A cartilagem articular é um tecido avascular, com baixa celularidade, composta principalmente de colágeno extracelular e proteoglicanos, com uma capacidade limitada de regeneração. Nos últimos anos, diversas abordagens clínicas e de pesquisa têm sido adotadas para reparar danos na cartilagem articular, como transplante de condrócitos, enxerto de periósteo, células-tronco mesenquimais e tecidos derivados dessas células. O isolamento de células-tronco mesenquimais foi relatado a partir de diferentes tecidos, incluindo medula óssea, tecido adiposo, sangue do cordão umbilical, sangue periférico e saco vitelino em equinos. As células-tronco mesenquimais derivadas de líquido e membrana sinoviais foram obtidas em humanos, cães, suínos e caprinos e são fontes promissoras para regeneração articular, já que são tecido-específicas e de fácil obtenção e cultivo. O objetivo deste trabalho foi estabelecer e caracterizar a linhagens de células obtidas de membrana e líquido sinoviais equinos. Os fragmentos foram obtidos por meio de artroscopias e cultivados em meios de cultura DMEM-H e MEM para obtenção das linhagens. Para análise da morfologia celular foi realizada a fotodocumentação das garrafas em microscopia invertida. A expressão de marcadores de células-tronco (CD45RO, OCT3/4, NANOG, CD105, CD90, CD34, CD117, CD133, TRA-1-81, VEGF-R1 e Ly6a), marcadores inflamatórios (COX-2, TNF-R1-r, CD11, CD1a e MCP-1) e marcadores envolvidos na checagem e progressão do ciclo celular (Caspase-3 fosforilada, HSP-47, P21, Ki67, Ciclina D1 e P53) mostraram diferencialmente expressos, sugerindo que podemos considerá-las uma possível fonte de células-tronco mesenquimais. Devido ao grande impacto que patologias na articulação têm sobre o desempenho atlético em cavalos, os resultados demonstrados neste trabalho será uma base para conduzir outros experimentos na avaliação das aplicações terapêuticas dessas células. / The articular cartilage is an avascular tissue with low cellularity composed of extracellular collagen and proteoglicans. It has a limited capacity of regeneration. Condrocyte transplantation, mesenchymal stem cells and tissues derived from these cells has been used by several researches to repair damage to the articular cartilage. The isolation of mesenchymal stem cells has been reported from different tissues such as bone marrow, adipose tissue, blood, umbilical cord, and yolk sac. The mesenchymal stem cells from synovial fluid and synovial membrane were obtained from humans, dogs, pigs and goats. These cells are tissue specific and easy to obtain and cultivate. The objective of this research is to obtain and characterize cells from equine synovial fluid and synovial membrane. The samples were obtained by arthroscopy and cultivated in the DMEM-H and MEM media. Cell morphology analyses were made by photodocumentation in inverted microscopy. The expression of stem cell markers (CD45RO, OCT3/4, NANOG, CD105, CD90, CD34, CD117, CD133, TRA-1-81, VEGF-R1 and Ly6a), inflammation markers (COX-2, TNF-R1-r, CD11, CD1a and MCP-1) and markers involved in checking and cell cycle progression (Caspase-3, HSP-47, P21, Ki67, Ciclina D1 and P53) showed differentially expressed. Mesenchymal stem cells from synovial membrane and synovial fluid provide promise for cell-based therapies for articular cartilage repair. These results may lead other experiments to use these cells to new therapeutic applications.

