Différentes techniques d'imagerie par contraste de phase des rayons X ont été récemment développées. Contrairement aux méthodes conventionnelles, qui mesurent les propriétés d'absorption des tissus, ces techniques donnent aussi le contraste du déphasage introduit par l'échantillon. Puisque le changement dans la phase peut être important même quand les différences en atténuation sont faibles ou absentes, le contraste d'image obtenable peut être considérablement augmenté, notamment pour les tissus mous biologiques. Ces méthodes sont donc très prometteuses pour une application dans le domaine médical. Cette Thèse a le but de contribuer à une compréhension plus profonde de ces techniques, en particulier la propagation-based imaging (PBI), la analyzer-based imaging (ABI) et la grating interferometry (GIFM), et d'étudier leur potentiel et la meilleure implémentation pratique pour les applications médicales. Une partie importante de cette Thèse est dédiée à l'utilisation d'algorithmes mathématiques pour l'extraction, à partir des images acquises, d'informations quantitatives (absorption, réfraction et diffusion) concernant l'échantillon. En particulier, cinq parmi les algorithmes les plus connus pour la technique ABI sont analysés théoriquement et comparés expérimentalement, dans les modalités planaire et tomographique, en utilisant des fantômes et des échantillons de tissu mammaire et d'os-cartilage. Une méthode semi-quantitative pour l'acquisition et la reconstruction d'images tomographiques dans les techniques ABI et GIFM est aussi proposée. Les conditions de validité sont analysées en détail et la méthode, permettant une simplification considérable de l'implémentation pratique, est vérifiée expérimentalement sur des fantômes et des échantillons humains. Enfin, une comparaison théorique et expérimentale des techniques PBI, ABI et GIFM est présentée. Les avantages et les désavantages de chacune des techniques sont mis en évidence. Les résultats obtenus par cette analyse peuvent être très utiles pour déterminer quelle technique est la plus adaptée à une application donnée.

<p>Articular cartilage is a highly specialized avascular tissue and consists of chondrocytes and two major components, a collagen-rich framework and highly abundant proteoglycans. The chondrocyte morphology and extracellular matrix properties vary with the depth of cartilage. Some past studies have defined the zonal distribution of a broad range of cartilage proteins in different layers. Based on the variations within each layer, the extracellular matrix can be further distinguished to pericellular, territorial and interterritorial regions. However, most of these studies used guanidine-HCl extraction that leaves an unextracted residual with a substantial amount of collagen. The high abundance of anionic polysaccharide molecules from cartilage adversely affects the chromatographic separation. Scatter oriented chondrocytes only account for the small proportion of the whole tissue protein extraction. However, the density of the cell varies with depth of cartilage as well. Moreover, the physiological status may also altered the extracellular matrix properties. Therefore, a comprehensive strategy to solve all these difficulties are necessary to elucidate the molecular structure of cartilage. </p><p>In this study, we used quantitative and qualitative proteomic analysis to investigate various cartilage tissue processing protocols. We established a method for removing chondrocytes from cartilage sections that minimized matrix protein loss. Quantitative and qualitative proteomic analyses were used to evaluate different cartilage extraction methodologies. The addition of surfactant to guanidine-HCl extraction buffer improved protein solubility. Ultrafiltration removed interference from polysaccharides and salts. The different extraction methods yielded different protein profiles. For instance, an overwhelming number of collagen peptides were extracted by the in situ trypsin digestion method. However, as expected, proteoglycans were more abundant within the guanidine-HCl extraction. </p><p>Subsequently we applied these methods to extract cartilage sections from different cartilage layers (superficial, intermediate and deep), joint types (knee and hip), and disease states (healthy and osteoarthritic). We also utilized lase capture microscopy (LCM) to harvest cartilage sample from individual subregions (territorial and interterritorial regions). The results suggested that there is more unique