A l’heure actuelle, les alliages à bases de titane sont les matériaux les plus utilisés en implantologie osseuse. Les procédés émergents de fabrication additive, tel que la fusion par faisceau d’électrons (EBM), permettent de fabriquer des structures architecturées sur-mesure en titane. Dans les cas cliniques difficiles, il est nécessaire de stimuler activement les cellules souches osseuses pour qu’elles produisent de l’os. Les protéines osseuses morphogénétiques (BMP-2, BMP-7) ont cette capacité d’ostéo-induction et sont utilisées en clinique. Cependant, leur délivrance par matrice de collagène est très mal contrôlée. Des revêtements de surface à base de polymères naturels, tels que la poly(L-lysine) et l’acide hyaluronique (PLL/HA), peuvent former des films biomimétiques servant de nanoréservoir pour ces protéines. L’objectif de cette thèse était de développer un implant innovant constitué de structures 3D en titane à la fois architecturées et ostéo-inductrices. Pour cela, des structures 3D poreuses en alliage de titane (Ti-6Al-4V) constituées de cellules cubiques ont été construites par EBM. La porosité a été bien contrôlée avec une différence par rapport aux modèles CAO de moins de 1%. La BMP-7 a été chargée et quantifiée dans les films biomimétiques. La capacité d’ostéo-induction des films a été évaluée avec des cellules souches mésenchymateuses de souris par leur expression de la phosphatase alcaline. L’expression de cette enzyme a augmenté de façon dose-dépendante avec la dose de BMP-7 initialement chargée. Le dépôt du film ostéo-inducteur sur les structures 3D architecturées a été caractérisé par microscopies optique et électronique. Les cellules souches cultivées au sein des structures 3D bioactives se différencient en cellules osseuses démontrant ainsi leur capacité ostéo-inductrice sur le court terme in vitro. Des tests préliminaires in vivo sont actuellement réalisés pour tester ces structures 3D bioactives dans un modèle fémoral de défaut osseux chez le rat. / To date, titanium-based alloys (Ti) remain the most used implantable materials for load-bearing applications. Emerging additive manufacturing techniques such as electron beam melting (EBM) enable to custom-build architectured scaffolds of controlled macroporosity. In very difficult clinical situations, potent bioactive signals are needed to boost stem cells: osteoinductive molecules such as bone morphogenetic proteins (BMP-2) are currently used for this purpose. However, one of their limitations is their inappropriate delivery with collagen sponges. Biomimetic surface coatings made of the biopolymers poly(L-lysine) and hyaluronic acid, (PLL/HA) polyelectrolyte films, have recently been engineered as nanoreservoirs for BMP proteins. The aim of this PhD thesis was to develop architectured and osteoinductive 3D titanium-based scaffolds as innovative synthetic bone grafts. To this end, we used the EBM additive manufacturing technique to engineer porous scaffolds with cubit unit-cells. Their surface was coated with biomimetic films containing the bone morphogenetic protein 7 (BMP-7). The porosity was well controlled with a difference from CAD models of less than 1%. The osteoinductive capacity of BMP-7 loaded films was assessed using murine mesenchymal stem cells (MSCs) by quantifying their alkaline phosphatase (ALP) expression, which increased in a dose-dependent manner. The coating of the 3D architectured scaffolds by the bioactive film was characterized using optical and electron microscopy techniques. Finally, the 3D architectured scaffolds coated with BMP-7-loaded films were proved to be osteoinductive at the early stage in vitro. Preliminary experiments are currently done to assess their performance in an in vivo model of a critical size femoral bone defect in rat.

