to evaluate the effectiveness of the instrumentation to restore the normal mechanical properties. Operated and intact situations are distinct and separate. Care should be taken when conclusions drawn from an operated situation are applied to an intact situation. It is generally assumed that the interaction between the removed and the remaining tissues is negligible.p Operative testing typically involves testing the specimen intact, then following operation, and again following repair or instrumentation. Care should be taken to retain the original loading alignment if the specimen is removed from the testing apparatus for the operative procedures to be carried out. A removable alignment device can be temporarily clamped to the two ends of the specimen to maintain the alignment of the specimen during simulated operation (Fig. 11). If the operative procedure is carried out with the specimen constrained in the testing apparatus, changes in loading should be considered.

2.2.4. Mechanical testing (1) Preconditioning •

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Preconditioning is also sometimes used to “warm up” the specimen. Results found from the first run of a test are often different from those found from following cycles, with the recorded values

The same caution applies to the progressive sectioning of various tissues. The order in which sectioning is performed may influence the various proportion of loads that are inferred to be supported by these tissues. The greater the extent of sectioning, the less likely the tissue is to reflect its in vivo mechanical function.

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Fig. 11. Interbody spinal fusion is simulated using bone cement. A jig has to be used to hold the specimen so as to maintain the anatomical alignment when the intervertebral disc is removed.

gradually approaching a consistent trend. Some controversy exists regarding preconditioning. While more consistent and reproducible results are found after preconditioning, this does not necessarily make the results more physiologically relevant (Aspden 1990); preconditioning will also cause hysteresis effects in the specimen. If preconditioning is to be carried out, it is strongly recommended that data from the first run (without preconditioning) are also recorded.q (2) Quasi-static loading •

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Static (stepwise) or quasi-static loading is probably the most common type of loading. The specimen is loaded either incrementally (stepwise) or continuously at a rate slow enough to obtain the same results as those by the application of a static load. Quasi-static

The data with and without preconditioning should be compared; and if large differences are found, careful consideration should be given to which data set is most appropriate for the aims of that particular series of biomechanical testing.

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loading is the most simple and convenient way to measure elastic material properties such as stiffness, ultimate strength, strain, etc., and is generally carried out on some sort of universal testing machine. Quasi-static loading to failure will give an indication of the failure mechanism, ultimate strain, etc., but attention should be paid to the clinical and physiological conditions. For example, ACL rupture of the knee is a common injury in many sports such as skiing and soccer, and the common load-to-failure mechanism in vivo is therefore a traumatic, high-speed load; in vitro quasi-static loading may not adequately resemble physiological failure conditions. Quasi-static loading using a testing machine should be under strain control (at a constant strain rate) as far as possible. Most musculoskeletal tissues are viscoelastic and, as such, the stiffness depends on the strain rate. If a specimen is tested at a constant stress (load) application rate, the strain rate will vary and therefore so will the stiffness. Viscoelastic effects may also need to be taken into consideration during static or quasi-static loading, as the values recorded under stepwise loading will “drift” with time after loading. Recording the value at a specific time after the load application will minimize errors caused by this effect. Pilot testing is recommended to determine the most appropriate time (Fig. 12).

(3) Viscoelastic properties •

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Most musculoskeletal materials are highly viscoelastic: apart from the amount of load or strain applied, the mechanical properties will depend as much upon the time over which the load or strain is applied. Stress relaxation (where the strain is held constant) and creep (where the stress is held constant) are the two most convenient and widely used methodologies for quantifying the viscoelastic properties of biomaterials (Fig. 13). For stress relaxation, the stress is recorded as a function of time; and for creep, the strain is recorded as a function of time. Viscoelastic properties can also be investigated by observing the hysteresis loop between loading and unloading, or by observing

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Fig. 12. Viscoelastic effects such as creep can complicate data collection during stepwise loading. Data should be recorded at a consistent time following loading, and an understanding of the nature of the viscoelastic effect is useful in determining the most appropriate time for data collection. (a) If the creep effect is slow but long-lived, recording data almost immediately after load application will be more efficient. (b) If, however, the creep is rapid but short-lived, then waiting until the strain value begins to plateau will help to minimize errors without causing unduly long experimentation times.

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Fig. 13. Typical viscoelastic phenomena for creep and stress relaxation testing. (a) In creep, a constant stress is applied between 0 and t. This produces an instant elastic deformation (0A), followed by the creep curve (AB). Removal of the stress results in instant elastic recovery (BC), followed by the creep recovery (CD). Note that there is often a permanent deformation (D) following testing. (b) In stress relaxation, a constant strain is applied, and the stress falls in an exponential fashion until the stress is removed at time t. If time t is sufficiently long, then the stress will typically reduce to a constant nonzero value.

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the response to cyclic loading of the specimen at higher frequencies. High-frequency cyclic loading of larger, stiffer specimens is often not possible simply due to the problems of generating sufficient load over such short time periods. Viscoelastic phenomena typically depend quite highly on temperature and humidity, and the full spectrum of viscoelastic relaxation or creep will often extend over hours, so variations in environmental conditions should be considered.r (4) Impact and fatigue testing •

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Stepwise and quasi-static loading are typically used to define the mechanical characteristics of a specimen. The aim of impact and fatigue testing is generally slightly different, in that the priority is normally to replicate the conditions under which mechanical failure of the tissues takes place in vivo. As such, impact and fatigue tests are often destructive, but not exclusively so. It is not normally possible to conduct impact testing using a testing machine, as the load application rate in these machines is generally limited. Some form of drop-weight system is generally the simplest mechanism for impact testing; and allows the impact energy to be estimated as the product of mass, height, and acceleration due to gravity (m × g × h). For realistic reproduction of impact injury, the direction of the load and constraints on the motion of the specimen need to be considered carefully. The complexity of impact testing varies widely. Simple impact testing is mainly qualitative, describing the type and extent of injuries found under different impact directions and different impact weights. Detailed quantitative analysis of impact testing requires synchronized high-speed data acquisition in conjunction with accelerometer data. While impact testing is not normally carried out on testing machines, these are very convenient for fatigue testing, which

It should also be noted that higher temperatures will tend to promote tissue decay in vitro, and so the testing lifetime of the specimen needs special consideration in such cases.

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consists of cycling a specimen through a repeat load until evidence of failure is found. Application of a consistent loading pattern over thousands of cycles or greater generally requires some automated testing machine and a well-controlled environment, as the specimen will deteriorate due to prolonged testing. Failure during fatigue is often gradual, and may not be obvious from visual inspection of the specimen. The mechanical properties should be monitored during fatigue (either continuously or at regular intervals of N cycles) to check for any signs of decreasing stiffness, etc. Fatigue failure is dependent on both the amplitude and frequency of loading applied and the number of cycles. Estimated physiological values should generally be used for the amplitude and frequency of loading, but the limited testing lifetime of the specimen generally means that the number of load cycles will be less than those experienced in vivo. Some pilot testing is often required to determine the relative load and maximum cycle numbers that will produce fatigue failure within a reasonable experimental time.

