Cheuk, Yau Chuk. / Thesis submitted in: Dec 2008. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 132-144). / Abstracts in English and Chinese. / ABSTRACT --- p.i / 論文摘要 --- p.iii / PUBLICATIONS --- p.v / ACKNOWLEDGEMENT --- p.vi / LIST OF ABBREBIVIATIONS --- p.vii / INDEX FOR FIGURES --- p.x / INDEX FOR TABLES --- p.xiv / TABLE OF CONTENTS --- p.xv / Chapter CHAPTER ONE - --- INTRODUCTION / Chapter 1.1 --- "Joint function, structure and biochemistry" / Chapter 1.1.1 --- Function of joint --- p.1 / Chapter 1.1.2 --- Types of cartilage --- p.1 / Chapter 1.1.3 --- Composition and structure of articular cartilage --- p.2 / Chapter 1.1.4 --- The subchondral bone --- p.3 / Chapter 1.1.5 --- Maturation of articular cartilage and subchondral bone --- p.3 / Chapter 1.2 --- Osteochondral defect / Chapter 1.2.1 --- Clinical problem --- p.6 / Chapter 1.2.2 --- Spontaneous repair --- p.7 / Chapter 1.2.3 --- Current treatment strategies --- p.7 / Chapter 1.2.4 --- Limitations of current treatment strategies --- p.8 / Chapter 1.2.5 --- Treatments under development --- p.11 / Chapter 1.2.6 --- Potential and limitations in cell therapies --- p.14 / Chapter 1.3 --- The 3-D scaffold-free cartilage / Chapter 1.3.1 --- Fabrication of scaffold-free cartilage --- p.16 / Chapter 1.3.2 --- Scaffold-free cartilage for chondral / osteochondral defect repair --- p.18 / Chapter 1.3.3 --- Scaffold-free bioengineered chondrocyte pellet from our group --- p.20 / Chapter 1.3.4 --- BCP as a possible treatment for OCD --- p.21 / Chapter 1.4 --- The objectives of the study --- p.22 / Chapter 1.5 --- The study plan / Chapter 1.5.1 --- Design of the study --- p.23 / Chapter 1.5.2 --- Choice of animal model --- p.23 / Chapter 1.5.3 --- Selection of evaluation time points --- p.24 / Chapter 1.5.4 --- Choice and modification of histological scoring system --- p.24 / Chapter CHAPTER TWO - --- METHODOLOGY / Chapter 2.1 --- Preparation of reagents and materials for tissue culture and histology --- p.26 / Chapter 2.2 --- Creation of osteochondral defect model --- p.28 / Chapter 2.3 --- Synthesis of scaffold-free cartilage using 3-D chondrocyte pellet culture / Chapter 2.3.1 --- Isolation of rabbit costal chondrocytes --- p.31 / Chapter 2.3.2 --- Three-dimensional chondrocyte pellet culture --- p.31 / Chapter 2.3.3 --- BrdU labeling for cell fate tracing --- p.32 / Chapter 2.4 --- Further characterization of the 3-D scaffold-free chondrocyte pellet / Chapter 2.4.1 --- Gross appearance --- p.35 / Chapter 2.4.2 --- Cell viability / Chapter 2.4.2.1 --- Alamar blue reduction assay --- p.35 / Chapter 2.4.3 --- Preparation of samples for histology --- p.36 / Chapter 2.4.4 --- General morphology and histomorphology / Chapter 2.4.4.1 --- H&E staining --- p.36 / Chapter 2.4.5 --- Cartilage properties / Chapter 2.4.5.1 --- Safranin O /Fast Green staining --- p.37 / Chapter 2.4.5.2 --- Immunohistochemistry of type II collagen --- p.37 / Chapter 2.4.5.3 --- Immunohistochemistry of type I collagen --- p.38 / Chapter 2.4.6 --- Angiogenic properties / Chapter 2.4.6.1 --- Immunohistochemistry of VEGF --- p.40 / Chapter 2.4.7 --- Osteogenic properties / Chapter 2.4.7.1 --- ALP staining --- p.40 / Chapter 2.5 --- Implantation of scaffold-free cartilage into osteochondral defect model / Chapter 2.5.1 --- Surgical procedures --- p.41 / Chapter 2.5.2 --- Experimental groups --- p.42 / Chapter 2.6 --- Assessment of osteochondral defect healing / Chapter 2.6.1 --- Macroscopic evaluation --- p.43 / Chapter 2.6.2 --- Preparation of samples for histology --- p.43 / Chapter 2.6.3 --- Histology for general morphology / Chapter 2.6.3.1 --- H&E staining --- p.45 / Chapter 2.6.4 --- Histological scoring / Chapter 2.6.4.1 --- Modification of the scoring system --- p.45 / Chapter 2.6.4.2 --- Procedures of scoring and validation --- p.45 / Chapter 2.6.5 --- Cell proliferation / Chapter 2.6.5.1 --- Immunohistochemistry of PCNA --- p.49 / Chapter 2.6.6 --- Cartilage regeneration / Chapter 2.6.6.1 --- Safranin O /Fast Green staining --- p.49 / Chapter 2.6.6.2 --- Immunohistochemistry of type II collagen --- p.49 / Chapter 2.6.6.3 --- Immunohistochemistry of type I collagen --- p.50 / Chapter 2.6.6.4 --- Polarized light microscopy --- p.50 / Chapter 2.6.7 --- Expression of angiogenic factor / Chapter 2.6.7.1 --- Immunohistochemistry of VEGF --- p.50 / Chapter 2.6.8 --- Bone regeneration / Chapter 2.6.8.1 --- μCT analysis --- p.50 / Chapter 2.6.9 --- Histomorphometric analysis of cartilage and bone regeneration --- p.53 / Chapter 2.6.10 --- BrdU detection for cell fate tracing --- p.55 / Chapter 2.6.11 --- Statistical analysis --- p.55 / Chapter CHAPTER THREE - --- RESULTS / Chapter 3.1 --- Further characterization of the 3-D chondrocyte pellet culture / Chapter 3.1.1 --- Gross examination --- p.57 / Chapter 3.1.2 --- Cell viability --- p.57 / Chapter 3.1.3 --- Cartilage properties --- p.61 / Chapter 3.1.4 --- Angiogenic properties --- p.63 / Chapter 3.1.5 --- Osteogenic properties --- p.64 / Chapter 3.2 --- Implantation of scaffold-free cartilage and assessment / Chapter 3.2.1 --- Gross examination --- p.65 / Chapter 3.2.2 --- General morphology --- p.67 / Chapter 3.2.3 --- Histological scores --- p.71 / Chapter 3.2.4 --- Cell proliferation --- p.75 / Chapter 3.2.5 --- Cartilage regeneration --- p.78 / Chapter 3.2.6 --- Expression of angiogenic factor --- p.90 / Chapter 3.2.7 --- Bone regeneration --- p.93 / Chapter 3.2.8 --- Histomorphometric analysis on cartilage and bone regeneration --- p.96 / Chapter 3.2.9 --- Cell fate tracing --- p.100 / Chapter CHAPTER FOUR - --- DISCUSSION / Chapter 4.1 --- Summary of key findings / Chapter 4.1.1 --- Further characterization of BCP and determination of implantation time --- p.102 / Chapter 4.1.2 --- Implantation of BCP in OCD --- p.102 / Chapter 4.2 --- Spontaneous healing in osteochondral defect / Chapter 4.2.1 --- Findings from the current study --- p.104 / Chapter 4.2.2 --- Comparison with other studies --- p.104 / Chapter 4.2.3 --- Factors affecting spontaneous healing --- p.105 / Chapter 4.3 --- Fabrication and further characterization of the 3-D chondrocyte pellet / Chapter 4.3.1 --- Comparison of different methods of producing scaffold-free cartilage construct --- p.106 / Chapter 4.3.2 --- Cartilage phenotype of the BCP --- p.107 / Chapter 4.3.3 --- Angiogenic and osteogenic potential of the BCP --- p.108 / Chapter 4.3.4 --- Role of mechanical stimulation on tissue-engineered cartilage --- p.109 / Chapter 4.4 --- Repair of osteochondral defect with allogeneic scaffold-free cartilage / Chapter 4.4.1 --- Advantages of the current scaffold-free chondrocyte pellet --- p.111 / Chapter 4.4.2 --- Remodeling of BCP after implantation --- p.111 / Chapter 4.4.3 --- Effect of BCP on cartilage repair --- p.112 / Chapter 4.4.4 --- Effect of BCP on bone regeneration / Chapter 4.4.4.1 --- Findings in the present study --- p.113 / Chapter 4.4.4.2 --- Possible reasons of slow bone repair --- p.114 / Chapter 4.4.4.3 --- Effect of BCP on bone region peripheral to defect --- p.115 / Chapter 4.4.5 --- Immunorejection-free properties of the BCP --- p.116 / Chapter 4.4.6 --- Comparison with other animal studies using scaffold-free cartilage --- p.117 / Chapter 4.4.7 --- Possibility of implanting a BCP cultured for shorter or longer period --- p.118 / Chapter 4.4.8 --- Scaffold-free cartilage construct and construct with scaffold for OCD repair --- p.119 / Chapter 4.4.9 --- Chondrocytes and stem cells for OCD repair --- p.120 / Chapter 4.5 --- Limitations of the study / Chapter 4.5.1 --- Animal model --- p.122 / Chapter 4.5.2 --- Histomorphometric analysis --- p.122 / Chapter 4.5.3 --- Lack of quantitative data analysis --- p.122 / Chapter 4.5.4 --- BrdU labeling of cells --- p.123 / Chapter 4.5.5 --- Lack of biomechanical test --- p.123 / Chapter 4.5.6 --- Small sample size --- p.123 / Chapter CHAPTER FIVE - --- CONCLUSION --- p.124 / Chapter CHAPTER SIX - --- FUTURE STUDIES / Chapter 6.1 --- Identification of factors affecting bone repair after OCD treatment --- p.125 / Chapter 6.2 --- Modifications of BCP treatment --- p.125 / Chapter 6.3 --- Alternative cell source --- p.126 / Chapter 6.4 --- Alternative cell tracking methods --- p.126 / Chapter 6.5 --- Inclusion of biomechanical test --- p.126 / APPENDICES / Appendix 1. Conference paper 1 --- p.129 / Appendix 2: Conference paper 2 --- p.130 / Appendix 3: Animal experimentation ethics approval --- p.131 / BIBLIOGRAPHY --- p.132