proteins existed in the superficial layer. By removing the chondrocytes, we were able to identify more extracellular matrix proteins. The phenotyping of cartilage subregions provided the chance to precisely localize the protein distribution, such as clusterin protein. We observed that the guanidine-HCl extractability (guanidine-HCl/ guanidine-HCl + in situ digestion extracts) of cartilage proteins. Proteoglycans showed high extractability while collagen and non-collagenous proteins had lower extractability. We also observed that the extractability might differ with depth of cartilage and also disease states might alter the characters as well. </p><p>Laser capture microscopy provides us the access to the cartilage subregions in which only few studies have investigated because of the difficulties to separate them. We established the proteomic analysis compatible-protocol to prepare the cartilage section for LCM application. The results showed that most of the proteoglycans and other proteins were enriched in the interterritorial regions. Type III and VI collagens, and fibrillin-1 were enriched in the territorial regions. We demonstrated that this distribution difference also varied with depth of cartilage. The difference of protein abundance between subregions might be altered because of disease states. </p><p>Last we were looking for the post-transliational modification existed in these subregions of cartilage. Deamidation is one of the modification without the enzyme involved. Previous studies have showed that deamidation may accumulated in the tissue with low turnover rate. Our proteomic analysis results suggests that abundance of deamidated peptides also varied in different layers and subregions of cartilage. </p><p>We have developed the monoclonal antibody based immunoassay to quantify the deamidated cartilage oligomeric matrix protein within cartilage tissue from different joints (hip and knee) and disease states (healthy, para-lesion, and remote lesion). The results suggests that the highest concentration of deamidated COMP was identified in arthritic hip cartilage. </p><p>The results of this study generated several reliable protocols to perform cartilage matrix proteomic analysis and provided data on the cartilage matrix proteome, without confounding by intracellular proteins and an overwhelming abundance of collagens. The discovery results elucidated the molecular architecture of cartilage tissue at different joint sites and disease states. The similarities among these cartilages suggested a constitutive role of some proteins such as collagen, prolargin, biglycan and decorin. Differences in abundance or distribution patterns, for other proteins such as for cartilage oligomaric matrix protein, aggrecan and hyaluronan and proteoglycan link protein, point to intriguing biological difference by joint site and disease state. Decellularization and a combination of extraction methodologies provides a holistic approach in characterizing the cartilage extracellular matrix. Guanidine-HCl extractability is an important marker to characterize the statue of cartilage; however it has not been fully understand. The protein distributions in matrix subregions may also serve as an index to characterize the metabolic status of cartilage in different disease states. A large sample cohort will be necessary to elucidate these characters.</p> / Dissertation

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Articular cartilage lining joints, such as in the knee, functions to reduce friction and absorb shock. Collagen type II is the largest constituent in the extracellular matrix of articular cartilage and its restoration is of the highest interest to tissue engineers. Cartilage has little ability to naturally regenerate due to the absence of vascularity and the inability of the chondrocytes to proliferate at a high rate. It would be ideal to create a mimicking extracellular matrix/scaffold from type II collagen that could possibly be used to replace damaged articular cartilage that has the same function and morphology. Three different groups of cartilage chips were utilized to extract type II collagen. The yield of the three groups was compared. The extracted type II collagen from the three groups was electrospun at the concentrations of 0.06, 0.08, 0.10 and 0.12 g/mL. Both the pore size and fiber diameter were analyzed. A SDS-Page was performed on the material to assure it was pure type II collagen and that no collagen type I contamination was present.

