Orientador: Maria Elvira Pizzigatti Correa / Dissertação (mestrado) - Universidade Estadual de Campinas. Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-15T16:05:51Z (GMT). No. of bitstreams: 1 Feio_PatriciadoSocorroQueiroz_M.pdf: 2401000 bytes, checksum: ec8461997196edfbdd60b22b52c1dc43 (MD5) Previous issue date: 2010 / Resumo: A hemofilia é uma doença hemorrágica hereditária ligada ao cromossomo X, decorrente da ausência ou da baixa quantidade no plasma dos fatores de coagulação Fator VIII (hemofilia A) ou do Fator IX (hemofilia B). Clinicamente, a hemofilia se caracteriza por episódios recorrentes de sangramentos profundos, que podem ocorrer espontaneamente ou em decorrência de traumatismos. O sistema músculo-esquelético é freqüentemente afetado pelos eventos hemorrágicos nos pacientes portadores de hemofilia. Dentro desse sistema, as articulações são alvos de episódios recorrentes de sangramentos, resultando em hemartroses. Estes episódios estimulam a hiperplasia da membrana sinovial articular, caracterizando o quadro das sinovites hemofílicas. O ciclo vicioso típico de hemartrose-sinovite-hemartrose quando estabelecido, tem como conseqüência a cronificação das alterações agudas articulares. Histologicamente, na sinovite é observada a proliferação de fibroblastos sinoviais e a presença de um infiltrado de células inflamatórias. Apesar da articulação temporomandibular (ATM) ser uma articulação do tipo sinovial são poucos os relatos sobre o seu envolvimento na sinovite hemofílica. Portanto, o objetivo deste estudo foi o de avaliar as características morfológicas da ATM de camundongos deficientes de fator VIII. Além disso, avaliar a indução de sinovite por hiper-extensão bucal. Para isso, as ATM de 30 camundongos deficientes de Fator VIII foram avaliadas. Cinco desses animais fizeram parte do grupo controle. E os outros 25 fizeram parte do grupo experimental para indução de sinovite pelo método de hiper-extensão bucal. Os animais do grupo controle foram sacrificados e as ATMs foram preparadas para avaliação histológica através da microscopia óptica. Os animais do grupo de estudo foram sacrificados após 2 e 5 dias e 2,4 e 6 semanas do procedimento. A seguir, as ATM foram preparadas para análise em microscopia e a membrana sinovial anterior inferior foi avaliada segundo os critérios descritos por Muto et al (1998b). Como resultado, as ATMs do grupo controle não apresentaram nenhuma alteração anatômica e a membrana sinovial anterior inferior apresentou entre 2-5 camadas de células sinoviais. No grupo de estudo o número de camadas de células sinoviais foram semelhantes ao grupo controle. Não foi observada dilatação capilar, adesão sinovial nem a presença de sangue na cavidade articular. Portanto, nossos resultados caracterizaram histologicamente a membrana sinovial anterior inferior de camundongos deficientes de Fator VIII e a metodologia empregada de hiper-extensão bucal não foi capaz de provocar sinovite na membrana sinovial avaliada / Abstract: Hemophilia is a hereditary hemorrhagic disease linked to chromosome X due to the deficiency of the activity of coagulation Factor VIII in hemophilia A, or Factor IX in hemophilia B. Clinically, hemophilia is characterized by recurrent severe bleedings, mostly in the musculoskeletal system. When the hemorrhage occurs inside the joint, it is called hemarthosis. Hemarthosis can stimulate the synovial membrane which results in synovial hyperplasia that can characterize the hemophilic synovitis. The typical vicious cycle haemartrosis-synovitis-haemarthrosis when established produces as a consequence, the chronification of joint acute changes. Histologically, synovitis is characterized by proliferation of the sinovial cells surrounded by an inflammatory infiltrate. Despite the fact that temporomandibular joint (TMJ) is a synovial joint there are few clinical reports in the literature regarding its involvement in hemophilic synovitis. Therefore, the goal of this study was to evaluate the morphological characteristics of TMJ in Factor VIII deficient mice. In addition, the second goal was to induce a TMJ synovitis using a forced jaw opening model. For this purpose, 30 Factor VIII deficient mice were enrolled in this study. Five of these animals were