Le cartilage est un tissu clé des articulations synoviales. Suite à un problème mécanique, traumatique ou inflammatoire, le cartilage est dégradé entrainant des douleurs articulaires et une perte de mobilité. Le cartilage étant un tissu non innervé et non vascularisé, son auto-réparation est très faible. De plus en plus de techniques sont développées pour la réparation des défauts cartilagineux mais aucune n’a encore permis d’obtenir un nouveau cartilage pleinement fonctionnel. En particulier, l’ingénierie tissulaire (IT) est une technique très prometteuse qui consiste à obtenir un greffon de cartilage dont les propriétés mécaniques et structurales soient satisfaisantes une fois implantée dans l’articulation. L’IT est basée sur l’association de cellules, d’un biomatériau et de facteurs de croissance. Le but de cette thèse est d’étudier l’effet de l’âge du donneur des cellules sur la synthèse du greffon par l’IT in vitro et sur la qualité du cartilage obtenu lors de l’implantation dans un modèle de rat NUDE. Puis dans une dernière partie, l’impact de l’environnement chimique et mécanique est étudié sur la qualité du greffon. Nos études montrent ainsi que l’âge du donneur aussi bien dans un contexte in vitro ou in vivo impacte la qualité du greffon et la réparation une fois implanté dans l’animal. En effet, les greffons issus des donneurs âgés ont des propriétés mécaniques légèrement plus élevées et une synthèse des protéines de la matrice extracellulaire (MEC) du cartilage significativement plus élevée que les greffons issus de donneurs jeunes. De plus, la réponse inflammatoire des greffons implantés dans un défaut cartilagineux chez le rat NUDE est plus faible pour les donneurs âgés. Enfin, nous montrons que le microenvironnement mécanique (compression ou pression hydrostatique) et chimique (liquide synovial (LS) ou TGF-β pur) joue un rôle important sur la réponse cellulaire. Par ailleurs, en fonction de l’âge, l’association de ces différents facteurs donnent des résultats différents. Par exemple, pour une sollicitation de type compression, c’est le LS qui est à favoriser pour obtenir les greffons de meilleure qualité dans le cas des donneurs âgés. Au contraire, pour la même sollicitation de type compression, c’est la présence de TGF-β1 qui conduit au greffon de meilleure qualité pour les donneurs jeunes. Ces études mettent en évidence l’importance de l’âge du donneur et montrent de plus qu’un protocole IT patient spécifique est la meilleure solution. / Cartilage is an important tissue of synovial joints. Following a mechanical problem, traumatic or inflammatory, the cartilage is degraded causing joint pain and loss of mobility. Because cartilage is a non-innervated and non-vascularized tissue, its self-repair is very weak. More and more techniques are being developed for the cartilage but none has resulted in a new fully functional cartilage. In particular, tissue engineering (TE) is a very promising technique that consists in obtaining a cartilage graft whose mechanical and structural properties are satisfactory once implanted in the joint. TE is based on the association of cells, biomaterial and growth factors. The aim of this thesis is to study the effect of cell donor’s age on graft synthesis by TE in vitro and on the quality of the cartilage obtained during implantation in a NUDE rat model. Then in a last part, the impact of the chemical and mechanical environment is studied on the quality of the graft. Our studies show that the age of the donor both in vitro and in vivo has an impact on graft quality and repair once implanted in the animal. In fact, grafts from older donors have slightly higher mechanical properties and significantly higher synthesis of extracellular matrix proteins (ECM) than grafts from younger donors. In addition, the inflammatory response of grafts implanted in a cartilage defect in the NUDE rat is lower for older donors. Finally, we show that the mechanical microenvironment (compression or hydrostatic pressure) and chemical microenvironment (synovial fluid (SF) or TGF-β) play an important role in the cellular response. Moreover, depending on age, the combination of these different factors gives different results. For example, for a compression solicitation, it is the SF that is to be favored to obtain better quality grafts in the case of elderly donors. On the contrary, for the same compression stress, it is the presence of TGF-β1 that leads to the best quality graft for young donors. These studies highlight the importance of donor age and further show that a specific patient protocol of TE is the best solution.