Over the years, different lubricants have been used to operate hip simulators. The current applicable ISO standard (ISO 14242-1:2002) recommends the use of 25% calf serum diluted with deionised water. The standard further recommends that the fluid be changed and the acetabular cup be weighed every 500 000 cycles. This procedure results in a loss of both the third body wear particles and the wear pattern. The purpose of this study was to develop a synthetic lubricant that would map the viscosity and lubricity properties of joint fluid (“synovial fluid”) over the whole duration of a simulator test, which is typically five million cycles. The first objective of this study was to find the effect of temperature increase on the viscous and lubricative properties of joint fluid retrieved from both primary and revision patients prior to surgery. The lubricity tests were done on a Linear-Oscillation Test Machine (SRV machine). Three test temperatures were used namely 38ºC, 50ºC and 60ºC. The load at failure and the average coefficient of friction were parameters measured during these tests. A decrease in the load at failure was found for an increase in test temperature, while the coefficient of friction stayed relatively stable. The viscosity tests were done using a Brookfield Viscometer. The three test temperatures mentioned above, were copied. The joint fluid tested showed pseudoplastic flow behaviour. An increase in the viscosity as a function of test temperature increase and a magnitude of shear rate was observed. The second objective of this study was to develop a synthetic lubricant that had the same average properties than that found for the retrieved joint fluid. A mixture of three different chemicals, namely Poloxamer 188, Xanthan Gum and Lube Boosterâ II was used to map the viscous and lubricative properties of the joint fluid. A comparative test using the synthetic lubricant and bovine serum was performed in a custom-built simulator. Wear debris was sampled at 500 000 cycle intervals up to 4 500 000 cycles. During these intervals the bovine serum stations were drained and washed with deionised water, but not stripped and weighed as specified in the ISO standard. This was done intentionally to preserve the wear pattern during the entire test. The synthetic lubricant stations were not stripped or drained during these intervals. This ensured that the wear pattern was maintained and that the effect of accumulative wear could be investigated throughout the duration of the test. The wear debris from the test was then compared to wear debris retrieved from scar tissue of revision patients. The wear debris that was found in the scar tissue retrieved from patients was similar in shape and size to that which was found in the simulator using bovine serum and the synthetic lubricant. It can thus be concluded that an acceptable lubricant had been developed to replace the current test medium in the simulators. / Dissertation (MEd (Mechanical Engineering))--University of Pretoria, 2006. / Mechanical and Aeronautical Engineering / unrestricted

Dentre as afecções que acometem as articulações dos equinos, a artrite séptica e a mais grave observada. A técnica da perfusão regional e um método comprovadamente eficiente para o tratamento de equinos acometidos por infecções sinoviais. O dimetilsulfóxido (DMSO) é um líquido orgânico, que possui a capacidade de penetrar, com extrema facilidade, em órgãos, tecidos e membranas celulares e intracelulares. Neste estudo, objetivou-se avaliar a concentração intra-articular da gentamicina administrada por perfusão regional intravenosa (PRI), associada ou não ao DMSO, bem como avaliar a influência do volume total perfundido o período de tempo onde a concentração inibitória mínima (CIM) foi mais eficiente e se a associação com o DMSO aumentou a CIM no líquido sinovial. Os animais foram distribuídos em quatro grupos experimentais, cada grupo recebeu gentamicina 6,6 mg/kg por PRI, o volume a ser administrado, após o cálculo da quantidade de gentamicina acrescida ou não de DMSO, era completado com solução de ringer com lactato estéril até o volume de 60mL nos grupos G60 e GD60, e até 250mL para os grupos G250 e GD250. As colheitas de líquido sinovial foram realizadas antes do início do experimento (T0), imediatamente depois da retirada do torniquete (T1) e após 4 (T2), 8 (T3), 12 (T4), 16 (T5) e 24 (T6) horas. O método para doseamento das concentrações de gentamicina empregado foi o de difusão em ágar. Destacamos que as concentrações de gentamicina no líquido sinovial