Experimental Research Methods in Orthopedics and Trauma E-Book

• Herausgeber: Thieme
• Kategorie: Fachliteratur
• Sprache: Englisch

Seitenzahl: 1278

Veröffentlichungsjahr: 2015

Beschreibung

<p><fontcolor="red"><strong><em>Covering all state-of-the-art experimental research methods in orthopedic surgery and trauma</em></strong></font></p><p>From bioinformatics to nanotechnology, advances in basic research ultimately drive advances in clinical care. This book provides a comprehensive summary of all current research methodologies for translational and pre-clinical studies in biomechanics and orthopedic trauma surgery. With this "roadmap" at hand, specialists and trainees will have the tools to conduct high-quality experimental research in any area of musculoskeletal science, with a solid understanding of how the findings can be applied in patient care. </p><p>Special Features:</p><ul><li>Utilizes the principles and methodology of modern, evidence-based medicine in pre-clinical musculoskeletal research <li> Offers a comprehensive analysis of in vivo models for studying different components of the musculoskeletal system<li> Demonstrates how principles of structural, functional, and numerical biomechanics can be utilized in well-defined experimental research studies – spanning topics from fracture fixation to gait analysis to bone remodeling<li> Covers the role of new macroscopic CT and ultrasound imaging techniques for assessing bone and cartilage function <li> Explores cutting-edge developments in cell culture research, molecular testing, and tissue engineering <li> Provides practical advice, a glossary of key terminology, and hundreds of illustrations to familiarize clinicians with every aspect of designing and interpreting an effective research study </li></ul><p>With 54 state-of-the-art chapters by orthopedic surgeons, musculoskeletal physicians, biologists, engineers, physicists, and mathematicians, <em>Experimental Research Methods in Orthopedics and Trauma</em> is the authoritative reference on the topic. It is essential for clinicians, basic researchers, and orthopedic surgical trainees who need to understand experimental research methodology

Leseprobe

Experimental Research Methods in Orthopedics and Trauma

Hamish Simpson, DM (Oxon), MA (Cantab), FRCS (Edinburgh & England)Professor of Orthopedic SurgeryDepartment of Orthopaedics and TraumaUniversity of EdinburghEdinburgh, United Kingdom

Peter Augat, PhDProfessor of BiomechanicsParacelsus Medical UniversitySalzburg, Austria

DirectorInstitute of BiomechanicsTrauma Center MurnauMurnau am Staffelsee, Germany

257 illustrations

ThiemeStuttgart · New York · Delhi · Rio de Janeiro

Library of Congress Cataloging-in-Publication Data

Simpson, A. Hamish R. W., author.

Experimental research methods in orthopedics and trauma / Hamish Simpson, Peter Augat.

p. ; cm.

Includes bibliographica references and index.

ISBN 978-3-13-173111-1 (alk. paper) – ISBN 978-3-13-173121-0 (eISBN)

I. Augat, Peter, author. II. Title.

[DNLM: 1. Biomedical Research. 2. Orthopedics–methods. 3. Biomechanical Phenomena. 4. Musculoskeletal Diseases. 5. Orthopedic Procedures–methods. WE 20]

RD732

616.7'027–dc23

2014019935

© 2015 by Georg Thieme Verlag KG

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ISBN 978-3-13-173111-1

Also available as an e-book:eISBN 978-3-13-173121-0

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Contents

Foreword

Endorsement by the International Combined Orthopaedic Research Societies (I-CORS) Member Organizations

Preface

Acknowledgments

Contributors

1 Why Do We Need Experimental Research?

1 Evidence-Based Research

2 Establishing a Basic Research Facility in Orthopedic Surgery

3 Good Laboratory Practice and Quality Control

4 How to Prepare for a Period in Research

2 Structural Biomechanics

5 Physiological Boundary Conditions for Mechanical Testing

6 Static, Dynamic, and Fatigue Mechanical Testing

7 Use of Human and Animal Specimens in Biomechanical Testing

8 Whole Bone Biomechanics

9 Biomechanics of Trabecular and Cortical Bone

10 Biomechanics of Fracture Fixation

11 Biomechanical Assessment of Fracture Repair

12 Biomechanics of Cartilage

13 Biomechanics of Joints

14 Spine Biomechanics

3 Functional Biomechanics

15 Musculokeletal Dynamics

16 Measurement Techniques

17 Clinical Assessment of Function

18 Functional Biomechanics with Cadaver Specimens

4 Numerical Biomechanics

19 Inverse Dynamics

20 Principles of Finite Elements Analysis

21 Validation of Finite Element Models

22 Computational Biomechanics of Bone

23 Numerical Simulation of Implants and Prosthetic Devices

24 Numerical Simulation of Fracture Healing and Bone Remodelling

5 Imaging

25 Micro-Computed Tomography Imaging of Bone Tissue

26 Imaging Bone

27 Ultrasound Techniques for Imaging Bone

28 In Vivo Scanning

29 Imaging of Cartilage Function

30 Histochemistry Bone and Cartilage

31 Immunohistochemistry

32 Molecular Imaging In Situ Hybridization

33 Laser Scanning Confocal Microscopy and Laser Microdissection

34 Image Analysis Histomorphometry Stereology

6 Cellular Studies

35 Cell Culture Research

36 Cartilage Explants and Organ Culture Models

37 Fluid Flow and Strain in Bone

38 Biomechanics of Bone Cells

7 Molecular Techniques in Bone Repair

39 Molecular Testing

40 Genetically Modified Models for Bone Repair

8 In Vivo Models

41 General Considerations for an In Vivo Model

42 Animal Models for Bone Healing

43 Models for Impaired Healing

44 In Vivo Models for Bone and Joint Infections

45 In Vivo Models for Articular Cartilage Repair

46 In Vivo Soft Tissue Models

9 Tissue Engineering

47 Scaffolds for Tissue Engineering and Materials for Repair

48 Use of Growth Factors in Musculoskeletal Research

49 Stem Cells for Musculoskeletal Repair

50 Biological Evaluation and Testing of Medical Devices

10 Statistics for Experimental Research

51 Study Design

52 Power and Sample Size Calculation

53 Nonparametric versus Parametric Tests

54 How to Limit Bias in Experimental Research

Index

Foreword

Advances in basic research ultimately drive advancements in clinical care. When we as orthopedic surgeons contemplate the treatments available to us today in comparison to those a generation ago we readily appreciate that the effectiveness of our diagnostic and therapeutic armamentarium greatly exceeds that of our predecessors. This is driven in large part by technological advances in imaging, biomechanics, molecular biology, nanotechnology, and bioinformatics. While progress has been real, it can be argued that orthopedic surgery is somewhat behind the curve in translating these basic advances to improvements in clinical practice. Thus, the publication of this book on experimental research methods is particularly timely. The editors have compiled a series of contributions from a group of interdisciplinary scientists with expertise in a broad array of scientific disciplines. This expertise has been brought to bear in developing a comprehensive volume on state-of-theart research methodologies that can be applied to improvement in the care of patients with musculo-skeletal injuries. This volume will be of value not only to basic researchers, but also to clinician scientists who are on the front lines of musculoskeletal research. In addition, this volume will be an important resource for orthopedic surgical trainees who need to learn the basics of experimental methodology so that they have the tools to interpret the orthopedic literature in their professional lives. Experimental Research Methods in Orthopedics and Trauma is a valuable contribution to our specialty.

Joshua J. Jacobs, MDProfessor and ChairmanDepartment of Orthopaedic SurgeryRush University Medical CenterChicago, Illinois, United States

Endorsement by the International Combined Orthopaedic Research Societies (I-CORS) Member Organizations

We are delighted that a book covering the spectrum of research methodologies in trauma and orthopaedics has been produced. The diverse specialties of the contributors and their wide geographical spread will ensure that the reader is presented with a comprehensive analysis of the available techniques. We consider that researchers commencing a musculoskeletal research project will find this book a very useful starting point.

