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Augmented soft tissue mobilization therapy (ASTM) is a newly developed massage technique. ASTM is applied with the aid of specifically designed, solid instruments and has been successfully used in the treatment of chronic tendinitis patients. In a study on collage nase -injure d rat Achilles tendons treated with ASTM, Davidson et. al (1997) reported gait improvement as well as fibroblasts proliferation and suggested ASTM may augment healing by the recruitment of fibroblast. The present study examined the morphological response of enzyme-induced rat Achilles tendons to 3 different ASTM pressure application: 1 newton; 2 newtons and 3 newtons. Collagenase-injured tendons exhibited disrupted and randomly arranged collagen fibers. Treatment applications were performed for 4 days for a total of 4 treatments. Morphological differences were demonstrated between groups in proportion to the ASTM treatment pressure application. The ASTM group treated with 3 newtons demonstrated the greatest mean fibroblast count (370.3 +/- 51.6). Further, electron microscopy revealed the presence of activated fibroblasts in the tendons of the 3 newtons, ASTM group. However, immunochemical staining comparisons of Type I and III collagen, fibroblast growth factor receptor, and insulin-like growth factor between groups were not remarkable. / Department of Physiology and Health Science

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

Functional integration of bone with soft tissues such as tendon is essential for joint motion and musculoskeletal function. This is evident in the rotator cuff of the shoulder, which consists of four muscles and their associated tendons that connect the humerus and scapula. The cuff functions to stabilize the shoulder joint, and actively controls shoulder kinematics. Rotator cuff injuries often occur as a result of tendon avulsion at the tendon-bone interface, with more than 250,000 cuff repair surgeries performed annually in the United States. However, these procedures are associated with a high failure rate, as re-tears often occur due to the lack of biological fixation of the tendon to bone post-surgery. Instead of regenerating the tendon-bone interface, current repair techniques and augmentation grafts focus on improving the load bearing capability of the repaired rotator cuff. Biologically, the supraspinatus tendon inserts into bone via a biphasic fibrocartilaginous transition, exhibiting region-dependent changes in its compositional, structural and mechanical properties, which enables efficient load transfer from tendon to bone as well as multi-tissue homeostasis. Inspired by the native tendon-bone interface, we have designed and evaluated a biomimetic bilayer scaffold, comprised of electrospun poly (lactide-co-glycolide) (PLGA) nanofibers seamlessly integrated with PLGA-hydroxyapatite (HA) fibers, in order to engineer tendon-bone integration. The objective of this thesis is to explore the key design parameters that are critical for integrative tendon-bone repair using this biphasic scaffold as a model. Specifically, intrinsic to the scaffold, effects of fiber alignment, fiber diameter, mineral distribution, and polymer composition on integrative rotator cuff tendon-bone healing were evaluated in vivo using a rat model. Results indicated that an aligned, nanofiber-based scaffold with a distinct order of non-mineralized and mineralized regions will lead to insertion regeneration and integrative tendon-bone repair. Additional tissue engineering design parameters such as healing time and animal model were also tested. It was observed that the biphasic scaffold exhibited a stable long term response, as the mechanical properties of rat shoulders repaired by this scaffold remained comparable to that of the control at 20 weeks post-surgery. This scaffold was also evaluated in a large animal model (sheep), in which a clinically-relevant rotator cuff repair procedure was implemented with the biphasic scaffold. Results demonstrated the scaffold lead to integrative rotator cuff repair through the regeneration of the enthesis in both small and large animal models. In summary, through a series of in vivo studies, the work of this thesis has identified the critical tissue engineering parameters for integrative and functional rotator cuff tendon repair. More importantly, the design principles elucidated here are anticipated to have a broader impact in the field of tissue engineering, as they can be readily applied towards the regeneration of other soft-hard tissue interfaces.

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.

Chan, Lai Shan. / Thesis submitted in: Dec. 2008. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 148-152). / Abstracts in English and Chinese. / ACKNOWLEDGEMENT --- p.I / PUBLICATIONS --- p.II / ABBREVIATION --- p.III-IV / INDEX OF FIGURES --- p.V-IX / INDEX OF TABLES --- p.X / ABSTRACT (English) --- p.XI-XIII / ABSTRACT (Chinese) --- p.XIV / Chapter CHAPTER 1 --- INTRODUCTION --- p.1-22 / Chapter CHAPTER 2 --- METHODOLOGY --- p.23-49 / Chapter CHAPTER 3 --- RESULTS --- p.50-97 / Chapter CHAPTER 4 --- DISCUSSION --- p.98-117 / BIBLIOGRAPHY / APPENDIX