na dose de 6.6.mg/Kg podem ser consideradas como adequadas, num período de até 24 horas após a administração. Nossos resultados apontam que o volume de 60 mL, pode ser considerado como o volume ideal de perfusão, bem como a associação do DMSO aumentou as concentrações de gentamicina (µg/mL) na articulação dos equinos e possivelmente reduziu a formação de edemas e aumentos de volume locais. / Among the diseases that affect the joints of horses, septic arthritis is the most serious observed. The regional perfusion technique is a well proven method for the treatment of horses affected by synovial infections. Dimethylsulfoxide (DMSO) is an organic liquid that has the ability to penetrate, with extreme ease on organs, tissues and cellular and intracellular membranes. This study aimed to evaluate the intra-articular concentration of gentamicin administered by intravenous regional perfusion (PRI), associated or not with DMSO, and assess the influence of the total volume infused the time period in which the minimum inhibitory concentration (MIC) It was more efficient and the association with DMSO increased the CIM in the synovial fluid. The animals were divided into four groups, each group received 6.6 mg gentamicin / kg per PRI, the volume to be administered, after calculating the amount of gentamicin plus or absence of DMSO was completed with Ringer\'s lactate solution with sterile until the volume in 60mL groups G60 and GD60, and up to 250 mL and G250 GD250 groups. The synovial fluid samples were collected before the start of the experiment (T0), immediately after removal of the tourniquet (T1) and after 4 (T2), 8 (T3), 12 (T4), 16 (T5) and 24 (T6) hours. The method for determination of gentamicin concentrations was employed the agar diffusion. We emphasize that the gentamicin concentrations in synovial fluid in 6.6.mg/Kg dose may be considered suitable, a period of up to 24 hours after administration. Our results indicate that the volume of 60 ml, can be considered as the ideal volume of the infusion, as well as the association of DMSO increased the gentamicin concentrations (µg / ml) in the joint of horses and possibly reduced edema formation and increases local volume.

The work deals with the lubrication of total knee replacement using fluorescence microscopy method, which allows unique insight into the contact between femoral and tibial component. The aim was to determine the effect of composition of synovial fluid (i.e. albumin, -globulin, hyaluronic acid and phospholipids) on film thickness and protein behaviour in contact, and also to determine changes of contact area during rotation. Since this is the first experimental work dealing with a knee replacement lubrication primarily, only simple rotation and load cycles were applied by the knee simulator. The output of the experiments was fluorescence intensity, which corresponds to dimensionless film thickness, over time. Another important output are the images directly showing the fluorescently labelled proteins in the contact area. The results show, there are fundamental differences in lubrication in different positions of rotation, due to changes of position, shape and behaviour of the contact area. The composition of the lubricant is also essential, since the proteins themselves form a relatively strong lubricating film and their mixture leads to a substantial reduction of film thickness, due to significant formation of clusters. Complex fluid, although it does not form the strongest layer, is able to create a quite continuous film.

The main goal of this diploma thesis is to determine the role of hyaluron acid and phospholipids on friction and lubrication of articular cartilage in regard to optimization of viscosupplements. This is carried out by measuring the coefficient of friction of the articular cartilage with several lubricants. Cartilage is lubricated particularly by a conventional viscosuplement, optimized viscosuplementation with phospholipids and model synovial fluid. In order to observe the function of those viscosuplements in the human body, both are mixed with the model synovial fluid, ubiquitous in human joints, in given ratio. Experiments revealed high friction when it comes to convectional viscosupplementation as opposed to low friction of the optimized viscosuplement with phospholipids. The same situation occurs when cartilage is lubricated with those viscosuplements mixed with model synovial fluid which might lead to development of a new, better, viscosupplementation based on hyaluron acid and phospholipids.