Constituent Members

Jiake Xu, MD, PhDAustralia & New Zealand Orthopaedic Research Society

Andrew McCaskie, MB, ChB, MMus, MD, FRCS, FRCS (T&O)British Orthopaedic Research Society

John Antoniou, MD, PhD, FRCSCCanadian Orthopaedic Research Society

Steven Boyd, PhDCanadian Orthopaedic Research Society

Ling Qin, PhDChinese Orthopaedic Research Society

Gang Li, PhDChinese Orthopaedic Research Society

Ting ting Tang, MD, PhDChinese Orthopaedic Research Society

Nicola Baldini, MD, PhDEuropean Orthopaedic Research Society

Nobuo Adachi, MDChairperson of Committee on International Affairs Japanese Orthopaedic Association

Gun-Il Im, MDKorean Orthopaedic Research Society

Theodore Miclau, MD, Chair, ICORSOrthopaedic Research Society

Je-Ken Chang, MDTaiwanese Orthopaedic Research Society

Oscar Kuang Sheng Lee, MDTaiwanese Orthopaedic Research Society

Associate Scientific Members

R. Geoff Richards, PhD, FBSEAO Foundation

X. Ed Guo, PhDInternational Chinese Musculoskeletal Research Society (ICMRS)

Candidate Members

Suresh Sivananthan, MDAsean Pacific Orthopaedic Research Society

Gautum Shetty, MSIndian Orthopaedic Research Society

Feza Korkusuz, MD,Turkish Orthopaedic Research Council

Preface

Medical science flourishes when it is carried out as a multi-disciplinary endeavour. To achieve this, medical scientists apply research methods from numerous scientific disciplines to unravel clinical problems. With the rapid development of science, the available research methods have become more varied, intricate and often more difficult to understand. Therefore, the medical scientist faces the challenge of choosing from a range of highly ingenious research tools and understanding their application for their own research endeavour. In addition, the interested reader of medical research papers is sometimes faced with mystifying descriptions of continuously developing research methodologies and need to have these elucidated.

This book on experimental research methodologies is compiled by scientists from numerous disciplines, but all of whom have a common interest in musculoskeletal science. Orthopaedic surgeons, musculo-skeletal physicians, biologists, engineers, physicists and mathematicians have composed a total of 54 chapters on research methodologies in musculoskeletal science. The general strategy for translational research involves defining a research question from a clinical problem, carrying out experiments to answer this question and then applying the findings back to the patient. The research question may be best answered with a patient investigation, an in vivo model, a cell culture experiment, a biomechanical measurement study, or a mathematical in silico model. This book provides a comprehensive summary of up-to-date research methodologies across this spectrum of types of study and is dedicated to the medical scientist interested in interdisciplinary research. While each chapter has been written by a specialist in the respective field, the aim has been to educate and teach the medical scientist who needs a comprehensive introduction. Thus, reading a chapter from this book will introduce you to the respective field and enable you to understand the research methodology and the findings obtained with its application, but will not necessarily enable you to accomplish a very specialized research task. Our book will also provide you with abundant practical advice and explanations of key terminology. This will familiarize you with each research area and will facilitate your participation in research activities.

Hamish SimpsonPeter Augat

Acknowledgments

The OTC Foundation would like to thank sincerely all contributors to this book: the OTC Research Committee who conceptualized its scope and content, the chapter authors who made available their knowledge and donated their time, and the editors and section editors who accompanied the shaping of the book from its beginnings to its final presentation. Volker Alt, Taco Blokhuis, Kurt Hankenson, Gabby Joseph, Thomas Link, Chuanyong Lu, and Esther van Lieshout are specially thanked for section editing of the book. The editors are greatly indebted to Vivien Gourlay and Sandra Augat who assisted them in managing the preparatory process with diligence, dedication, and patience.

The OTC Foundation is also grateful to Stryker for a research grant, which made this book possible.

Richard HelmerManaging EditorOTC Foundation

Contributors

Rana Abou-Khalil

Senior Research Fellow

INSERM UMR1163

Université Paris Descartes-Sorbonne Paris Cité

Institut Imagine

Hôpital Necker-Enfants Malades

Paris, France

Kishan Victoria Aldridge, BSc(Hons), MBChB(Hons), MRCS(Ed), MSc

ECAT Clinical Lecturer University of Edinburgh

MRC Human Genetics Unit

The MRC Institute of Genetics and Molecular Medicine

The University of Edinburgh

Western General Hospital

Edinburgh, Scotland, United Kingdom

Roland Aldridge, PhD

Clinical Lecturer and Honorary Special Registrar in General Surgery

Department of Clinical Surgery

The University of Edinburgh

Edinburgh, Scotland, United Kingdom

Volker Alt, MD, PhD

Deputy Clinic Director

Department of Orthopaedic Trauma Surgery University

Hospital Giessen-Marburg GmbH, Campus Giessen

Justus-Liebig-University Giessen

Giessen, Germany

Andrew A. Amis, Prof. FREng, DSc(Eng)

Professor of Orthopaedic Biomechanics

Department of Mechanical Engineering

Imperial College London

London, United Kingdom

Richard van Arkel, MEng

Imperial College London

Department of Mechanical Engineering

London, United Kingdom

Peter Augat, PhD

Professor of Biomechanics

Paracelsus Medical University

Salzburg, Austria

Director

Institute of Biomechanics

Trauma Center Murnau

Murnau am Staffelsee, Germany

Astrid D. Bakker, PhD

Associate Professor

Department of Oral Cell Biology

Academic Centre for Dentistry Amsterdam (ACTA)

University of Amsterdam and VU University Amsterdam

MOVE Research Institute Amsterdam

Amsterdam, The Netherlands

Spencer Behr, MD, PhD

Assistant Professor

Department of Radiology and Biomedical Imaging

University of California San Francisco (UCSF)

