Background: The impairment of articular cartilage caused by trauma or degenerative pathology is one of the most challenging issues in clinical Orthopedics because of the limited intrinsic regenerative capability of this tissue. Hypoxia is a major stimulus to initiate gene programs in regulating chondrogenic lineage cell functions during cartilage development and regeneration. Hypoxia-inducible factor-1α (HIF-1α), the key transcription factor to sense oxygen fluctuations of cells, is abundantly expressed in the cartilage and considered as a potential therapeutic target for cartilage tissue homeostasis or repair. However, the molecular mechanisms and therapeutic efficacy of targeting the HIF-1α pathway remain to be well defined. / Methods: Osteochondral defect mouse model was generated to examine the hypoxia states during articular cartilage repair with the Hypoxyprobe. Specific HIF-1α deletion in the repairing tissue was established to determine its regulatory role during cartilage restoration. Deferoxamine (DFO), stabilizing HIF-1α from proteolysis by inhibiting the prolyl hydroxylases (PHDs), was investigated systemically on the function of chondroprogenitors and mesenchymal stem cells (MSCs) in vitro. Alcian blue staining determined the proteoglycan synthesis. HIF components, chondrogenic related genes and proteins were examined by quantitative PCR, western blotting and immunohistochemistry, respectively. The proliferation, differentiation and migration assays were performed to determine the influence of DFO onchondroprogenitors and MSCs. The recruitment or engraftment of MSCs in the injured site was traced by transplantation of GFP-labeled MSCs adjacent to the defect region, and examined by immunofluorescence staining. DFO incorporated in a 3D alginate-gelfoam scaffold was analyzed for its therapeutic effects on the articular cartilage regeneration. At 6 and 12 weeks following surgery, the cartilage tissue repair was scored and the expression of proliferating cell nuclear antigen (PCNA), Sox9 and collagen typeⅡ(Col2) was examined by immunohistochemistry. / Results: Hypoxia states and the expression of HIF-1α in the repair tissue were ubiquitously existed in the osteochondral defect model. DFO significantly upregulated HIF-1α expression and nuclear localization, and increased the levels of PHDs. DFO increased chondroprogenitor cell proliferation as visualized by colony forming unit assay, which was in accord with the upregulation of cyclin D1. DFO significantly induced chondrogenic differentiation indexed by increased Col2 and Sox9 protein expression and elevated proteoglycan synthesis. With sustained upregulation of HIF-1α DFO was supposed to effectively promote chondrogenesis in mimic of hypoxic microenvironment. DFO also increased the migration of MSCs, and elevated the expression of tissue inhibitor metalloproteinase-3 (TIMP3) through transcriptional control by HIF-1α. Furthermore, DFO initiated MSCs membrane protrusion through regulating the expression and interaction of the key focal adhesion proteins vinculin and paxillin. In vivo study showed that DFO dramatically facilitated the recruitment and functional engraftment of MSCs to the lesion site compared with the controls. Alginate-gelfoam scaffold incorporated with DFO enhanced articular cartilage repair through increasing chondrogenic cell proliferation, differentiation and proteoglycan synthesis. The enhanced therapeutic effect of DFO on articular cartilage repair was eliminated following HIF-1α deletion in the repairing cells of the cartilage lesion. The results indicate that the positive effect of DFO on articular cartilage repair is at least partially mediated by HIF-1α. / Conclusion: HIF-1α is an essential mediator during articular cartilage repair. Activation of HIF-1α by PHD inhibitor DFO increases chondroprogenitor cell proliferation, differentiation and migration in vitro. DFO enhances articular cartilage repair through coordinating MSCs migration, chondrogenic differentiation and functional engraftment. The results provide proof of principle that targeting the HIF-1α pathway may serve as a novel approach for promoting articular cartilage regeneration. / 背景：关节软骨自愈能力非常有限，由创伤或退行性病变引起关节软骨损伤的治疗是骨科领域的一大难题。