O cavalo tem sido há milhares de anos um dos animais de maior utilidade para o homem, tanto no trabalho quanto no esporte. A integridade de sua estrutura física é determinante para a qualidade de seu desempenho, sendo alvo importante das estratégias terapêuticas e preventivas da atualidade. Diversos estudos têm esclarecido parte da cascata de eventos que ocorre em ambiente intra-articular, revelando os principais mediadores nocivos e ampliando as opções para tratamentos mais eficientes. Experimentos utilizando a citometria de fluxo foram capazes de demonstrar que a adição de plasma às células de líquido sinovial (LS) previamente inflamadas diminui a produção de espécies reativas de oxigênio durante o burst oxidativo, conferindo capacidade antioxidante a este hemoderivado. Pouco se sabe, no entanto, sobre seu potencial pró ou anti-inflamatório, pois não existem relatos na literatura investigando tal atividade. Assim, desenvolvemos este estudo com o objetivo de acompanhar os efeitos exercidos pelo plasma autólogo condicionado (ACP) sobre os tecidos articulares, reportando as alterações clínicas e no LS das articulações metacarpofalangeanas hígidas que receberam este tratamento. Foram administrados 4 ml de ACP em 10 articulações metacarpofalangeanas de equinos saudáveis, enquanto as articulações contralaterais receberam 4 ml de solução fisiológica, como controle. LS foi coletado antes de cada aplicação e nos momentos 3, 6, 24, 48 e 168 horas após. Foi realizada avaliação física diária, e as amostras coletadas foram submetidas imediatamente a análise física (volume, cor, aspecto e viscosidade), teste da qualidade do precipitado de mucina, contagem total e diferencial das células nucleadas. O sobrenadante foi congelado para dosagem posterior de ureia, proteína total, ácido hialurônico (AH), condroitim sulfato (CS), prostaglandina E2 (PGE2) e das citocinas fator de necrose tumoral alfa (TNF-α), interleucina 1 beta (IL-1β) e antagonista do receptor de IL-1 (IL-1ra). A avaliação física dos animais detectou presença de claudicação nas articulações metacarpofalangeanas tratadas com ACP nos momentos 24 e 48 horas após sua administração. A análise física do LS das mesmas articulações revelou maior contaminação das amostras com sangue às 6 horas e maior contagem de células nucleadas às 3, 6, 24 e 48 horas, com predomínio de leucócitos polimorfonucleares. Ainda, o ACP induziu maiores concentrações de proteína total e PGE2 às 3 e 6 horas, e maior concentração de CS às 24 horas. Houve também tendência ao aumento de TNF-α às 24 horas. Às 168 horas, todavia, nenhuma alteração foi observada em quaisquer dos itens avaliados. Não ocorreram alterações nos demais aspectos físicos do LS nem na qualidade do precipitado de mucina, tampouco nas concentrações de ureia e AH. Estes resultados indicam que em articulações hígidas o ACP possui ação pró-inflamatória transitória, com maiores concentrações de PGE2 induzindo o catabolismo dos PGs da matriz, aumentando particularmente o CS. / History has connected human and equine species, for work and pleasure purposes and in both scenarios, horses\' physical integrity is of paramount importance for adequate performance. Soundness has been the target of many studies in orthopedic therapeutics and preventive equine medicine. Several of these studies have thrown light on the sequence of deleterious intra-articular events that take place after an insult, revealing key mediators of joint destruction and widening treatment options. Experiments evaluating the effects of plasma on reactive oxygen species production by chemically stimulated synovial fluid (SF) cells revealed a potent antioxidant effect. Little is known, however about plasma´s anti or pro inflammatory activities, still an unexplored property of plasma. This study was to designed to observe the effects of autologous conditioned plasma (ACP) on articular components, reporting findings of serial SF analysis and clinical evaluations, before and after its administration in healthy metacarpophalangeal joints. Four mililiters of ACP were injected in 10 healthy metacarpophalangeal joints, and the contralateral joints were injected with 4 ml of saline, serving as controls. SF was obtained for analysis before, and then 3, 6, 24, 48 and 168 hours after treatment injection. Horses were subjected to daily clinical evaluations and synovial fluid samples were immediately analyzed for color, viscosity, volume, aspect, quality of mucin clot and total and differential nucleated cell counts. The supernatant was frozen and stored for posterior dosages of urea, total protein, hyaluronic acid (HA), chondroitin sulphate (CS), prostaglandin E2 (PGE2), tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL- 1β), and interleukin 1 receptor antagonist (IL-1ra). Physical evaluation of ACP treated subjects detected mild lameness at 24 and 48 hours observation points. SF analysis of ACP treated joints revealed blood contamination and higher total nucleated cell counts at 3, 6, 24 and 48 hours, with predominance of polymorphonuclear cells. ACP treatment has also induced higher protein concentrations and PGE2 levels at 3 and 6 hours and higher CS levels at 24 hours in synovial fluid analysis. At 24 hours, TNFα concentrations were higher, although not significantly. At 168 hours post ACP treatment, however, no change was observed in any parameter of synovial fluid analysis. No alterations were detected in the remaining items evaluated, nor in the quality of the mucin clot, urea concentration or HA. These results indicate that, when injected into healthy joints, ACP elicits a transient inflammatory response, characterized by higher PGE2 concentrations, matrix catabolism, with particular increase in CS.