San Francisco, California, United States

Pinaki Bhattacharya, PhD

KU Leuven - University of Leuven

Department of Mechanical Engineering

Leuven, Belgium

Taco Johan Blokhuis, PhD

Department of Surgery/Traumatology

Utrecht University Medical Center

Utrecht, The Netherlands

Torsten Blunk, PhD

Department of Trauma, Hand, Plastic and Reconstructive Surgery

University of Würzburg

Würzburg, Germany

Gordon William Blunn, PhD

John Scales Centre for Biomedical Engineering

Institute of Orthopaedics and Musculoskeletal Science

Division of Surgery and Interventional Science

University College London

Royal National Orthopaedic Hospital

Middlesex, United Kingdom

Joseph Borrelli, Jr, MD

Orthopedic Surgeon

Texas Health Physicians Group

Texas Health Arlington Memorial Hospital

Arlington, Texas, United States

Nathalie Bravenboer

Senior Researcher

Department of Clinical Chemistry

MOVE Research Institute Amsterdam

VU University Medical Center

Amsterdam, The Netherlands

Wing-Hoi Cheung, PhD

Associate Professor

Department of Orthopaedics and Traumatology

The Chinese University of Hong Kong

Shatin, Hong Kong, China

Simon Kwoon Ho Chow, BSc, MSc, PhD

Department of Orthopaedics and Traumatology

The Chinese University of Hong Kong

Shaitin, Hong Kong, China

Nicholas Clement

Department of Orthopaedics and Trauma

Edinburgh University

Edinburgh, Scotland, United Kingdom

Melanie Jean Coathup

Senior Lecturer in Orthopaedics

John Scales Centre for Biomedical Engineering

Institute of Orthopaedics and Musculoskeletal Science

Division of Surgery and Interventional Science

University College London

Royal National Orthopaedic Hospital

Middlesex, United Kingdom

Céline Colnot, PhD

INSERM UMR1163

Université Paris Descartes-Sorbonne Paris Cité

Institut Imagine

Hôpital Necker-Enfants Malades

Paris, France

Luca Cristofolini, PhD

Professor of Biomechanics

Department of Industrial Engineering

School of Engineering and Architecture

University of Bologna

Bologna, Italy

Dieter R. Dannhorn, PhD

Owner and General Manager

Dr Dannhorn Consulting and More

Erolzheim, Germany

Hannah Darton

PhD Student

Imperial College London

Department of Mechanical Engineering

South Kensington Campus

London, United Kingdom

Marcel Dijkgraaf, PhD

Consultant in Clinical Research Methodology and Statistics

Clinical Research Unit

Academic Medical Center Amsterdam

Amsterdam, The Netherlands

Lutz Dürselen, PhD

Professor of Biomechanics

Head of Joint Biomechanics Research Group

Institute of Orthopaedic Research and Biomechanics

Centre of Musculoskeletal Research Ulm

Ulm University

Ulm, Germany

Sebastian Eberle, PhD

Computational Engineer

KRP-Mechatec Engineering GbR

Munich, Germany

Regina Ebert, PD PhD

Orthopedic Center for Musculoskeletal Research

University of Würzburg

Würzburg, Germany

Nan van Geloven, PhD

Biostatistician

Clinical Research Unit

Academic Medical Center Amsterdam

Amsterdam, The Netherlands

Allen Edward Goodship, BVSc, PhD, MRCVS

Emeritus Professor of Orthopaedics

John Scales Centre for Biomedical Engineering

Institute of Orthopaedics and Musculoskeletal Science

Division of Surgery and Interventional Science

University College London

Royal National Orthopaedic Hospital

Middlesex, United Kingdom

Rob de Haan, PhD

Professor of Clinical Epidemiology

Clinical Research Unit

Academic Medical Center Amsterdam

Amsterdam, The Netherlands

Camilla Halewood, MEng, MBiomedE

Biomechanical Engineer

Department of Mechanical Engineering

Imperial College London

London, United Kingdom

David Hamilton, PhD

Department of Orthopaedics

University of Edinburgh

Edinburgh, Scotland, United Kingdom

Kurt D. Hankenson, DVM, PhD

Associate Professor

Department of Physiology

Associate Director

Laboratory for Comparative Orthopedic Research

Michigan State University

East Lansing, Michigan, United States

Markus O. Heller, PhD

Professor of Biomechanics

University of Southampton

Bioengineering Science Research Group

Faculty of Engineering and the Environment

Southampton, United Kingdom

Christoph Henkenberens

Department of Trauma Surgery

University Hospital Giessen-Marburg GmbH,

Campus Giessen, Germany

Laboratory of Experimental Trauma Surgery,

Justus-Liebig-University

Giessen, Germany

Safa Herfat, PhD

Orthopaedic Trauma Institute

Department of Orthopaedic Surgery

University of California at San Francisco

San Francisco General Hospital

San Francisco, California, United States

Paul Hindle, MBChB, MRCS (Eng), RAF

Trauma and Orthopaedic Registrar

The Royal Infirmary of Edinburgh

Edinburgh, Scotland, United Kingdom

Timothy M. Jackman

Department of Biomedical Engineering

Boston University

Boson, Massachusetts, United States

Franz Jakob, Prof. Dr.

Orthopedic Center for Musculoskeletal Research

Orthopedic Department

University of Würzburg

Würzburg, Germany

Ineke D.C. Jansen, PhD

Department of Periodontology

Academic Centre for Dentistry Amsterdam (ACTA)

University of Amsterdam and VU University Amsterdam

MOVE Research Institute Amsterdam

Amsterdam, The Netherlands

Richard T. Jaspers, PhD

Assistant Professor

Laboratory for Myology

MOVE Research Institute Amsterdam

Faculty of Human Movement Sciences

VU University Amsterdam

Amsterdam, The Netherlands

Paul J. Jenkins, MBChB, MRCS(Ed)

Consultant Orthopaedic Surgeon

Glasgow Royal Infirmary

Glasgow, Scotland, United Kingdom

Gabby B. Joseph, PhD

Musculoskeletal and Quantitative Imaging Research Group

Department of Radiology and Biomedical Imaging

University of California San Francisco

San Francisco, California, United States

Petra Juffer, PhD

Department of Oral Cell Biology

Academic Centre for Dentistry Amsterdam (ACTA)

University of Amsterdam and VU University Amsterdam

MOVE Research Institute Amsterdam

Amsterdam, The Netherlands

Christian Kaddick, PhD

EndoLab

Mechanical Engineering GmbH

Rosenheim, Germany

Grace Kim, PhD

Sibley School of Mechanical & Aerospace Engineering

Cornell University

Ithaca, New York, United States

Jenneke Klein-Nulend, PhD

Professor

Department of Oral Cell Biology

Academic Centre for Dentistry Amsterdam (ACTA)

University of Amsterdam and VU University Amsterdam

MOVE Research Institute Amsterdam

Amsterdam, The Netherlands

Barbara Klotz

Orthopedic Center for Musculoskeletal Research

University of Würzburg

Würzburg, Germany

Kwong-Man Lee, PHD

Scientific Office

Lee Hysan Clinical Research Laboratories

Chinese University of Hong Kong

Shatin, Hong Kong, China

G. Harry van Lenthe, PhD

KU Leuven - University of Leuven

Department of Mechanical Engineering

Leuven, Belgium

Xiaojuan Li

Musculoskeletal and Quantitative Imaging Research Group

Department of Radiology and Biomedical Imaging

University of California San Francisco

San Francisco, California, United States

Esther M.M. Van Lieshout, MSc PhD

Associate Professor

Erasmus MC, University Medical Center Rotterdam

Trauma Research Unit, Department of Surgery

Rotterdam, The Netherlands

Thomas M. Link, MD, PhD

Professor of Radiology

Chief, Musculoskeletal Imaging

Clinical Director

Musculoskeletal and Quantitative Imaging Research

Department of Radiology and Biomedical Imaging

University of California San Francisco

San Francisco, Califorrnia, United States

Chuanyong Lu, MD

Orthopaedic Trauma Institute

Department of Orthopaedic Surgery

University of California at San Francisco

San Francisco General Hospital

San Francisco, California

Department of Pathology

SUNY Downstate Medical Center

Brooklyn, New York, United States

Punyawang Lumpaopong, PhD

Imperial College London

Department of Mechanical Engineering

London, United Kingdom

Thomas J. MacGillivray

Senior Research Fellow

Clinical Research Imaging Centre

Queen's Medical Research Institute

University of Edinburgh

Edinburgh, Scotland, United Kingdom

Ralph Marcucio, PhD

Orthopaedic Trauma Institute

Department of Orthopaedic Surgery

University of California at San Francisco

San Francisco General Hospital

San Francisco, California, United States

Gabriel McDonald

Engineer

Saint-Gobain

Boston, Massachusetts, United States

Jeffrey Meier, MD

Department of Radiology

University of Colorado, Denver

Aurora, Colorado, United States

Birgit Mentrup

Orthopedic Center for Musculoskeletal Research

University of Würzburg

Würzburg, Germany

Marjolein C. H. van der Meulen, PhD

Sibley School of Mechanical & Aerospace Engineering

Cornell University

Ithaca, New York

Hospital for Special Surgery

New York, New York, United States

Theodore Miclau, MD

Orthopaedic Trauma Institute

Department of Orthopaedic Surgery

University of California at San Francisco

San Francisco General Hospital

San Francisco, California, United States

Leanora Anne Mills, FRCS Orth&Tr

Consultant Orthopaedic Surgeon

Royal Aberdeen Childrens Hospital

Aberdeen, Scotland

Elise F. Morgan, PhD

Orthopaedic & Developmental Biomechanics Lab

Departments of Mechanical and Biomedical Engineering

Boston University

Boston, Massachusetts, United States

Iain R. Murray, BMedSci, MRCSEd, Dip SEM, PhD

ECAT Clinical Lecturer, Scottish Centre for Regenerative Medicine

The University of Edinburgh

Edinburgh, Scotland, United Kingdom

Frank Niemeyer, PhD

Scientific Computing Centre Ulm (UZWR)

