ShcA (Src Homology and Collagen A) est une protéine adaptatrice qui se lie à la partie cytoplasmique de LRP1 (Low Density Lipoprotein-related receptor 1), un récepteur transmembranaire qui protège contre l'athérosclérose. La calcification vasculaire est une complication majeure de cette maladie et ses mécanismes ressemblent au processus d’ostéochondrogenèse. Nous avons étudié le rôle de ShcA endothélial dans la formation des lésions d’athérosclérose ainsi que les rôles de ShcA et LRP1 dans la chondrogenèse. ShcA endothélial participe à la formation des lésions d’athérosclérose in-vivo. En inhibant la NOS endothéliale et activant l’expression de ICAM-1 via ZEB1, ShcA favorise l’adhésion des monocytes. La suppression de ShcA dans les chondrocytes a conduit au développement de souris présentant un phénotype de nanisme par une inhibition de la différenciation hypertrophique des chondrocytes. Ceci conduit également à une diminution du développement de l’arthrose liée au vieillissement. La suppression de LRP1 dans les chondrocytes conduit également à un phénotype de nanisme chez la souris, mais par des mécanismes différents. / ShcA (Src Homology and Collagen A) is an adaptor protein that binds to the cytoplasmic tail of the Low Density Lipoprotein-related receptor1 (LRP1), a trans-membrane receptor that protects against atherosclerosis. Vascular calcification is a major complication of this disease and its mechanisms highly resemble the process of osteochondrogenesis. We studied the role of endothelial ShcA in atherosclerotic lesion formation as well as the roles of ShcA and LRP1 in chondogenesis. Endothelial ShcA participates in the formation of atherosclerotic lesions in-vivo. By inhibiting endothelial NOS and activating the expression of ICAM-1 via ZEB1, ShcA enhances monocyte adhesion. The deletion of ShcA in chondrocytes led to the development of mice with a dwarfism phenotype by inhibiting chondrocyte hypertrophic differentiation. This also led to a decrease in the development of age-related osteoarthritis. The deletion of LRP1 in chondrocytes also led to a dwarfism phenotype in our mouse model, but trough different mechanisms.

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Fracture repair is a regenerative event that involves the precise coordination of a variety of cells for successful healing process. Within the microstructure hierarchy of bone repair, the predominant cells involved include the chondrocytes, osteocytes, osteoblasts, and osteoclasts. Although the role of osteoblasts during fracture healing has been previously shown, their role during the initiation phase of endochondral fracture repair remains unclear. In order to study the role of osteoblasts during fracture repair, we used a transgenic mouse model expressing the herpes simplex virus thymidine kinase gene in early differentiating osteoblasts, which allows conditional ablation of cells in osteoblastic lineage upon treatment with the Gancicolvir drug. Results from this study suggest that not only are osteoblasts required in later stages of fracture repair as the medium for bone synthesis, and osteoclast activation during bone remodelling, but could also be required for the initiation and advancement of the endochondral ossification process.

Fracture repair is a regenerative event that involves the precise coordination of a variety of cells for successful healing process. Within the microstructure hierarchy of bone repair, the predominant cells involved include the chondrocytes, osteocytes, osteoblasts, and osteoclasts. Although the role of osteoblasts during fracture healing has been previously shown, their role during the initiation phase of endochondral fracture repair remains unclear. In order to study the role of osteoblasts during fracture repair, we used a transgenic mouse model expressing the herpes simplex virus thymidine kinase gene in early differentiating osteoblasts, which allows conditional ablation of cells in osteoblastic lineage upon treatment with the Gancicolvir drug. Results from this study suggest that not only are osteoblasts required in later stages of fracture repair as the medium for bone synthesis, and osteoclast activation during bone remodelling, but could also be required for the initiation and advancement of the endochondral ossification process.

