Esta pesquisa teve como objetivo estudar as alterações da cartilagem articular dos côndilos femural de ratos, provocadas pela variação da amplitude de movimento e da frequência, utilizando-se uma máquina de movimentação de flexo-extensão contínua passiva. Foram utilizados 24 ratos machos Wistar, divididos em quatro grupos: G1 (exercitados com 80 ciclos/minuto em grande amplitude), G2 (exercitados com 80 ciclos/minuto em pequena amplitude), G3 (exercitados com 40 ciclos/minuto em grande amplitude) e G4 (exercitados com 40 ciclos/minuto em pequena amplitude). Os animais pertencentes aos quatro grupos realizaram exercícios de flexo-extensão contínuas, diariamente, por 1 hora, através de um equipamento que atende aos princípios do mecanismo biela-manivela, o qual produz movimentos contínuos de flexo-extensão com grande e pequena amplitude, em frequência de 80 e 40 ciclos por minuto. Após 15 dias os animais foram sacrificados e de cada um a cartilagem articular do côndilo lateral do joelho direito foi retirada, incluída, cortada, corada com hematoxilina-eosina e analisada em fotomicroscópio. Pela análise histológica observou-se aumento do número de células na área da cartilagem articular definida para o estudo, nos quatro grupos exercitados, quando comparados ao respectivo controle. Os animais exercitados à velocidade de 80 ciclos por minuto e grande amplitude apresentaram um aumento no número de células quando comparados aos animais exercitados à velocidade de 40 ciclos por minuto em grande e pequena amplitude. O mesmo ocorreu com os animais exercitados com velocidade de 80 ciclos por minuto e pequena amplitude, comparados aos animais exercitados com velocidade de 40 ciclos por minuto e pequena amplitude. Quanto à análise das medidas de espessura foi encontrado aumento em todos os grupos em relação ao respectivo controle. Os animais exercitados à velocidade de 80 ciclos por minuto e grande amplitude apresentaram um aumento da medida de espessura quando comparados aos animais exercitados à velocidade de 40 ciclos por minuto e pequena amplitude, o mesmo ocorrendo com os animais exercitados com velocidade de 40 ciclos por minuto e pequena amplitude. Os dados demonstram que os exercícios de flexo-extensão contínua passiva estimulam aumento do número de células e aumento da espessura, não podendo ser atribuída como processo de lesão e, sim, como aumento de atividade metabólica e fator de proteção e adaptação. / This research had as objective to study the alterations of the articular cartilage of the knee of mices provoked by the variation of the movement width and of the frequency, using a machine of movement of passive continuous flexo-extension. 24 Wistar male mices were used, divided in four groups: G1 (exercised with 80 cycles/minute in great width), G2 (exercised with 80 cycles/minute in small width), G3 (exercised with 40 cycles/minute in great width) and G4 (exercised with 40 cycles/minute in small width). The animals belonging to the four groups accomplished flexo-extension exercises daily for 1 hour, through a equipment that assists to the principles of the \"rod-cranck\" mechanism, which produces continuous movements of flexo-extension with great width and small width in frequency of 80 and 40 cycles per minute. After 15 days the animals were sacrificed, the articular cartilage was collected, included, cut, colored with hematoxiline-eosine and analyzed in a photomicroscope. For the histologic analysis an increase of the number of cells was observed in every area of the articular cartilage in the 4 exercised groups when compared to the respective control, that the exercised animals at the speed of 80 cycles/minute and great width presented an increase in the number of cells when compared the exercised animals at the speed of 40 cycles/minute in great and small width, the same thing happening with the animals exercised with speed of 80 cycies/minute and small width, when compared to the exercised animals of 40 cycles/minute and small width. As the analysis of the measures of thickness it was found an increase in all the groups in relation to the respective control. The exercised animals at the speed of 80 cycles/minute and great width presented an increase of the measure of thickness when compared the exercised animals the speed of 40 cycles/minute and small width, the same happening with the animals exercised with speed of 40 cycles/minute and small width. The data demonstrate that the exercises of passive continuous flexo-extension stimulate an increase of the number of cells and increase of the thickness, not allowing us to attribute it as a Iesion process, but as an increase of metabolic activity as a protection and adaptation factor.