University of Ulm

Ulm, Germany

Geert von Oldenburg

Director Research and Development

Biomechanics & Systems Integration

Stryker Trauma & Extremities

Schönkirchen, Germany

Thomas R. Oxland, PhD

Director

ICORD

Blusson Spinal Cord Centre

Vancouver, BC, Canada

Bidyut Pal, PhD

Research Associate

Department of Mechanical Engineering

Imperial College London

London, United Kingdom

Pankaj Pankaj, PhD

Reader

Institute for Bioengineering

School of Engineering

The University of Edinburgh

King's Buildings

Edinburgh, Scotland, United Kingdom

Janak L. Pathak

Department of Oral Cell Biology

Academic Centre for Dentistry Amsterdam

University of Amsterdam and VU University Amsterdam

MOVE Research Institute Amsterdam

Amsterdam, The Netherlands

Catherine Jane Pendegrass

John Scales Centre for Biomedical Engineering

Institute of Orthopaedics and Musculoskeletal Science

Division of Surgery and Interventional Science

University College London

Royal National Orthopaedic Hospital

Middlesex, United Kingdom

John Rasmussen, Prof. PhD

Department of Mechanical and Manufacturing Engineering

Aalborg University

Aalborg, Denmark

Ines L.H. Reichert, FRCS (Tr & Orth), MD, PhD

Consultant Orthopaedic and Trauma Surgeon

Hon Sen Lecturer

King's College

London, United Kingdom

Johannes B. Reitsma, MD, PhD

Associate Professor

Julius Center for Health Sciences and Primary Care

University Medical Center Utrecht

Utrecht, The Netherlands

Jim Richards, Prof. of Biomechanics, PhD, MSc, BEng

Allied Health Research Unit

University of Central Lancashire

Preston, Lancashire, United Kingdom

Dieter Rosenbaum, Prof. PhD

Institut für Experimentelle Muskuloskelettale Medizin

Universitätsklinikum Münster

Westfälische Wilhelms-Universität Münster

Münster, Germany

Erin Ross

Edinburgh Orthopaedic Engineering Centre

The University of Edinburgh

Edinburgh, Scotland, United Kingdom

Reinhard Schnettler

Department of Trauma Surgery

University Hospital Giessen-Marburg GmbH Campus

Giessen, Germany

Laboratory of Experimental Trauma Surgery

Justus-Liebig-University

Giessen, Germany

Andreas Martin Seitz, PhD

Institute of Orthopaedic Research and Biomechanics

Centre of Musculoskeletal Research Ulm

Ulm University

Ulm, Germany

James Selfe, Prof. of Physiotherapy, PhD, MA, GDPhys, FCSP

Allied Health Research Unit

University of Central Lancashire

Preston, Lancashire, United Kingdom

Scott Ian Kay Semple, PhD SRCS

Reader

Clinical Research Imaging Centre

Queen's Medical Research Institute

Edinburgh, Scotland, United Kingdom

Ulrich Simon, PhD

Scientific Computing Centre Ulm (UZWR)

University of Ulm

Ulm, Germany

Hamish Simpson, DM (Oxon) MA (Cantab), FRCS (Edinburgh & England)

Professor of Orthopedic Surgery

Department of Orthopaedics and Trauma

University of Edinburgh

Edinburgh, United Kingdom

Innes D M Smith

Orthopaedic Research UK Clinical Research Fellow

Musculoskeletal Research Unit

Department of Orthopaedic and Trauma Surgery

The University of Edinburgh

Edinburgh, Scotland, United Kingdom

Andre F. Steinert, Prof., Dr. med.

Department of Orthopaedic Surgery

Orthopedic Center for Musculoskeletal Research

Julius-Maximilians-University Würzburg

Würzburg, Germany

J.M. Stephen, PhD

Imperial College London

Department of Mechanical Engineering

London, United Kingdom

Michael Tanck, PhD

Assistant Professor

Department of Clinical Epidemiology, Biostatistics and Bioinformatics

Academic Medical Center, University of Amsterdam

Amsterdam, The Netherlands

David Volkheimer

Institute of Orthopaedic Research and Biomechanics

University of Ulm

Ulm, Germany

Robert James Wallace, PhD

Department of Orthopaedics

University of Edinburgh

Edinburgh, Scotland, United Kingdom

Christopher C. West, MBChB, BMedSci (Hons), MRCS (Eng)

Clinical Research Fellow and Honorary Registrar in Plastic Surgery

The Centre for Regenerative Medicine

The University of Edinburgh

Edinburgh, Scotland, United Kingdom

Hans-Joachim Wilke

Institute of Orthopaedic Research and Biomechanics

University of Ulm

Ulm, Germany

Zohar Yosibash, DSc

Hans Fischer Senior Fellow

Institute for Advanced Study

Technical University of Munich

Munich, Germany

Professor of Mechanical Engineering

Computational Mechanics Laboratory

Department of Mechanical Engineering

Ben-Gurion University of the Negev

Beer-Sheva, Israel

Yan-Yiu Yu, PhD

Research Scientist

Orthopaedic Trauma Institute

Department of Orthopaedic Surgery

University of California at San Francisco

San Francisco General Hospital

San Francisco, California, United States

Part 1

Why Do We Need Experimental Research?

1 Evidence-Based Research

2 Establishing a Basic Research Facility in Orthopedic Surgery

3 Good Laboratory Practice and Quality Control

4 How to Prepare for a Period in Research

1 Evidence-Based Research

Hamish Simpson

Poor quality research has little or no value; therefore, it is essential that we ensure that any research we carry out is to a high standard. Guidelines have been published by several bodies, such as the Wellcome Trust and the UK Medical Research Council for Good Research Practice; these cover the ethical and data protection aspects of good research. For clinical research, several frameworks have been suggested in order to ensure a high standard of research. These include the CONSORT statement for the reporting of clinical trials and the principles of evidence-based medicine. Many of the requirements in these clinical frameworks can be considered in a preclinical research setting and if followed will ensure that the pre-clinical research is carried out to the highest standards.

1.1 Lessons to Be Learned from Evidence-based Medicine for Preclinical Research

In 1996, David Sackett wrote that “Evidence-based medicine is the conscientious, explicit and judicious use of current best evidence in making decisions about the care of individual patients.”1

Evidence-based medicine is the integration of best research evidence with clinical expertise and patient values. Evidence-based medicine asks questions, finds and appraises the relevant data, and harnesses that information for everyday clinical practice. Evidence-based medicine follows five steps encompassed within the five “A”s:

1. Ask as answerable question

2. Find the relevant Articles (the evidence)

3. Critically Appraise the evidence (validity, impact, applicability)

4. Apply

5. Assess

The same steps can be applied to translational research. In particular, it is very important to formulate a clear clinical question from a patient's problem.

Asking the right question can be difficult, yet it is fundamental to carrying out relevant translational research. One framework that has been suggested to help formulate the question for evidence-based medicine is “PICO.” This framework states that a “well-built” question should include four parts, referred to as PICO, that identify the patient problem or population (P), intervention (I), comparison (C), and outcome(s) (O). Not all translational research can be fitted into this framework, but it does stress the importance of starting with the right question and the necessity of having appropriate control groups.

The next two steps of evidence-based medicine are also entirely relevant to preclinical research, namely finding the relevant previous publications and critically appraising this literature to ensure that the experimental design is optimized. In addition, it is important that the model, whether it is biomechanical, in vitro, in silico, or in vivo, is valid for the question being addressed. For instance, although muscle structure is similar in different mammals, the structure of bone and its propensity for remodeling vary in different mammals, and it is essential this is taken into account in ensuring the model is valid (see Chapter 42).

1.2 Lessons to Be Learned from Clinical Trial Design for Preclinical Experiments

The second framework described for the reporting of clinical trials but also of relevance to preclinical research is the CONSORT statement outlined in Table 1.1. The items of particular relevance to preclinical research are outlined in Table 1.2.

