APPLICATIONS OF HUMAN BONE MATERIALS AND SYNTHESIZED BIOMATERIALS FOR BONE-RELATED TISSUE ENGINEERING A Dissertation Presented to The Graduate Faculty of The University of Akron In Partial Fulfillment Of the requirements for the Degree Doctor of Philosophy Qing Yu December, 2016 APPLICATIONS OF HUMAN BONE MATERIALS AND SYNTHESIZED BIOMATERIALS FOR BONE-RELATED TISSUE ENGINEERING Qing Yu Dissertation Approved: Accepted: ______________________________ ______________________________ Advisor Department Chair Dr. William J. Landis Dr. Coleen Pugh ______________________________ ______________________________ Committee Member Dean of the College Dr. Nita Sahai Dr. Eric J. Amis ______________________________ ______________________________ Committee Member Dean of the Graduate School Dr. Coleen Pugh Dr. Chand Midha ______________________________ ______________________________ Committee Member Date Dr. Marnie Saunders ______________________________ Committee Member Dr. Ge Zhang ii ABSTRACT Engineered bone grafting has been considered as one of the alternative methods for bone regeneration in both fundamental research and clinical applications to address bone disorders. Bone graft materials, autologous bone, allogeneic bone and synthetic polymer scaffolds have been commonly utilized surgically as substrates for bone grafting. In this dissertation, periosteum, a thin membrane in which progenitor cells can develop into osteoblasts to regenerate bone tissue, has been applied in three different studies to determine its capability to induce new bone formation. In the first study, human periosteum-wrapped bone allografts were implanted subcutaneously in athymic mice followed by sample harvest and gene expression analysis and histological assessment. The second study developed a tissue-engineering approach to generate a functional tendon-to-bone enthesis. In this instance, the constructs were fabricated from human periosteum-wrapped allograft bone and tenocyte- and chondrocyte-seeded biomaterials. The constructs were then implanted with and without mechanical force by either tethering them to the trapezius and gluteus maximus muscles of athymic mice or not tethering them at all. Biomechanical, histological, and histochemical properties of these tendon-to-bone enthesis models were analyzed following their iii implantation. The third study was designed to determine the possible effects of bromine- or silicon-functionalized poly(lactic acid) (Si-PLA) scaffolds on skeletal development. To examine initially the cytotoxicity of bromine on periosteal cells, a PrestoBlue® assay was performed on human periosteal cell-seeded brominated PLA scaffolds over a 21-day time period. With application of histological and gene expression analysis, new bone formation and resorption were detected in human-periosteum allografts implanted for different time periods. Correlated histological and gene data showed that periosteum has the capability of inducing bone regeneration in allografts and in tendon-to-bone enthesis models. Tissue-engineered enthesis models fabricated with periosteum-allograft and chondrocyte- and tenocyte- seeded scaffolds provide a novel method for healing enthesis defects in regenerative medicine. In addition, results from bromine cytotoxicity studies of human periosteal cells imply subsequent Si-PLA experiments with minimal numbers of bromine residues on the backbone of PLA. These tissue engineering investigations suggest that both allograft bone and biosynthetic polymers have great potential in regenerative medicine applications for bone. iv DEDICATION To my mother, who has been the most supportive and loveliest person in the world. v ACKNOWLEDGMENTS Firstly, I would like to express my deep and sincere appreciation to Dr. William J. Landis, my dearest advisor, for his continuous support of my Ph.D. study and related research, for being a tremendous mentor, for his patience, encouragement, motivation, and omniscience. His guidance in not only my writing and research but also my life is invaluable. Besides work, he and his wife Jane Landis treat me like their family member. I could not have imagined a better advisor and mentor for my graduate study. A special thanks to Mrs. Robin Jacquet for her help with the implantation of the constructs into nude mice, the analysis of gene expression, the amount of time and effort she devoted in ensuring my success in laboratory research. She also showed me the path to faith in God with her love. My sincere thanks also go to my committee members, Dr. Coleen Pugh (Department of Polymer Science) for her assistance in polymer synthesis, Dr. Nita Sahai (Department of Polymer Science) for her insights into my research, Dr. Marnie Saunders (Department of Biomedical Engineering) for providing instrument