Orientador: Orival Andries Júnior / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Educação Física / Made available in DSpace on 2018-08-22T22:03:57Z (GMT). No. of bitstreams: 1 Lima_CarolinaMartins_M.pdf: 1429127 bytes, checksum: 645e26f53455ecb88a9e0f3771a1876b (MD5) Previous issue date: 2013 / Resumo: A Hipermobilidade Articular Generalizada é uma disfunção que dá ao indivíduo capacidade de realizar movimentos articulares exagerados, e na natação é necessário grande amplitude para uma performance eficiente. Sendo assim, o estudo teve como objetivo, verificar a influência da hipermobilidade articular, na performance e no índice de lesões de atletas voluntários na natação. Para isso, foram avaliados 20 atletas do estado de São Paulo, de ambos os sexos, de 15 a 20 anos de idade, e separados em três grupos, atletas com HAG (grupo 1), atletas sem HAG (grupo 2) e todos os atletas (grupo 3). Os dados foram obtidos através dos critérios de Carter e Wilkinson, parcialmente modificados por Beighton e Horan para diagnosticar HAG e flexiteste nas articulações de ombro e tornozelo para complementar o estudo. O Inquérito de Morbidade Referida adaptado foi utilizado para análise da incidência de lesão. Ainda, realizou-se filmagem em campo aberto de um estimulo máximo de 50 metros no nado crawl para avaliação da performance, sendo analisados tempo e frequência de braçadas. Observou-se que os atletas que apresentaram HAG consequentemente tiveram maior flexíndice. Quando analisado o desempenho, o grupo 1 apresentou menor frequência de braçadas, maior comprimento de braçadas e maior velocidade. Além do que, o tempo para execução do nado foi menor para o grupo 1, corroborando a hipótese de a HAG melhorar a performance do atleta de natação, apesar de não apresentar significância estatística. Com relação à incidência de lesão, o grupo 1 apresentou maior incidência de lesões osteoarticulares, percebidas no treino aquático, e principalmente no joelho, ombro e tornozelo, e o grupo 2 apresentou menor incidência de lesão, acometendo mais lesões do tipo musculares, percebidas na musculação e principalmente no ombro. Sendo assim, pôde-se concluir que os atletas com HAG apresentaram maior incidência de lesão e melhor performance que os atletas de natação sem HAG / Abstract: The Generalized Joint Hypermobility (GJH) is a disorder which gives the individual the ability to perform joint exacerbated movements, and the swimming uses movements of large amplitude for efficient performance. Thus, the study aims to verify the influence of GJH in the occurrence of injuries and overall performance of swimmers volunteers, since little has been reported on the influence of hypermobility and swimming. To do so, we evaluated 20 athletes in the state of São Paulo,, of both sexes, 15-20 years, and separated into three groups, athletes with GJH (group 1), GJH athletes without (group 2) and all athletes (group 3). The data has been obtained using the criteria of Carter and Wilkinson, partially modified by Beighton and Horan to diagnose and GJH flexitest in joints of shoulder and ankle. The Morbidity Survey adapted was used for analysis of injury incidence. More filming in the open for a stimulus of 50 meters freestyle at full speed for performance analysis, and analysis time and stroke rate. It has been observed that the incidence of GJH is provided above and Flexindex increases when there are GJH. When analyzing the performance, it was noted that the stroke rate, stroke length and speed were higher in the group 1. In addition to the time for running the swimming was lower for the group 1, corroborating the hypothesis that the GJH improve athletic performance swimming, despite not being statistically significant. Regarding the incidence of injury, the group 1 showed more osteoarticular lesions, seen in the water training, and especially in the knee, shoulder and ankle, and the group 2 showed a lower incidence of injury, most common injuries like muscle, and mainly perceived in bodybuilding and particularly the shoulder. To sum up, it is possible to conclude that athletes with GJH have had a higher incidence of injury and better performance than the swimmers without GJH / Mestrado / Biodinamica do Movimento e Esporte / Mestre em Educação Física