Of particular note are the statements about minimizing bias (see also Chapter 54). Frequently, this is not done in preclinical research,2,3 even when it adds little to the complexity of the design. For example, (1) randomization: Ideally the allocation of specimens (for biomechanical or in vitro work) or animals (for in vivo studies) should be randomized in a similar manner to patient randomization for clinical trials. (2) The assessments should be carried out in a blinded manner. For instance, if the number of positive cells on a histological section are being counted, the assessor should be unaware of which group the histological section has come from. (3) Multiple observers should be used if possible.

If the steps outlined for clinical research, which are relevant to preclinical research, are applied, the standard of the preclinical research will rise and with this the degree to which the preclinical studies can be applied clinically will increase.

For in vivo preclinical studies, an excellent fuller reporting guideline has been produced by Kilkenny and co-authors4: The ARRIVE guidelines (Table 1.3).

1.3 Levels of Evidence

For clinical research, studies should be graded for quality and weight according to five levels of evidence, the hierarchy of clinical evidence:

Table 1.1 The CONSORT Framework

Title and abstract

1a

Identification as a randomized trial in the title

1b

Structured summary of trial design, methods, results, and conclusions (for specific guidance, see CONSORT for abstracts)

Introduction

Background and objectives

2a

Scientific background and explanation of rationale

2b

Specific objectives or hypotheses

Methods

Trial design

3a

Description of trial design (such as parallel, factorial) including allocation ratio

3b

Important changes to methods after trial commencement (such as eligibility criteria), with reasons

Participants

4a

Eligibility criteria for participants

4b

Settings and locations where the data were collected

Interventions

5

The interventions for each group with sufficient details to allow replication, including how and when they were actually administered

Outcomes

6a

Completely defined prespecified primary and secondary outcome measures, including how and when they were assessed

6b

Any changes to trial outcomes after the trial commenced, with reasons

Sample size

7a

How sample size was determined

7b

When applicable, explanation of any interim analyses and stopping guidelines

Randomization

Sequence generation

8a

Method used to generate the random allocation sequence

8b

Type of randomization; details of any restriction (such as blocking and block size)

Allocation concealment mechanism

9

Mechanism used to implement the random allocation sequence (such as sequentially numbered containers), describing any steps taken to conceal the sequence until interventions were assigned

Implementation

10

Who generated the random allocation sequence, who enrolled participants, and who assigned participants to interventions

Blinding

11a

If done, who was blinded after assignment to interventions (e.g., participants, care providers, those assessing outcomes) and how

11b

If relevant, description of the similarity of interventions

Statistical methods

12a

Statistical methods used to compare groups for primary and secondary outcomes

12b

Methods for additional analyses, such as subgroup analyses and adjusted analyses

Results

Participant flow (a diagram is strongly recommended)

13a

For each group, the numbers of participants who were randomly assigned, received intended treatment, and were analyzed for the primary outcome

13b

For each group, losses and exclusions after randomization, together with reasons

Recruitment

14a

Dates defining the periods of recruitment and follow-up

14b

Why the trial ended or was stopped

Baseline data

15

A table showing baseline demographic and clinical characteristics for each group

Numbers analyzed

16

For each group, number of participants (denominator) included in each analysis and whether the analysis was by original assigned groups

Title and abstract

Outcomes and estimation

17a

For each primary and secondary outcome, results for each group, and the estimated effect size and its precision (such as 95% confidence interval)

17b

For binary outcomes, presentation of both absolute and relative effect sizes is recommended

Ancillary analyses

18

Results of any other analyses performed, including subgroup analyses and adjusted analyses, distinguishing prespecified from exploratory

Harms

19

All important harms or unintended effects in each group (for specific guidance, see CONSORT for harms)

Discussion

Limitations

20

Trial limitations, addressing sources of potential bias, imprecision, and, if relevant, multiplicity of analyses

Generalizability

21

Generalizability (external validity, applicability) of the trial findings

Interpretation

22

Interpretation consistent with results, balancing benefits and harms, and considering other relevant evidence

Other information

Registration

23

Registration number and name of trial registry

Protocol

24

Where the full trial protocol can be accessed, if available

Funding

25

Sources of funding and other support (such as supply of drugs), role of funders

Table 1.2 Components of the CONSORT Framework of Particular Relevance to Preclinical Research

Abstract

1

Structured summary of trial design, methods, results, and conclusions

Background and objectives

2

Specific objectives or hypotheses

Methods

3

The interventions for each group with sufficient details to allow replication, including how and when they were actually administered

Outcomes

4

Completely defined prespecified primary and secondary outcome measures, including how and when they were assessed

Sample size

5

How sample size was determined

Randomization

6

Type of randomization; details of any restriction (such as blocking and block size)

7

Mechanism used to implement the random allocation sequence (such as sequentially numbered containers), describing any steps taken to conceal the sequence until interventions were assigned

Blinding

8

If done, who was blinded after assignment to interventions (e.g., researchers or those assessing outcomes) and how

Statistical methods

9

Statistical methods used to compare groups for primary and secondary outcomes

Results

10

For each group, losses and exclusions after randomization, together with reasons (an experimental flow diagram should be considered)

Harms

11

All important harms or unintended effects in each group

Discussion

12

Interpretation consistent with results, balancing benefits and harms, and considering other relevant evidence

Funding

13

Sources of funding and other support (such as supply of drugs), role of funders

Table 1.3 Animal Research: Reporting in Vivo Experiments: The ARRIVE Guidelines

Item

Recommendation

Title

1

Provide as accurate and concise a description of the content of the article as possible.

Abstract

2

Provide an accurate summary of the background, research objectives including details of the species or strain of animal used, key methods, principal findings, and conclusions of the study.

Introduction

Background

3

a. Include sufficient scientific background (including relevant references to previous work) to understand the motivation and context for the study, and explain the experimental approach and rationale.

b. Explain how and why the animal species and model being used can address the scientific objectives and, where appropriate, the study's relevance to human biology.

Objectives

4

Clearly describe the primary and any secondary objectives of the study, or specific hypotheses being tested.

Methods

Ethical statement

5

Indicate the nature of the ethical review permissions, relevant licenses (e.g., Animal [Scientific Procedures] Act 1986), and national or institutional guidelines for the care and use of animals, that cover the research.

Study design

6

For each experiment, give brief details of the study design, including:

a. The number of experimental and control groups.

b. Any steps taken to minimize the effects of subjective bias when allocating animals to treatment (e.g., randomization procedure) and when assessing results (e.g., if done, describe who was blinded and when).

c. The experimental unit (e.g. a single animal, group, or cage of animals).

A timeline diagram or flow chart can be useful to illustrate how complex study designs were carried out.

Experimental procedures

7

For each experiment and each experimental group, including controls, provide precise details of all procedures carried out. For example:

a. How (e.g., drug formulation and dose, site and route of administration, anesthesia and analgesia used [including monitoring], surgical procedure, method of euthanasia). Provide details of any specialist equipment used, including supplier(s).

b. When (e.g., time of day).

c. Where (e.g., home cage, laboratory, water maze).

d. Why (e.g., rationale for choice of specific anesthetic, route of administration, drug dose used).

Experimental animals

8

a. Provide details of the animals used, including species, strain, sex, developmental stage (e.g., mean or median age plus age range), and weight (e.g., mean or median weight plus weight range).

b. Provide further relevant information such as the source of animals, international strain nomenclature, genetic modification status (e.g., knockout or transgenic), genotype, health/immune status, drug- or test-naïve, previous procedures, etc.

Housing and husbandry

9

Provide details of:

a. Housing (e.g., type of facility, specific pathogen free; type of cage or housing; bedding material; number of cage companions; tank shape and material, etc., for fish).

b. Husbandry conditions (e.g., breeding program, light/dark cycle, temperature, quality of water, etc., for fish, type of food, access to food and water, environmental enrichment).

c. Welfare-related assessments and interventions that were carried out before, during, or after the experiment.