(5) Destructive and nondestructive testing •

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Destructive testing simply involves testing the tissue to failure. This can be done under any of the testing conditions, but the loading mechanism should be relevant to the clinical situation. Impact (trauma) and fatigue (repeat stress fractures) are common modes of tissue failure in vivo. Particular care should be taken with the load application rate when testing viscoelastic tissues to failure.s Nondestructive testing simply involves testing the specimen to stress or strain values below those that will cause tissue failure.

s As mentioned, viscoelastic tissues will show different mechanical properties when tested at different load rates, but they will also show very different failure mechanisms. For example, a ligament tested to failure at a slow loading rate is likely to show a ductile failure mechanism by fiber pull-out, whereas the same specimen tested under impact is likely to show a brittle failure mechanism with crack propagation and fiber–matrix debonding. Complex loading patterns are often involved during tissue failure in vivo and may sometimes be difficult to replicate in vitro; careful comparison should thus be made between the in vitro and in vivo injury patterns.

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Approximations to physiological loading are generally used, but determining what constitutes a realistic in vitro representation of in vivo loading is not always straightforward. Load or stress values can generally be found in the literature, based on various methods including analysis of electromyogram (EMG) activity, theoretical models, and in vivo measurements (Bloebaum et al. 1997). The in vivo strains are sometimes easier to measure (by radiography), and these may also be used as the criteria for physiological loading. Some degree of preloading may be required to represent the in vivo condition. For example, most ligaments and tendons are prestrained in situ, and lumbar spinal joints are subject to a compressive force of approximately 50% of body weight. Preloading should preferably be calculated using data or measurements made prior to testing in order to replicate the in vivo situation for individual specimens. The stiffening effect of constraining the proportions of the tissue should also be considered. If the dimensions of an extended tissue are constrained along directions perpendicular to the strain axis, this will have the effect of stiffening the tissue. Such a constraint may or may not be in place in vivo. If more than one loading test is applied to a single specimen, then the testing sequence should be noted. Viscoelastic effects, fatigue, and microdamage may all have an effect on the results; and if at all possible, the testing sequence should be randomized to avoid such effects from skewing the results.

2.2.5. Mechanical testing on bone specimens •

Fracture healing research and osteoporosis studies involve mechanical testing on bone specimens. Our recent summary on testing bone specimens provided comprehensive basic knowledge on bone biomechanics as well as testing techniques for bone specimens at organ, tissue, and matrix levels using both destructive and nondestructive or noncontact methods (Qin and Zhang 2005). In particular, we specified how to obtain the most up-to-date

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information on testing methods that are described in homepages of some relevant or known research societies and device suppliers. In addition, Qin and Zhang (2005) mentioned practical tips for the preparation and preservation of bone specimens. Factors affecting testing results or data interpretations were also summarized to avoid inconsistent and incomparable testing results.

3. Data Evaluation 3.1. Results and interpretations It is important that specimens should be retained after testing. If spurious results are found, the specimen can then be examined to establish potential causes for the inconsistency in results. Posttest examination of the physical properties of the specimen to obtain some understanding of the relationship between structure, mechanical properties, and function is a common feature of experimental biomechanics. A vast majority of the biomechanical testing carried out is aimed at investigating the human condition; but at the same time the majority of testing is performed on animal specimens, as human specimens are limited. The effects of different species should not be neglected, and studies have been carried out to investigate the mechanical properties of human connective tissues as compared to those of different laboratory animals (Yingling et al. 1999).

3.2. Influence of biological variability of specimens Biological variability often dominates the results of mechanical testing. For example, the bone of a young, active male donor is likely to be several times stiffer and stronger than that of an aged female donor. This biological variability arises at the macroscopic tissue level. For instance, the tissue properties of cortical, cancellous, and osteoporotic bone are similar at the material level, but vary widely at the structural (tissue) level, mainly due to differences in porosity. Careful distinction should therefore be made between the material properties and the structural properties of the entire tissue. For example,

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measurement of ligament stiffness in terms of stress and strain will produce consistent results that take the dimensions of the tissue into account; the material parameters of many tissues (elastic modulus, Poisson’s ratio, etc.) have been comprehensively documented in standard texts. However, the actual dimensions of the material may vary widely depending on the donor, and as such recording forces and displacements (rather than stresses and strains) may give a better idea of the properties of the entire tissue. The calculation of material parameters such as stress and strain will require conversion by the dimensions of the material. This is not always straightforward, as the cross-sectional areas of many tissues vary widely along their lengths. Specimens may be prepared to uniform areas, or mean values for the cross-sectional area can be used. The most significant effect of biological variability is in the quantitative results of mechanical testing. The qualitative results should therefore be examined critically for consistency between specimens, as different qualitative results are generally a better indicator of differences between specimens (e.g. experimental vs. normal/control groups). A large variation in the results may not be simply due to biological variability, but may also indicate a poor experimental design or setup. Results should always be examined critically with this in mind. Because of biological variability, a nondestructive load may result in damage to tissues that are somewhat weaker than the average. The specimen should be observed carefully during loading, and any noises or sudden “jumps” in displacement noted, as these often indicate failure. If damage is suspected, the specimen should be examined carefully, by dissection if necessary. If no signs of damage can be found in the specimen and the data show no clear evidence of a failure point (sudden drop in load), then the data must be included in the analysis. If data sets are to be excluded from analysis (because of damage, technical problems, etc.), the decision to exclude the data should be made before any analysis is carried out. If the data are included in the analysis and give very different results, these results should stand unless overwhelming evidence can be found to validate its exclusion. Mathematical equations for determining the mechanical properties of specimens for various standard biomechanical tests — such as

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three-point bending, four-point bending, and torsional and pure shear tests — are available in standard handbooks (An and Draughn 2000). As musculoskeletal tissues are usually of irregular geometry, caution should be taken in applying equations with assumptions of regular geometrical dimensions. Moreover, correct units should be used: most equations use meter and Newton; while most dimensions are measured in millimeter, kilogram, or even non-SI units.

3.3. The role of statistical analysis Results of biomechanical testing often require statistical analysis to determine whether any significant changes have been found (e.g. between experimental and control groups). All statistical testing should be included in the experimental design and decided prior to testing. Decisions on which statistical tests to use after the results have been reviewed will tend to be biased in favor of a positive result. If it becomes apparent that statistical testing is required after the initial analysis of results, the choice of testing should be made by a statistician familiar with the aims and methods of testing but blinded to the results obtained.

4. Summary The mechanical testing of musculoskeletal tissues can be divided into three major stages: (1) tissue storage, handling, preparation, and mounting; (2) testing methodologies; and (3) data analysis. The specimens should be stored to minimize autolysis and drying effects when they cannot be tested within 24 hours of excision from the donor. Mounting is a critical step for testing; and great care should be paid to avoid damaging, overstraining, or misalignment of the specimen. Custom-made mounting jigs and frames are commonly used to attach specimens to the test equipment. The specimens can be tested using conventional testing machines to examine tissue properties such as load, displacement, torque, etc. Alternative simple loading jigs via deadweight and spring can also apply a force to the unconstrained end of the specimen for

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measurement. A more advanced technique of robotics allows specimen testing without motion constraint, while at the same time automatically recording the force and displacement data. Importantly, the viscoelastic properties of the tissue as well as the experimental design, testing sequence, loading frequency, and amplitude should be noted. Specimens should be retained after testing and examined to establish potential causes for any inconsistency in results. Posttest examination of the physical properties of the specimen to obtain some understanding of the relationship between structure, mechanical properties, and function is a common feature of experimental biomechanics. During data analysis, biological variability often dominates the results of mechanical testing, but can be minimized by a better experimental design or setup. Finally, it is reminded that if any data included in the analysis gives very different results, these results should stand unless overwhelming evidence can be found to validate its exclusion. We hope that this chapter will help in the choice of an appropriate strategy, and indicate areas where particular care should be taken.