Dada sua natureza cursorial, a espécie equina depende da livre movimentação para o desempenho de atividades diárias e para sobrevivência. Esta habilidade natural para o movimento é explorada em alto grau durante a prática esportiva, razão da existência de grande parte dos criatórios e centros de treinamento. Enfermidades articulares, como a osteoartrite e a osteocondrose, que reduzam ou causem prejuízo permanente à mobilidade e ao desempenho atlético são um desafio terapêutico. Os hemoderivados autólogos surgiram como opções acessíveis e seguras para o tratamento destas afecções articulares e apesar de amplamente difundido, seu emprego acontece a despeito de lacunas no conhecimento dos métodos ideais de preparação e administração, bem como dos mecanismos de ação destes produtos derivados do sangue. As propriedades antioxidantes do plasma processado autólogo demonstradas in vitro, em estudo utilizando células de líquido sinovial equino previamente estimuladas, motivaram a realização deste estudo com o objetivo de verificar os efeitos deste hemoderivado, in vivo, em articulações de equinos com osteoartrite ou osteocondrose. Tais propriedades despertaram também o interesse em verificar a ocorrência de eventos relacionados ao estresse oxidativo nestas enfermidades, o que justificaria seu emprego na clínica ortopédica. Para tal fim, líquido sinovial foi obtido antes da abordagem cirúrgica de articulações acometidas pela osteoartrite ou osteocondrose, para análise do perfil oxidante/antioxidante, físico, celular, molecular e inflamatório. Após o término da artroscopia, um grupo de animais recebeu injeção intra-articular do plasma processado autólogo e outro grupo não recebeu nenhuma medicação intra-articular, servindo como controle. Quarenta e oito horas após a cirurgia, nova amostra de líquido sinovial foi coletada, e as mesmas análises foram realizadas. As análises físicas e a contagem de células totais foram realizadas imediatamente após as coletas. O sobrenadante resultante da centrifugação do líquido sinovial foi congelado a -80º C para análises posteriores. Elas consistiram nas dosagens da catalase, superóxido dismutase, glutationa peroxidase, capacidade antioxidante total, vitamina E, proteína carbonil, condroitim sulfato, ácido hialurônico, prostaglandina E2, proteína total e ureia. Em relação à caracterização das enfermidades, as articulações acometidas por osteoartrite apresentaram maiores concentrações de catalase e de vitamina E, refletindo maior ativação dos sistemas de defesa antioxidante enzimáticos e não enzimáticos nesta enfermidade. A osteoartrite apresentou, ainda, natureza mais inflamatória, quando comparada à osteocondrose, com concentrações mais elevadas de prostaglandina E2 e proteína total, além de maiores contagens de células totais no líquido sinovial das articulações afetadas. No entanto, a osteocondrose apresentou caráter mais destrutivo em relação aos componentes da matriz extracelular e do líquido sinovial, com concentrações superiores de condroitim sulfato e ácido hialurônico, respectivamente, no líquido sinovial destas articulações. Os resultados demonstraram a ocorrência de estresse oxidativo no período pós-operatório através da ativação das defesas antioxidantes enzimáticas, com aumento da concentração de catalase. O tratamento com o plasma processado autólogo levou a aumento das concentrações de vitamina E no líquido sinovial, incrementando o sistema de defesa antioxidante não enzimático. Não foram observados efeitos do tratamento sobre o metabolismo da cartilagem articular ou biomarcadores inflamatórios. Pode-se inferir que o plasma processado autólogo apresenta potencial terapêutico no combate ao estresse oxidativo. / As a cursorial animal, the horse relies on soundness to fully accomplish daily life activities and survival. Horses natural ability for movement justifies breeding and training investments, and is explored to its maximum during athletic activities. Articular disorders, such as osteoarthritis and osteochondrosis, with their potential to reduce physical performance, are a therapeutic challenge. Autologous haemoderivates have appeared as safe and accessible options for the management of these conditions, but their widespread use has outpaced scientific evidence regarding mechanisms of action and ideal methods for preparation and administration. The antioxidant properties of autologous processed plasma, previously demonstrated in vitro on stimulated equine synovial fluid cells, posed a question on whether