Sample size

10

a. Specify the total number or animals used in each experiment and the number of animals in each experimental group.

b. Explain how the number of animals was decided. Provide details of any sample size calculation used.

c. Indicate the number of independent replications of each experiment, if relevant.

Allocating animals to experimental groups

11

a. Give full details of how animals were allocated to experimental groups, including randomization or matching if done.

b. Describe the order in which the animals in the different experimental groups were treated and assessed.

Experimental outcomes

12

Clearly define the primary and secondary experimental outcomes assessed (e.g., cell death, molecular markers, behavioral changes).

Statistical methods

13

a. Provide details of the statistical methods used for each analysis.

b. Specify the unit of analysis for each dataset (e.g., single animal, group of animals, single neuron).

c. Describe any methods used to assess whether the data met the assumptions of the statistical approach.

Results

Baseline data

14

For each experimental group, report relevant characteristics and health status of animals (e.g., weight, microbiological status, and drug- or test-naïve) before treatment or testing (this information can often be tabulated).

Numbers analyzed

15

a. Report the number of animals in each group included in each analysis. Report absolute numbers (e.g., 10/20, not 50%).

b. If any animals or data were not included in the analysis, explain why.

Outcomes and estimation

16

Report the results for each analysis carried out, with a measure of precision (e.g., standard error or confidence interval).

Adverse events

17

a. Give details of all important adverse events in each experimental group.

b. Describe any modifications to the experimental protocols made to reduce adverse events.

Discussion

Interpretation/scientific implications

18

a. Interpret the results, taking into account the study objectives and hypotheses, current theory, and other relevant studies in the literature.

b. Comment on the study limitations including any potential sources of bias, any limitations of the animal model, and the imprecision associated with the results.

c. Describe any implications of your experimental methods or findings for the replacement, refinement, or reduction (the 3Rs) of the use of animals in research.

Generalizability/translation

19

Comment on whether, and how, the findings of this study are likely to translate to other species or systems, including any relevance to human biology.

Funding

20

List all funding sources (including grant number) and the role of the funder(s) in the study.

1. Level 1

a) Systematic reviews of randomized control trials

b) Randomized control trials

2. Level 2 Cohort studies

3. Level 3

a) Case-controlled trials (comparisons made but not randomized)

b) Observational studies (including surveys and questionnaires)

4. Level 4

a) Case series

b) Case reports

5. Level 5 Editorials, expert opinion

In a similar way, preclinical research can be graded into different levels depending on the quality of the experimental design and the number of “dropouts” (Fig. 1.1).

The higher the level of evidence of a piece of research, the more notice is taken of the conclusions in applying them to the patient. Preclinical research has been considered by some to come at the bottom of this pyramid of evidence; however, if the preclinical research is carefully designed so that it represents the clinical scenario accurately and performed to a high standard with steps taken to avoid bias, then it should be placed higher up the pyramid and potentially above the level of case series (Fig. 1.2). In some clinical areas, this may mean that it is the highest level of evidence available and as such should have a major influence on patient management.

Fig. 1.1 There is a spectrum of quality of preclinical studies, ranging from poorly conducted studies in models that do not resemble the human situation to well-designed experiments in clinically relevant models of the disease.

Fig. 1.2 Well-conducted research in relevant clinical models can have a greater impact on guiding the treatment for a patient than low-level clinical research. Therefore, top-level preclinical studies can slot in the hierarchy of evidence toward the middle of the pyramid rather than at the bottom of it.

In addition to informing patient care directly, preclinical studies also have a role in the design of clinical trials for complex interventions. According to Campbell et al,5 complex interventions are “built up from a number of components, which may act both independently and interdependently.” Evaluating complex interventions can pose a considerable challenge. Preclinical studies inform the preliminary phase of the design of studies for the evaluation of complex interventions (see following box) and as such they have a crucial role in ensuring that clinical trials address the right question with the right design.

Medical Research Council Framework for Design and Evaluation of Complex Interventions

Stepwise approach (on paper)

Phase 0—Preclinical or theoretical (why should this intervention work?)

Phase 1—Modelling (how does it work?)

Phase 2—Exploratory or pilot trial (optimizing trial measures)

Phase 3—Definitive randomized controlled trial

Phase 4—Implementation

References

[1] Sackett DL, Rosenberg WM, Gray JA, Haynes RB, Richardson WS. Evidence based medicine: what it is and what it isn't. BMJ 1996; 312: 71–72

[2] Kilkenny C, Parsons N, Kadyszewski E et al. Survey of the quality of experimental design, statistical analysis and reporting of research using animals. PLoS ONE 2009; 4: e7824

[3] Landis SC, Amara SG, Asadullah K et al. A call for transparent reporting to optimize the predictive value of preclinical research. Nature 2012; 490: 187–191

[4] Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG. Improving bioscience research reporting: The ARRIVE guidelines for reporting animal research. J Pharmacol Pharmacother 2010; 1: 94–99

[5] Campbell NC, Murray E, Darbyshire J et al. Designing and evaluating complex interventions to improve health care. BMJ 2007; 334: 455–459

Further Reading

van der Worp HB, Howells DW, Sena ES et al. Can animal models of disease reliably inform human atudies? PLoS Med 2010;7(3): e1000245

2 Establishing a Basic Research Facility in Orthopedic Surgery

Chuanyong Lu, Safa Herfat, Céline Colnot, Ralph Marcucio, and Theodore Miclau

The initiation of research programs requires complex decision-making as directional, logistical, and financial considerations must be evaluated. The greatest barriers to the development of new basic research facilities include available technical expertise, space, and finances. The establishment of an orthopedic laboratory should be based on research interest, expertise, funding, and the surrounding research environment. Collaboration between multiple disciplines and centers is key to the success of a research program. Here, we outline some of the major research directions in orthopedics and list the basic equipment to set up a laboratory for cellular and molecular biology, biomechanics, and tissue engineering of musculoskeletal tissues.

Because our population is aging and the rate of automobile accidents worldwide is increasing, musculoskeletal problems continue to represent a significant source of death and disability, with growing societal and economic burdens. Although our knowledge about musculoskeletal diseases has been greatly improved in the last decades, we are still facing challenges and have limited options to treat fracture nonunions, large segmental bone/joint defects, degenerative joint diseases, failed implants, and ligament injuries. Research into the normal biology of musculoskeletal tissues, the diseases and injuries associated with these tissues, and the underlying mechanisms of musculoskeletal tissue regeneration continue to gain importance. These investigations often require a multidisciplinary approach including basic cellular and molecular biology, bioengineering, biomechanics, and clinical research. Collaboration between disciplines and centers with different expertise is essential to continue to advance the field. A team of self-motivated scientists and experienced technicians is key to the success of a laboratory. The purpose of this chapter is to address issues that may be of interest to the development of new basic science research programs and initiatives. A brief review of the current and developing areas of orthopedic research is included, and the resources required for establishing a new biology, tissue engineering, or biomechanics research laboratory are described.

2.1 Development of a Basic Biology Laboratory

2.1.1 Research Directions for a Biology Laboratory

The goal of orthopedic research is to develop better treatments to prevent, cure, or slow down the progress of musculoskeletal disorders. Research into basic cellular and molecular mechanisms of skeletogenesis, skeletal tissue regeneration, and musculoskeletal disorders is critical to the accomplishment of this goal.

Embryonic Skeletal Development

The human skeleton forms through two distinct processes: endochondral ossification and intramembranous ossification. During endochondral ossification, progenitor cells differentiate into chondrocytes and form cartilage first, which is then replaced by bone. All the long bones in our body (except for the clavicle) develop through this process. Intramembranous ossification occurs through direct bone formation; examples are the flat craniofacial bones. Bone development is a highly regulated process that involves multiple transcription factors, such as SOX-9 and cbfa-1, and their downstream molecules. Since bone regeneration in adults largely recapitulates the process that occurs during embryonic skeletal development and shares similar regulating mechanisms, the findings in skeletogenesis frequently lead to novel therapeutic targets for bone repair. For example, molecules such as matrix metalloproteinase-9, matrix extracellular phosphoglycoprotein, and wnt proteins are expressed during both skeletogenesis and adult bone repair. Further research demonstrated that matrix extracellular phosphoglycoprotein and wnt proteins could promote osteogenesis in vitro or in vivo.