References An YH, Draughn RA (eds.). Mechanical Testing of Bone and the Bone–Implant Interface. CRC Press, Boca Raton, FL, 2000. Ashman RB. Ultrasonic determination of the elastic properties of cortical bone: techniques and limitations. PhD thesis, Tulane University, New Orleans, LA, 1982. Aspden RM. The effect of boundary conditions on the results of mechanical tests (letter to editor). J Biomech 23(6):623, 1990. Black J. Dead or alive: the problem of in vitro tissue mechanics. J Biomed Mater Res 10(3):377–389, 1976. Bloebaum RD, Skedros JG, Vajda EG et al. Determining mineral content variations in bone using backscattered electron imaging. Bone 20(5): 485–490, 1997. Bonfield W, Tully AE. Ultrasonic analysis of the Young’s modulus of cortical bone. J Biomed Eng 4(1):23–27, 1982.

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Burstein AH, Currey JD, Frankel VH, Reilly DT. The ultimate properties of bone tissue: the effects of yielding. J Biomech 5(1):35–44, 1972. Burstein AH, Wright TM (eds.). Fundamentals of Orthopaedic Biomechanics. Williams & Wilkins, Baltimore, MD, 1994. Chae Y, Aguilar G, Lavernia EJ, Wong BJ. Characterization of temperature dependent mechanical behavior of cartilage. Lasers Surg Med 32(4):271–278, 2003. Choi K, Kuhn JL, Ciarelli MJ, Goldstein SA. The elastic moduli of human subchondral, trabecular, and cortical bone tissue and the size-dependency of cortical bone modulus. J Biomech 23(11):1103–1113, 1990. Evans FG (ed.). Mechanical Properties of Bone. Thomas, Springfield, IL, 1973. Helvatjoglu-Antoniades M, Papadogiannis Y, Lakes RS et al. The effect of temperature on viscoelastic properties of glass ionomer cements and compomers. J Biomed Mater Res B Appl Biomater 80(2):460–467, 2007. Hukins DW, Kirby MC, Sikoryn TA et al. Comparison of structure, mechanical properties, and functions of lumbar spinal ligaments. Spine 15(8):787–795, 1990. Pelker RR, Friedlaender GE, Markham TC et al. Effects of freezing and freeze-drying on the biomechanical properties of rat bone. J Orthop Res 1(4):405–411, 1984. Pietrucha K. Changes in denaturation and rheological properties of collagenhyaluronic acid scaffolds as a result of temperature dependencies. Int J Biol Macromol 36(5):299–304, 2005. Qin L, Zhang M. Mechanical testing for bone specimens. In: Deng HW, Liu YZ (eds.), Current Topics of Bone Biology, World Scientific, Singapore, pp. 177–212, 2005. Smeathers JE, Joanes DN. Dynamic compressive properties of human lumbar intervertebral joints: a comparison between fresh and thawed specimens. J Biomech 21(5):425–433, 1988. Turner CH, Burr DB. Basic biomechanical measurements of bone: a tutorial. Bone 14(4):595–608, 1993. Yingling VR, Callaghan JP, McGill SM. The porcine cervical spine as a model of the human lumbar spine: an anatomical, geometric, and functional comparison. J Spinal Disord 12(5):415–423, 1999.

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Chapter 48

Motion Analysis in Musculoskeletal Research Zong-Ming Li

Motion analysis is increasingly used to study the complex kinematics of the musculoskeletal system. In this chapter, both the theory and applications of motion analysis are presented. After the definition of a Cartesian coordinate frame is introduced, a description of transformations between multiple coordinate frames is given; the decomposition of a transformation matrix into anatomical joint motion parameters (e.g. Euler angles) is then explained. Kinematic analysis in musculoskeletal research is illustrated by several examples. The first example describes a reaching-and-grasping task in which mathematical transformations are applied to position the hand with respect to an object during grasping. The second example demonstrates the utility of motion analysis in revealing the coupling motion of the wrist between flexion-extension and radial-ulnar deviation. The third example shows the application of the motion analysis technique to the study of thumb kinematics, providing insight into the complex movements of thumb joints generated by individual muscles. The last example illustrates the study of knee biomechanics, including a description of knee joint kinematics during functional activities and determination of in situ ligament forces using robotic technology. It is hoped that the theoretical knowledge and biomechanical examples will help readers apply the motion analysis technique to various research problems associated with the musculoskeletal system. Keywords:

Motion analysis; kinematics; joint; hand; wrist; thumb; ligament.

Corresponding author: Zong-Ming Li. Tel: +1-412-6481494; fax: +1-412-6488548; E-mail: [email protected]

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1. Introduction An important aspect of musculoskeletal system research is the quantification of segment and joint kinematics in three-dimensional (3D) space. Kinematics of human motion is a valuable method for assessing the function of the musculoskeletal system. A complete and accurate description and understanding of human movement are important in diagnosing musculoskeletal disorders and in assessing disease severity and treatment efficacy. Analytically, kinematic data are needed as inputs for inverse dynamics analyses, where joint forces and moments are calculated. Stereophotogrammetric methods have been used to acquire human movement data. The general principle of this approach involves tracking the locations of reference points by multiple two-dimensional (2D) views of the points. This tracking is followed by reconstruction of the 3D coordinates of each point (Abdel-Aziz and Karara 1971). The reference points are typically established using skin surface markers or sensors attached to palpable bony landmarks; alternatively, coordinates of reference points can be measured by magnetic tracking systems or linkage mechanisms. The 3D coordinates are then used for the calculation of relative motion between two segments of a joint (An and Chao 1984). To do this, mathematical techniques are employed, including the helical axis representation (Blankevoort et al. 1990; Woltring et al. 1994), the joint coordinate system (Chao 1980; Grood and Suntay 1983), and the matrix method (Zatsiorsky 1998). All of these approaches are capable of completely and accurately describing relative translations and rotations between two segments. In the author’s view, the matrix method is a particularly powerful tool to relate spatial relationships among body segments, as well as between body segments and external objects. In this chapter, the theory of motion analysis using the matrix method is first presented. Then, four examples of how motion analysis may be applied are shown. The first example uses the task of grasping an object to demonstrate how multiple coordinate frames are defined and shows simple transformation matrix manipulations. The second example shows how the range of motion of the wrist joint can be calculated from experimental data obtained with relatively simple equipment. The third example illustrates the application of motion

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analysis to the elucidation of the complex kinematic roles of individual thumb muscles. The final example demonstrates how the motion analysis method can be combined with advanced technology to solve complicated research questions related to the knee joint.

2. Mathematical Background 2.1. Formation of coordinate frames with body segments In the study of human kinematics, the global coordinate frame and local coordinate frames are commonly fixed with the external environment and body segments, respectively. When three nonlinear points within a body segment are defined in the global coordinate frame, the position and orientation of the segment can be specified by a local coordinate frame (Fig. 1). Two vectors, r1 and r2, are created between these points, and the cross-products of these vectors are calculated to define the local coordinate frame. The unit vectors of local coordinate frame 1 — I1, J1, and K1 — are I1 = r1/|r1| K1 = (r1 × r2 )/|r1 × r2| J1 = K1 × I1.

Z1

Y1

O1

r2

r1

Z0 O0

X1 X0

Y0

Fig. 1. The orientation and location of a body segment in global coordinate frame 0 are given by local coordinate frame 1. Three points within the segment are used to define the axes of local coordinate frame 1.

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The orientation and location of a local coordinate frame relative to a global coordinate frame can be expressed via a transformation matrix.

