by Pau Hon-Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 146-179). / Abstracts in English and Chinese. / ACKNOWLEDGEMENT --- p.i / ABBREVIATIONS --- p.iii / ABSTRACT (English) --- p.v / ABSTRACT (Chinese) --- p.ix / FLOW CHART --- p.xi / INDEX OF FIGURES --- p.xxi / INDEX OF TABLES --- p.xxiv / PUBLICATION LIST --- p.xxv / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Basic Structure of Tendons --- p.3 / Chapter 1.2 --- Anatomy of Patellar Tendon --- p.9 / Chapter 1.3 --- Cellular Characteristics of Fibroblasts --- p.11 / Chapter 1.4 --- Healing Process in Tendons --- p.13 / Chapter 1.5 --- Chronic Tendon Disorder --- p.19 / Chapter 1.6 --- Aims and Objectives --- p.25 / Chapter 1.7 --- Study Plan --- p.26 / Chapter CHAPRER 2 --- MATERIALS AND METHODOLOGY --- p.35 / Chapter 2.1 --- Collection of human Tissues Samples --- p.38 / Chapter 2.2 --- Tissue Culture --- p.40 / Chapter 2.3 --- Preparation of Conditioned Medium --- p.46 / Chapter 2.4 --- Proliferation Response of Tendon Fibroblasts --- p.48 / Chapter 2.5 --- Invasion Assay --- p.53 / Chapter 2.6 --- Chick Chorioallantonic Membrane Model --- p.56 / Chapter 2.7 --- SDS-PAGE --- p.58 / Chapter 2.8 --- Enzyme Linked Immunosorbent Assay --- p.63 / Chapter 2.9 --- Immunocytochemical Staining --- p.68 / Chapter 2.10 --- Statistical Analysis --- p.69 / Chapter CHAPTER 3 --- RESULTS --- p.74 / Chapter 3.1 --- Patient's Information --- p.76 / Chapter 3.2 --- Primary Explant Cell Culture --- p.77 / Chapter 3.3 --- Proliferation Response of Tendon Fibroblasts --- p.79 / Chapter 3.4 --- Invasion Assay --- p.82 / Chapter 3.5 --- Chick Chorioallantonic Membrane Model --- p.83 / Chapter 3.6 --- SDS-PAGE --- p.85 / Chapter 3.7 --- Enzyme Linked Immunosorbent Assay --- p.86 / Chapter 3.8 --- Immunocytochemical Staining --- p.88 / Chapter CHAPTER 4 --- DISCUSSION --- p.116 / Chapter 4.1 --- Higher Proliferation Capacity of Tendinosis Fibroblasts --- p.118 / Chapter 4.2 --- Tendinosis Fibroblasts Secrete Angiogenesis Factors to Enhance Angiogenesis --- p.123 / Chapter 4.3 --- Tendinosis Fibroblasts Secrete More Proteolytic Enzyme --- p.126 / Chapter 4.4 --- Proinflammatory Characteristics in Tendinosis Fibroblasts --- p.129 / Chapter 4.5 --- Comparison of in vitro and Histological Study --- p.132 / Chapter 4.6 --- Tendinosis Fibroblasts Seems to be Transformed --- p.134 / Chapter 4.7 --- Limitation of Study --- p.136 / Chapter CHAPTER 5 --- FUTURE RESEARCH DIRECTION --- p.140 / Chapter 5.1 --- Further Exploration of the Factors Secreted in Tendinosis Fibroblasts --- p.141 / Chapter 5.2 --- Proteoglycan Synthesis in Tendinosis Fibroblasts --- p.142 / Chapter 5.3 --- Tendinosis Animal Model and Clinical Study --- p.143 / Chapter CHAPTER 6 --- CONCLUSION --- p.144 / BIBLIOGRAPHY --- p.146 / APPENDIX --- p.180

Dissertation (MHSc--Health Science) -- Auckland University of Technology, 2005. / Also held in print (82 leaves, 30 cm.) in North Shore Theses Collection (T 612.76 POT)

by Wang Wen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 113-124). / Abstracts in English and Chinese. / ABSTRACT --- p.i / FLOWCHART --- p.vi / ACKNOWLEDGEMENT --- p.x / ABBREVIATIONS --- p.xi / INDEX FOR FIGURES --- p.xii / INDEX FOR TABLES --- p.xv / TABLE OF CONTENTS --- p.xvi / Chapter 1. --- INTRODUCTION --- p.1 / Chapter 1.1 --- PATELLAR TENDINOSIS --- p.1 / Chapter 1.1.1 --- Introduction --- p.1 / Chapter 1.1.2 --- Epidemiology of Patellar Tendinosis --- p.3 / Chapter 1.1.3 --- Etiology of Patellar Tendinosis --- p.3 / Chapter 1.1.4 --- Manifestations of Patellar Tendinosis --- p.4 / Chapter 1.1.5 --- Imaging Examination on Patellar Tendinosis --- p.4 / Chapter 1.1.6 --- Clinical Diagnosis of Patellar Tendinosis --- p.6 / Chapter 1.1.7 --- Management of Patellar Tendinosis … --- p.6 / Chapter 1.2 --- ANATOMY AND HISTOLOGY OF PATELLAR TCNDON --- p.7 / Chapter 1.3 --- STRUCTURE AND METABOLISM OF TENDON --- p.9 / Chapter 1.3.1 --- Tenocytes --- p.9 / Chapter 1.3.2 --- Extra-cellular Matrix --- p.11 / Chapter 1.3.2.1 --- Collagen --- p.11 / Chapter 1.3.2.2 --- Proteoglycans --- p.12 / Chapter 1.4 --- ROLES OF GROWTH FACTORS TENDON HEALING AND REPAIR --- p.14 / Chapter 1.4.1 --- Platelet-Derived Growth Factor --- p.14 / Chapter 1.4.2 --- Transforming Growth Factor-beta --- p.15 / Chapter 1.5 --- HISTOPATHOLOGY OF PATELLAR TENDINOSIS --- p.16 / Chapter 1.6 --- STUDY PLAN --- p.17 / Chapter 1.6.1 --- Characterization on Hypercellularity --- p.18 / Chapter 1.6.2 --- Characterization on Disorganization and Loosening of Collagen --- p.18 / Chapter 1.6.3 --- Characterization on Inflammatory Trace --- p.20 / Chapter 1.6.4 --- Characterization on Growth Factors in Tendinosis --- p.21 / Chapter 1.7 --- OBJECTIVES --- p.22 / Chapter 2. --- MATERIALS AND METHODS --- p.27 / Chapter 2.1 --- HUMAN TISSUES --- p.27 / Chapter 2.1.1 --- Patellar Tendinosis Tissues --- p.27 / Chapter 2.1.1.1 --- Diagnosis of patellar tendinosis --- p.27 / Chapter 2.1.1.2 --- Recruitment of patients --- p.27 / Chapter 2.1.4 --- Healthy Patellar Tendon tissues --- p.28 / Chapter 2.2 --- TISSUES COLLECTION AND PREPARATION --- p.28 / Chapter 2.3 --- HISTOLOGICAL STUDY ON HUMAN SPECIMENS --- p.28 / Chapter 2.3.1 --- Haematoxyline and Eosin Staining --- p.29 / Chapter 2.3.2 --- Safranin O Staining --- p.29 / Chapter 2.3.2.1 --- Reagents preparation --- p.29 / Chapter 2.3.2.2 --- Experimental procedure --- p.30 / Chapter 2.3.5 --- Polarization Microscopy --- p.30 / Chapter 2.4 --- IMMUNOHISTOCHEMICAL STAINING --- p.30 / Chapter 2.4.1 --- Reagents Preparation --- p.31 / Chapter 2.4.2 --- Experimental Procedure --- p.33 / Chapter 2.5 --- IMAGE ANALYSIS --- p.35 / Chapter 2.5.1 --- Equipment --- p.35 / Chapter 2.5.2 --- Procedures --- p.35 / Chapter 2.6 --- IN SITU ZYMOGRAPHY --- p.37 / Chapter 2.6.1 --- Reagents Preparation --- p.37 / Chapter 2.6.2 --- Experimental Procedure --- p.38 / Chapter 2.7 --- STATISTIC ANALYSIS.… --- p.39 / Chapter 3. --- RESULTS --- p.42 / Chapter 3.1 --- HUMAN SAMPLES --- p.42 / Chapter 3.1.1 --- Patellar tendinosis patients --- p.42 / Chapter 3.1.2 --- Healthy control group --- p.43 / Chapter 3.2 --- HISTOLOGICAL STUDY ON HUMAN SPECIMENS --- p.43 / Chapter 3.2.1 --- Gross Morphology --- p.43 / Chapter 3.2.2 --- Haematoxyline and Eosin Staining --- p.44 / Chapter 3.2.3 --- Safranin O Staining --- p.44 / Chapter 3.2.4 --- Polarization Microscopy --- p.44 / Chapter 3.3 --- IMAGE ANALYSIS --- p.45 / Chapter 3.3.1 --- Immunohistochemistry of PCNA --- p.45 / Chapter 3.3.2 --- Immunohistochemistry of hsp47 --- p.46 / Chapter 3.3.3 --- Immunohistochemistry of Procollogen Type I --- p.47 / Chapter 3.3.4 --- Immunohistochemistry of MMP1 --- p.47 / Chapter 3.3.5 --- Immunohistochemistry of TIMP1 --- p.48 / Chapter 3.3.6 --- Immunohistochemistry of COX-2 --- p.49 / Chapter 3.3.7 --- Immunohistochemistry of TGFP --- p.49 / Chapter 3.3.8 --- Immunohistochemistry of PDGFbb --- p.50 / Chapter 3.3.9 --- Immunohistochemistry of PDGFRβ --- p.51 / Chapter 3.3.10 --- Summary of Image