Nitric oxide is a small free radical generated by family of enzymes, the nitric oxide synthases. In a series of experiments performed over the last 15 years we showed that nitric oxide is induced by all three isoforms of nitric oxide synthase during tendon healing and that it plays a crucial beneficial role in restoring tendon function. In normal tendon we found very little nitric oxide synthase activity while in injured rat and human tendons nitric oxide synthase activity was expressed in healing fibroblasts in a temporal fashion. In healing rat Achilles tendon fibroblasts the first isoform to be expressed was endothelial nitric oxide synthase (eNOS), followed by inducible nitric oxide synthase (iNOS), and then brain or neuronal nitric oxide synthase (bNOS). Systemic inhibition of nitric oxide synthase activity decreased the cross sectional area and mechanical properties of the healing rodent Achilles tendons. Addition of nitric oxide via NO-flurbiprofen or NO-paracetamol enhanced rat Achilles tendon healing. Addition of nitric oxide to cultured human tendon cells via chemical means and via adenoviral transfection enhanced collagen synthesis, suggesting that one mechanism for the beneficial of nitric oxide on tendon healing might be via matrix synthesis. The final part of the work involved three randomized, double-blind clinical trials which evaluated the efficacy of nitric oxide donation via a patch in the management of the tendinopathy. In all three clinical trials there was a significant positive beneficial effect of nitric oxide donation to the clinical symptoms and function of patients with Achilles tendinopathy, tennis elbow and Achilles tendonitis.

The aim of this study was to investigate which protocols Occupational Therapists (OT’s) use when rehabilitating clients after flexor tendon repairs, and to investigate the therapist’s knowledge regarding these protocols, to guide therapists and institutions in using effective methods within the South African context. A questionnaire was sent to OT’s in South Africa. Of the 32 responses, 50% had more than 10 years experience and 50%, less experience. 81.2% were private practitioners and 28% worked in government. The trend of protocol use was as follows: 18.8% used a Duran-type passive mobilization protocol, 25% used a Kleinert-type protocol - a passive flexion protocol (but labelled an active mobilization protocol in literature as it allows active extension of the fingers), 28.1% used Early Active Mobilization and 3.1% used an Immobilization-type protocol. 64.5% of the sample used static splints, 9.7% used dynamic splints and 25.8% used a combination. Most (83.3%) continued the splint at 4 weeks but only 26.6% were using the splint at 6 weeks. At week 1, 30% allowed active flexion of the fingers, whereas at week 4 and 5, 60% allowed active flexion. The referring doctor and confidence in one’s own skills were the main factors influencing protocol choice. Resources available influenced the protocol choice, which can be problematic in South Africa. Access to literature was mostly through textbooks (90.6%), although journal articles were accessed (internet - 50%, hard copy - 62.5%). More than half of the sample attended courses regularly. Most therapists were happy with their outcomes, regardless of which protocol used. Therapists need to build their confidence, realising the efficacy of various protocols is similar, according to research. Thus whatever factors influence protocol choice, they will likely not be critical to good outcomes. / Thesis (M.O.T.)-University of KwaZulu-Natal, Durban, 2012.

The glenohumeral (GH) joint is the most mobile joint in the human body, but its mobility inherently increases the risk of instability. The humeral head sits in a shallow glenoid in the scapula like a golf ball sitting on a tee. The stability in this joint is provided by the rotator cuff muscles and tendons that actively pull the humerus back into the socket to prevent dislocation, especially during overhead motions. However, the rotator cuff is prone to tears, resulting in pain, loss of mobility, and recreational limitations. Surgical reattachment of the tendon to the bone is challenging due to the mechanical disparity between the two tissues, resulting in stress concentrations and a high risk of retear. Notably, the specialized tissue at the tendon-to-bone attachment, which facilitates stress transfer between tendon and bone in healthy joints, does not regenerate after surgical reattachment and healing, making tendon-to-bone repairs prone to re-tears. A comprehensive understanding of GH joint biomechanics is essential for developing early interventions to prevent rotator cuff injuries. Furthermore, improving tendon-to-bone fixation during rotator cuff repair is critical to improve post-surgery outcomes. In the last decade, bioinspired solutions have shown considerable promise for addressing several biomedical problems. This thesis draws bioinspiration from two animals that have evolved unique mechanical functions: (i) the bat shoulder joint, which facilitates repetitive overhead motions during flight and may offer insights into rotator cuff pathology and (ii) the curvature of python snake teeth, which enables secure grasping of prey without soft tissue tearing. In the first part of the thesis, the bat shoulder was studied for its unique characteristics relative to mice. Overhead motions in humans often lead to shoulder injuries, partly because the bony anatomy of the unstable GH joint places greater stress on the joint's surrounding soft tissues to stabilize these motions. Traditional animal models used to study shoulder pathology are quadrupeds, which lack the capacity for overhead motion. In contrast, bats consistently engage in overhead motion during flight, subjecting their shoulders to substantial loading throughout their relatively long lifespan. Remarkably, the biomechanical demands placed on a bat's shoulder are estimated to exceed those of a competitive swimmer’s by 45-fold, despite sharing similar coracoacromial arch anatomy with humans. We were inspired to study functional adaptations in the shoulders of bats that enable this overhead motion. We performed comparative anatomy studies of the shoulders of bats and mice, similarly-sized quadrupeds. By quantifying the constraints imposed by the bony anatomy, we identified adaptations of the shoulder, including the rotator cuff tendons, that allow bats to sustain overhead motion in a high stress, repeated loading environment, without injury. In the second part of the thesis, python teeth were used as inspiration to develop a repair device optimized to grasp the rotator cuff without tearing. Rotator cuff repair surgeries fail frequently, with 20-94% of the 600,000 repairs performed annually in the United States resulting in retearing of the rotator cuff. The most common cause of failure is sutures tearing through tendons at grasping points. To address this issue, we examined the specialized teeth of snakes of the Pythonoidea superfamily, which effectively grasp soft tissues without tearing. To apply this non-damaging and effective gripping approach to the surgical repair of tendons, we developed and optimized a python-tooth inspired array as an adjunct to current rotator cuff suture repair, and found that it nearly doubled repair strength. Integrated simulations, 3D printing, and ex vivo experiments revealed a relationship between tooth shape and grasping mechanics, and enabled optimization of a tooth array device to enhance rotator cuff repair to distribute stresses and increase tendon-bone contact. The efficacy of the approach was demonstrated via human cadaver tests, suggesting an alternative to traditional suturing paradigms that may reduce tendon re-tearing. Collectively, these studies contribute to a better understanding of the biomechanics of the GH joint and offer novel, bioinspired approaches for rotator cuff repair. The functional adaptations of bats provide insight into developing new approaches to treat GH joint instability, and a clinically relevant python-tooth inspired device can ultimately reduce the high rates of re-rupture currently observed in rotator cuff repair.