在软骨发育和再生过程中，低氧条件对启动基因表达及调控软骨系细胞功能至关重要。低氧诱导因子-1α（HIF-1α）作为关键的转录因子可感应细胞外氧含量变化，广泛存在于软骨组织中，并被认为对维持软骨组织内稳态及促进软骨修复有重要作用。然而，以HIF-1α 通路为靶点的小分子靶向药物的分子机制与治疗效果尚不明确。 / 方法：本课题系统性地研究了HIF 信号通路激活剂去铁胺（DFO）对软骨损伤的作用。我们构建了骨软骨缺损模型，应用缺氧探针检测了软骨缺损过程中修复组织的低氧状态，并特异性敲除软骨修复组织中HIF-1α 表达，研究其在软骨再生过程中的调节作用。我们用阿利新蓝染色检测软骨细胞蛋白多糖的合成及分泌。通过实时荧光定量聚合酶链式反应，免疫印迹以及免疫组化等方法检测了HIF 家族成员和软骨分化标志物的基因和蛋白含量变化。通过增殖及迁移实验检测了DFO 对软骨细胞或者骨髓间充质干细胞（MSC）功能的影响。另外，我们还将GFP 标记的MSC 注射到与小鼠软骨缺损区域相邻的软骨下骨中，观察其在软骨缺损模型中的募集及功能性植入。我们以藻酸盐和明胶海绵复合物为给药系统，包载DFO 并作用于关节软骨缺损部位。术后6 周及12 周取材，以番红O 染色检测DFO 对小鼠关节软骨缺损的修复效果，并通过免疫组化检测增殖细胞核抗原（PCNA），Sox9 以及Col2 等蛋白的表达。 / 结果：低氧状态和HIF-1α 在骨软骨缺损模型中的软骨缺损区域广泛存在和表达。DFO 显著提高了HIF-1α 蛋白表达及转运入核，增加了脯氨酸羟化酶（PHD）表达。在软骨祖细胞中，DFO 可提高其增殖、克隆能力，并增加周期蛋白D1的表达。同时，DFO 能明显促进软骨祖细胞分化，增加软骨分化标志物基因以及Sox9 和Col2 蛋白表达，提高蛋白多糖分泌。通过持续性激活HIF-1α，DFO可模仿低氧微环境来提高软骨细胞增殖、分化能力。分子机制研究发现，DFO激活HIF-1α 后，HIF-1α 作用在靶基因金属蛋白酶组织抑制剂-3 启动子上，增加其转录和蛋白表达，进而提高MSC 的迁移能力。另外，激活HIF-1α 蛋白可增加黏着斑蛋白，桩蛋白表达以及它们的相互作用，促进MSC 伪足延伸。体内实验中，通过追踪小鼠体内GFP 标记的MSC 发现， DFO 可在软骨损伤早期（7 天及14 天）提高受损部位MSC 募集数量，并促进其向软骨细胞谱系分化。通过增加软骨系细胞增殖、分化、蛋白多糖合成，包载DFO 的藻酸盐明胶海绵给药系统显著提高了软骨缺损组织的修复效果。而在软骨修复组织中特异性敲除HIF-1α 蛋白后，明显降低了DFO 对软骨缺损的治疗效果，提示DFO对软骨修复的作用至少部分由HIF-1α 介导。 / 结论：HIF-1α 是关节软骨修复过程中的重要调控因子。PHD 抑制剂DFO 可以激活HIF-1α 表达，增加软骨祖细胞增殖、分化和迁移。DFO 通过调控MSC 募集、软骨细胞谱系分化以及功能性植入，明显改善关节软骨再生修复的效果。本研究为HIF-1α 信号通路作为一种新的治疗靶点促进关节软骨再生提供了重要证据。 / Shu, Yinglan. / Thesis Ph.D. Chinese University of Hong Kong 2015. / Includes bibliographical references (leaves 155-181). / Abstracts also in Chinese. / Title from PDF title page (viewed on 09, September, 2016). / Shu, Yinglan. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

Cartilage repair and/or replacement is necessary for many orthopaedic conditions including fissures from blunt trauma, autograft or allograft transplantation, and replacement of focal defects with biological or synthetic constructs. In cartilage repair, initial integration between the host tissue and repair site is desirable to allow for nutrient transport, molecular deposition to enhance fixation, and eventual stress transmission across the interface. It has been postulated that effective transport and crosslinking of newly synthesized collagen molecules across a repair site may be vital to the process of integrative repair, and recent experiments have correlated collagen deposition with the strength of such repair. Other investigations have shown that enzymatic degradation of the cartilage surface may enhance integrative repair and can increase bond strength of an adhesive to cartilage. This study explored a novel approach involving photochemical bonding of cartilage tissue samples through collagen crosslinking as a means to achieve rapid and effective initial fixation, with the goal of enhancing biological integration. Photosensitized collagen gels were first analyzed via FTIR to determine the crosslinking effects with respect to collagen type and photochemical mechanism. Using the photogellation FTIR results as a parametric guide, in vitro mechanical testing of photochemically bonded meniscal fibrocartilage and hyaline articular cartilage tissues was performed using a modified single-lap shear test. Finally, the cellular viability and bond stability of a photochemically bonded cartilage interface was evaluated over seven days of in vitro culture, where the bond strength was assessed by pushout of cores from annular defects. Results of this study have demonstrated the potential of combining enzymatic surface modification with photodynamic techniques to directly bond cartilage tissues for initial fixation.