these effects would occur in vivo. Therefore, the present study aimed to verify autologous processed plasma´s effects in osteoarthritic and osteochondritic joints, when administered at the end of the arthroscopic procedure, with particular emphasis on possible antioxidant attributes. The study also aimed to detect evidences of oxidative stress in these disorders, what could further justify its use. For this purpose, synovial fluid was obtained immediately before the beginning of the surgery, and analyzed to yield an oxidant/ antioxidant, physical, cellular, molecular and inflammatory profile of osteoarthritis and osteochondrosis. After the end of the procedure, one group of horses received the intraarticular medication and another group of untreated horses served as controls. Forty-eight hours after surgery synovial fluid was retrieved from operated joints, and a second analysis was performed. Physical and cellular analyses were conducted immediately after sample collection and the supernatant was stored at -80º C for further evaluations. These consisted in catalase, superoxide dismutase, glutathione peroxidase, total antioxidant capacity, vitamin E, protein carbonyl groups, chondroitin sulphate, hyaluronic acid, prostaglandin E2, total protein and urea determinations. Osteoarthritic joints revealed greater activation of antioxidant defense systems, enzymatic and non-enzymatic, with greater activity of catalase, and greater concentrations of vitamin E, respectively, when compared to osteochondritic joints. Additionally, these joints had higher total nucleated cell counts and prostaglandin E2 and total protein concentrations, revealing a more inflammatory profile when compared to that of osteochondritic joints. Nonetheless, in osteochondritic joints synovial fluid and matrix components degradation was more pronounced, as there were higher concentrations of chondroitin sulphate and hyaluronic acid in synovial fluid obtained from these joints. Results also showed the occurrence of oxidative stress, 48 hours after the arthroscopic procedure, through higher catalase activity in synovial fluid, reflecting activation of antioxidant defenses. Treatment with autologous processed plasma increased vitamin E concentrations, incrementing non-enzymatic antioxidant defenses. No effects were observed on metabolic or inflammatory biomarkers. It was possible to infer that autologous processed plasma has the therapeutic potential to lessen oxidative stress effects on articular components in the postoperative period.

Na espécie equina, as enfermidades osteoarticulares causam prejuízo econômico e impacto negativo no desempenho atlético, devido aos danos causados na cartilagem articular. A regeneração da cartilagem hialina e a manutenção da integridade das estruturas que a compõe norteiam a busca do tratamento ideal. Neste contexto, este estudo foi delineado com o objetivo de investigar a presença de células-tronco mesenquimais (CTMs) no líquido sinovial (LS) e na membrana sinovial (MS) de equinos com articulações hígidas, com osteocondrite dissecante (OCD) e com osteoartrite (OA) e compará-las, visando estabelecer qual fonte celular possui melhor característica fenotípica e capacidade de diferenciação celular, mais especificamente, aquela que seja superior em relação à capacidade condrogênica. Foram utilizados equinos machos e fêmeas de diferentes idades, totalizando 97 articulações. O LS e MS foram coletados durante artroscopia e as células foram cultivadas, e avaliadas por citometria de fluxo com os anticorpos CD44, CD90, CD105, CD34; e por imunocitoquímica com os anticorpos nanog, oct4, PGP 9.5, lisozima, vimentina e citoqueratina. Adicionalmente, o potencial de diferenciação das células foi avaliado para as linhagens condrogênica, osteogênica e adipogênica. Foi realizado teste de tumorigenicidade em camundongos Balb-Cnu/nu, para comprovar aplicabilidade clínica, e posteriormente, as CTMs provenientes de LS de articulações hígidas foram aplicadas em articulações de equinos. A identidade das células foi comprovada durante o cultivo demonstrando características de adesão ao plástico e morfologia fibroblastóide. A média percentual das populações positivas para CD90 foi de 64,9% (LS-H), 48,3% (LS-OCD), 48,1% (LS-OA), 66,6% (MS-H), 40,2% (MS-OCD) e 40,3% (MS-OA). A porcentagem de células positivas para CD44 foi de 1,18% (LS-H), 3,98% (LS-OCD), 14,2% (LS-OA), 1,9% (MS-H), 2,17% (MS-OCD) 8,56% (MS-OA). Não foi observada expressão dos anticorpos CD34 e CD105. Na análise