2.2. Transformation matrix between coordinate frames When local coordinate frame 1 is related to global coordinate frame 0 by translation and rotation, and the position of a point P is given by its vector components in frame 1 (Fig. 2), the location of this point in frame 0 can be expressed if the rotation matrix and location of frame 1 with respect to frame 0 are known: Ê x 0 ˆ È I 0 ◊ I 1 I 0 ◊ J 1 I 0 ◊ K 1 ˘ Ê x1 ˆ Ê a01 ˆ Áy ˜ = Í J ◊I J 0 ◊ J 1 J 0 ◊ K 1 ˙˙ ◊ Á y1 ˜ + Á b01 ˜ , 0 0 1 Í Á ˜ Á Á ˜ ˜ ÁË z ˜¯ ÍK ◊ I K ◊ J K ◊ K ˙ ÁË z ˜¯ ÁË c ˜¯ 0 1˚ 0 1 0 1 1 0 01 Î
 

(1)

[R01 ]

Z0

P Y2 O2 X2

Z2 Z1

O1 O0

Y1

X1 Y0

X0

Fig. 2. Three coordinate frames: global coordinate frame 0 and local coordinate frames 1 and 2. Frame 1 is related to the global frame by rotations around frame 0 and a translation from O0 to O1. An arbitrary point P in space can be expressed as a vector with respect to any of the frames.

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where (x0, y0, z0)T and (x1, y1, z1)T are the position vectors of point P measured in frame 0 and frame 1, respectively, and (a01, b01, c01)T is the origin of frame 1 measured in frame 0. The elements in the rotation matrix [R] are direction cosines of the X1-, Y1-, and Z1-axes with respect to global coordinate frame 0. (I0, J0, K0) and (I1, J1, K1) are unit vector sets associated with frames 0 and 1, respectively. The nine elements in [R01] are direction cosines among the vectors, giving the orientation of frame 1 with respect to frame 0. Using a 4 × 4 transformation matrix [T01], the location of point P in frame 0 can be determined by one matrix multiplication:

Ê x 0 ˆ È I 0 ◊ I 1 I 0 ◊ J 1 I 0 ◊ K 1 a01 ˘ Ê x1 ˆ Á y ˜ Í J 0 ◊ I 1 J 0 ◊ J 1 J 0 ◊ K 1 b01 ˙ Á y ˜ ˙ ◊ Á 1˜ . Á 0˜ = Í Í I K J K K c ◊ ◊ ◊ K z 0 1 0 1 0 1 01 ˙ Á z1 ˜ Á 0˜ ˙ ÁË 1 ˜¯ Í 0 0 0 1 ˙ ÁË 1 ˜¯ ÍÎ
  ˚

(2)

[T01 ]

Now consider three coordinate frames: global coordinate frame 0 and local coordinate frames 1 and 2. Several typical problems are encountered that require matrix manipulation among the three coordinate frames. Given the transformation matrices of frame 1 and frame 2 with respect to global coordinate frame 0, [T01] and [T02], respectively, the relative transformation matrix from frame 2 to frame 1 is [T12 ] = [T01]-1[T02 ].

(3)

Given the transformation matrix from frame 1 to frame 0, [T01], and the relative transformation of frame 2 to frame 1, [T12], the transformation matrix from frame 2 to frame 0, [T02], is [T02 ] = [T01][T12 ].

(4)

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Without loss of generality, the transformation of frame n to the global frame from the composition of a series of frames — frame 0, frame 1, frame 2, …, frame n — is given by chain multiplication of the corresponding transformation matrices: [T0n ] = [T01][T12 ][T(n -1)n ].

(5)

2.3. Angular orientation and the transformation matrix In many applications, it is desirable to derive the relative angular orientation of two coordinate frames based on a known transformation matrix. The method of Euler angles is a convenient way to describe the overall orientation of one frame with respect to the other. When a transformation matrix between two coordinate frames is given, the Euler angles may be determined from the elements of the rotational submatrix [R], assuming a certain order of rotations. There are a number of rotational conventions for Euler angles; that is, a given transformation matrix can be decomposed into different rotational angles, depending on which order they are applied and about which axes the rotations are performed. However, it is possible to define Euler angles in such a way that they are sequence-independent (Chao 1980; Grood and Suntay 1983). Consider two coordinate frames 1 and 2 (Fig. 2). Frame 2 can be obtained from frame 1 by a translation from O1 to O2 and successive rotations of α, β, and γ with respect to the X1-axis and the resulting Y- and Z- axes, respectively. First, translate frame 1 to O2, rotate the translated frame around the X1-axis through an angle α, and indicate this intermediate frame as O2XαYα Zα. Then, rotate frame O2XαYα Zα around the Yα-axis through an angle β and indicate this intermediate frame as O2XβYβ Zβ. Finally, rotate frame O2XβYβ Zβ around the Zβ-axis through an angle γ to give O2Xγ Yγ Zγ , which coincides with frame 2. Frame 2 can be related to

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frame 1 in terms of the Euler angles by the following transformation calculation: [T12 ] = [T1g ] = [T1Ta ][T1Ra ][Tab ][Tbg ] È1 Í0 =Í Í0 Í ÍÎ0

0 1 0 0

0 0 a12 ˘ È1 0 b12 ˙ Í0 cos a ˙Í 1 c12 ˙ Í0 sin a ˙Í 0 0 1 ˙˚ ÍÎ0

0 - sin a cos a 0

cos b cos g È Ícos a sin g + sin a sin b cos g =Í Ísin a sin g - cos a sin b cos g Í 0 ÍÎ

0˘ È cos b 0˙ Í 0 ˙Í 0˙ Í- sin b ˙Í 1˙˚ ÍÎ 0

0 sin b 1 0 0 cos b 0 0

- cos b sin g cos a cos g - sin a sin b sin g sin a cos g + cos a sin b sin g 0

0˘ Ècos g 0˙ Í sin g ˙Í 0˙ Í 0 ˙Í 1˙˚ ÍÎ 0

- sin g cos g 0 0

0 0 1 0

0˘ 0˙ ˙ 0˙ ˙ 1˙˚

sin b a12 ˘ - sin a cos b b12 ˙ , ˙ cos a cos b c12 ˙ ˙ 0 1 ˙˚

(6) where [T 1Tα] and [T R1α] stand for the translation and rotation parts of the transformation from frame 1 to frame α, respectively; and a12, b12, and c12 are the coordinates of the origin of frame 2 measured in frame 1. For a given transformation matrix, the elements of the matrix can be interpreted in terms of the Euler angles α, β, and γ. Angles α, β, and γ can be computed by equating each element in the rotational submatrix with the corresponding element in the known transformation matrix. The XYZ sequential rotation described above is convenient to use, since it matches clinical definitions of joint motion such as flexion-extension, abduction-adduction, and internal-external rotation. Thus, a particular joint coordinate system was developed to facilitate anatomical relevance during human movement (Grood and Suntay 1983). The joint coordinate system is established based on three nonorthogonal axes, with two of the axes fixed to different segments and the other being the common perpendicular. The first axis is the fixed body axis and is perpendicular to the sagittal plane of the proximal segment; the third axis is the long axis of the distal segment; and the second axis is the floating axis, generated by the cross-product of the first and third axes. To provide the body-fixed axes, local coordinate frames can be defined for the two segments.