Introdução: Materiais com modificações na formulação original do MTA, incluindo o NeoMTA Plus, têm sido desenvolvidos buscando melhorar suas propriedades físicoquímicas. O objetivo do presente estudo foi analisar a resposta do tecido ósseo de ratos Wistar frente ao implante de NeoMTA Plus em comparação com o MTA Angelus. Metodologia: 24 ratos foram anestesiados e uma broca de baixa rotação foi empregada para confeccionar três cavidades cirúrgicas no fêmur direito. As cavidades cirúrgicas foram preenchidas aleatoriamente com os materiais teste NeoMTA Plus e MTA Angelus ou deixada vazia (controle negativo). Após 7, 30 e 90 dias as características do reparo ósseo foram classificadas em escores com o auxilio de microscópio óptico. As comparações entre os grupos foram realizadas pelo teste de Friedman e as comparações entre os três períodos experimentais em cada grupo foram realizadas pelo teste de Kruskal-wallis, seguido pelo teste de Dunn. O nível de significância foi estabelecido em P<.05. Resultados: Não foram observadas diferenças estatísticas entre os materiais e o grupo controle nos 3 tempos experimentais. Em todos os grupos houve diferença estatisticamente significante entre os tempos de 7 e 30 dias e nenhuma diferença foi observada entre os tempos de 30 e 90 dias. Além disso, o reparo em 90 dias foi significativamente melhor do que em 7 dias nos grupos NeoMTA Plus e controle. Conclusões: O NeoMTA Plus demonstrou comportamento biológico satisfatório em contato direto com o tecido ósseo de ratos Wistar, apresentando resultados semelhantes aos grupos MTA Angelus e controle. / Introduction: Materials that alter the original formulation of MTA have been developed to improve physical-chemical properties, including NeoMTA Plus (NMTAP). The aim of the present study was to analyze bone tissue reactions to NMTAP compared to MTA Angelus (MTA-A). Methods: 24 animals were anesthetized and a slow-rotation bur was used to create three surgical cavities in their right femur. Surgical cavities were randomly designated to NMTAP, MTA-A and negative control (empty cavity) groups. After 7, 30, and 90 days histological analysis of the characteristics of bone repair were classified in scores. Friedman’s test performed intergroup comparisons and Kruskal-Wallis test, followed by the Dunn’s post hoc test, compared the three experimental periods in each group. The significance level was set at P<.05. Results: No differences were found between the materials and control group in each experimental period. In all groups, the bone tissue repair improved from 7 to 30 days and there was no difference from 30 to 90 days. Moreover, the repair at 90 days was significantly better than at 7 in NMTAP and control groups. Conclusions: NeoMTA Plus showed satisfactory biological behavior when in direct contact with rat bone tissue, presenting repair characteristics similar to those produced by MTA Angelus and by the control group.

Bone undergoes constant turnover throughout life and has the capacity to regenerate itself. However, the repair of critical size defects, caused by bone diseases such as osteoporosis, can be more problematic. Therefore, there is a clinical need for a bone graft substitute that can be used at sites of surgical intervention to enhance bone regeneration. Poly(vinylphosphonic acid-co-acrylic acid) (PVPA-co-AA) has recently been identified as a potential candidate for use in bone tissue scaffolds. It is hypothesised that PVPA-co-AA can mimic the action of bisphosphonates – a class of drugs used in the treatment of osteoporosis – by binding to calcium ions from bone mineral surfaces. In this way, bisphosphonates can affect bone turnover by increasing the activity of osteoblasts and reducing osteoclast activity. Although PVPA-co-AA has been shown to improve bone formation, the mechanism of action has so far not been fully elucidated. Therefore, this work aims to understand the effect of copolymer composition on the properties of PVPA-co-AA, and thus to determine its effect on osteoblast adhesion and proliferation. PVPA-co-AA copolymers have been synthesised with a range of monomer feed ratios. It was found that a VPA content of 30 mol % led to the greatest calcium binding affinity of the copolymer and is thus expected to lead to enhanced bone formation and mineralisation of the matrix produced by osteoblast cells. The release profile of PVPA-co-AA from electrospun PCL scaffolds was investigated. It was shown that all of the PVPA-co-AA was released into aqueous media within 8 h of immersion. It was also found that the calcium chelation from osteogenic differentiation media significantly increased within the first 8 h. Therefore, it was concluded that PVPA-co-AA is released from the scaffolds, where it can then bind to calcium ions from the bone mineral surface to promote mineralisation, thus acting as a mimic of non-collagenous proteins, which are present in the extracellular matrix (ECM) of bone. Hydrogels of PVPA-co-AA have been produced and the effect of monomer feed ratio (0-50 mol % VPA) on the properties of the gels was explored. It was found that an increase in VPA content led to greater hydrogel swelling and increased porosities. Hydrogels that contained 30 and 50 mol % VPA were shown to have similar morphologies to the native ECM of bone. Rheological testing showed that hydrogels with higher VPA contents were more flexible and could be deformed to a large extent without permanent deformation of their structure. An increase in osteoblast adhesion and proliferation was observed for hydrogels with 30 and 50 mol % VPA content as well as superior cell spreading. Osteoblast cell metabolic activity also increased as a function of VPA content in the hydrogels. This work indicates that hydrogels of PVPA-co-AA, with VPA contents of 30 or 50 mol %, are ideal for use as bone tissue scaffolds. Furthermore, the mechanical and cell adhesion properties of the gels can be tuned by altering the copolymer composition. Finally, composite hydrogels of PVPA-co-AA and hydroxyapatite (HA) have been produced and investigated for their ability to remove fluoride ions from groundwater. It was found that the fluoride uptake ability of PVPA-HA hydrogels was significantly enhanced when compared with HA powder alone. Furthermore, the fluoride uptake was dependent on many factors, including pH, contact time and the presence of competing ions. It was possible to regenerate the hydrogel to remove the fluoride ions, and thus it was shown that the material can be used a number of times with only a slight reduction in its fluoride uptake capacity.