<p>Selenium (Se) is an essential trace element and metalloid involved in several key metabolic activities: protection against oxidative damage, regulation of immune and thyroid function, and fertility. Several recent lines of evidence from epidemiology, genetic, and transgenic animal studies suggest that Se may play a protective role in Osteoarthritis (OA). However, the exact protective mechanism of Se is still unclear. </p><p>In this study, we hypothesized that Se exerts its chondroprotective benefit via an anti-oxidative and anti-inflammatory effect mediated by specific selenoproteins that neutralize cytokine-induced inflammatory responses in chondrocytes. We established an in vitro system for studying the effect of Se in the chondrosarcoma cell line SW-1353 and in human primary chondrocytes. Selenomethionine (SeMet) induced gene expression and enzyme activity of both antioxidative enzymes glutathione peroxidase (GPX) and thioredoxin reductase (TR) in SW-1353 cells. Our data suggest that Se may be protective against oxidative stress through regulation of the activity of these antioxidative enzymes.</p><p>As IL-1&beta; is one of the primary pro-inflammatory cytokines contributing to the progression in OA, we next investigated the effect of Se on the gene expression induced by physiological doses of IL-1&beta;. SeMet inhibited IL-1&beta; induced catabolic gene expression of matrix metalloproteinase 1 (MMP1) and MMP13 as well as total MMP activity in chondrocytes. Similarly, SeMet inhibited chondrocyte gene expression of IL-1&beta; induced nitric oxide synthase (iNOS) and cyclooxygenase (COX2) with corresponding reductions in nitric oxide (NO) and prostaglandin E2 (PGE2) production. In addition, SeMet pretreatment attenuated the IL-1&beta; induced activation of p38 MAPK but not the ERK, JNK or NFkB pathways. Taken together, our results suggest that Se inhibits IL-1&beta; induced expression of inflammatory and catabolic genes, partly through inhibition of IL-1&beta; cell signaling. </p><p>Since Se may function through selenoproteins, we evaluated the role of three specific major selenoproteins, GPX1, TR1 and DIO2, in modifying the inflammatory response stimulated by IL-1&beta; in chondrocytes by RNA interference. Based on RNA interference results, DIO2 and TR1 mediated the inhibitory effect of SeMet on IL-1&beta; induced COX2 gene expression, while GPX1 did not show a significant inhibitory effect on Se. Depletion of DIO2 increased the IL-1&beta; induced COX2 gene expression. This suggests that DIO2 may negatively modulate the IL-1&beta; response. Our data also suggest that part of this inhibitory effect of DIO2 could be through regulation of IL-1&beta; gene expression itself. These results highlight a potential new role of DIO2 in modulating the inflammatory response in chondrocytes </p><p>In summary, the result of this study suggests that Se may exert its chondroprotective effect through specific selenoproteins which neutralize oxidative stress and modify the inflammatory response in chondrocytes.</p> / Dissertation

In pathological states such as osteoarthritis, the complex metabolic balance of cartilage is disrupted, leading to a loss in the integrity and biomechanical function of cartilage. Osteoarthritis affects more than 20 million Americans, costing the United States economy over $60 billion yearly. Risk factors for osteoarthritis include age, excessive joint loading, and joint injury. Tissue engineering offers a potential solution for the replacement of diseased and/or damaged cartilage. Unfortunately, plentiful donor cell populations are difficult to assemble, as chondrocytes have a well characterized lack of expansion potential. Mesenchymal progenitor cells offer an alternative with a high expansion potential capable of supplying large quantities of cells. Using an immature bovine model, the chondrogenic differentiation of articular chondrocytes and bone marrow stromal cells was found to be scaffold, media and mechanical stimulation dependent. TGF-beta signaling participated in the response of articular chondrocytes to dynamic compressive loading, as well as enhanced the chondrogenesis of bovine BMSCs, through interactions between loading and TGF-beta/Smad signaling. Also, dynamic loading altered gene expression, matrix synthesis rates and intracellular phosphorylation for bovine BMSCs. However the response of the cells to dynamic loading depends on both media supplementation and the duration of unloaded culture. These studies establish signaling through the TGF-beta pathway as a mechanotransduction pathway for chondrocytes and chondroprogenitors in 3D culture.