included in the control group and their TMJ was evaluated using light microscopy to establish the regular morphology of this joint. The other 25 were submitted to a forced jaw opening, for 5 minutes per day, during 10 days. These animals were sacrificed after 2 and 5 days and 2, 4 and 6 weeks of the procedure. TMJ was prepared for optical microscopy analysis and the anterior inferior synovial membrane was studied using Muto et al (1998) criteria. As a result, the anatomic characteristic of the TMJ was similar with other mice and the anterior inferior synovial membrane of this group presented 2-5 cell layers. In the study group, no anatomic changes were observed. No difference was observed regarding the thickness of sinovial cell layer of the studied group and the control group. Capillaries dilatation, synovial adhesion and blood in the joint chambers were not observed. Therefore, our results have histologically characterized the anterior inferior synovial membrane of TJM in Factor VIII deficient mice. The methodology used for inducing sinovites was not capable of inducing sinovites in the TMJ of Factor VIII deficient mice / Mestrado / Estomatologia / Mestre em Estomatopatologia

INTRODUÇÃO: A prática de exercício físico e esporte tem sido recomendada para pacientes com hemofilia com o intuito de melhorar a qualidade de vida e reduzir a morbidade ocasionada por episódios recorrentes de sangramento no sistema musculoesquelético. A natação e os exercícios aquáticos são amplamente indicados para essa população por ocasionar uma menor sobrecarga articular e um menor risco de sangramento. Porém, ainda não se sabem os efeitos que o exercício aeróbico aquático pode ocasionar na inflamação articular e densidade mineral óssea após episódios de hemartrose de repetição. MÉTODOS: 26 ratos wistar foram divididos em Grupo-controle (C; n=8), Grupo Hemartrose (H; n=8) e Hemartrose Exercício (HE; n=10). A hemartrose foi induzida na articulação do joelho direito semanalmente por 8 semanas no Grupo H e HE por meio de infusão de sangue autólogo (0.1ml). O Grupo HE realizou um protocolo de exercício aeróbico aquático com um peso de 5% do peso corporal fixado na cauda com sessões diárias de 1 hora, 5x por semana por 8 semanas. A densidade mineral óssea (DMO) foi avaliada pré e pós-protocolo nas seguintes regiões: corpo total, fêmur, tíbia e articulação do joelho. Ao final do experimento, o plasma e o lavado do líquido sinovial foram avaliados para as concentrações das seguintes citocinas: interleucina (IL)-1alfa, IL-4, IL-6, IL-10, IL-17A, proteína quimiotática de monócitos 1 (MCP-1), fator de crescimento endotelial vascular (VEGF), fator de necrose tumoral alfa (TNF-alfa) e interferon y (IFN-y). A concentração plasmática dos hormônios: adrenocoticotrófico (ACTH), corticosterona e melatonina, e os marcadores plasmáticos do metabolismo ósseo pró-peptídeo aminoterminal do pró-colágeno tipo I (P1NP) e telopeptídeo carboxiterminal do colágeno tipo I (CTX) foram avaliados também no final do estudo. RESULTADOS: O protocolo de exercício reduziu os níveis de MCP-1 (p=0,036) e VEGF (p=0,014), e aumentou os níveis de IL-4 (p < 0,02) e IL-10 (p < 0,01) no líquido sinovial em relação ao Grupo H. A análise plasmática mostrou um aumento da concentração de IL-4 (p=0,002) e melatonina (p=0,04), e uma redução de MCP-1 (p=0,02) e TNF-alfa (p=0,04) no Grupo HE quando comparado com o H. O grupo H mostrou uma redução da DMO no fêmur, tíbia e articulação do joelho quando comparado com os Grupos C e HE (p < 0,0001 para todas as comparações vs. C e HE). O nível de P1NP foi maior no Grupo HE do que no H (p=0,002) e de CTX menor no Grupo H quando comparado com o HE (p=0,001). CONCLUSÕES: Em conjunto, nossos resultados sugerem a capacidade do exercício aeróbico aquático em reduzir a inflamação articular, dor e prevenir a perda óssea local em modelo experimental de hemartrose / INTRODUCTION: Physical exercise and sports has been recommended for patients with hemophilia with the aim to improve the quality of life and co-morbid related to repetitive episodes of bleeds into musculoskeletal system. Swimming and aquatic exercises has been widely indicated for this population given the fact that it promote less joint overload and a decreased