imunocitoquímica foi detectada expressão positiva para os anticorpos: lisozima, PGP 9.5, PCNA e vimentina, e negativa para nanog, oct4 e citoqueratina. A multipotência (osteogênica, condrogênica e adipogênica) das células foi confirmada através da coloração Alizarin Red para detecção de matriz de cálcio, Oil Red O para detecção de gotículas de gordura e azul de toluidina, alcian blue e hematoxilina eosina para detecção de matriz de proteoglicanos. Com relação aos resultados do teste tumorigênico, nenhum órgão dos camundongos foi afetado, assegurando a aplicabilidade das células estudadas. Ainda, as articulações de equinos tratadas, não apresentaram quaisquer sinais de reação inflamatória após aplicação de células alogênicas. Por fim, concluímos que, a fenotipagem positiva de CD44 e CD90 somada à capacidade de diferenciação nas linhagens osteogênica e condrogênica confirma a presença de CTMs nas populações celulares obtidas de LS e MS de equinos. Também foi observado que as células de LS provenientes de articulações hígidas, são as de melhor utilização clínica, uma vez que apresentaram maior expressão de CD90 e demonstraram melhor capacidade de diferenciação celular em relação às células derivadas de articulações enfermas. Além disso, possuem método mais fácil de colheita em relação à colheita de MS, visando futura terapia celular na rotina clínica / In the equine species, osteoarticular diseases cause significant economic losses and negative impact on equine athletic performance. The hyaline cartilage regeneration and the maintenance of integrity of its components guide the search for the ideal treatment. In this scenario, this study aimed to investigate the presence of mesenchymal stem cell (MSCs) in the synovial fluid (SF) and in the synovial membrane (SM) of healthy equine joints, osteoarthritic (OA) and osteochondritic joints (OCD), comparing their potential as cellular sources, according to their differentiation ability, in particular with superior chondrogenic potential and the phenotypic characteristics of the MSCs. Ninety-seven equine joints from males and females of different ages were used to harvest cells. SF and SM were obtained during arthroscopy and the cells SF and SM were cultured and assessed for CD90, CD44, CD105 and CD34 markers by flow cytometry, and nanog, oct4, PGP 9.5, lyzozyme, vimentin and cytokeratin were assessed by immunocytochemistry. Additionally, cells were evaluated in vitro for their osteogenic, adipogenic and chondrogenic differentiation potential. The tumorigenicity test was carried in Balb-C nu/nu mice, to verify the safety of cell sources and, later, mesenchymal stem cells harvested from healthy equine joints were injected into equine joints. The identity of these cells was confirmed during cell growth, through properties of plastic adhesion and fibroblastoid morphology. The mean percentage of CD90 positive cells was 64.9% (SF-H), 48.3% (SF-OCD), 48.1% (SF-OA), 66.6% (SM-H), 40.2% (SM- OCD) and 40.3% (SM-OA). The percentage of CD44 positive cells was 1.18 % (SF-H), 3.98% (SF-OCD), 14.2% (SF-OA), 1.9% (SM-H), 2.17% (SM-OCD) and 8.56% (SM-OA). The expression of CD34 and CD105 antibodies was not observed. Through immunocytochemical analysis, expression for lysozyme, PGP9.5, PCNA e vimentin antibodies was detected and negative expression for nanog, oct4 e cytokeratin was observed. The multipotent capacity of mesenchymal stromal cells for lineage differentiation (osteogenic, chondrogenoic and adipogenic) was confirmed with different staining techniques: Alizarin Red enabled detection of the calcium matrix, Oil Red O enabled the detection of fat droplets and Toluidin Blue, Alcian Blue and haematoxylin eosin enabled detection of proteoglycan matrix. Results of tumorigenic tests in mice showed no compromise of any internal organ, assuring applicability of the studied cells. Furthermore, equine joints treated with MSC harvested from healthy joints did not show any signs of an inflammatory reaction after injection of the allogeneic cells. The presence of cells with positive CD44 and CD90 phenotypes and with the ability to differentiate into osteogenic and chondrogenic lineages confirms the presence of MSCs in equine SF and SM. Cells obtained from healthy SF were more suitable for clinical application, for they presented higher CD90 expression and demonstrated greater differentiation capabilities, when compared to that of cells retrieved from compromised joints. In addition to that, SF derived cells are easier to obtain when compared to SM cells, aiming their future application clinical