Patient expectations have been shown to be a major predictor of outcomes. Furthermore, fulfilled expectations have been linked to increased patient satisfaction and rehabilitation adherence. Expectations may be influenced by a variety of factors, including patient characteristics, pre-operative function, or disease characteristics. However, it is currently unknown what factors and to what degree they may influence patient expectations prior to knee surgery. Furthermore, understanding the importance and values of those expectations for recovery using qualitative methods has not previously been conducted in this patient population. A mixed methods design was used. Twenty-one participants scheduled to undergo cartilage repair of the knee, including autologous chondrocyte implantation, osteochondral allograft transplantation, or meniscal transplant were included. During their pre-operative visit, participants completed an expectations survey (Hospital for Special Surgery (HSS) Knee Surgery Expectations Survey) and the Knee Injury and Osteoarthritis Outcome Score (KOOS) as a measure of functional ability. At their first post-operative visit, patients completed the Self-Efficacy for Rehabilitation Scale (SER). Rehabilitation adherence was collected by the participant’s rehabilitation provider. A selected sample of 6 participants participated in a semi-structured interview 6 months following surgery to better understand their expectations for recovery. Pearson correlation coefficients were used to determine relationships between expectations and KOOS scores, SER scores, and measures of adherence. Results demonstrated that patients have moderate expectations for recovery and these expectations were positively associated with pre-operative pain, activities of daily living, and knee-related quality of life as measured by the KOOS. In addition, a negative relationship was found between patient expectations and adherence with home exercises, use of a brace, and weight-bearing restrictions. Four qualitative themes emerged as participants’ described how previous recovery experiences shaped their recovery following cartilage repair of the knee. Patient education, pre-habilitation, and the use of psychological skills during rehabilitation may help to manage patient expectations, improve rehabilitation adherence, and assist clinicians in providing more focused and individualized patient care.

京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第23834号 / 薬科博第149号 / 新制||薬科||16(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 竹島 浩, 教授 金子 周司, 教授 土居 雅夫 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

[Truncated abstract] Osteoarthritis (OA) is the most common form of arthritis, characterized by progressively degeneration of articular cartilage. Chondrocyte is the only cell type in articular cartilage tissue and responsible for cartilage matrix turnover. This thesis focuses on the biological and genetic behaviors of human chondrocyte and potential therapeutic strategies that target on chondrocyte. Chondrocytes have been used for the tissue-engineered cartilage construction, especially in articular cartilage repair. The technique of chondrocyte-base tissue engineering utilizes in vitro propagated chondrocytes combined with several manufactured biomaterials to regenerate cartilage tissue. Although these technologies have been successfully applied in clinic, the biological characteristics of chondrocyte during in vitro propagation and after implantation remain unclear. This thesis reviewed the present studies of chondrocyte biology and its potential uses in tissue engineering. Particularly, chondrocytes have been shown to de-differentiate into fibroblastic-cells when they are exposed to inflammatory conditions or cultured on monolayer in vitro. This thesis investigated the gene expression profile of chondrocytes when they are cultured and serially passaged on monolayer in vitro. Human chondrocytes obtained from OA patients were cultured up to passage 6. Twenty-eight chondrocyte associated genes were measured by Real-time PCR. The results showed that a number of genes were changed in expression levels at various stages of passage as indications of chondrocyte de-differentiation. Chondrocytes derived from OA patients or normal donors exhibited a very similar gene expression pattern. Interestingly, transcription factor Sox-9, which plays a key role in chondrogenesis remained unchanged with increasing passage number, indicating that the de-differentiation process of chondrocyte is reversible. This thesis also focused on the development of novel pharmacological approaches for OA that target on articular chondrocyte. The clinical feature, etiology, pathogenesis, diagnostic approaches, conventional and potential future treatments for OA were briefly reviewed in this thesis. ... The effects of natural compounds on chondrocyte gene expression, proteoglycan degradation and nitric oxide production were measured. The results showed that parthenolide, a NF-kB inhibitor, regulated chondrocyte function by suppressing the up-regulation of gene expression of inflammatory factors and matrix proteinases induced by lipopolysaccharide, and down-regulating COX-2 expression. Parthenolide was able to reduce proteoglycan degradation in human chondrocytes, but had no effect on nitric oxide production. These results suggest that parthenolide mediates inflammatory-activated NF-kB pathway, and subsequently reduces inflammatory response, prevents cartilage destruction and relieves pain, and hence may be useful for OA treatment.