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Z1

Y1 X1

Z2

Femur O1

AbductionAdduction

F

X2

FlexionExtention

O2 Tibia

Z0

Y2 O0 Internal-External Rotation

X0

Y0

Fig. 3. Joint coordinate system of the knee joint adopted from Grood and Suntay (1983). Cartesian coordinate frames are defined in each segment. Coordinate frame 1 is attached to the femur, and frame 2 is attached to the tibia. For both frames, the Z-axis is positive in the proximal direction, the Y-axis is positive in the anterior direction, and the X-axis is positive to the right. A floating axis (F ) is defined as a common perpendicular to the two axes fixed to the femur (X1) and the tibia (Z2). The joint coordinate system is formed by two independent body-fixed axes (X1 and Z2) and the common perpendicular (F ). Joint angles are defined by rotation occurring about the three axes of the joint coordinate system. Flexion-extension is about the femur-fixed axis (X1), external-internal rotation is about the tibia-fixed axis (Z2), and abduction-adduction is about the floating axis (F ).

For example, consider the knee joint as an example (Fig. 3). The flexion-extension axis (X1) is fixed to the distal femur. The plane of flexion-extension is fixed to the femur. The axis of internal-external rotation (Z2) is along the longitudinal axis of the tibia and moves with the tibia. The common perpendicular of X1 and Z2 defines the floating axis (F ) for abduction-adduction, which is fixed neither to the femur nor to the tibia. This definition of rotations around the axes of flexion-extension (α), abduction-adduction (β), and axial rotation (γ) is coincident with the above Euler angles, whereas the sequence of the three rotations remains independent in the joint coordinate system. In addition, the rotation angles can be easily determined by direction

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angles between the floating axis and the local coordinate frame axes as follows: cos a = J 1. e Êp ˆ cos Á - b ˜ = I 1. K 2 Ë2 ¯ cos g = J 2 . e ,

(7)

where e, I1 , J1 , J2 , and K2 are the unit vectors of the floating axis, local X1-axis, Y1-axis, Y2-axis, and Z2-axis, respectively.

3. Examples of Application 3.1. Reaching and grasping In human reaching and robotics problems, it is useful to define multiple coordinate frames. In a simple grasping example, four coordinate frames are attached to various parts (Fig. 4): frame 0 to the globe, frame 1 to the body, frame 2 to the hand, and frame 3 to the object the hand must grasp. Frame 1 is used to describe the location of the hand with respect to the body, which is the role of the proprioceptive

Fig. 4. Coordinate frames attached to the globe (0), the body (1), the hand (2), and the object (3).

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nervous system. Frame 2 is used to assess the relative distances between the object and the hand, and frame 3 is used to locate points on the object. The location and orientation of the object relative to the global coordinate frame or the body frame are often known, but the central nervous system may need to know the relative location and orientation of the object with respect to the hand so that it can be moved correctly to pick up the object. Assuming coordinate frame 3, attached to the object, is positioned relative to the global coordinate frame by the transformation matrix 0.39 0.87 3.00 ˘ È0.30 Í0.75 -0.67 0.04 10.00˙ ˙ [T03 ] = Í Í0.60 0.64 -0.49 18.00˙ ˙ Í 0 0 1 ˙ ÍÎ 0 ˚ and the body frame is positioned relative to the global frame by È 0.95 0.32 -0.03 1.00 ˘ Í -0.32 0.95 0.00 5.00 ˙ ˙, [T01] = Í Í 0.03 0.01 1.00 9.00˙ ˙ Í 0 1 ˙ 0 ÍÎ 0 ˚ what is the transformation matrix [T12] that allows successful grasping given the hand frame relative to the body frame? Chain matrix transformation from the global frame to the body frame to the hand frame can be expressed as [T02] = [T01][T12]. In order to put the hand on the object, frame 2 and frame 3 must be aligned such that [T02] = [T03]; therefore, [T03] = [T01][T12]. Solving this matrix equation for [T12] yields

[T12 ] = [T03 ][T01]-1

0.61 0.79 0.53˘ È0.06 Í0.81 -0.50 0.32 5.46 ˙ ˙. =Í Í0.59 0.62 -0.52 8.94˙ ˙ Í 0 0 1 ˙ ÍÎ 0 ˚

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The three columns of the 3 × 3 rotational submatrix [R] and the last column give the orientation and location of the hand frame in terms of the body frame, respectively. Positioning of the hand frame as such allows successful grasping of the object.

3.2. Wrist kinematics The wrist is capable of an arc of active angular motion that results from interplay among the complex articular contour of carpal bones, a highly developed arrangement of ligament meshwork, and the intricate action of the muscular system. Wrist kinematic data provide important information for investigating underlying pathologies. Conventional wrist joint goniometry in clinics concerns the range of motion in isolated planes, such as flexion-extension in the sagittal plane and radialulnar deviation in the frontal plane. In this example, we demonstrate the utility of kinematic principles in describing wrist motion. A subject was asked to move the wrist in circumduction. A 3D MicroScribe digitizing device (Immersion Corp., San Jose, CA, USA) was used to record the location of six surface points of the upper extremity in a global coordinate frame (Fig. 5). The local X1-axis was oriented normal to the plane determined by points 2, 3, and 4.

Fig. 5. Experimental setup for wrist motion and definition of local coordinate frame 2. The points for digitization are the lateral humeral epicondyle (1), three arbitrary points on the dorsal surface of the distal forearm (2, 3, 4), the ulnar styloid (5), and the head of the third metacarpal (6).

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The local Y1-axis, representing the lateral direction, was formed by calculating the cross-product of the local X1-axis and a vector between points 1 and 6. The local Z1-axis was determined by calculating the cross-product of the local X1- and Y1-axes. The origin of frame 1 was defined to be at the wrist joint center, which was determined by fitting a sphere to the position data of point 6 using a least squares algorithm. Local coordinate frame 2 was defined by translating frame 1 along the X1-axis, so that the origin of frame 2 was on the dorsal surface. The transformation matrix between the global coordinate frame and local coordinate frame 2, [T02], was then calculated. The recorded coordinates of point 6 in the global coordinate frame were converted to frame 2 (Fig. 6). The projection angles of the position vector of point 6 in frame 2 were calculated, representing the flexionextension and radial-ulnar deviation angles. The ulnar styloid was found to be located 19.4 mm dorsal, 22.2 mm medial, and 18.0 mm proximal to the wrist joint center. It was observed that extension was coupled with radial deviation, and that

Fig. 6. Surface plot of point 6 on the third metacarpal head in local frame 2 located dorsal to the wrist joint center (units are in mm).

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Fig. 7. Wrist joint flexion-extension vs. radial-ulnar deviation (units are in degrees).

flexion was coupled with ulnar deviation. A maximum flexion of 40° occurred at ~40° of ulnar deviation (Fig. 7). A maximum extension of 60° was reached at a radial deviation of 20°. Simultaneous extension and ulnar deviation, as well as flexion and radial deviation, were very limited.