risk of bleeding. However, we still don´t know how the aquatic aerobic exercise can influence the joint inflammation and bone mineral density after repetitive episodes of hemarthrosis. METHODS: 26 male rats wistar were divided in Control Group (C; n=8), hemarthrosis Group (H; n=8) and hemarthrosis exercise Grup (HE; n=10). hemarthrosis was weekly induced via autologous blood injections (0.1 mL) over 8 weeks. The HE Group was subjected to a swimming exercise protocol that included a weight attached to the tail (5% of body weight), and it was performed in 1-hour sessions 5 times a week for 8 weeks. The bone mass density (BMD) was evaluated at the femur, tibia, knee joint regions and total body at baseline and after the haemarthrosis protocol. At end of exercise protocol plasma and synovial fluid concentration of interleukin (IL)-1alfa, IL-4, IL-6, IL-10, IL-17A, monocyte chemoattractant protein-1 (MCP-1), vascular endothelial growth factor (VEGF), tumor necrosis factor alfa (TNF-alfa) and interferon interferon (IFN-y). Hormone plasmatic concetration were assesed for adrenocorticotropic (ACTH), corticosterone and melatonin and bone turnover markers Procollagen Type 1 N-Terminal Propeptide (P1NP) and collagen type 1 cross-linked C-telopeptide (CTX) were also assessed at the end of the study. RESULTS: A decrease in concentration of MCP-1 (p=0,036) and VEGF (p=0,014) as well as a increase in IL-4 (p < 0,02) and IL-10 (p < 0,01) levels were observed at HE group when compared to H group. Plasmatic analysis showed a increase in IL-4 (p=0,002) and melatonin (p=0,04) and a reduction in MCP-1 (p=0,02) and TNF-alfa (p=0,04) in HE Group in relation to H group. The H Group showed significantly reduced gains in BMD at the femur, tibia and knee joint compared with that of the HE Group (p < 0.0001 for all comparisons vs. the H). The P1NP concentrations were higher in the HE Group than in the H (p=0.002), and the CTX levels were lower in the HE Group than in the H (p=0.001). CONCLUSIONS: Taken together, our results suggests that aquatic aerobic exercise has the capacity to reduce the joint inflammation, pain and prevent bone loss in an experimental-induced hemarthrosis model

INTRODUÇÃO: A prática de exercício físico e esporte tem sido recomendada para pacientes com hemofilia com o intuito de melhorar a qualidade de vida e reduzir a morbidade ocasionada por episódios recorrentes de sangramento no sistema musculoesquelético. A natação e os exercícios aquáticos são amplamente indicados para essa população por ocasionar uma menor sobrecarga articular e um menor risco de sangramento. Porém, ainda não se sabem os efeitos que o exercício aeróbico aquático pode ocasionar na inflamação articular e densidade mineral óssea após episódios de hemartrose de repetição. MÉTODOS: 26 ratos wistar foram divididos em Grupo-controle (C; n=8), Grupo Hemartrose (H; n=8) e Hemartrose Exercício (HE; n=10). A hemartrose foi induzida na articulação do joelho direito semanalmente por 8 semanas no Grupo H e HE por meio de infusão de sangue autólogo (0.1ml). O Grupo HE realizou um protocolo de exercício aeróbico aquático com um peso de 5% do peso corporal fixado na cauda com sessões diárias de 1 hora, 5x por semana por 8 semanas. A densidade mineral óssea (DMO) foi avaliada pré e pós-protocolo nas seguintes regiões: corpo total, fêmur, tíbia e articulação do joelho. Ao final do experimento, o plasma e o lavado do líquido sinovial foram avaliados para as concentrações das seguintes citocinas: interleucina (IL)-1alfa, IL-4, IL-6, IL-10, IL-17A, proteína quimiotática de monócitos 1 (MCP-1), fator de crescimento endotelial vascular (VEGF), fator de necrose tumoral alfa (TNF-alfa) e interferon y (IFN-y). A concentração plasmática dos hormônios: adrenocoticotrófico (ACTH), corticosterona e melatonina, e os marcadores plasmáticos do metabolismo ósseo pró-peptídeo aminoterminal do pró-colágeno tipo I (P1NP) e telopeptídeo carboxiterminal do colágeno tipo I (CTX) foram avaliados também no final do estudo. RESULTADOS: O protocolo de exercício reduziu os níveis de MCP-1 (p=0,036) e VEGF (p=0,014), e aumentou os níveis de IL-4 (p < 0,02) e IL-10 (p < 0,01) no líquido sinovial em relação ao Grupo H. A análise plasmática mostrou um aumento