A longitudinal study was designed to characterize developmental patterns of plasma (PL) and synovial fluid (SF) total insulin-like growth factor-I (IGF-I) concentrations, as well as their association with measurements of skeletal growth in Thoroughbred horses. Horses were randomly assigned to one of two dietary treatment groups and fed diets with either a high or low starch content to examine the effects of dietary energy source on PL and SF IGF-I. At 3, 6, 9, 12 and 15 mo of age, PL and carpal SF samples were collected for analysis of total IGF-I. Body weight gain, wither height gain and forearm length gain were calculated for the 90 day periods between SF and PL sampling. No influence of diet on PL or SF IGF-I was detected (P > 0.05). Average SF IGF-I concentrations were 30.1 ± 1.8% of that found in PL, and PL and SF IGF-I were positively correlated (r = 0.48, P = 0.0003) There was an effect of month of age on both PL and SF IGF-I concentrations (P < 0.05). There was a positive correlation between all measures of gain except forearm length gain with PL and SF IGF-I (r = 0.41 to 0.55, P < 0.05). In our second study, we evaluated the use of a liquid-nitrogen cooled mortar and pestle, motorized freezer mill and rotor-stator homogenizer for homogenization of small (<50mg) articular cartilage samples. The rotor-stator homogenizer produced quanitfiable RNA yields, and was used to evaluate three different RNA isolation protocols. Two of the protocols were commercially available RNA extraction kits, with the third a modified guanidinium isothiocyanate/acid-phenol extraction procedure. The combined average yield for all protocols was 91.9 ng RNA/mg of cartilage. All protocols yielded a sufficient quantity of quality RNA suitable for gene expression analysis. / Master of Science

Osteoarthritis is a debilitating joint disease characterized by the degradation of articular cartilage due to long term wear or acute injury. OA can lead to pain, limited mobility, and stiffness in the joint, and current treatment options often require invasive surgery or are limited to corrective attempts at mitigating pain. Due in part to the complexity of the disease and lack of holistic understanding of its advancement, there is no known treatment to halt or reverse the effects of OA progression in the joint. In order to address this need, the underlying mechanisms that drive the mechanical degradation of cartilage structure in its progression must be determined. The objective of this dissertation is to (1) investigate the mechanical breakdown of cartilage through fatigue failure in physiologically relevant models and (2) to introduce a minimally invasive method for increasing the mechanical integrity of cartilage in an effort to reverse the effects of OA. In order to classify the mechanical mediation of wear in OA disease pathology, wear progression in human articular cartilage must be fully characterized. Human articular cartilage exhibits a remarkable resilience to wear during frictional sliding, making it difficult to induce damage in the tissue in experimental models. Previous work established reciprocal compressive stresses, and not frictional stresses, as the primary initiator of delamination fatigue wear in immature bovine cartilage. In Chapter 2, we tested the hypothesis that reciprocal compressive stresses could induce fatigue wear in human articular cartilage and thus establish a reproducible and characterizable model of wear induction in human tissue. Human articular cartilage was subjected to 24 hours of frictional sliding in two contact configurations: stationary contact area (SCA), and migrating contact area (MCA). Five samples were tested in the SCA configuration, which induces frictional stresses, and five were tested in the MCA configuration, which induces reciprocal compressive stresses and frictional stresses. The SCA samples showed no conclusive damage after 24 hours of sliding, and recovered 99.3% ± 2.34% of their original thickness after testing. Three out of five MCA samples showed conclusive signs of damage, one in the form of tissue splitting, one in the form of blister formation, and one in the form of complete tissue tearing. The average friction coefficient in the SCA group (μ_SCA= 0.090 ± 0.008) was higher than the average friction coefficient in the MCA group (μ_MCA= 0.066±0.020; p=0.03). Although conducted as two separate studies, the results in Chapter 2 provide a preliminary data set to suggest that reciprocal compressive stresses are responsible for fatigue failure in human tissue, coherent with the results in the immature bovine model. Additionally, results of Chapter 2 establish a reproducible and physiologically relevant protocol for damage induction in human tissue. Future work will investigate this hypothesis with directly paired SCA and MCA human articular cartilage tissue samples of similar OA grade. To further understand cartilage damage mechanics in physiologically relevant conditions, Chapter 3 and 4 investigate the role of synovial fluid in fatigue failure of immature bovine cartilage. Synovial fluid is often incorrectly identified as the source of low friction in cartilage sliding. In fact, it has an effect