3.3. Thumb kinematics The thumb plays a critical role in human hand function, and its motion is complex in three dimensions (Li and Tang 2007). Our knowledge of the function of thumb muscles has been dominated by unidirectional, simplified nomenclature such as flexors or extensors. In our laboratory, we investigated 3D thumb joint movements produced by individual extrinsic thumb muscles using cadaveric hands. 3D thumb kinematics was obtained by optically tracking surface markers attached to the hand. T-shaped plates with twelve 5-mmdiameter reflective markers were attached to the hand (Fig. 8) to

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Fig. 8. A specimen (with surface markers) mounted in the fixation apparatus for muscle loading and thumb motion recording.

establish local coordinate frames (Fig. 9). Markers 1 and 2, 4 and 5, and 7 and 8 were attached to the dorsal aspect of the first metacarpal, proximal phalanx, and distal phalanx, respectively, and aligned with their respective long axes. The coordinate frame of the first metacarpal was formed by markers 1, 2, and 3. The Z-axis pointed proximally from marker 2 to marker 1; the Y-axis was perpendicular to the plane formed by markers 1, 2, and 3, and pointed dorsally; and the X-axis was orthogonal to the Y- and Z-axes and pointed towards the medial border. The coordinate frame for the proximal phalanx was defined by markers 4, 5, and 6 in a manner similar to that used for the metacarpal, as was the coordinate frame for the distal phalanx defined by markers 7, 8, and 9. An additional plate with markers 10, 11, and 12 was attached to the dorsal aspect of the second and third metacarpals to establish a reference coordinate frame representative of the stationary hand and forearm. The Z-axis pointed proximally from marker 11 to marker 10; the X-axis was perpendicular to the plane formed by markers 10, 11, and 12, and pointed to the dorsal hand; and the Y-axis was orthogonal to the Z- and X-axes and pointed in the radial direction. The cadaver specimens were dissected to expose the musculotendinous junctions of the extrinsic thumb muscles (Fig. 8). Each

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Y X Y X

Z

Z X Y

Z X

Y Z

Fig. 9. Coordinate frames of the thumb and forearm established by the surface markers. Extension (+) and flexion (−) occur about the X-axis, abduction (+) and adduction (−) occur about the Y-axis, and pronation (+) and supination (−) occur about the Z-axis.

muscle/tendon was loaded to 10% of its maximal force capability, while the motion of the markers was recorded by a motion system (Vicon 460; Vicon UK, Oxford, UK). Three-dimensional angular movements of the carpometacarpal, metacarpophalangeal, and interphalangeal joints were calculated from the marker data and subsequent transformations. The results showed that each extrinsic muscle produced unique joint angular trajectories in multiple directions. Figure 10 shows an example of the complex movements generated by the extensor pollicis longus. This muscle generated six movements, including extension, adduction, and supination at the carpometacarpal joint; extension and adduction at the metacarpophalangeal joint; and extension at the interphalangeal joint. The motion

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Fig. 10. Ranges of motion (ROM, in degrees) at the carpometacarpal (CMC), metacarpal (MCP), and interphalangeal (IP) joints produced by the extensor pollicis longus.

analysis technique thus provides a novel insight into the biomechanical role of the extrinsic muscles of the thumb.

3.4. Knee kinematics Injuries to the knee, in particular anterior cruciate ligament (ACL) tears, frequently occur during various activities. Determining knee kinematics and force carried by the ACL during different activities will allow diagnostic exams, reconstruction techniques, and rehabilitation protocols to be improved. Researchers have developed a robotic testing system with a force-moment sensor mounted on the end-effector to study knee joint biomechanics using cadaveric specimens (Woo et al. 2000). The robotic manipulator is designed to move its end-effector to a desired position and orientation with respect to its fixed base, i.e. to achieve a specified [TBE] (Fig. 11). However, knee kinematics is defined by a transformation matrix of the local coordinate frame attached to the tibia with respect to the local coordinate frame attached to the femur, [TFT]. To allow the robotic system to reproduce

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Fig. 11. Schematic diagram of the robotic testing system showing a knee mounted for testing.

the known [TFT], the robot is programmed to reproduce the following transformation matrix: [TBE ] = [TBF ][TFT ][TTE ], where [TBF] and [TTE] are constant transformation matrices between the robot base and femur, and between the tibia and robot endeffector, respectively. [TBF] and [TTE] are determined using local coordinate frames derived from digitizing points on the corresponding parts. Prior to testing, the tibia is attached to the end-effector of the robot and the femur is rigidly mounted with respect to the robot base coordinate frame. With the joint moved to the target position and orientation, the forces and moments applied to the joint are recorded. The same knee kinematics is again reproduced with the ACL transected, and a new set of force-moment data is recorded. Because the robot reproduces identical knee kinematics before and after the ligament is transected, the in situ force in the ligament is the difference

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between the two sets of force-moment data in accordance with the principle of superposition. This robotic procedure can be extended to examine a knee with various ligament reconstruction techniques. Similar studies can be performed in other ligaments of the knee and in ligaments of other joints. In addition to the motion control mode in which forces and moments are recorded in response to known kinematics, the robot system can be operated in force control mode in which resulting kinematics is measured when a known force/ moment is applied to the intact/transected knee; testing in this mode is similar to clinical examinations for diagnosing ligament injury or deficiency. Knee joint kinematics used as input parameters to the robot movement can be collected during a functional activity (e.g. jumping) or a clinical examination (e.g. anterior drawer test). A series of coordinate frames is necessary in order to determine [TFT] (Fig. 12). First, magnetic sensors 1 and 2 (or sets of markers) are attached to the femur and tibia to record the position and orientation of the two segments, allowing for the determination of transformation matrices

Fig. 12. Coordinate frames used to record knee kinematics. Coordinate frames 1 and 2 give sensor locations and orientations, and frames F and T provide the location and orientation of the anatomical features of the femur and tibia, respectively.

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[T01] and [T02]. A digitizing probe using a third magnetic sensor is used to locate anatomical landmarks and define anatomical coordinate frames of the femur and tibia, establishing transformation matrices [T0F] and [T0T]. The constant transformation matrices between the sensors and anatomical coordinate frames, i.e. [T1F] and [T2T], are calculated as follows: [T1F ] = [T01]-1[T0 F ] [T2F ] = [T02 ]-1[T0 T ]. The knee is then subjected to a functional activity or a clinical examination while [T01] and [T02] are recorded. The transformation matrices of the anatomical coordinate frames with respect to the global coordinate frames [T0F] and [T0T] are determined using the recorded [T01] and [T02] and the known constant transformations [T1F] and [T2T]:

[T0 F ] = [T01][T1F ] [T0 T ] = [T02 ][T2T ]. The derived kinematics of the anatomical femur and tibia frames in the global frame provides the location and orientation of the tibial anatomic frame with respect to the femoral frame, i.e. [TFT] = [T0F]−1 [T0T]. In addition to being used as an input to the robotic testing device, this transformation matrix between the tibia and femur can be further decomposed to clinically relevant knee kinematics in the joint coordinate system.

4. Discussion and Summary The motion analysis described in this chapter is a convenient and powerful tool for human kinematics in comparison with other currently available measurement techniques such as electrogoniometry and accelerometry. Although the goniometric method has the

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advantage of direct measurement of angles between adjacent segments, it is limited in examining motion in multiple planes simultaneously or analyzing multiple joints at the same time; in addition, relative motion between two segments does not provide segment kinematics with respect to the inertial reference frame. With accelerometers, body segment acceleration data are obtained, and velocity and displacement information of body segments can be obtained by integrating these acceleration data with appropriate boundary conditions. This calculation provides kinematics of body segments in an inertial reference frame from which joint kinematics may be obtained; however, complex computation and cumbersome instrumentation prohibit its use for many practical applications (An and Chao 1984). The 3D coordinates of points on objects of interest can be easily obtained using commercial devices such as spatial digitizers, magnetic tracking devices, or stereophotogrammetric recording systems. In particular, modern motion recording systems provide convenient reconstruction of 3D coordinates of reference locations on multiple body segments, thus subsequently allowing a description of complex human motion using matrix transformation and decomposition. With the rapid development of motion recording technology using fast-rate and high-resolution cameras, as well as more automated algorithms, motion analysis has empowered researchers to investigate the musculoskeletal system. The matrix method is the focus of motion analysis in this chapter because it offers several particular advantages: (1) The motion of an object can be described in multiple orthogonal planes, which can be strategically defined by the user. (2) The method provides spatial relationships among multiple body segments, as well as between body segments and remote external objects. The position and orientation of any segment can be described with respect to any other segment. (3) Joint rotations and translations in anatomical terms can be easily obtained from the transformation matrix by appropriately defined coordinate frames in the adjacent segments.