Made available in DSpace on 2016-06-02T20:19:17Z (GMT). No. of bitstreams: 1 4141.pdf: 1483003 bytes, checksum: 97d4909d05c961c3bb73e05056aee7d3 (MD5) Previous issue date: 2012-02-29 / Financiadora de Estudos e Projetos / The aim of this study was to evaluate the effect of a strength exercise protocol on articular cartilage in animal model of osteoarthritis (OA). Thirty-six rats were divided in 6 groups: Control (C, n = 6), Osteoarthritis (OA, n = 6), Sham (S, n = 6), Control with exercise (E n = 6), Osteoarthritis with exercise (OAE, n = 6) and Sham with exercise (SE, n = 6). The animal model of osteoarthritis was anterior cruciate ligament transection (ACLT) in rats. After 2 weeks of ACLT, groups E, OAE and SE started the strength exercise protocol, three times a week for 8 weeks. We used the Mankin Histologic Grading System, measured the density of chondrocytes, the density of collagen fibers and the expression of collagen type II, chondroitin sulfate and fibronectin. The groups subjected to strength exercise protocol, OAE and SE, had lower values for the Mankin score, chondrocyte density and fibronectin expression and higher values for collagen fibers density, type II collagen expression and chondroitin sulfate expression when compared with OA and S, respectively. Group E was different from group C only in chondrocyte density and fibronectin expression. Therefore, strength exercise changes the content and expression of different articular cartilage constituent, having influence on our animal model of osteoarthritis and provides benefits to the cartilaginous tissue. / O objetivo desse estudo foi avaliar o efeito de um protocolo de exercício resistido na cartilagem articular de modelo animal de osteoartrite (OA). Trinta e seis ratos foram divididos em 6 grupos: Controle (C), Osteoartrite (OA); Sham (S), Exercício (E), Osteoartrite e Exercício (OAE), Sham e Exercício (SE). Os grupos OA, OAE, S e SE foram submetidos à transecção cirúrgica do ligamento cruzado anterior (LCA) do joelho esquerdo, sendo que somente os grupos OA e OAE tiveram o LCA seccionado. Após 2 semanas da cirurgia, os grupos E, OAE e SE iniciaram o protocolo de exercício resistido, 3 vezes por semana, durante 8 semanas. Foi aplicado o sistema de graduação histológica de Mankin, mensurado a densidade de condrócitos, a densidade de fibras colágenas e a expressão de colágeno tipo II, sulfato de condroitina e fibronectina. Os grupos submetidos ao protocolo de exercício resistido, OAE e SE, apresentaram menores valores para o sistema de graduação de Mankin, densidade de condrócitos e expressão de fibronectina e maiores valores para densidade de fibras colágenas, expressão de colágeno tipo II e sulfato de condroitina quando comparados aos grupos OA e S, respectivamente. O Grupo E apresentou diferença do grupo C somente na avaliação da densidade de condrócitos e na expressão de fibronectina. Portanto, o exercício resistido promove modificações no conteúdo e na expressão de diferentes constituintes da cartilagem articular, exercendo influência em nosso modelo de osteoartrite e trazendo benefícios para o tecido cartilaginoso.