da concentração de IL-4 (p=0,002) e melatonina (p=0,04), e uma redução de MCP-1 (p=0,02) e TNF-alfa (p=0,04) no Grupo HE quando comparado com o H. O grupo H mostrou uma redução da DMO no fêmur, tíbia e articulação do joelho quando comparado com os Grupos C e HE (p < 0,0001 para todas as comparações vs. C e HE). O nível de P1NP foi maior no Grupo HE do que no H (p=0,002) e de CTX menor no Grupo H quando comparado com o HE (p=0,001). CONCLUSÕES: Em conjunto, nossos resultados sugerem a capacidade do exercício aeróbico aquático em reduzir a inflamação articular, dor e prevenir a perda óssea local em modelo experimental de hemartrose / INTRODUCTION: Physical exercise and sports has been recommended for patients with hemophilia with the aim to improve the quality of life and co-morbid related to repetitive episodes of bleeds into musculoskeletal system. Swimming and aquatic exercises has been widely indicated for this population given the fact that it promote less joint overload and a decreased risk of bleeding. However, we still don´t know how the aquatic aerobic exercise can influence the joint inflammation and bone mineral density after repetitive episodes of hemarthrosis. METHODS: 26 male rats wistar were divided in Control Group (C; n=8), hemarthrosis Group (H; n=8) and hemarthrosis exercise Grup (HE; n=10). hemarthrosis was weekly induced via autologous blood injections (0.1 mL) over 8 weeks. The HE Group was subjected to a swimming exercise protocol that included a weight attached to the tail (5% of body weight), and it was performed in 1-hour sessions 5 times a week for 8 weeks. The bone mass density (BMD) was evaluated at the femur, tibia, knee joint regions and total body at baseline and after the haemarthrosis protocol. At end of exercise protocol plasma and synovial fluid concentration of interleukin (IL)-1alfa, IL-4, IL-6, IL-10, IL-17A, monocyte chemoattractant protein-1 (MCP-1), vascular endothelial growth factor (VEGF), tumor necrosis factor alfa (TNF-alfa) and interferon interferon (IFN-y). Hormone plasmatic concetration were assesed for adrenocorticotropic (ACTH), corticosterone and melatonin and bone turnover markers Procollagen Type 1 N-Terminal Propeptide (P1NP) and collagen type 1 cross-linked C-telopeptide (CTX) were also assessed at the end of the study. RESULTS: A decrease in concentration of MCP-1 (p=0,036) and VEGF (p=0,014) as well as a increase in IL-4 (p < 0,02) and IL-10 (p < 0,01) levels were observed at HE group when compared to H group. Plasmatic analysis showed a increase in IL-4 (p=0,002) and melatonin (p=0,04) and a reduction in MCP-1 (p=0,02) and TNF-alfa (p=0,04) in HE Group in relation to H group. The H Group showed significantly reduced gains in BMD at the femur, tibia and knee joint compared with that of the HE Group (p < 0.0001 for all comparisons vs. the H). The P1NP concentrations were higher in the HE Group than in the H (p=0.002), and the CTX levels were lower in the HE Group than in the H (p=0.001). CONCLUSIONS: Taken together, our results suggests that aquatic aerobic exercise has the capacity to reduce the joint inflammation, pain and prevent bone loss in an experimental-induced hemarthrosis model

Post-traumatic osteoarthritis (PTOA) of the knee is a complication resulting from direct injury to the joint, such as anterior cruciate ligament and meniscus tears, and accounts for approximately 12% of all OA cases. The economic and clinical impact of PTOA is also greater than idiopathic OA, as patients are younger and often more active, requiring treatments for symptomatic OA over a greater fraction of their lifetime. A common strategy to manage pain and inflammation associated with PTOA is the intraarticular administration of corticosteroids. However, these injections are limited due to the requirement of high-doses imposed by synovial joint clearance rates and their resulting systemic side effects. In addition, currently used broad-spectrum corticosteroids are palliative and not curative, stemming from incomplete knowledge of specific mechanisms that drive cartilage degeneration and other joint pathologies. Thus, most patients with PTOA eventually undergo surgical procedures such as osteochondral graft transplantation for focal defects and