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(4) The matrix method can be easily implemented in conjunction with modern motion recording systems, which provide 3D coordinates of numerous reference points. (5) While this chapter primarily concerns kinematics, the matrix method also has applications in kinetic analysis, for example, converting forces and moments measured in one coordinate frame to another. The utilities of motion analysis were illustrated with several examples of biomechanical applications. The example of reaching and grasping demonstrated the attachment of coordinate frames to various body segments and external objects, which facilitates quantitative analyses of motor tasks. The second example described wrist joint motion based on coordinates of anatomical landmarks using a simple digitizing device; here, motion analysis helped reveal motion coupling of the wrist complex in multiple planes in addition to the information obtained in isolated single planes. The third example of motion analysis of the thumb helped us gain insight into the complex kinematic role of individual thumb muscles. The last example highlighted the use of the motion analysis method for the study of knee biomechanics, including expression of knee joint kinematics during functional activities and determination of in situ ligament forces using robotic technology. Motion analysis has thus proven to be indispensable for numerous complex biomechanical studies. We have described the method of motion analysis and provided a few examples of its utility. In the future, motion analysis will remain a valuable tool in many research settings, such as in the development of interactive testing environments where real-time kinematic feedback is needed. Robotic technology — with its high accuracy, precision, and automation — will be increasingly incorporated with the motion analysis method to study joint biomechanics and assist rehabilitation. It is hoped that the theoretical underpinnings and practical applications of motion analysis were amply and concisely illustrated in this chapter, and that readers will use this method to study a wide spectrum of biomechanics problems.

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Acknowledgments The author thanks Jesse A. Fisk and Savio L.-Y. Woo for their contribution to the application example on knee kinematics, and acknowledges the support of the Frank E. Raymond Memorial Research Grant from the Orthopaedic Research and Education Foundation (OREF).

References Abdel-Aziz Yl, Karara HM. Direct linear transformation from comparator coordinates into object space coordinates in close-range photogrammetry. In: Proceedings of the ASP/IU Symposium on Close-Range Photogrammetry, American Society of Photogrammetry, Falls Church, Urbana, IL, pp. 1–18, 1971. An KN, Chao EY. Kinematic analysis of human movement. Ann Biomed Eng 12:585–597, 1984. Blankevoort L, Huiskes R, de Lange A. Helical axes of passive knee joint motions. J Biomech 23:1219–1229, 1990. Chao EY. Justification of triaxial goniometer for the measurement of joint rotation. J Biomech 13:989–1006, 1980. Grood ES, Suntay WJ. A joint coordinate system for the clinical description of three-dimensional motions: application to the knee. J Biomech Eng 105:136–144, 1983. Li ZM, Tang J. Coordination of thumb joints during opposition. J Biomech 40:502–510, 2007. Woltring HJ, Long K, Osterbauer PJ, Fuhr AW. Instantaneous helical axis estimation from 3-D video data in neck kinematics for whiplash diagnostics. J Biomech 27:1415–1432, 1994. Woo SL, Debski RE, Zeminski J et al. Injury and repair of ligaments and tendons. Annu Rev Biomed Eng 2:83–118, 2000. Zatsiorsky VM. Kinematics of Human Motion. Human Kinetics, Champaign, IL, 1998.

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3D culture

165, 166

binarization threshold 301, 310 biomaterials 441–443, 448, 453 biomechanical test 422 bioreactor 39, 40, 43, 49, 50, 57, 58, 60 bisphosphonate 231, 232, 241, 243–245 bone 21, 23, 25, 26, 35, 36, 179, 181–189, 191–193, 195, 196, 198, 200–203, 205, 207–210, 216, 217, 219–222, 225, 227, 228, 313, 314, 317, 321–323, 326, 327, 477, 478, 482, 483, 485, 487–492, 583–588, 590–594, 602, 691–723 bone adaptation 63 bone bank 201 bone density 331 bone ingrowth 511, 512, 517–520, 522, 527–529 bone lengthening 381, 383, 384, 393, 397, 605, 609, 610, 612, 613

ACL reconstruction 511–516, 527, 529, 530 adipogenesis 39, 55, 56 alkaline phosphatase (ALP) 99, 100, 110, 127, 129–131 analgesia 349, 350, 354–365 angiography 301 animal model 421, 422, 435, 559, 560, 563, 564, 749, 754, 755, 763 animals 635, 636, 647 anterior cruciate ligament transection (ACLT) 559, 560 apoptosis 113, 116, 118, 121 apparent anisotropic elastic properties 671, 672, 674 atrophic nonunion 401–403, 408, 415, 416 avascular necrosis (AVN) 495, 496 891

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bone loss 457, 458, 463, 465, 469–471 bone microarchitecture 635, 636, 647 bone mineral density (BMD) 279, 280, 285, 286, 291, 421–423, 427–429, 431, 433, 435, 535, 545, 649–652, 654, 656–658, 660–663, 668 volumetric 635, 636 bone mineralization 619, 620, 627–629 bone remodeling 63, 231, 232, 245 bone remodeling process 671, 673, 685 bone repair 63 bone–tendon (B-T) junction 535 calcium phosphate ceramics 249, 250, 265 callotasis 381 callus 301, 302, 304, 307, 309–311 calvarium 369, 370 canalicular system 135, 136, 140–142, 144, 145 cartilage 21, 165–168, 173, 179, 219, 223, 749–765 casting 457, 469, 470 cell traction force (CTF) 773–777, 781–785 cell traction force microscopy (CTFM) 773–775, 778, 781–785 centrifugation 165, 169, 172, 173 chondrocytes 153–161, 165–170, 173

coculture 99–101, 104, 105 confocal microscope 135, 140, 141 contact microradiography 231, 232, 234, 236, 241, 243, 245 cortical porosity 692 culture 153–156, 158, 159, 161 cytostaining 127, 130, 131 data analysis 3, 16, 17 DCE-MRI 729–733, 736–738, 740, 742–745 decalcification 179, 180, 182, 184–186, 199, 301, 305, 307, 309 decalcification method 201 denervation 477–479, 492 differentiation 75–78, 82, 84, 85, 89 distraction 619–623, 625–629 distraction osteogenesis 381, 382, 384, 387–390, 398 disuse 691, 692, 695, 716–721 disuse model 457, 463, 467, 469, 470 disuse osteoporosis 478 DNA microarray 3–6, 8, 9, 16 double labeling 135 DXA 381, 388, 390, 605–607, 611, 612, 619–629 dynamic MRI 495, 499–501 ECM 63 endochondral ossification 535 ethics 349–353 explant culture 127–129, 131, 132 extracellular matrix 153, 154