[Truncated abstract] Articular cartilage has no vascular, neural, or lymphatic supply, and hence no intrinsic capacity to self-repair following injury. These physiological limitations, combined with the inability of local chondrocytes to contribute to the repair process, translate to poor structural and functional outcomes in these troublesome defects, and osteoarthritic deterioration with time. Subsequently, many surgical therapies have been trialed to stimulate cartilage repair, but none have produced reliable outcomes. Hence, cartilage repair research has been broadened, with many investigators now focused on cell-based treatment. Smith began a revolution of autologous cell research when in 1965 she isolated chondrocytes from articular cartilage and transplanted them into fresh cartilage nodules (Smith, 1965). Since, new technologies and improved techniques have seen autologous chondrocyte implantation (ACI) widely accepted for use in clinical orthopaedics (Bentley et al., 2003; Brittberg et al., 1994; Grande et al., 1989; Peterson et al., 2002). At present, matrix-induced autologous chondrocyte implantation (MACI) is the most surgically simple form of ACI, boasting clinical outcomes comparable to any technique on the market, and far less complications compared to the first generation of ACI - periosteal ACI (Bartlett et al., 2005; Behrens et al., 2006; Gigante et al., 2006; Henderson et al., 2004; Marlovits et al., 2005; Minas, 2001; Willers et al., 2007; Zheng et al., 2007). But whilst MACI has been adopted by the orthopaedic surgeon for articular cartilage repair, many of the molecular, histological, and clinical factors governing patient outcomes are still largely understudied. Firstly we assessed the bioactivity of fibrin sealant (FS - Tisseel®), a critical component of MACI, on the migration and proliferation of human articular chondrocytes in vitro. We also looked to elucidate the associated molecular mechanisms of thrombin, a key active ingredient in FS, by examining the expression and activation of proteaseactivated receptors (PARs), established thrombin receptors. All four PAR isoforms were detected in human chondrocytes, with PAR-1 being the major isoform expressed. '...' This thesis has demonstrated biological, histological, and clinical features of the MACI technique. Our in vitro has supported the use of fibrin sealant and collagen membrane as the major material components of MACI, illustrating improved chondrocyte proliferation, migration, and chondrogenic differentiation. We have evidenced that MACI stimulates successful production of hyaline-like cartilage by 6 months, while also showing that revised and clinically failed repair tissues are predominantly hyaline-like and fibrocartilage with inferior composition. Clinically, we have documented significant improvements in patient repair structure, function, symptoms, quality of life, and satisfaction, whilst concurrently confirming sentiment within the literature regarding the importance of exercise/ rehabilitation for maximising MACI outcome. In summary, the findings presented in this thesis suggest that MACI is a biologically sound and clinically efficacious cell-based treatment option for repairing articular cartilage defects.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.

Strategien zur Gewebsreparatur durch Zelltransplantate erfordern die Verfügbarkeit einer ausreichenden Menge von Zellen, die Schaffung konduktiver Mikrokulturbedingungen für die Integration und die Entwicklung des Implantats und die Entwicklung reproduzierbarer chirurgischer Technik für die klinische Anwendung des kultivierten Transplantats. In der vorliegenden Arbeit wurden mehrere Techniken der Zelltransplantation entwickelt und tierexperimentell erprobt. Unter Verwendung von Alginat wurde eine neue sequentielle Zellkulturtechnik für Knorpeltransplantate entwickelt. Der optimale Kompromiß zwischen der Matrixstabilisierung und einer ausreichenden Diffusionskapazität für die Zellfunktion wurde bei einer Mischung aus 0,6 % Alginat und 4,5 % Fibrin gefunden. Weitere untersuchte Matrixstrukturen zur Transplantation von Chondrozyten, wie die bioresorbierbaren Polymere, das Kollagen-Fibrin-Gel besitzen gegenüber der gegenwärtig kommerziell genutzten Methode hinsichtlich des chirurgischen Prozederes bei vergleichbaren histologischen Ergebnissen Vorteile. Die histomorphologischen Veränderungen und die Entwicklung des Transplantats in vivo werden durch die spezifischen Bedingungen der Transplantatumgebung beeinflußt. Dabei ist ein vollständiges zonales und sequentielles Remodeling von Knorpel-Knochendefekten nur bei nicht ausdifferenzierten Zellen (embryonale Chondrozyten, periostale Zellen) zu erkennen, da diese Zellen ein exzellentes chondrogenes und osteogenes Potential besitzen. Transplantate unter Verwendung von Chondrozyten zeigen dagegen nur eine sehr geringe Rekonstruktion des subchondralen Knochens. Periostale Zellen sind in vitro ohne Verlust des Phänotyps amplifizierbar und stellen daher eine optimale Zellquelle für das Tissue Engineering dar. Für das Bone Engineering ist die Kombination der osteokonduktiven Eigenschaften unterschiedlicher Trägermaterialien mit Zellen, die ein osteogenes Potential besitzen ein neuer Weg zur Optimierung des Prozesses der knöchernen Rekonstruktion, wie in Versuchen zur Rekonstruktion segementaler Ulnadefekte bei Kaninchen