Articular cartilage is an extraordinary tissue, allowing frictionless movements of articulated joints, and acting as a load-bearing cushion to protect joints from damage. Breakdown of articular cartilage may result in crippling diseases such as osteoarthritis (OA) and, since articular cartilage has a limited repair capacity, a greater understanding of the mechanisms of joint homeostasis and its response to injury are of great clinical need. In this project the hypothesis that endogenous mesenchymal stem cells (MSCs) may contribute to the healing process of a full-thickness articular cartilage defect was investigated by combining a mouse model of joint surface injury and repair with a nucleoside analogue labelling scheme in DBA/1 mice. Following injury, proliferative responses of nucleoside analogue-retaining cells were detected between 4 and 12 days post injury (dpi) in both the bone marrow and the synovial membrane of the knee joint. Phenotypic analysis of these label-retaining cells using immunofluorescence staining revealed an MSC-compatible phenotype (CD44+, CD105+, CD146+, PDGFRα+ and p75NGFR+), with differences observed between the two tissues in expression of CD105 and CD146. The response of the label-retaining cells to the injury was associated with early activation of Notch signalling (4 dpi), followed by BMP signalling at 8 dpi and TGF-β at 12 dpi. Conversely, canonical Wnt signalling, which was active in uninjured knee joints and in injured knee joints up to 8 dpi, was attenuated at 12 dpi. The contribution of nerve growth factor (NGF), known as a pain mediator in OA, to the repair process was then investigated in vitro. NGF was released by both cartilage explants and femoral head cultures following injury. Using a Transwell-based cell migration assay, NGF was revealed to have a chemotactic effect on human bone marrow derived MSCs, but not synovial membrane derived MSCs. High-density micromass cultures also revealed NGF had a potent stimulatory effect on the chondrogenic differentiation of mesenchymal cells. The data presented here demonstrate a contribution of endogenous MSCs to the repair of articular cartilage in vivo and suggest a possible new therapeutic strategy: stimulation of in vivo recruitment of MSCs by modulating signalling pathways activated during the healing process. Furthermore, a novel role for NGF as a factor involved in migration and the chondrogenic differentiation of MSCs is suggested.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

A osteoartrose (OA) de joelho é uma das doenças osteomioarticulares mais comuns no mundo, afetando 2693 em cada 100.000 mulheres e 1770 em cada 100.000 homens. Embora o treinamento de força (TF) seja amplamente recomendado para a melhoria das debilidades físicas encontradas em pacientes com OA, o uso de cargas entre 70-85% da força dinâmica máxima (FDM) pode ser limitado não somente pela dor, mas também pela própria etiologia da doença, representando uma limitação para esta prática. O treinamento de força associado à oclusão vascular (TFOV) baseia-se na execução do TF em intensidades entre 20 e 50% da FDM, combinado à oclusão do fluxo sanguíneo através do uso de torniquetes. Estudos têm mostrado que a magnitude das mudanças na força e massa musculares após um período de treinamento com esta técnica é similar as causadas pelo TF de alta intensidade (70-85% FDM) sem adição da oclusão vascular. O objetivo do presente trabalho foi investigar a eficácia da associação da oclusão vascular ao TF como modelo de intervenção não farmacológica para melhoria da dor, força muscular, funcionalidade e qualidade de vida em pacientes diagnosticadas com OA de joelho. Diante disso, 48 participantes mulheres foram randomicamente distribuídas em três grupos: treinamento de força de baixa intensidade (TFB), treinamento de força de alta intensidade (TFA) ou treinamento de força de baixa intensidade associado à oclusão vascular (TFOV) e receberam treinamento duas vezes por semana durante doze semanas. No período basal e após a intervenção, as pacientes passaram por avaliações físicas (testes de funcionalidade e força), responderam questionários de qualidade de vida e de dor (índice WOMAC \"Western Ontario and McMaster Universities Osteoarthritis Index\" e SF-36 \"The Short Form 36 Health Survey\") e exame de imagem da área da secção transversa (AST) do músculo quadríceps por meio de tomografia computadorizada. Durante o período de treinamento, quatro pacientes do grupo TFA foram excluídas do protocolo por dor no