Analysis of Immunohistochemical staining --- p.51 / Chapter 3.4 --- IN SITU ZYMOGRAPHY --- p.52 / Chapter 4. --- DISCUSSION --- p.93 / Chapter 4.1 --- DIAGNOSIS OF PATELLAR TENDINOSIS --- p.93 / Chapter 4.2 --- HYPERCELLULARITY IN PATELLAR TENDINOSIS --- p.95 / Chapter 4.3 --- COLLAGEN DISTURBANCE IN PATELLAR --- p.97 / Chapter 4.4 --- INFLAMMATORY RESPONSE IN PATELLAR TENDINOSIS --- p.100 / Chapter 4.5 --- THE EXPRESSION OF GROWTH FACTORS IN PATELLAR TENDINOSIS --- p.102 / Chapter 4.6 --- PROPOSED PATHOGENESIS FOR PATELLAR TENDINOSIS --- p.105 / Chapter 4.7 --- LIMITATION OF THIS STUDY --- p.108 / Chapter 4.8 --- FUTURE STUDY --- p.109 / Chapter 5. --- CONCLUSION --- p.111 / BIBLIOGRAPHY --- p.113

An attempt to reproduce tendon sheaths using autologous venous grafts has been undertaken in Cercopithecus aethiops (Blue Vervet Monkey). Ten venous grafts were tested. Five were placed around sutured tendons in paratenon, and the remainder around tendons in sheath formation. In no instance did synovial-like sheaths form. In the latter series, the experiment was controlled, and the results obtained expressed in terms of function. The functional results of the venous ensheathed tendons were worse than those of the controlled tendons.The controlled experiment was confined to the digital sheath, an area notoriously liable to adhesion formation, and the one which offered the most critical test of operative technique.A method for the evaluation of function, following the repair of divided tendons in the experimental animal has been presented. Voluntary movement of joints in the experimental animal was obviously impossible, but the method employed in this experiment, namely, the electrical stimulation of muscles, and the photographic recording of the range of joint movement, presented no disadvantages.The experimental animal used in this study was ideal, in that anatomical studies of the hand of this species, revealed features both structurally and functionally comparable to those in man.The anatomical and physiological aspects of tendon action, the mechanics of tendon gliding, and the healing processes in divided tendons have been studied.The fate of human autologous venous grafts has been investigated.A review of previous methods employed to prevent peritendinous adhesions has been presented.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 6 uppsatser.

Diss. (sammanfattning) Umeå : Univ., 2006. / Härtill 4 uppsatser.

No description available.

Li Chi Kei. / Thesis (M.Phil.)--Chinese University of Hong Kong,1995. / Includes bibliogrpahical references (leaves 54-68). / Abstract --- p.1 / Acknowledgments --- p.4 / Lists of Figures --- p.5 / List of Tables --- p.8 / Chapter Chapter 1 --- Introduction --- p.9 / Chapter 1.1 --- Ligamental Injury --- p.9 / Chapter 1.2 --- ACL Injury --- p.10 / Chapter 1.3 --- Patellar Tendon and Anterior Cruciate Ligament --- p.11 / Chapter 1.4 --- Patellar Tendon Bone Graft --- p.13 / Chapter 1.41 --- Strength of the Patellar Tendon Bone Graft --- p.13 / Chapter 1.42 --- Use of the Patellar Tendon Bone Graft --- p.14 / Chapter 1.5 --- Patellar Tendon Bone Graft in Anterior Cruciate Ligament Reconstruction --- p.14 / Chapter 1.6 --- Complications of Anterior Cruciate Ligament Reconstruction after the Use of Patellar Tendon Bone Graft --- p.16 / Chapter 1.7 --- Healing of the Patellar Tendon Donor Site --- p.17 / Chapter 1.8 --- Objective of the Study --- p.22 / Chapter 1.9 --- Significance of the Study --- p.22 / Chapter