Studies in the Development of a Xenograft-Derived Bone
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STUDIES IN THE DEVELOPMENT OF A XENOGRAFT-DERIVED BONE SCAFFOLD BY DANIEL NICHOLAS BRACEY A Dissertation Submitted to the Graduate Faculty of WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Molecular Medicine and Translational Sciences May 2017 Winston-Salem, North Carolina Approved By Thomas L. Smith, Ph.D., Advisor Thomas C. Register, Ph.D., Chair Jeffrey S. Willey, Ph.D. L. Andrew Koman, M.D. Mark E. Van Dyke, Ph.D. DEDICATION I dedicate this dissertation to my family. To my wife, Mary, for her love and endless patience while I gave myself to this research, To my daughters, Lucy and Stirling, who have shown me joy, To my Mother, Jeri, for raising me to appreciate the value of education and hard work. ii ACKNOWLEDGEMENTS I would like to thank the Department of Orthopaedic Surgery for giving me the opportunity to pursue my training at Wake Forest. I thank Dr. Tom Smith, Dr. Beth Smith, Dr. Jason Lang and Dr. Andrew Koman for believing in my potential as an orthopaedic surgeon and extending me the physician scientist residency position. I thank my committee members for listening and thinking with me. I especially thank Dr. Jeff Willey for opening his lab and office to me at any hour of the day, and his generosity with time and resources. I thank Dr. Mark Van Dyke and Dr. Tom Register for challenging my understanding of scientific principles and broadening my knowledge. Thank you to my co-residents and fellow physician scientists for your support of my research interests. I am especially grateful to Dr. Patrick Whitlock and Dr. Thorsten Seyler for imparting their mentorship in my pursuit of this project. I thank Dr. Ian Hutchinson for his enthusiasm and teaching me cell culture principles. I thank Dr. Johannes Plate and Dr. Austin Stone for their mentorship ahead of me in the orthopaedic research lab. Lastly, I thank Dr. Robert Booth for inviting me into his operating room and opening my eyes to the field of orthopaedic surgery. His support, motivation and guidance have dramatically changed the course of my career. iii TABLE OF CONTENTS Dedication .......................................................................................................................... ii Acknowledgements ......................................................................................................... iii Table of Contents ............................................................................................................ iv List of Figures and Tables .............................................................................................. vi List of Abbreviations ..................................................................................................... vii Abstract ............................................................................................................................ ix CHAPTER 1 ...................................................................................................................... 1 An Introduction to Bone Biology and Bone Grafting Principles References ..................................................................................................................... 21 CHAPTER 2 .................................................................................................................... 26 The Development of a Xenograft-Derived Bone Scaffold: A Critical Evaluation of Scaffold Biology and Structure Abstract ........................................................................................................................ 27 Introduction .................................................................................................................. 29 Methods ........................................................................................................................ 31 Results ........................................................................................................................... 38 Discussion .................................................................................................................... 48 Conclusions .................................................................................................................. 52 References .................................................................................................................... 53 CHAPTER 3 .................................................................................................................... 61 Assessing the Biologic Safety of a Xenograft-Derived Bone Scaffold: In Vitro Cytocompatibility Studies and Quantification of the Alpha-Gal Epitope Abstract ........................................................................................................................ 62 Introduction .................................................................................................................. 64 Methods ........................................................................................................................ 66 Results ........................................................................................................................... 76 Discussion .................................................................................................................... 84 Conclusions .................................................................................................................. 89 References .................................................................................................................... 90 CHAPTER 4 .................................................................................................................... 96 Evaluation of the Osteoinductive Potential in a Xenograft-Derived Bone Scaffold Abstract ........................................................................................................................ 97 Introduction ................................................................................................................ 100 Methods ...................................................................................................................... 103 Results ......................................................................................................................... 110 Discussion .................................................................................................................. 124 iv Conclusions ................................................................................................................ 128 References .................................................................................................................. 130 CHAPTER 5 .................................................................................................................. 137 Summary of Dissertation ............................................................................................ 138 Future Directions ........................................................................................................ 140 References ................................................................................................................... 144 Appendix ........................................................................................................................ 147 Funding Sources.......................................................................................................... 147 Curriculum Vitae .......................................................................................................... 148 v LIST OF FIGURES AND TABLES Chapter 2 Figures and Tables Figure 1 Page 38 Figure 2 Page 39 Figure 3 Page 40 Figure 4 Page 43 Figure 5 Page 44 Table 1 Page 44 Table 2 Page 45 Table 3 Page 47 Chapter 3 Figures and Tables Figure 1 Page 71 Figure 2 Page 74 Figure 3 Page 76 Figure 4 Page 78 Figure 5 Page 78 Figure 6 Page 79 Figure 7 Page 81 Figure 8 Page 83 Figure 9 Page 84 Chapter 4 Figures and Tables Figure 1 Page 105 Figure 2 Page 112 Figure 3 Page 113 Figure 4 Page 115 Figure 5 Page 116 Figure 6 Page 119 Figure 7 Page 120 Figure 8 Page 121 Figure 9 Page 123 Chapter 5 Figures and Tables Figure 1 Page 142 vi LIST OF ABBREVIATIONS 2d Two-dimensional 3d Three-dimensional α-Gal Alpha-Gal Epitope (Galalpha1-3Galbeta1-(3)4GlcNAc-R) ACL Anterior Cruciate Ligament ALK Alkaline Phosphatase ANOVA Analysis of Variance ATCC American Type Culture Collection BMP Bone Morphogenic Protein BSA Bovine Serum Albumin CFU Colony Forming Unit Col1 Collagen-1 CT Computer tomography DAPI 4',6-diamidino-2-phenylindole DBM Demineralized Bone Matrix DICOM Digital Imaging and Communications in Medicine DMEM Dulbecco’s Modified Eagle’s Medium DNA Deoxyribonucleic Acid EDTA Ethylenediaminetetraacetic acid FBS Fetal Bovine Serum FEA Finite Element Analysis GF Gelfoam H&E Hematoxylin and Eosin vii HRP Horse Radish Peroxidase LPS Lipopolysaccharide MPa Megapascal M-PER Mammalian Protein Extraction Reagent MS Mass Spectrometry MSC Mesenchymal Stem Cell N Newton NHP Non-Human Primate OM Osteogenic Media PAA Peracetic Acid PCR Polymerase Chain Reaction RNA Ribonucleic Acid RUNX2 Runt-Related Transcription Factor 2 SAL Sterility Assurance Level SEM Scanning Electron Microscopy SV5 Simian Virus 5 TNFα Tumor Necrosis Factor Alpha viii ABSTRACT Bone grafts are used in nearly one half of all musculoskeletal surgeries and represent an enormous health care expenditure. Tissue-engineered bone graft substitutes have garnered considerable attention to