joelho. Após a intervenção, no WOMAC, apenas os grupos TFOV e TFB apresentaram diminuições significativas na dor (p=0,0358 e p=0,0044, respectivamente), nos demais domínios, o único grupo que apresentou diminuições significativas de escore foi o TFOV (rigidez: p=0,0167 e funcionalidade p=0,0358). Nos testes de funcionalidade, os grupos TFOV e TFA apresentaram aumentos significativos no desempenho do \"Timed-stands test\" (p=0,0251 e p=0,003), no \"Timed-up-and-go\" não foram encontradas melhoras significantes nos grupos. Com relação a força, apenas os grupos TFOV e TFA aumentaram significativamente os valores no leg-press (p<0,0001) e na extensão de joelhos (p<0,0001). Comportamento similar foi encontrado no aumento da AST, grupos TFOV e TFA apresentaram aumentos significativos (p<0,0001). A melhora de qualidade de vida foi significante nos três grupos quando analisamos a somatória dos domínios do WOMAC (TFOV: p=0,0173; TFA: p=0,0438; TFB: p=0,0301), porém o SF-36 não foi capaz encontrar melhoras significativas nos grupos. Dessa forma, concluímos que o TFOV apresenta-se como uma estratégia relevante e segura de intervenção não farmacológica para mulheres acometidas por OA sintomática de joelhos, constituindo um modelo de tratamento capaz de induzir adaptações funcionais e morfológicas de grande interesse para esta população / Osteoarthritis (OA) of the knee is one of the most common articular disease worldwide, affecting 100,000 women in 2693 and 1770 in every 100,000 men. Although strength training (ST) is widely recommended for improving the physical weaknesses found in patients with OA, using loads between 70-85% of maximal dynamic strength (MDS) can be limited not only by pain, but also by the own etiology of the disease, representing a limitation of this practice. Strength training associated with vascular occlusion (STVO) is based on the execution of the ST at intensities between 20 and 50% of MDS, combined with the occlusion of blood flow through the use of tourniquets. Studies have shown that the magnitude of changes in strength and muscle mass after a period of training this technique is similar to those caused by high-intensity ST (MDS 70-85%) without the addition of the vascular occlusion. The objective of this study was to investigate the efficacy of the combination of vascular occlusion to the ST as non pharmacologic intervention model for improving pain, muscle strength, functionality and quality of life in patients diagnosed with knee OA. Thus, 48 women participants were randomly divided into three groups: strength training low intensity (STL), strength training, high intensity (STH) or low-intensity strength training associated with vascular occlusion (STVO) and trained two times per week for twelve weeks. At baseline and after the intervention, the patients underwent physical assessments (tests of functionality and strength), answered questionnaires on quality of life and pain (WOMAC index \"Western Ontario and McMaster Universities Osteoarthritis Index\" and SF-36 \"The Short form 36 Health Survey \") and the cross section area (CSA) of the quadriceps muscle was assessed using computed tomography. During the training period the STH group, four patients were excluded from the protocol due to knee pain. After the intervention, the WOMAC, only the STVO and STL groups showed significant decreases in pain (p=0.0358 and p=0.0044, respectively), in other domains, the only group that showed significant decreases in score was the STVO (stiffness: p= 0.0167 and p = 0.0358 functionality). In functionality testing, the STVO and STH groups showed significant increases in performance \"Timed-stands test\" (p=0.0251 and p=0.003), the \"Timed-up-and-go\" were not significant improvements found in groups. Regarding strength, only the STVO and STH groups significantly increased values in leg press (p<0.0001) and knee extension (p<0.0001). Similar behavior was found in increased AST and STH STVO groups showed significant increases (p<0.0001). The improvement of quality of life was significant in all three groups when analyzing the sum of the domains of WOMAC (STVO: p=0.0173; STH: p=0.0438; STL: p=0.0301), but the SF-36 was not able to find significant improvements in groups. Thus, we conclude that the STVO presents itself as a relevant and safe strategy of non-pharmacological intervention for women suffering from symptomatic knee OA, constituting a model of treatment capable of inducing functional and morphological adaptations of great interest to this population