on the friction coefficient that is far secondary to interstitial fluid load support. Further, reciprocal compressive stresses, not frictional stresses, have been shown to be responsible for fatigue failure. It is imperative to understand the function of synovial fluid in wear mechanics. We tested the hypothesis that synovial fluid reduces the rate of fatigue failure in immature bovine articular cartilage due to the protective effects of its molecular constituents. Eight paired medial and lateral tibial plateaus were tested in MCA sliding in phosphate buffer saline (n=8) or synovial fluid (n=8) to directly compare fatigue rate in synovial fluid versus phosphate buffer saline. An additional study evaluated the effect of molecular constituents on wear rate by testing medial and lateral tibial plateaus in 50% (n=8) and 25% (n=8) synovial fluid diluted with phosphate buffer saline. All eight samples tested in phosphate buffer saline damaged after 24 hours of reciprocal sliding, and none of the samples tested in pure synovial fluid became damaged over the same duration. After an additional two days of sliding, two of eight samples tested in pure synovial fluid got damaged. In the samples tested in 50% and 25% synovial fluid-phosphate buffer saline dilutions, one sample and five samples got damaged after 72 hours of sliding respectively. The results of this study confirmed the hypothesis that synovial provides a protective effect against fatigue failure. The study also suggests that dilution of the synovial below a critical value reduces the concentration of molecular constituents available to protect the cartilage against damage. Chapter 4 investigates the mechanism of synovial fluid’s protective effect further, by examining its potential to extend the duration of elevated fluid load support under compression and thereby reduce cartilage susceptibility to fatigue. The results of Chapter 4 illustrated that synovial fluid had no effect on the stress relaxation response of the cartilage to unconfined compression, disproving the presented hypothesis. Therefore, future work will investigate the function of synovial fluid in reducing the rate of fatigue, independent of its effect on friction. The final two studies of this dissertation present a novel treatment modality to induce collagen crosslinks that enhance the cartilage equilibrium modulus. The technique introduced is presented as a minimally invasive alternative to current surgical interventions and proposes to increase the integrity of early-OA tissue. In Chapter 5, we investigate the hypothesis that low-level femtosecond laser treatment of cartilage can increase the stiffness of the equilibrium modulus without damaging tissue integrity or cell viability. In the first experiment, six immature bovine cartilage samples were treated with the laser and the equilibrium modulus was found to increase in stiffness (p<10⁻³). The technique was also applied to human articular cartilage tissue with “low” and “high” OA, and tissue was found to have an increase in equilibrium modulus (p=0.003 and p=0.03, respectively). Cell viability was preserved under these treatment conditions. Chapter 6 further outlines a safe envelope of laser treatment parameters through evaluation of the effect of thermal heating on the equilibrium modulus of cartilage samples. The results of this study found that temperatures above 65 ℃ (p<10⁻³) increase the tissue modulus, but no change in modulus occurs below 65 ℃ (p=1.00). The results of Chapter 6 provide an insight to the mechanical effect of thermal exposure, and informed the laser treatment parameters presented in Chapter 5, which were confirmed to produce thermal heating far below temperatures that result in thermal stiffening. Through the results presented in Chapter 5 and 6, preliminary data is provided to introduce a novel method for crosslink induction in the superficial zone of articular cartilage. In future work, this technique can be applied as a potential strategy to increase fatigue wear resistance, and to reduce the progression of OA in diseased tissue. The work presented in this dissertation seeks to contribute to the understanding of fatigue wear in articular cartilage under physiologically relevant conditions, as well as introduce a method for enhancing cartilage tissue properties with laser treatment. In the first half of the dissertation, the effect of reciprocal compressive stresses was evaluated in human articular cartilage tissue and in immature bovine cartilage immersed in synovial fluid in an effort to understand the mechanism of delamination fatigue failure in OA progression. In the second half, a laser treatment modality was shown to increase tissue equilibrium modulus stiffness without compromising tissue viability.

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

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-&#945;), interleucina 1 beta (IL-1&#946;) 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-&#945; 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-&#945;), interleukin 1 beta (IL- 1&#946;), 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, TNF&#945; 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.