gezeigt werden konnte. Die Herstellung eines präossären stabilen aber formbaren Transplantats mit vielfältigen klinischen Einsatzmöglichkeiten ist unter Verwendung von biodegradierbaren Polymeren und von Fibrinbeads realisierbar. Der Einsatz von Wachstumsfaktoren, wie TGF-?1 und die zunehmenden Erkenntnisse zu den Zell-Zell- und Zell-Matrix-Interaktionen ermöglichen die verbesserte Generation ortsständigen Gewebes durch multipotente Zellen. Die immer komplexere und umfassendere Wiederholung der sich in der Ontogenese abspielenden Vorgänge durch die Techniken des Tissue Engineering, ermöglicht die Schaffung therapeutischer Optionen zur Behandlung von Knochen- und Knorpeldefekten, wo bisher keine existierten oder nur unzulänglich vorhanden waren. / Strategies for tissue repair by cell transplants require the availability of a sufficient amount of cells, the creation of conductive microculture conditions for the integration and development of the implant and the development of reproducible surgical techniques for the clinical application of the cultivated transplant. Within the frame of the present work, several techniques of cell transplantation were developed and tested by way of experiment. By using alginate, a new sequential cell culture technique was developed for cartilage transplants. The optimum compromise between the matrix stabilization and a sufficient diffusion capacity for the cell function was found with a mixture of 0.6 % of alginate and 4.5 % of fibrin. Further investigated matrix structures for the transplantation of chondrocytes, such as the bio-absorbable polymers, the collagen fibrin jelly show advantages compared with the method commercially applied at present regarding the surgical procedure with the gained histological results being comparable. The histomorphological changes and the development of the transplant within the living body are influenced by the specific conditions of the transplant environment. In this connection, a complete zonal and sequential remodeling of osteochondrodefects can only be detected for non-outdifferentiated cells (embryonic chondrocytes, periosteal cells) as these cells have an excellent chondrogenic and osteogenic potential. When using chondrocytes for transplants, however, the transplant only shows a very little restoration of the subchondral bone. Periosteal cells can be amplified in the living body without losing the phenotype, thus constituting an optimum cell source for tissue engineering. For the bone engineering, the combination of the osteoconductive properties of different carrier materials with cells having an osteogenic potential is a new way for optimizing the process of bone restoration as it was demonstrated in tests for the restoration of segmental ulnar defects occurring with rabbits. The generation of a preosteal stable, but mouldable transplant with manifold clinical possibilities of utilization can be realized by using biodegradable polymers and fibrin beads. The use of growth factors, such as TGF-?1, and the increasing knowledge of cell-cell and cell-matrix interactions enable the improved generation of stationary tissue by multipotent cells. The more and more complex and comprehensive repetition of processes going on in the ontogenesis by way of tissue engineering enables the creation of therapeutic options for the treatment of osteochondrodefects where hitherto none existed or just in a too small number.

Scar-free wound healing and regeneration are uncommon phenomena permitting the near complete restoration of damaged tissues, organs and structures. Although rare in mammals, many lizards are able to undergo scarless healing and regeneration following loss of the tail. This study investigated the spontaneous and intrinsic capacity of the leopard gecko (Eublepharis macularius) tail to undergo scar-free wound healing and regeneration following two different forms of tail loss: autotomy, a voluntary and evolved mechanism of tail shedding at fracture planes; and surgical amputation, involuntary loss of the tail outside the fracture planes. Furthermore, I investigated the ability of the regenerate tail to regenerate by amputating a regenerate tail (previously lost by autotomy). To investigate these phenomena I imaged wound healing and regenereating tails daily (following autotomy and amputation) to document gross morphological changes. I used histochemistry to document tissue structure and immunohistochemistry to determine the tissue/cellular location of my five proteins of interest (PCNA, MMP-9, WE6, α-sma, TGF-β3). Each of these proteins of interest has been previously documented during wound healing and/or regeneration in other wound healing/regeneration model organisms (e.g. mice, urodeles, lizards, zebrafish). Scar-free wound healing and regeneration occurred following autotomy, amputation of the original tail and amputation of the regenerate tail, indicating that the leopard gecko tail has an instrinsic scar-free wound healing and regenerative capacity that is independent of the mode of tail loss (autotomy or amputation). Furthermore immunohistochemistry revealed a conserved sequence and location of the expression of the five proteins of interest following both forms of tail loss. These results provide the basis for further studies investigating scar-free wound healing and regeneration in a novel amniote model, the leopard gecko. / NSERC