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
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328177 |
| Date | January 2012 |
| Contributors | Ni, Ming, Chinese University of Hong Kong Graduate School. Division of Orthopaedics & Traumatology. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource (xx, 131 leaves) : ill. (some col.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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