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / An experimental synovitis model of the radio-carpal joint was induced in 8 male intact ponies with an intra-articular injection of 0.25ml of Freund s complete adjuvant. Four ponies had the affected joint irradiated with a 4J/cm2 of laser in a cranio-caudal direction. The control group received the same technique with the device turned off. The heart rate was lower (p<0.05) in the treated group (37±6.9) than in the control group (41.2±5.7). The carpal angle of maximum flexion was significantly (p <0.05) lower in the treated group (94.4±27.2) than in the control group (125.6±5.3). The results of this study show that treatment with laser of gallium arsenide, in the dose of 4J/cm2, presented analgesic effect and improved joint movement in this model of Freund s adjuvant induced acute synovitis. / Um modelo experimental de sinovite aguda rádio-carpiana foi induzido em oito pôneis machos adultos com uma injeção intra-articular de 0,25 ml de adjuvante completo de Freund para avaliar o efeito da terapia laser nesta lesão. Um grupo (n= 4) teve a articulação afetada tratada com laser de arseneto de gálio na dose diária de 4J/cm2 na incidência crânio-caudal por 15 sessões. O grupo controle (n= 4) não foi tratado. A freqüência cardíaca foi estatisticamente (p<0,05) menor no grupo tratado (37±6,9) do que no grupo controle (41,2±5,7). O ângulo de flexão carpal máximo foi significativamente (p<0,05) menor no grupo tratado (94,4±27,2) do que no grupo controle (125,6±5,3). Os resultados deste estudo mostram que a terapia com laser de arseneto de gálio na dose de 4J/cm2 apresentou efeito analgésico e melhorou a mobilidade do membro afetado pela sinovite aguda induzida por Adjuvante Completo de Freund.

This diploma thesis deals with an experimental analysis of lubrication of total hip joint replacement using fluorescence microscopy, focusing on so-called soft bearing pairs. Experiments were realized using a hip joint simulator, which was modified and refined for fluorescence microscopy. The aim of this diploma thesis was to determine the effect of the individual components of the synovial fluid on the lubrication mechanisms. For this purpose, different model fluids were designed to represent the composition of proteins and other synovial fluid constituents to elucidate the effect of albumin, -globulin and hyaluronic acid on the resulting lubrication performance. The experiments were separate into two categories different in type of load, dynamic (representative walking) and combined (representing standing with short walk). The experimental results were time-dependent fluorescence intensity values, representing dimensionless film thickness parameters. The values were recorded on the graphs and supplemented with fluorescence images taken during the characteristic sections of the measurements. The measured data was subsequently confronted with previously published articles. It was found that the dominant constituent responsible form film thickness is albumin, while -globulin and hyaluronic acid forms thin stable layer enhancing adsorption ability of albumin which contributes to increase of film thickness eventually.

This diploma thesis deals with an experimental analysis of knee joint replacement lubrication. The experiments were realized at a knee joint simulator which can apply conditions according to certain standard and survey the phenomena by using fluorescence microscopy. The aim of thesis is to clarify the influence of particular components of synovial fluid on the lubrication process. The intensity of fluorescence expresses dimensionless parameter of a lubrication film thickness. There was a fundamental study with mineral oils before the experiments with the synovial fluid. The study allows to have a look at contact transformation during walk. Results are shown in graphs as dependency of intensity on time, including pictures showing phenomena in the contact zone. Experiment results show that protein -globulin creates a layer on the surface. There is albumin on the layer and it makes the lubricating film thicker. The protein interaction is supported by hyaluronic acid and fosfolipids which stabilizes the created structure. According to lubrication is behaviour of film related to a complex structure of synovial fluid. Thesis gives more information about behaviour of synovial fluid and can be used for future development of knee replacements.