Chapter 2 --- Material & Method --- p.24 / Chapter 2.1 --- Animal Model --- p.24 / Chapter 2.2 --- Grouping --- p.24 / Chapter 2.3 --- Operative Procedure --- p.24 / Chapter 2.4 --- Method of Assessment --- p.26 / Chapter 2.41 --- Gross Morphology --- p.26 / Chapter 2.42 --- Biomechanical Testing --- p.27 / Chapter 2.43 --- Biochemical Assay --- p.28 / Chapter 2.43.1 --- Water Content --- p.29 / Chapter 2.43.2 --- Collagen Content --- p.29 / Chapter 2.44 --- Histology --- p.30 / Chapter 2.45 --- Immunohistochemistry --- p.31 / Chapter 2.5 --- Statistics --- p.32 / Chapter Chapter 3 --- Results --- p.33 / Chapter 3.1 --- Operation Complications --- p.33 / Chapter 3.2 --- Gross Morphology --- p.33 / Chapter 3.21 --- Dimension of the Patellar Tendon --- p.33 / Chapter 3.22 --- Dimension of the Patellar Tendon Bone Graft --- p.33 / Chapter 3.23 --- Dimension of the Remain Patellar Tendon --- p.34 / Chapter 3.24 --- Gross Appearance of the Patellar Tendon Donor Site --- p.35 / Chapter 3.3 --- Biomechanical Testing --- p.36 / Chapter 3.31 --- Failure Load --- p.36 / Chapter 3.32 --- Ultimate Stress --- p.37 / Chapter 3.33 --- Stiffness --- p.37 / Chapter 3.34 --- Energy Absorbed before Failure --- p.38 / Chapter 3.4 --- Biochemical Assay --- p.38 / Chapter 3.41 --- Water Content --- p.38 / Chapter 3.42 --- Collagen Content --- p.39 / Chapter 3 .5 --- Histology & Immunohistochemistry --- p.39 / Chapter 3.51 --- Morphology of Control Tendon --- p.40 / Chapter 3.52 --- Morphology of Experimental Tendon with 'Open' Procedure --- p.40 / Chapter 3.53 --- Morphology of Experimental Tendon with 'Close' Procedure --- p.41 / Chapter 3.54 --- Healing at the Patellar and Tibial Insertion Site --- p.41 / Chapter 3.55 --- Morphology of the Cartilage Surface --- p.42 / Chapter Chapter 4 --- Discussion & Conclusion --- p.43 / Chapter 4.1 --- Gross Morphology --- p.43 / Chapter 4.2 --- Biomechanical Testing --- p.45 / Chapter 4.3 --- Biochemical Assay --- p.47 / Chapter 4.4 --- Histology & Immunohistochemistry --- p.49 / Chapter 4.5 --- Conclusion --- p.51 / References --- p.54 / Figures / Tables / Appendix

肌腱損傷發生率高，並且癒合結果很不理想，因為少量的肌腱細胞缺乏有效的修復能力，僅僅通過瘢痕形成來癒合, 肌腱瘢痕癒合難以恢復原本的肌腱組織結構及力學特性。目前，國內外臨床上治療肌腱損傷的方法很多，包括藥物、物理治療、手術等，這些並不能獲得滿意的療效。因此，如何採用肌腱組織工程技術迅速、安全、有效的修復肌腱損傷已成為運動醫學領域急需解決的重要問題。 / 有研究表明，骨髓間充質幹細胞、表皮成纖維細胞、肌腱細胞和胚胎幹細胞通過肌腱組織工程技術用於肌腱修復及再生取得了不錯的療效。但是，這些來源的細胞存在分化效率低，形成畸胎瘤和異位骨化等風險。近來，有研究報導可從人、小鼠、大鼠和兔的肌腱組織中分離培養出幹細胞，可作為肌腱組織工程種子細胞的一種新選擇，用於肌腱修復和再生。對於間充質幹細胞的成肌腱分化，有研究報導結締組織生長因子（CTGF）和抗壞血酸（維生素C的一種形式）在膠原及細胞外基質合成、調節細胞成肌腱分化方面扮演者重要的角色。 / 本研究的旨在：（1）在大鼠髕腱損傷模型中，證實肌腱幹細胞可作為一種新的幹細胞來源用於肌腱修復；（2）檢驗結締組織生長因子和抗壞血酸能在體外促進肌腱幹細胞的成肌腱分化；（3）嘗試通過肌腱幹細胞的成肌腱分化過程在體外構建不含外源性支架的肌腱樣組織；（4）探索該肌腱樣組織在大鼠髕腱損傷模型中是否可以促進肌腱癒合。 / 在大鼠急性髕腱損傷動物模型中，與對照組相比，肌腱幹細胞組具有更好的膠原排列，顯著增高的最大張力和楊氏模量，表明肌腱幹細胞可作為一種新的幹細胞來源用於肌腱損傷的修復。結締組織生長因子和抗壞血酸體外誘導肌腱幹細胞2周後，可顯著增加Tenomodulin, Scleraxis, Thbs4, I型膠原等肌腱相關基因的表達以及膠原蛋白的合成，說明結締組織生長因子和抗壞血酸可促進肌腱幹細胞的成肌腱分化。被結締組織生長因子和抗壞血酸誘導兩周後，肌腱幹細胞可形成了細胞膜樣結構，將這種細胞膜纏繞在迴紋針上，構建成肌腱樣組織，其具有相對疏鬆的細胞外基質和雜亂排列其中的肌腱幹細胞，以及表達Tenomodulin，I型膠原和III型膠原。將該肌腱樣組織移植到裸鼠體內8周和12周可形成新生肌腱組織，梭形細胞縱行分佈在平行的膠原纖維之間，並表達Tenomodulin，I型膠原和III型膠原蛋白。在大鼠髕腱損傷動物模型中，與對照組相比較，該肌腱樣組織可通過恢復肌腱組織結構及生物力學特性來促進肌腱癒合。 / 總的來說，本研究證實肌腱幹細胞可作為一種新的幹細胞來源用於肌腱組織工程促進肌腱再生。結締組織生長因子和抗壞血酸可調控肌腱幹細胞的成肌腱分化，並形成細胞膜結構。該細胞膜結構可在體外構建出不含外源性支架的肌腱樣組織，進而在裸鼠體內形成新生肌腱，並且在大鼠髕腱損傷模型中可有效的促進損傷肌腱的癒合。這種不含外源性支架的肌腱樣組織有希望成為肌腱組織工程技術的新手段，在肌腱再生和肌腱修復的臨床應用及基礎研究方面有廣泛的前景。 / Tendon injuries are common and tendon healing outcome is poor, because tendon contains few cells with limited capacities for self-repair/regeneration. The current treatments on tendon injuries including drugs, physiotherapy, and surgery are not ideal and there is a need for the development of novel tissue-engineering strategies for tendon repair. / Previous studies have shown positive effects of bone marrow-derived mesenchymal stem cells (BMSCs), dermal fibroblast, tenocytes, and embryonic stem cells-derived MSCs for tendon repair/regeneration. However, these cells have limitations including insufficient differentiation; risk of teratoma and ectopic bone formation etc. Recently, stem cells have been isolated from tendons of human, mouse, rat and rabbit and considered as a new alternative cell source for tendon tissue engineering (TDSCs). For tenogenic differention of MSCs, connective tissue growth factor (CTGF) and ascorbic acid (one form of vitamin C) are reported to play important roles in promoting collagen and other extracellular matrixes (ECM) production, and regulating the MSCs differentiation towards tenogenic pathway. / The aims of the current study are: (1) To investigate the use of TDSCs in tendon repair in a rat acute patellar tendon injury model; (2) To test the effects of CTGF and ascorbic acid on tenogenic differentiation of TDSCs in vitro; (3) To construct scaffold-free tendon-like tissues in vitro using tenogenically differentiated TDSCs; (4) To promote tendon healing by engineered tendon-like tissues in a rat acute patellar tendon injury model. / In the rat acute patellar tendon injury model, in contract to control group, TDSCs treated group showed better alignment of collagen fibers and the significantly higher ultimate stress and Young’s modulus, indicating TDSCs may be an alternative cell source for tendon repair. The effects of CTGF and ascorbic acid on tenogenic differentiation of TDSCs were also confirmed with higher expression of tendon related markers such as Tenomodulin, Scleraxis, Thbs4, Type I Collagen, etc; with higher production of collagenous proteins. After treatment with CTGF and ascorbic acid for 2 weeks, TDSCs can form cell sheets, which can be harvested, rolled up on a U-shaped spring to form tendon-like tissues in culture, which had loose extracellular matrices and randomly distributed TDSCs and also expressed Tenomodulin, Type I & III collagen. Following transplantation of the engineered tendon-like tissue in nude mice for 8 and 12 weeks, neo-tendon tissues were formed, with thin and parallel collagen fibrils and extracellular matrices of Tenomodulin, Type I & III collagen. Finally in the rat patellar tendon window injury model, data suggested that the engineered tendon-like tissue could promote tendon healing with significantly improved histological features and biomechanical properties comparing to the control group. / In conclusion, our study has indicated that TDSCs can be an alternative cell source in tendon tissue engineering for tendon regeneration. The tenogenic differentiation of TDSCs, induced by CTGF and ascorbic acid in vitro, produces cell sheets, which can be constructed tendon-like tissues in vitro; to form neo-tendon and repair tendon injuries in vivo. The use of engineered scaffold-free tendon tissue for tendon tissue engineering has potentials in clinical application for tendon repair/regeneration. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Ni, Ming. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 107-126). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / DEDICATION --- p.I / ACKNOWLEDGEMENT --- p.II-III / TABLE OF CONTENTS --- p.IV-IX / PUBLICATIONS --- p.X-XII / ABBREVIATION --- p.XIII-XV / ABSTRACT (ENGLISH) --- p.XVI-XVIII / ABSTRACT (CHINESE) --- p.XIX-XX / Chapter CHAPTER 1 --- Introduction --- p.1 / Chapter 1.1 --- Epidemiology of tendon injury --- p.1 / Chapter 1.2 --- Healing process of tendon injury --- p.1 / Chapter 1.3 --- Tendon tissue engineering for tendon repair --- p.2 / Chapter 1.4 --- Stem cells in tendon repair --- p.2 / Chapter 1.5 --- Tenogenic differentiation of tendon derived stem cells --- p.7 / Chapter 1.6 --- Growth factors for tenogenic differentiation --- p.8 / Chapter 1.7 --- Vitamin C for tenogenic differentiation --- p.9 / Chapter 1.8 --- Summary --- p.10 / Chapter CHAPTER 2 --- Hypothesis, Objectives and Study Design --- p.11 / Chapter 2.1 --- Hypothesis --- p.11 / Chapter 2.1.1 --- Overall hypothesis --- p.11 / Chapter 2.1.2 --- Specific hypothesis --- p.11 / Chapter 2.2 --- Objectives --- p.12 / Chapter 2.3 --- Study design --- p.12 / Chapter 2.3.1 --- Study I --- p.12 / Chapter 2.3.2 --- Study II --- p.14 / Chapter 2.3.3 --- Study III --- p.14 / Chapter 2.3.4 --- Study IV --- p.17 / Chapter CHAPTER 3 --- Tendon-derived Stem Cells (TDSCs): A New Cell Source for Tendon Repair (Study I) --- p.19 / Chapter 3.1 --- Materials and Methods --- p.19 / Chapter 3.1.1 --- Isolation and characterization of rat GFP-TDSCs --- p.19 / Chapter 3.1.2 --- Animal surgery --- p.20 / Chapter 3.1.3 --- Ultrasound imaging --- p.25 / Chapter 3.1.4 --- Histology --- p.27 / Chapter 3.1.5 --- Biomechanical test --- p.27 / Chapter 3.1.6 --- Ex vivo fluorescence imaging --- p.28 / Chapter 3.1.7 --- Data analysis --- p.29 / Chapter 3.2 --- Results --- p.29 / Chapter 3.2.1 --- Gross observation of the injured knee and patellar tendon --- p.29 / Chapter 3.2.2 --- Histology of regenerated tendon tissue --- p.30 / Chapter 3.2.3 --- Biomechanical test of regenerated tendon tissue --- p.32 / Chapter 3.2.4 --- Ex vivo fluorescence imaging of GFP-TDSCs --- p.33 / Chapter 3.2.5 --- Ultrasound imaging of wound gap volume --- p.34 / Chapter 3.3 --- Discussion --- p.35 / Chapter 3.4 --- Conclusion --- p.50 / Chapter CHAPTER 4 --- Tenogenic Differentiation of Tendon-derived Stem Cells (TDSCs) (Study II) --- p.51 / Chapter 4.1 --- Materials and Methods --- p.51 / Chapter 4.1.1 --- Tenogenic differentiation of tendon-derived stem cells (TDSCs) --- p.51 / Chapter 4.1.2 --- Quantification of collagenous proteins --- p.51 / Chapter 4.1.3 --- Quantitative Real Time PCR (qRT-PCR) --- p.52 / Chapter 4.1.4 --- Data analysis --- p.54 / Chapter 4.2 --- Results --- p.55 / Chapter 4.2.1 --- Quantification of collagenous proteins --- p.55 / Chapter 4.2.2 --- Tenogenic, osteogenic and chondrogenic markers mRNA expression --- p.57 / Chapter 4.2.3 --- Tendon extracellular matrix markers mRNA expression --- p.57 / Chapter 4.3 --- Discussion --- p.59 / Chapter 4.4 --- Conclusion --- p.66 / Chapter CHAPTER 5 --- Engineered Scaffold-free Tendon Tissue Produced by Tendon-derived Stem Cells (TDSCs) Cell Sheet (Study III) --- p.67 / Chapter 5.1 --- Materials and Methods --- p.67 / Chapter 5.1.1 --- In vitro engineered scaffold-free tendon tissue by TDSCs cell sheet --- p.67 / Chapter 5.1.2 --- In vivo neo-tendon formation using engineered scaffold-free tendon tissue in nude mouse model --- p.67 / Chapter 5.1.3 --- Histology and immunohistochemistry staining --- p.68 / Chapter 5.1.4 --- In vivo fluorescence imaging --- p.69 / Chapter 5.1.5 --- Data analysis --- p.70 / Chapter 5.2 --- Results --- p.70 / Chapter 5.2.1 --- Gross observation of TDSCs cell sheet and engineered scaffold-free tendon tissue --- p.70 / Chapter 5.2.2 --- Histological and immunohistochemical characteristics in engineered scaffold-free tendon tissue --- p.71 / Chapter 5.2.3 --- Gross observation and in vivo fluorescence imaging of neo-tendon tissue --- p.74 / Chapter 5.2.4 --- Histology of neo-tendon tissue --- p.75 / Chapter 5.2.5 --- Immunohistochemistry staining in neo-tendon tissue --- p.76 / Chapter 5.3 --- Discussion --- p.78 / Chapter 5.4 --- Conclusion --- p.82 / Chapter CHAPTER 6 --- Use of Engineered Scaffold-free Tendon Tissue for Tendon Repair (Study IV) --- p.83 / Chapter 6.1 --- Materials and methods --- p.83 / Chapter 6.1.1 --- Animal surgery --- p.83 / Chapter 6.1.2 --- Ex vivo fluorescence imaging --- p.84 / Chapter 6.1.3 --- Histology and immunohistochemistry staining --- p.85 / Chapter 6.1.4 --- Biomechanical test --- p.86 / Chapter 6.1.5 --- Ultrasound imaging --- p.87 / Chapter 6.1.6 --- Data Analysis --- p.87 / Chapter 6.2 --- Results --- p.88 / Chapter 6.2.1 --- Gross observation of the injured knee and patellar tendon --- p.88 / Chapter 6.2.2 --- Histology of regenerated tendon tissue --- p.89 / Chapter 6.2.3 --- Tendon specific and ECM markers expression in regenerated tendon tissue --- p.91 / Chapter 6.2.4 --- Osteogenic and chondrogenic specific markers expression in neo-tendon tissue --- p.93 / Chapter 6.2.5 --- The fate of the transplanted engineered scaffold-free tendon tissue --- p.93 / Chapter 6.2.6 --- Biomechanical test of regenerated tendon tissues --- p.94 / Chapter 6.3 --- Discussion --- p.96 / Chapter 6.4 --- Conclusion --- p.102 / Chapter CHAPTER 7 --- General Conclusions --- p.103 / Chapter 7.1 --- General discussion --- p.103 / Chapter 7.2 --- General conclusions --- p.105 / FUNDING --- p.106 / REFERENCES --- p.107 / APPENDIX --- p.127

肌腱修復一直是一個難題，因為依靠現在的治療很難將肌腱功能恢復到正常水平，近年來肌腱幹細胞的分離和發現為肌腱修復提供了新的策略。但是在利用肌腱幹細胞修復肌腱之前，我們應該瞭解肌腱幹細胞的哪些方面呢？ / 不同來源的成體幹細胞雖然具備相似的幹細胞特性，但是他們仍然具有組織特異性和功能的差異。這就意味選擇合適的細胞來源對於肌腱再生和肌腱組織工程有特殊意義。所以我們認為與骨髓間充質幹細胞相比，肌腱幹細胞具備特殊的幹細胞特性。迄今為止，還沒有研究比較肌腱幹細胞和骨髓間充質幹細胞的幹細胞特性。臨床應用要求幹細胞在體外增殖培養，體外的微環境也會影響幹細胞的幹性和治療潛能，所以我們還並不清楚肌腱幹細胞的幹性在體外培養中維持多久。成功的幹細胞治療需要深入理解組織特異性幹細胞的體內特徵和他們在組織修復中的作用。肌腱幹細胞的体内特徵还有没详细研究过，而且也不知道這些內源性幹細胞是否參與肌腱修復。 / 所以為了更好地利用肌腱幹細胞進行肌腱修復，本研究的總體目標是比較肌腱幹細胞和骨髓間充質幹細胞的幹細胞特性，同時從臨床角度考慮研究肌腱幹細胞體外幹性的維持。進一步研究鑒定肌腱幹細胞的體內特徵，並且探索他們在肌腱癒合中的作用。本研究將會探討我們應該瞭解關於肌腱幹細胞的體內和體外特性。 / 在第一部分研究中， 我們從同一隻GFP大鼠中分離出肌腱幹細胞和骨髓間充質幹細胞。經過比較，我們發現肌腱幹細胞与骨髓間充質幹細胞相比具备更高的克隆形成能力，增殖速度，更強的多向分化能力和更高的肌腱相关的基因表达。所以肌腱幹細胞表現出更好的幹性，可能是比骨髓间充质干细胞更好的用于肌腱再生的细胞来源。 / 在第二部分研究中，我們發現肌腱幹細胞伴隨體外傳代培養細胞衰老β-半乳糖苷酶活性增高，而同時間充質幹細胞標誌物和多向分化能力降低，所以研究人員和臨床醫生在利用肌腱幹細胞進行組織工程時需要考慮在體外傳代培養中他們的幹性的變化。 / 在第三部分研究中，IdU標記滯留細胞方法用於在體內標記幹細胞。我們發現休眠的幹細胞以IdU標記滯留細胞的形式存在於肌腱中，相比肌腱本體更多標記滯留細胞位於和肌腱腱鞘和肌腱骨結合部位。其中我們發現在肌腱腱鞘中的標記滯留細胞位於血管周圍的微環境血管，所以血管周圍的微環境可能是肌腱幹細胞來源之一。肌腱損傷后，位於損傷區域的標記滯留細胞的數量，增殖標誌物，肌腱相關標誌物， 多能性標誌物，和微血管相關標誌物都有明顯增加，意味著標記滯留細胞可能通過遷移，增殖和分化參與肌腱修復。 / 綜上所述，我們的結果為理解肌腱幹細胞的體外幹性特徵和在體外培養中的幹性變化以及体内肌腱幹細胞的鑒定提供了新的解釋，這有利于未來促進肌腱幹細胞的組織工程應用於肌腱修復。 / Tendon repair remains a great challenge due to current therapies cannot restore normal tendon function. Tendon-derived stem cells (TDSCs) have been isolated from tendon tissues and characterized in vitro in recent studies and provide new strategies for tendon repair. But what should we know about tendon stem cells before we use them to repair injured tendon? / Although stem cells that originate from different tissues share some common stem cell characteristics, they might also exhibit some tissue unique properties and hence functional differences. Therefore, we hypothesized that TDSCs have unique stemness properties compared with bone marrow-derived stem cells (BMSCs). There has been no study to compare the stemness properties of TDSCs and BMSCs. Clinical applications often require the in vitro expansion of stem cells. In vitro microenvironment also affects the stemness properties and therapeutic potential of stem cells. It is not clear if the stemness properties of TDSCs can be maintained and how long that they can be preserved during in vitro expansion. Moreover, successful stem cell-based repair therapies will require an understanding of tissue specific stem cells in vivo and their roles in the tissue repair. Tendon stem cells have not been described in details in vivo and it is unknown whether these endogenous stem cells participate in the tendon healing. / Therefore, in order to better make use of TDSCs for tendon repair, the objective of this study is to characterize the stemness properties of TDSCs compared with BMSCs and also to investigate the stemness limitation of TDSCs during culture in vitro for clinical use purpose. Furthermore, this study aims to identify the putative tendon stem cells in vivo and their role in tendon healing. This study would tell how much we should know about tendon stem cells in vitro and in vivo. / In the first part of the study, TDSCs and BMSCs were isolated from the same GFP Sprague-Dawley rat. TDSCs showed higher mensenchymal and pluripotent stem cell makers; clonogenicity; proliferative capacity; and tenogenic, osteogenic, chondrogenic, and adipogenic differentiation markers and multi-lineage differentiation potential than BMSCs. Compared with BMSCs, TDSCs shows great stemness properties and might be an alternative cell source for tendon regeneration. / In the second part of this study, the senescence-associated β-galactosidase activity of TDSCs increased while their stem cell-related marker expression and the multi-lineage differentiation potential decreased during in vitro passaging. It suggests that researchers and clinicians need to consider the changes of stemness properties of TDSCs when multiplying them in vitro for tissue engineering. / In the third part of the study, IdU label-retaining method was used for the labeling of stem cells in vivo. We have identified quiescent stem cells as IdU label retaining cells (LRCs) at the peritenon, tendon mid-substance and tendon-bone junction. More LRCs were found at the peri-tenon and tendon-bone junction compared to the mid-substance. Some LRCs could be identified in the peri-vascular niche in the peri-tenon, suggesting that peri-vascular niche is one source of tendon stem cells. After injury, The LRC number and the expression of proliferative, tendon-related, pluripotency and pericyte-related markers in LRCs in the window wound increased, indicating that LRCs might be involved in tendon repair via cell migration, proliferation and differentiation. / In conclusion, our results have provided new findings about the understanding of tendon-derived stem cells including their stemness properties and their changes during the in vitro culture, as well as in vivo identity of tendon stem cells, which might facilitate the application of TDSCs in tissue engineering for tendon repair in the future. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Tan, Qi. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 130-162). / Abstracts also in Chinese.