Bone Regeneration

Bone repair is initiated by an acute inflammatory response, which is followed by the recruitment of skeletal progenitor cells, differentiation of chondrocytes and osteoblasts, formation of bone and cartilage, and remodeling of the callus. Inflammation plays an important role in fracture healing by modulating angiogenesis, stem cell recruitment, and callus remodeling. Numerous cytokines, growth factors, and molecules are involved in fracture healing. Expression of tumor necrosis factor-α, interleukin (IL)-1, IL-6, IL-10, IL-18, vascular endothelial growth factor, and bone morphogenetic proteins (BMPs) are detected in fracture calluses.1,2,3 The contribution of these molecules, cytokines, and inflammatory cells to fracture healing is a focus of current orthopedic research. Other important research topics of bone regeneration are determining the sources of repairing cells, improving bone regeneration in patients with diabetic mellitus or peripheral vascular diseases, and treating large bone defects.

Cartilage Repair

Although we have been studying cartilage repairs for decades, it still remains a challenge to regenerate cartilage with the morphology, chemical compositions, biomechanical properties, and long-term functions comparable to native cartilage. Articular cartilage has a poor capacity to repair itself after injury and tends to heal through the formation of fibrocartilage, which has inferior biomechanical characteristics to resist compression stress compared to normal articular hyaline cartilage. Further understanding of the differences between hyaline cartilage and fibrocartilage, as well as the molecular mechanisms that govern chondrocyte differentiation and extracellular matrix production, could provide clues on how to direct cells to produce hyaline cartilage instead of fibrocartilage. In this field, growth factors such as transforming growth factor (TGF)-β, insulinlike growth factor-1, fibroblast growth factor-2, and BMP-7 may be exploited to improve cartilage regeneration.

Fibrocartilaginous tissues, such as meniscus and intervertebral disk, are equally challenging to regenerate because of the lack of adequate blood supply and the high demand for mechanical loading. The intervertebral disk is composed of three tissues: the cartilage endplate, nucleus pulposus, and annulus fibrosus. These three types of tissue create architecture with a greater level of complexity than that of articular cartilage. Successful regeneration of intervertebral disk may require the regeneration of all three tissues in one implant. Tissue engineering will play an important role in cartilage regeneration, and the right scaffolds, cells, and growth factors need to be tested.

Soft Tissue Regeneration

Muscles, tendons, and ligaments along with blood vessels and nerves are closely associated with bone. The basic biology of muscle and muscle repair is relatively well understood compared to other soft tissues. However, further advances are needed to treat devastating diseases such as Duchenne muscular dystrophy and to improve muscle repair. The biology of tendons and ligaments is now being better understood with the identification of key molecular pathways and cells involved in these tissues. Like muscle and bone healing, tendon and ligament healing is initiated by an inflammatory response that may be modulated to stimulate repair. The key issue with current tendon repair strategies is the difficulty in achieving functionality that is equal to that of the preinjured state, which makes it necessary to explore new growth factors and cells to accomplish the task. In addition, we need to optimize the mechanical stimuli and postsurgical rehabilitation. Tissue engineering may provide promising solutions to tendon or ligament repair in difficult cases such as anterior cruciate ligament (ACL) rupture.

Arthritis

Rheumatoid and degenerative arthritis affects a large proportion of the population and are leading causes of disability. The role of inflammation in the pathogenesis of arthritis has been well established, and several groups of drugs have been developed to tackle inflammation and thus modify the progression of disease. One example is the use of tumor necrosis factor-α antagonists in rheumatoid arthritis. Recent studies have also focused on the role of stem cells in the pathogenesis of arthritis and on the therapeutic effects of mesenchymal stem cells (MSCs) on arthritis, taking advantage of the immune-regulatory effects of MSCs.

Mesenchymal Stem Cells

Stem cell biology is a hot topic in the field of orthopedic research. Experimental studies have shown that stem cells that contribute to fracture healing could derive from peripheral circulation and bone marrow. Transplanted MSCs can differentiate into osteoblasts and chondrocytes in fracture calluses. In addition, MSCs have paracrine effects by expressing potent growth factors and cytokines that modulate angiogenesis, inflammation, and stem cell recruitment/differentiation. The paracrine effects of MSCs in fracture healing have not been fully determined. There is evidence showing that MSC-conditioned medium can improve fracture healing.4 Current research on MSCs is hindered by the lack of specific surface markers and the inefficiency of in vivo cell tracking techniques.

One recent major achievement in stem cell research is the discovery of induced pluripotent stem (iPS) cells. The iPS cells were first established by retrovirus-mediated transduction of four transcription factors (c-Myc, Oct3/4, SOX2, and Klf4) into mouse fibroblasts or human fibroblasts. The current trend is to use fewer transcription factors or small chemical compounds to reprogram somatic cells. iPS cells have the potential to differentiate toward osteoblasts,5 indicating their value in bone regeneration. However, the risk of tumorogenesis of iPS cells needs to be addressed prior to clinical application.

2.1.2 Infrastructure and Equipment of a Biology Laboratory

The infrastructure required to run an orthopedic basic research laboratory is similar to any other biological laboratory. Fume hoods are required to vent noxious and dangerous chemicals. An animal housing facility is necessary if work will be performed on any in vivo model. If work is to be performed on established or primary cell lines, then a separate cell culture room should be considered. By isolating cell culture facilities, reduced foot traffic around the incubators and hoods will aid in keeping cultures free of bacteria and mold. Another part of the laboratory should be set aside for processing, sectioning, and staining of histological specimens. This area should be located in a “dust-free” area away from drafts that will create difficulty handling ribbons of histology sections. Work with radioactive materials can be made safer by defining and restricting use of these materials to dedicated areas of the laboratory. Similarly, a dedicated imaging suite that contains all of the microscopes that will be used for documentation and analysis of data will allow undisturbed specimen viewing, will allow the room to be darkened for specialized imaging such as epifluorescence, and will reduce the amount of dust that accumulates on working parts of the microscope. Basic equipment for a biology laboratory is listed in Table 2.1.

2.2 Development of a Tissue Engineering Laboratory

2.2.1 Research Directions of a Tissue Engineering Laboratory

Tissue engineering is a science of tissue regeneration using combinations of scaffolds, cells, and growth factors. A tissue engineering research team should have expertise in biomaterials, cell biology, molecular biology, and animal surgeries. Advances in the research of novel biomaterials, scaffold fabrication, and growth factors, in combination with improved understanding of the cellular and molecular mechanisms of musculoskeletal tissue regeneration, will further progress this field.

Biomaterials and Scaffolds

The most commonly used biomaterials for bone and cartilage regeneration include (1) natural materials such as collagen, gelatin, fibrin gel, silk, chitosan, and demineralized bone matrix; (2) synthetic materials such as poly(Llactic acid), poly(glycolic acid), and polycaprolactone; and (3) mineral components such as hydroxyapatite, tricalciumphosphate, and bioglass. Scaffolds can be fabricated synthetic materials by electrospinning, thermally induced phase separation, or three-dimensional (3D) computer-assisted printing. To facilitate mineralization, mineral components can be mixed with other materials before fabrication or deposited on the surface of scaffold. Scaffolds should have the right compositions and 3D structure to facilitate cell adhesion, stem cell recruitment, proliferation, and differentiation. Optimization of the porosity and components has been extensively explored. Recent advances in 3D printing technique allow the fabrication of scaffold with multiple layers that recapitulate the biomechanical property and environment for bone, cartilage, or ligament regeneration in the same construct.