Disorders affecting articular cartilage are amongst the most common problems in orthopaedics. Osteoarthritis, the end stage of the disease of articular cartilage, reduces the quality of life for tens of millions of people in the world, and has a profound impact on the economics of industrialized countries. Despite progress in articular cartilage research, the problem is still far from being defeated. Various models e.g. in vitro cartilage explants and in vivo animal models, have been established for cartilage research, but each has its own limitations. Thus, a novel ex vivo isolated joint organ culture model was developed. Bovine metatarsophalangeal joints were chosen as a suitable synovial joint because it consists of a hinge-type joint that is similar to the human knee joint, and has a large cartilage surface that provides enough space for multiple sampling in the same joint. The joints were isolated aseptically and placed into culture media. The viability of chondrocytes, glycosaminoglycan (GAG) content of cartilage matrix, cartilage morphology and water content of matrix were evaluated under different culture conditions, i.e. static, static with flowing media, and dynamic with different durations of the movement period. The model was used to investigate the effect on the sharp scalpel cartilage injury of adding serum to the culture medium by culturing the whole joint explants in serum-supplied or serum-free media. The feasibility of investigating the early phases of chondrocyte implantation in this model was also studied: circular holes of 2.5 mm diameter were created by making a pilot hole with a 2.0 mm drill followed by using a fresh 2.5 mm biopsy punch. Allogeneic isolated chondrocytes at different passages were aggregated as cell pellets and implanted in the holes to evaluate their integration ability and the response from the recipient cartilage. Results from the static model showed that, after 28 days culture, the chondrocytes were still alive with 66.5%, 80.9% and 46.9% viability in the superficial, middle and deep zones, respectively. The GAG content of the static model decreased 19.2% after the first week of culture and then lost another 15.0% during the third week. Paradoxically, at end of the 4th week the GAG level rebounded to some extent and increased 19.0% relative to the previous week. Interestingly, the cell viability of all three zones improved if the culture fluid was flowing as seen with the experiments carried out with stirred media or dynamic movement of the articular surfaces. (e.g. for the stirred media after 28 days of culture the chondrocyte viability was 80.6%, 92.4% and 70.4% for the superficial, middle and deep zones respectively.) The GAG content was maintained at a constant level in the contact area of the dynamic model, but decreased as in the media-stirred model and non-contact area of the dynamic model to a similar extent to that observed with the static model. In the injury model, the GAG content fell approximately 10.8% straight after the scalpel cut, but no further loss was observed if the joint was cultured in the serum-supplied media. In contrast, if the injured joint was cultured in the serum-free media, the GAG content continued to fall week by week and finally dropped by 41.7% at the end of the 4th week. In the chondrocyte implantation model, the majority of the host chondrocytes around the circular defect were alive (78.5 % viability). Viewed from the surface, the dead cells were all within 20 μm from the cut edge. The implanted chondrocytes, which were aggregated as cell pellets, began to transform their shapes and spread to the surrounding surface of the recipient cartilage, but did not appear to integrate with the host tissue during the first 2 weeks of culture. The results supported the validity of this ex vivo joint model and demonstrated that the chondrocytes subjected to flow of the media or dynamic loads survived well over a 4 week period. Of importance was the finding that there was no measured loss of the matrix GAG content when the joints were under dynamic load compared to all of the non-loaded conditions. This whole joint model could be of value in providing a more natural and controllable platform where research involving the normal processes or pathologic mechanisms of articular cartilage can be investigated, as well as the early response to newly developed pharmacological agents and cartilage tissue engineering constructs.