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893

failure behavior 671, 683–685 fat 313, 315, 317, 326, 327 fibrocartilage 535, 536, 551, 553, 554 fixatives 179–181, 187 fluorescence-activated cell sorter 127, 131 fluoride ion 331, 337–341, 344 fluorine 331 fracture 301–305, 307–310, 401–403, 406–408, 410–416 fracture healing 279, 283, 285, 286, 477–480, 492 fracture risk 457, 458

image scanning 3, 14 immunohistochemistry 21, 32, 35, 36 implant 583–594, 602 in situ hybridization 21–23, 28, 32, 33, 35 in vitro 635, 636, 646, 647 in vitro study 839 in vivo 313–318, 635, 636, 641–644, 647, 671–673, 683–686 innervation 477–479, 492 interstitial bone 813, 815, 816, 819

generation 75–77, 79 giant cell tumor 147 goats 349, 350, 353–355, 357, 358, 361, 362, 364, 365, 421–427, 430, 431, 433–435 growth plate 153–155, 569–572, 574, 576

joint 869–871, 875, 876, 879–881, 883–889 joint space narrowing 749, 750, 765

hand 869, 877–879, 881, 882 high resolution 789, 790 high-resolution pQCT 635, 636 hindlimb suspension 457, 459, 460, 462, 463 hip arthroplasty 605, 610, 613 histology 559, 562 humans 635, 636, 641–643, 647 hybridization 3–7, 13, 14, 16 hypertrophy 154 icariin 39, 47, 48, 51, 54–56 image registration 649–651, 654, 664

kinematics 869–871, 879, 881, 884–890 knee 559–562, 565, 749–751, 753–765 knee arthroplasty 605, 610–613 ligament 869, 879, 884–886, 889 limb lengthening 619, 627–629 linear elastic analysis 671, 677 loading 789–791, 796, 798, 800, 802, 803, 805 loading conditions 671, 673, 677, 684, 685 long digital extensor tendon 512, 513, 515 macrophage 147–150 manual palpation 441, 450, 451

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mapping 789, 790, 797 material property 789 mechanical testing 381, 387, 390, 395, 839, 840, 856, 863–867 mesenchymal stem cells (MSCs) 39, 40, 46–52, 54, 55, 58–60, 63, 64 extraction 63 proliferation 63 methods 583, 584, 587, 602 microangiography 495, 501, 502, 505 microcarrier 39, 43, 49, 50, 57, 58, 60 microcirculation 729, 730, 743 micro-computed tomography 313–315 micro-CT 279, 280, 297, 301, 302, 307, 309–311, 441, 448, 452, 495, 501, 502, 504, 505, 511, 512, 517–519, 527, 529, 583–588, 591, 593, 595, 597, 598, 600–602 microdamage 691, 692, 694, 695, 708, 709, 712, 713, 716, 721 micro-FE 671–680, 683–686 micromechanical test 813–815, 822, 827, 830–834 middle suture 369, 374–377 mineralization 301, 309, 789, 805, 807 degree of 231, 232, 235, 239–245, 671, 684 mineralized bone 219, 225, 227, 228 mineralized nodules 99, 106, 111

molecular imaging 331, 332 motion analysis 869–871, 887–889 mouse model 369 MRI 749–752, 754–757, 760–765 multinuclear giant cells 147, 149, 150 multinucleate giant cells 147 muscle 313, 315, 319, 327 muscle atrophy 457, 458, 469 musculoskeletal tissues 839, 840, 853, 858, 866 myofibroblasts 773, 783–785 nanoindentation 789–797, 802–807 neovascularization 301, 302, 304, 310 neurectomy 457, 470 NMR 691, 692, 694–706, 708–723 NMR system 692, 697 nonlinear analysis 671, 683, 684 nonunion 401–403, 405–417 normal and delayed healing 535 operative care 349, 353, 355, 356 osteoarthritis (OA) 559, 749, 750, 758, 759, 764 osteoblasts 75–77, 87, 88, 90, 99, 100, 105–111 osteocalcin 127, 131 osteoclast 113, 114, 116–122, 124, 147, 148 osteoclastogenesis 369, 370, 374 osteocyte 76, 135–137, 139–142, 144, 145

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osteogenesis 39, 46, 301, 308, 309, 535, 545, 549, 553, 619–622, 629 osteoid 219, 225, 226, 228 osteointegration 583, 584, 587 osteolysis 369, 370, 372, 374, 375 osteonal bone 813, 816 osteoporosis 279, 280, 297, 421–423, 434, 435, 649, 650, 659 osteosarcoma 730–732, 740–743 osteotomy 569, 570, 573 ovariectomy 421, 422, 425, 433, 434 partial patellectomy 535–539, 541, 542, 546–548, 550, 553, 554 patella–patellar tendon complex 535 peak enhancement percentage 495, 500, 501 pellet culture 165–167, 170–173 perfusion function 495, 498, 499 periosteal cell 127–132 periosteum 401, 405, 406, 414, 415 pharmacokinetic model 729–731, 736, 737, 739, 740, 743, 744 physeal closure 569, 570, 574 physeal injury 569 pore size 691–698, 701–704, 711, 713, 718–722 porosity 583, 584, 586, 587, 593–595, 597, 599, 601, 602 positron emission tomography 331, 332

895

posterior spinal fusion 441, 442, 444, 448, 449, 453 pQCT 381, 388, 390, 392–394, 441, 448, 512, 524, 525, 529 prethrombotic disorders 495, 496 probe 8, 11, 13 proliferating cell 147, 148 proliferation 153, 154, 158 proximal femur 649, 650, 652, 653, 659, 661, 662, 664, 666, 667 quantitative computed tomography (QCT) 649–652, 654, 659–662, 664–668 rabbits 349, 350, 353–355, 357, 360–365, 401, 403, 405, 407, 408, 410–416, 535–537, 540, 543, 554 radiographs 749, 750, 765 rat 301–304, 309, 310, 349, 357, 360–362, 364, 559–562, 565 regeneration 75–77, 79 renal tubular epithelial cells 99 resorption pit 113, 117, 119, 121, 122, 124 rheumatoid arthritis 729–731, 744 Salter–Harris fracture 569 sawing 249, 251, 263, 264, 273 scaffold 583, 584, 586, 588, 593, 595–602 scanning resolution 301, 310 Scion Image 231, 232, 235, 236, 241, 243–245

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sectioning 249, 251, 256, 261, 263–266, 273 small animal models 313, 315 small animal PET 331, 333–337, 343 spaceflight 649, 650, 651, 659, 661, 662, 664, 665, 667 spinal fusion 279, 286 spine 649–651, 659, 660, 662, 663, 666–668 spin-spin relaxation 692, 695, 708, 721 SPIO 39, 40, 44, 50, 51, 59, 60 staining 249, 251, 262, 264, 266–268, 270 stem cells 75–79, 441 steroid-associated osteonecrosis (ON) 495–497, 508 stromal-like mononuclear cells 147 surface interaction 63 surgery 349, 350, 353, 354, 356–359, 362–365

thumb 869, 871, 881–884, 889 tissue culture 147, 151 tissue engineering 441 trabecular bone 813–815, 827–830 trabecular microarchitecture 421, 422, 428, 433 TRAP 113, 115, 117–122

tendon fibroblasts 773, 781 tensile strength 535, 552 tension 813, 815, 821, 823

X-gal staining 21, 35, 37 XtremeCT 635, 636, 638, 641, 643–647

ultrasound 201, 205–207, 209, 210, 217 undecalcified histology 249–251, 256, 259, 262, 274 vasculature 279 voxel conversion 671, 674 washing 3, 10, 13, 14 water distribution 691–695, 697, 704, 707, 708, 717, 718, 721 wear debris 369, 370, 372 wrist 869, 870, 879–881, 889