For example, scaffolds for articular cartilage are made with two layers, one desirable for cartilage regeneration and another for subchondral bone formation. To facilitate the attachment of ligament to bone, a stratified or multiphasic scaffold is designed for interface tissue engineering.6 Stem cells or growth factors can be added to the scaffolds to achieve better results.

Table 2.1 Infrastructure and Equipment of a Biology Laboratory

Infrastructure and equipment

Personnel

Cell biologist, molecular biologist, developmental biologist

Space

Dedicated space for tissue processing and sectioning, RNA extraction, cell culture, and radioactive materials

Chemicals

Fume hoods, inflammable cabinet

Cell culture

Dissecting microscope, cell culture hood, incubators, water bath, low-speed centrifuge, revert microscope with fluorescence capability, liquid nitrogen tank, access to fluorescence-activated cell sorting equipment

Molecular biology

Reverse transcription–polymerase chain reaction, polymerase chain reaction, electrophoresis, nano drop spectrophotometer, spectrometer, western blotting equipment, setup for in situ hybridization, centrifuges, access to microarray equipment

Histology

Tissue processing equipment; microtomes for paraffin sectioning, frozen sectioning, and undecalcified tissue sectioning; microscopes; histomorphometry equipment (stereology or BioQuant system; BioQuant Inc., San Diego, CA); equipment for immunostaining

Surgery

Surgery room, surgery table, fracture apparatus, drill, saw, general surgical instruments, anesthesia machine (isoflurane), dissecting microscope, animal facility

Bone analysis

X-ray machine, microcomputed tomography, dual-energy X-ray absorptiometry, biomechanical equipment, Fourier transform infrared spectrometer

Stem Cells

Research on stem cells is a major component for tissue engineering. Currently, most studies are using stem cell transplantation to improve tissue regeneration, in which stem cells are collected, cultured, and expanded in vitro with or without differentiation before being transplanted in vivo. Cell transplantation provides repairing cells to the site and may employ the paracrine effects of transplanted cells. However, cell transplantation is limited by several disadvantages including an additional surgery to collect cells for culture, expensive cell culture procedures, and risk of contamination. Recently, scientists have explored the efficiency of cell homing, which uses growth factors to recruit host cells into the scaffolds, to improve tissue regeneration. As a proof of concept, anatomically correct scaffolds for rabbit humeral condyle were fabricated, incorporated with TGF-β3, and implanted in rabbits. Without delivering exogenous stem cells with the scaffold, this approach of cell homing was able to regenerate a functional humeral condyle in these animals, which exhibited an articular surface of hyaline cartilage.7 In the direction of cell homing, further research is necessary to find out the most potent growth factors for MSCs recruitment in both in vitro and in vivo settings.

Growth Factors

Numerous growth factors are capable of improving skeletal tissue regeneration, among them are BMPs, TGF-β, platelet-derived growth factor, fibroblast growth factors, vascular endothelial growth factor, and stromal cell–derived factor-1, etc. As our understanding of stem cell biology improves, this list will get longer. To regenerate different types of tissue, specific growth factors should be chosen. For examples, BMPs are potent for bone regeneration, whereas TGF-β3 is useful for cartilage repair.7 The safety of these growth factors in humans has not been fully established. Controlled release may lower the dose of growth factors, thus decreasing their side effects and cost.

Large Tissue Regeneration and Vascularization

One of the goals and challenges of tissue engineering is to repair large tissue defects. Large skeletal defects have a limited healing capability due to multiple factors. One factor is the lack of repairing cells or growth factors, which can be corrected by transplanting or homing MSCs. Another important factor is inadequate blood supply or inefficient revascularization. Adding proangiogenic factors to scaffolds facilitates tissue regeneration. Bioreactors allow the regeneration of large anatomically shaped tissues in vitro with scaffold and seeded cells; however, currently there is no available technique to build a working vascular system into these regenerates. The viability of a large regenerate produced in a bioreactor would therefore be significantly compromised, which hinders the clinical application of in vitro organ regeneration.

2.2.2 Infrastructure and Equipment of a Tissue Engineering Laboratory

A laboratory focusing on skeletal tissue engineering shares similar equipment as a biology laboratory for cell culture, histological, cellular, and molecular analyses. In addition, a tissue engineering laboratory should have special equipment for scaffold fabrication, as well as those for in vitro and in vivo tissue regeneration. Table 2.2 lists some of the basic equipment for tissue engineering.

Table 2.2 Infrastructure and Equipment of a Tissue Engineering Laboratory

Infrastructure and Equipment

Personnel

Stem cell biologist, chemist, molecular biologist, collaboration with veterinarians for large animal models

Space

Dedicated space for cell culture and scaffold fabrication

Chemicals

Fume hoods, inflammable cabinet

Scaffold fabrication

Electrospinning setup (a spinneret, a high-voltage direct current power supply, a syringe pump, and a grounded collector), freeze drying machine, 3D-Bioplotter (EnvisionTEC, Dearborn, MI), computers, bioreactors

Cell culture

Dissecting microscope, cell culture hood, incubators, water bath, low-speed centrifuge, revert microscope with fluorescence capability, access to fluorescence-activated cell sorting equipment, access to in vivo fluorescence imaging system

Histology

Tissue processing equipment; microtomes for paraffin sectioning, frozen sectioning, and undecalcified tissue sectioning; microscopes; histomorphometry equipment (stereology or BioQuant systems; BioQuant Inc., San Diego, CA)

Molecular biology

Reverse transcription–polymerase chain reaction, polymerase chain reaction, electrophoresis, nano drop spectrophontometer, spectrometer, western blotting equipment, setup for in situ hybridization, centrifuges, access to microarray equipment

Surgery

Surgical room, surgical table, general surgical instruments, fracture apparatus, drill, saw

Bone analysis

X-ray machine, microcomputed tomography, dual-energy X-ray absorptiometry, biomechanical equipment, Fourier transform infrared spectroscopy

2.3 Development of an Orthopedic Biomechanics Laboratory

2.3.1 Research Directions of a Biomechanics Laboratory

Orthopedic biomechanics is an area of orthopedic research that focusses on applying theoretical and experimental mechanics to study the musculoskeletal system. The role of biomechanics is critical in establishing design parameters and evaluation criteria for orthopedic treatments, surgical techniques, devices, and implants. Orthopedic biomechanics researchers are currently studying the musculoskeletal system using a variety of approaches that include experimental biomechanical analysis (in vivo and in vitro) and computational analysis (modeling and simulation).

Together, experimental and computational approaches continue to increase our knowledge of normal biomechanics, as well as having implications for orthopedic injuries, conditions, and treatments. Although most laboratories may focus on either experimental or computational approaches, some employ both. For example, a sports biomechanics laboratory may acquire kinematic measurements for an athlete and then use these measurements to build musculoskeletal models for further biomechanical analysis. Both computational and experimental approaches are currently being used for sports medicine research investigating the biomechanical effects of ACL injuries, evaluating ACL reconstruction techniques and grafts types, determining how ACL injuries lead to long-term complications such as osteoarthritis, and establishing functional tissue engineering parameters for artificial ACL replacements.

Orthopedic biomechanics researchers are expanding the technical applications of imaging, modeling and simulation, material testing, robotics, and measurement instrumentation. Recent advances in technology now allow a level of accuracy in motion measurement that was previously not attainable. These new technologies have made it possible to measure in vivo motion more accurately and reproduce the in vivo condition more accurately when conducting in vitro biomechanical testing. Advanced modeling software (e.g., Mimics; Materialise, Leuven, Belgium) now allows investigators to build accurate 3D models from data acquired from various imaging modalities. These 3D models can then be imported into finite element software to perform computational biomechanics analysis of musculoskeletal tissues, surgical techniques, and devices. Sensors needed for in vivo and in vitro measurement of joint and tissue deformations and loads also continue to evolve.