Memoria para optar el título de Bioquímico / No autorizada por el autor para ser publicada a texto completo en el Portal de Tesis Electrónicas / Las estrategias clínicamente usadas para el tratamiento de lesiones articulares se enfocan en aliviar el dolor y la inflamación, porque el cartílago adulto tiene una mínima capacidad regenerativa. Así, frente a un daño severo el cartílago hialino es remplazado por cartílago fibroso, que se distingue del primero en la composición de su MEC y en consecuencia, en sus propiedades biomecánicas. El objetivo del presente trabajo es desarrollar una estrategia de terapia celular que permita regenerar el cartílago articular. La hipótesis de trabajo es que células troncales mesenquimáticas (MSC) autólogas, embebidas en ácido hialurónico (HA) e implantadas en una lesión condral grave, en presencia de dexametasona como anti-inflamatorio e inductor de diferenciación, regeneran in vivo cartílago hialino y no fibrocartílago. Para evaluar dicha hipótesis, se produjo quirúrgicamente una lesión de 20,25 mm2 x 1,5 mm de profundidad (aproximadamente 30 mm3) en el cartílago patelar de conejos New Zealand. Dos semanas después, en dicha lesión se implantaron 1x106 cMSC, previamente aisladas de la médula ósea del mismo animal, embebidas en HA. A partir de ese momento, se inyectó intra-articularmente 0,25 mg/Kg de dexametasona una vez a la semana. A las 6 semanas post-implante, los conejos fueron sometidos a eutanasia, se disectaron sus cartílagos articulares y se analizaron molecularmente en función de la expresión de col1, col2, vers, agg y gapdh, para discriminar entre cartílago hialino y fibroso. Los resultados obtenidos en este trabajo muestran que es factible obtener MSC a partir de aspirados de médula ósea de conejo, dado que las células adherentes aisladas proliferan en presencia de un medio definido suplementado con suero fetal bovino (SFB) y se diferencian in vitro a adipocitos, osteocitos y condrocitos. Por su parte y como era de esperar, se observó que las lesiones condrales de espesor completo cicatrizan a expensas de tejido fibroso, que expresa altos niveles de col1 y vers y bajos niveles de col2 y agg. En cambio, si a 2 semanas post-lesión, en ella se implantan cMSC embebidas en HA y luego se inyecta intra-articular y post-operatoriamente dexametasona, 6 semanas post-implante se genera un cartílago que expresa altos niveles de col2 y agg y bajos niveles de col1 y vers. Estos resultados sugieren que es posible regenerar cartílago hialino utilizando MSC en presencia de HA y dexametasona en un plazo de 6 semanas. Lo cual debe corroborarse con estudios histológicos y funcionales, para poder afirmar que se dispone de una estrategia terapéutica que garantiza la regeneración del cartílago hialino

This thesis deals with the study of the effect of the constituents of the model synovial fluid on the frictional properties of articular cartilage. The influence of constituents, concentration, speed and load is observed. Experiments were performed on a commercial tribometer at configuration pin-on-plate. Several types of lubricants containing synovial fluid constituents have been selected for the experiments. Lubricants were prepared at two concentrations, the concentration of healthy individuals and at a concentration typical of for osteoarthritic patients. Speeds 5 and 10 mm/s and 5 and 10 N loads were used for all experiments. It is shown that when using only lubricant containing proteins, no difference in the coefficient of friction is observed and the effect of concentration is also not observed. The addition of hyaluronic acid has a synergistic effect with -globulin, however in the case of lubricants containing albumin, the effect is opposite. After the addition of phospholipids, no significant effect on friction is observed in -globulin containing lubricants. No significant effect of the composition and concentration of the lubricants is observed with the load change.