This thesis deals with the experimental study of lubrication of hip joint replacements. The influence of the synovial fluid composition and the head material to the lubricating film thickness were asssesed by using a pendulum hip joint simulator. Both of these effects were assesed under static and dynamic loading conditions. The experimental results showed that the movement of the head against the cup is unnecessary for the separation of the rubbing surfaces by a layer of adsorbed proteins. Composition of the synovial fluid then significantly influences the thickness of the lubricating film. Two opposite extremes have been observed during tests with two model synovial fluids. The first fluid formed a very strong lubricating film. The second sample almost prevent adsorption of proteins and consequently formed only very thin lubricating film. The influence of the material is noticeable when comparing the results obtained with a metal head and ceramic heads. Metal head formes under the same conditions thicker lubricating film. This evidence may be partly influenced by different diametrical clearances, which were in the case of ceramic heads higher.

L’arthrite juvénile idiopathique (AJI) est un groupe hétérogène de rhumatismes inflammatoires chroniques affectant les enfants de moins de 16 ans. Cette atteinte inflammatoire d’origine inconnue est caractérisée par une arthrite persistant plus de 6 semaines en l’absence de traitements.Afin de mettre en évidence des mécanismes impliqués dans la physiopathologie de l’AJI, une inclusion de patients atteints d’une autre forme d’arthrite juvénile, à savoir l’arthrite septique, a été effectuée. En parallèle, des études comparatives entre le sang périphérique (SP) et le liquide synovial (LS) des patients atteints d’AJI ont été réalisées afin de rechercher des mécanismes spécifiques aux perturbations articulaires. Un intérêt particulier a été porté sur les sous-populations de monocytes et de cellules dendritiques (DCs) ainsi que les profils d’expression des microARNs (miARNs) dans le sang périphérique et le LS des patients. Ces différents marqueurs biologiques ont été choisis car ils jouent un rôle majeur à la fois dans la régulation de l’inflammation et la pathogénèse des maladies inflammatoires.L’analyse de l’expression des miARNs par une approche de séquençage à haut débit suivie d’une validation par RT-qPCR a mis en évidence des miARNs dérégulés de façon spécifique dans l’AJI par rapport à l’AS. De plus, la caractérisation phénotypique des sous-populations de cellyles myéloïdes a montré une accumulation et une activation cellulaire propre à l’AJI. Dans l’ensemble, ce projet m’a permis d’identifier différents acteurs cellulaires et moléculaires pouvant être impliqués dans la physiopathologie de l’AJI. / Juvenile idiopathic arthritis (JIA) is a heterogeneous group of chronic inflammatory rheumatism affecting children under 16 years of age. This inflammatory disorder of unknown origin is characterized by arthritis lasting more than 6 weeks in the absence of treatments.In order to highlight mechanisms involved in the pathophysiology of JIA, an inclusion of patients suffering from septic arthritis, another form of juvenile arthritis, was performed. In parallel, comparative studies between the peripheral blood (PB) and the synovial fluid (SF) of patients with JIA were carried out in order to search for mechanisms specific of joint disturbances.We focused on monocytes and dendritic cells (DCs) subsets as well as the expression patterns of microRNAs (miRNAs) in the PB and SF of patients. These different biological markers are known to play a major role both in the regulation of inflammation and the pathogenesis of inflammatory diseases.Analysis of miRNA expression by a high-throughput sequencing approach followed by RT-qPCR validation revealed specifically deregulated miRNAs in JIA compared to AS. In addition, the phenotypic characterization of myeloid cell subpopulations showed an accumulation and activation profile specific of JIA cells. Overall, this project allowed me to identify different cellular and molecular actors that might be involved in the pathophysiology of JIA.

The excellent lubrication present within mammalian synovial joints attracted scientific interest, and some close-to-realistic models were applied to study the mechanism in vitro. In this project, the synergistic lubrication of synovial fluid was investigated by using 1,2-Dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), hyaluronic acid (HA), and phosphate-buffered saline (PBS buffer) to mimic the synovial fluid. Lubrication by the model synovial fluid was studied using borosilicate glass specimens in Mini-Traction Machine (MTM). The experiments proved that the DPPC vesicle solution and mixed DPPC/HA solution had excellent lubrication ability, stemming both from adsorption of a lubricious layer at the surface of glass specimens and from presence of material reservoir available for repair of wear defects in the lubricious layer. Comparing the macroscale results obtained in this project by MTM with the results in previous studies on microscale by using AFM, we concluded that the microscale study of synergistic lubrication could predict macroscale results, even though some differences were detected due to limited possibilities for exact replication of experimental conditions at the two scales.