in more severe cases, total knee arthroplasty. As such, the studies presented in this dissertation (i) offer specific insights into mechanisms by which traumatic injury can drive joint degeneration and (ii) present novel strategies to modulate joint responses to pathological factors by leveraging sustained drug-delivery platforms. In Part I, mechanistic assessments of human cartilage and synovium responses to insults are conducted to identify novel pathways that may lead to impaired joint homeostasis. First, a direct consequence of traumatic injury, hemarthrosis, is explored as a potential contributor to the development of PTOA specifically through contributions by red blood cells. We demonstrate for the first time the differential roles of erythrocytes in their intact and lysed states through measures of oxidative stress and changes to metabolomic profiles in the context of ferroptosis. Furthermore, we demonstrate the therapeutic potential of Ferrostatin-1, a lipophilic radical scavenger in inhibiting pathological changes to cartilage and its crosstalk with the neighboring synovium in an in vitro model of hemophilic arthropathy. Second, a strategy to prevent an indirect consequence of traumatic injury, arthrofibrosis, is presented in an in vitro model of joint contraction. Fibrosis and the presence of hyperplastic synovium are implicated in the progression of OA through pathological shifts in tissue composition as well as secreted factors that promote cartilage degeneration and the maintenance of a pro-inflammatory joint environment. A type I transforming growth factor beta-1 receptor inhibitor, SB-431542, is encapsulated in polymeric microspheres for the prophylactic treatment of arthrofibrosis through sustained low-dose drug delivery to circumvent the challenges associated with resident joint clearance rates. Utilizing human-based in vitro models of cartilage and synovium pathology, we present novel mechanisms and therapeutic strategies to prevent pathological changes following traumatic joint injury that may contribute to the development of PTOA. In Part II, the sustained delivery platform introduced in Part I is extended to the treatment of PTOA. Osteochondral graft transplantation is currently the clinical gold standard for large focal cartilage lesions. However, allograft procedures are limited due to the lack of available donor tissues and autografts are associated with complications due to donor-site morbidity. In both cases, grafts are subject to failure, potentially in part due to the continual presence of pro-inflammatory factors following surgical procedure. In this section, we present cellular agarose hydrogels embedded with dexamethasone-releasing microspheres that are integrated with a titanium base as a functional tissue-engineered alternative to native osteochondral allografts. These allogenic tissue-engineered grafts were assessed in an in vivo preclinical canine model in their ability to maintain clinical function and to modulate the inflammatory response over the course of 12 months. We successfully demonstrated the feasibility of using engineered grafts by comparing clinical measures of range of motion, function, lameness, and pain, as well as modified cartilage graft scores, against native osteochondral allograft controls. In addition, improvements in the histopathological scoring of neighboring synovial and meniscal tissues indicate the therapeutic capacity of dexamethasone released from within the joint to modulate the inflammatory response up to one-year post-implantation. Taken together, the studies presented in this dissertation identify novel mechanisms behind pathological changes to the cartilage and synovium that may contribute to the development of PTOA following injury. Potential therapeutic targets, inhibitory compounds, and delivery strategies are also assessed using human-based in vitro models of disease and further validated in an in vivo canine model through a clinically relevant timeframe. Ultimately, we demonstrate for the first time, the use of dual-function tissue-engineered grafts in a weight-bearing region of the knee joint to circumvent limitations associated with the clinical gold standard for the treatment of large focal cartilage defects.