Articular cartilage is a highly organized, anisotropic tissue lining the ends of bones within synovial joints. Composed primarily of water, collagens, proteoglycans and chondrocytes which synergistically give rise to the tissue's mechanical and tribological properties. Fluid pressurization and resistance to fluid flow within the porous extracellular matrix of cartilage, coupled with the low hydraulic permeability of the tissue endow the tissue with a viscoelastic response to loading and aid to reduce the coefficient of friction between articulating surfaces, with the pressurized fluid supporting 95% of applied loads. Experiencing millions of articulations throughout an average lifetime, articular cartilage possesses distinct biotribological properties. These require effective lubrication, mediated by the synergistic interaction between fluid and boundary lubricants, to provide a low coefficient of friction and prevent wear at the cartilage surface. Osteoarthritis is the progressive deterioration of articular cartilage and synovial joint structure and function, leading to softer and wear prone tissue on account of altered biochemical composition of the extracellular matrix. Plain radiography remains the most accessible tool and the current standard of care to visualize musculoskeletal diseases and injuries (e.g., osteoarthritis), but cannot directly visualize soft tissues or cartilage, and diagnoses are based solely on boney changes, which occur in the later stages of the disease. Coupled with no way to quantitatively assess tissue health prior to irreversible deterioration, there remains no cure for osteoarthritis. Integral to OA pathology are concomitant changes in the biochemical composition of synovial fluid that result in deterioration of rheological properties, contributing to increased cartilage wear. To address both the lack of quantitative diagnosis methods and lack of chondroprotective therapies, this dissertation presents a dual faceted approach to quantitatively image articular cartilage health, coupled with lubrication strategies to improve cartilage lubrication, and preserve cartilage tissue. This dissertation describes the synthesis of tantalum oxide nanoparticles of varying surface charges for use as contrast agents for rapid, minimally invasive, non-destructive, and quantitative contrast-enhanced computed tomography to assess both the biochemical content and biomechanical integrity of articular cartilage. Ex vivo contrast enhanced computed tomography attenuation using the nanoparticle contrast agent reveals correlations between attenuation and the mechanical and biochemical properties of the tissue. The lubrication strategy described within this dissertation involves introducing a rolling ball element between two surfaces to reduce friction. In this strategy, either single, globular macromolecules or nanoparticles are employed as ball bearings between articulating surfaces to reduce friction when asperities on the surfaces are in direct contact. Rheological characterization and construction of classical Stribeck curves using the lubricant formulations reveal that introducing the rolling element reduces the coefficient of friction during boundary lubrication, while leaving the rheological properties of the base fluid intact. Ex vivo cartilage mechanical testing involving shear deformation under varying speeds and loads reveal improved biotribological performance compared to pure synovial fluid or saline.

First, an in vitro growth experiment was performed to test the hypothesis that applying dynamic unconfined compression during culture produces het- erogeneous remodeling in newborn bovine articular cartilage explants. Het- erogeneous measures of cartilage microstructure were obtained by biochemical assays and quantified polarized light microscopy. Significant differences were measured between the GAG content in the inner and outer portions of the sam- ples stimulated with dynamic unconfined compression. The COL fiber network was found to be more highly aligned in the inner portion of the sample than in the peripheral region. Next, a poroelastic finite element model with a remodeling subroutine was developed to test the hypothesis that the magnitude of relative interstitial fluid velocity and maximum principle strain stimulate GAG and COL fiber network remodeling, respectively, in articular cartilage during culture with dynamic unconfined compression. The GAG remodeling law was successful in predicting the heterogeneous changes in GAG content. The collagen remodeling law was not successful in predicting the changes in the COL network microstructural orientation, suggesting another mechanical cue is responsible for stimulating the remodeling of the COL fiber network.