access to perform tensile testing, and Dr. Ge Zhang (Department of Biomedical Engineering) for her encouragement and guidance in my career and my life. I thank Dr. John Elias (Department of Research, Akron General Medical vi Center) for his help with preliminary tensile testing and Gunze, Ltd. (Kyoto, Japan) for supplying the polymer materials. I would like to thank Dr. Hitomi Nakao, Dr. Philip McClellan, Dr. Narihiko Hirano, and Mr. Josh Bundy (the Landis Laboratory, Department of Polymer Science) for their assistance with sample preparation and implantations and Dr. Colin Wright, Dr. Xiang Yan, and Mr. Haidong Zhu (Department of Polymer Science) for their assistance in polymer synthesis. I gratefully acknowledge support from the National Science Foundation (DMR-1006195; Dr. Coleen Pugh, PI, Department of Polymer Science, University of Akron) for aspects of this dissertation concerning polymer synthesis. I would especially like to thank the Northeast Ohio Medical University Comparative Medicine Unit for animal housing and care, the Gift of Hope Organ & Tissue Donor Network (Itasca, IL) and Dr. Susan Chubinskaya (Rush University, Chicago) for human donor tissue, and the Musculoskeletal Transplant Foundation (Jessup, PA) for donation of allograft bone. I also would like to express respect and gratitude to the families of donors for access to tissues. Words cannot express how grateful I am to my mom and dad, my grandparents, and my aunts and uncles -- all my sweetest family members -- for their support and love as always. Xing and my friends, Tianyi, Shuo, Jing, Fan, Sharon, and Chris have encouraged me to walk through the most difficult time. The love, prayers, compassion, and generosity of my church people have made me a stronger person. “The Lord is my shepherd, I lack nothing.” (Psalm 23:1, NIV) vii TABLE OF CONTENTS Page LIST OF FIGURES …………………………………………………………………….xii LIST OF TABLES …………………………………………………………………..…xvii LIST OF SCHEMES……………………………………………………………..…...xviii CHAPTER I. A HUMAN PERIOSTEUM-BONE ALLOGRAFT ............................................. 19 1.1 Introduction .............................................................................................. 19 1.1.1 Basic Concepts of Periosteum .......................................................... 19 1.1.1.a Anatomical Aspects ................................................................... 20 1.1.1.b Microscopic Considerations ....................................................... 23 1.1.2 Basic Concepts of Bone .................................................................... 24 1.1.2.a Components of Bone ................................................................. 26 1.1.2.b Functions of Bone ...................................................................... 27 1.1.2.c Bone Remodeling ...................................................................... 28 1.1.2.d Mechanisms of Bone Remodeling ............................................. 33 1.1.3 Application of Polymerase Chain Reaction Analysis in Osteogenesis Studies .............................................................................................. 36 1.1.4 Role of Periosteum in Bone Healing ................................................. 39 1.1.5 Utilization of Periosteum in Tissue Engineering of Bone Grafting ..... 41 1.2 Materials and Methods ............................................................................. 48 1.2.1 Experimental Materials...................................................................... 48 viii 1.2.2 Experimental Methods ...................................................................... 49 1.2.2.a Fabrication of Periosteum-bone Allografts ................................. 49 1.2.2.b Specimen Implantation, Harvesting, and Processing ................. 50 1.2.2.c Specimen Staining ..................................................................... 53 1.2.2.d Quantitative Polymerase Chain Reaction Assessment .............. 54 1.2.2.e Statistical Analysis ..................................................................... 60 1.3 Results and Discussion ............................................................................ 60 1.3.1 Histological Results ........................................................................... 60 1.3.2 RT-qPCR Results ............................................................................. 69 1.3.3 Discussion ......................................................................................... 77 II. A HUMAN TENDON-TO-BONE ENTHESIS MODEL .................................... 83 2.1 Introduction .............................................................................................. 83 2.1.1 Basic Concepts of an Enthesis ........................................................