In summary, the biomechanical stimulations can augment osteotendinous healing processes by facilitating better fibrocartilagious transitional zone regeneration as well as the restoration of proprioceptions, and the early application showed the more beneficial effects. However, further experimental and clinical studies are still needed to explore the optimal timing, intensity, frequency, and duration of the proposed postoperative biomechanical stimulation protocols. / LIPUS is a "non-contact" biomechanical stimulation, which can provide a direct mechanical stimulation through cavitation and acoustic microstreaming effects to improve tissue healing in a less-than-rigid biomechanical environment. So the mechanical stimulation induced from LIPUS could be applied immediately after surgery without worrying about the mechanical strain exceed the structural property at the osteotendinous healing interface in the early phase of repair. In this part of study, we also examined the effects of the regime of biomechanical stimulations applying immediately after repair on the osteotendinous healing interface. By using the same healing junction model, forty-two female New Zealand white rabbits were randomly divided into two groups; daily mechanical stimulation was applied immediately after surgery lasting up to post-operative 12 weeks on the healing interface in the treatment group. The regime of mechanical stimulations included by LIPUS was 20 minutes, 5 days per week for 4 weeks, followed by cyclic mechanical stimulation generated from quadriceps muscles induced by FES for 8 weeks. Results showed that early application of biomechanical stimulations on the osteotendinous healing interface were significantly better radiologically, histologically and biomechanically than that of not any or later application of the biomechanical stimulations during the osteotendinous healing processes when assessing at the same healing time point. In addition, the early application of biomechanical stimulations showed the better functional recovery in terms of the restoration of the proprioceptions, which an increased numbers of sensory nerve endings labeled by calcitonin gene-relate peptide (CGRP) was detected in the whole osteotendinous healing complex. / Sports or trauma injuries around osteotendinous junctions are common; treatments usually require surgical reattachment of the involved tendon to bone. Restoration of osteotendinous junction after repair is slow and difficult due to regenerating the intermitted fibrocartilage zone to connect two different characteristic tissues, tendon to bone. Although the factors influencing fibrocartilage zone regeneration and remodeling during osteotendinous repair are poorly understood, however, is believed that the mechanical environment plays an important role in such healing process. In present study, the effects of mechanical stimulation on osteotendinous healing process were examined, in the way of mechanical stimulations induced by biophysical stimulations, surface functional electric stimulation (FES) and low intensity pulsed ultrasound (LIPUS), applying on the patellar tendon to patellar bone healing interface in an established partial patellectomy model in rabbits. / The mechanotransductive stimulation linked to the transmission of forces across osteotendinous junction can be generated from its muscle contraction induced by FES. In the partial patellectomy model, thirty-five female New Zealand white rabbits were randomly divided into two groups with initial immobilization for 6 weeks, daily FES was applied to quadriceps muscles for 30 minutes, 5 days per week for 6 weeks in treatment group and compared with non-treatment control group at postoperative week 6, 12 and 18, radiologically, histologically and biomechanically. Results showed that FES-induced cyclic mechanical stimulation significantly increased new bone formation and its bone mineral density. An elevated expression of tenascin C and TGFbeta1; an increased proteoglycant stainability; mature fibrocartilage zone formation with better resumptions of biomechanical properties also observed on the osteotendinous healing interface, indicating that the post-operative programmed cyclic mechanical stimulation generated from its muscle contraction has beneficial effects on osteotendinous healing processes by facilitating the fibrocartilagious transitional zone regeneration. / by Wang Wen. / Advisers: Kai Ming Chan; Ling Qin. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1550. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 159-175). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.