Joint disease is detrimental to basic quality of life. Articular cartilage is responsible for reducing friction and distributing loads in joints as they undergo large, repetitive load cycles each day, but damaged tissue has very limited intrinsic regenerative ability. Osteoarthritis (OA), the most common joint disease, affects over 500 million people worldwide, contributes more than $27 billion dollars in annual healthcare expenditures, and has increased in prevalence by nearly 50% since 1990 with our aging population. In spite of all this, OA remains a chronic degenerative condition lacking in effective treatment strategies. For cartilage repair in late-stage disease, synthetic joint replacements carry risk of altered loading and metal hypersensitivity, while clinically approved autografts or autologous chondrocyte implantation procedures suffer from lack of donor tissue and donor site morbidities. Prior to surgical intervention, OA management is focused on analgesia rather than preventing or slowing early-stage disease. Disease-modifying OA drugs are yet to successfully complete clinical trials, in part due to the widespread use of animal models for therapeutic discovery rather than high-fidelity human models. Alleviating the burden of cartilage damage will require improvements in both early-stage therapeutic interventions and late-stage repair. Tissue engineering has the potential to offer more biologically faithful cartilage derived with minimal invasiveness, but the resulting cartilage currently lacks the organization or maturity of native tissue. Thus, the central concept of my thesis work was to introduce biologically inspired spatiotemporal cues to guide engineered cartilage formation, establishing novel methods for cartilage tissue engineering that would provide (i) cartilage-bone grafts for regenerative implantation and (ii) advanced in vitro models for studying osteochondral disease. United by the central theme of cartilage, this dissertation spanned three complementary and interacting areas of tissue engineering: regenerative medicine in Aim 1, tools and technological development in Aim 2, and organs on a chip in Aim 3. In Aim 1, we created patient-specific cartilage-bone constructs with native-like features at a clinical scale, using decellularized bone matrix, autologous adipose-derived stem/stromal cells, and dual-chamber perfusion bioreactors to recapitulate the anatomy and zonal organization of the temporomandibular ramus-condyle unit with its fibrocartilage. We validated key tissue engineering strategies for achieving in vivo cartilage regeneration, with the cartilage-bone grafts serving as templates for remodeling and regeneration, rather than providing direct replacements for the native tissue. To enable precise in vitro manipulation of TGF-β signaling, a key pathway in cartilage development, in Aim 2 we developed an optogenetic system in human induced pluripotent stem cells and used light-activated TGF-β signaling to direct differentiation into smooth muscle, tenogenic, and chondrogenic lineages. This optogenetic platform served as a versatile tool for selectively activating TGF-β signaling with precise spatiotemporal control. Using optogenetic recapitulation of physiological spatiotemporal gradients of TGF-β signaling in Aim 3, we formed stratified human cartilage integrated with subchondral bone substrate, towards in vitro engineering of native-like, zonally organized articular cartilage. Collectively, these studies established novel cartilage tissue engineering approaches which can be leveraged to alleviate the burden of joint disease.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21019号 / 医博第4365号 / 新制||医||1028(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 別所 和久, 教授 安達 泰治, 教授 妻木 範行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Articular cartilage degeneration, called osteoarthritis, in the hip joint is a serious condition that affects millions of individuals yearly, with limited clinical solutions available to prevent or slow progression of damage. Additionally, the effects of high-risk factors (e.g. obesity, soft and hard tissue injuries, abnormal joint alignment, amputations) on the progression of osteoarthritis are not fully understood. Therefore, the objective of this thesis is to generate a finite element model for predicting osteochondral tissue stress and strain in the human hip joint during gait, with a future goal of using this model in clinically relevant studies aimed at prevention, treatment, and rehabilitation of OC injuries. A subject specific finite element model (FEM) was developed from computerized tomography images, using rigid bones and linear elastic isotropic material properties for cartilage as a first step in model development. Peak contact pressures of 8.0 to 10.6 MPa and contact areas of 576 to 1010 mm2 were predicted by this FEM during the stance phase of gait. This model was validated with in vitro measurements and found to be in good agreement with experimentally measured contact pressures, and fair agreement with measured contact areas.