A deficiência óssea requer procedimentos restauradores como uso de enxertos e substitutos ósseos para a reabilitação estética e funcional. Com o desenvolvimento dos biomateriais, a biocerâmica à base de fosfato de cálcio tornou-se alternativa promissora para a recomposição de estruturas ósseas, entretanto não apresenta potencial de osteoindução. O material hidróxido de cálcio (Ca(OH)2) demonstra propriedades antibacterianas e capacidade de induzir a formação de tecido ósseo. O objetivo deste trabalho foi avaliar o processo de reparo ósseo proporcionado pelo BTCP e Ca(OH)2 , isolados e associados. Realizou-se experimento em tíbias posteriores de dezoito coelhos. Dois animais receberam marcadores ósseos para fluorescência (alizarina, calceína e tetraciclina), e para cada tíbia foi utilizado um tipo de material de preenchimento (BTCP, Ca(OH)2, BTCP com Ca(OH)2 e sangue como controle); sendo eutanasiados após 56 dias para a análise de fluorescência e histomorfometria. Os 16 coelhos restantes foram aleatoriamente selecionados para receber os quatro materiais de preenchimento. Estes foram eutanasiados nos tempos de 14, 28, 42 e 56 dias para a análise morfológica microscópica com coloração em hematoxilina e eosina. Observou-se formação óssea em todos os grupos, e os que utilizaram BTCP apresentaram atraso para o início da reparação. O sítio preenchido com sangue ocorreu apenas o reparo da estrutura lesada, consistindo na reconstituição da cortical óssea e tecido medular em 28 dias. Aos 56 dias, o grupo de BTCP com Ca(OH)2 apresentou maior formação de trabéculas no interior da tíbia. Na avaliação histomofométrica, o marcador calceína apresentou maiores valores de deposição óssea em relação à alizarina e tetraciclina. Conclui-se que os biomateriais BTCP e Ca(OH)2 estão diretamente envolvidos na formação de tecido ósseo no interior do defeito; a combinação do Ca(OH)2 com BTCP mostrou aumento do potencial de formação óssea; e houve maior deposição óssea no período entre quinta e sexta semana de reparação indicado pelo marcador calceína. / Insufficient bone volume requires restorative procedures such as use of grafts and bone substitutes for cosmetic and functional rehabilitation. The development of biomaterials made available bioceramic based on calcium phosphate that is showing to be promising alternatives for the restoration of bony structures, however it do not has osteoinductive potential. Calcium hydroxide (Ca(OH)2) shows antibacterial properties and induce bone tissue formation as well. The objective of this research was to evaluate the bone healing process promoted by BTCP and Ca(OH)2 isolated or conjugated. The experiment was conducted in posterior rabbit tibiae of 18 animais. Two animais received bone markers (alizarin, calcein, tetracycline) and each of their tibias received a type offiller material (BTCP, Ca(OH)2, BTCP plus Ca(OH)2 and blood as control group), being euthanized after 56 days for the histomorphometry and fluorescence analysis. The remaining 16 rabbits were randomly selected to receive one of the 4 filling materiais. These rabbits were euthanized on 14,28,42 and 56 days for the micromorphological analysis in hematoxylin and eosin slides. The bone formation was observed in ali groups, and who used BTCP had delay to the start of repair. The site filled with blood occurred only repair of the damaged structure, consisting of the reconstitution of the bone cortical and medullary tissue in 28 days. After 56 days, the BTCP group with Ca(OH)2 showed higher trabecular formation inside the tibia. In histomorfometric evaluation, the marker calcein had higher bone deposition values in relation to alizarin and tetracycline. It was concluded that the biomaterials BTCP and Ca(OH)2 are directly involved in the formation of bone tissue; the conjugation of Ca(OH)2 with BTCP increased bone formation potential; and there was greater bone deposition in the period between the fifth and sixth week of repairs indicated by calcein marker.

Made available in DSpace on 2017-07-10T14:17:12Z (GMT). No. of bitstreams: 1 Poliana - cd.pdf: 2397137 bytes, checksum: 9e02b7d7572ba82c2026b9255476d132 (MD5) Previous issue date: 2015-05-26 / Obesity has been the subject of several controversies, whether in scientific circles or among the general population. Few subjects call so much attention to society and researchers, whether due to the huge number of obese people worldwide, or due to its potential complications to the health of individuals. Several chronic diseases have been associated with obesity among them periodontal diseases, which may be associated with bone resorption and have its mechanisms intensified in obesity. Nowadays, it is demonstrated that the bone tissue undergoes influence of obesity, so that female sex hormones seem an important link between obesity and bone mass gain in obese patients. Given the importance of obesity and its considerable relation to periodontal disease and bone tissue the aim of this study was to evaluate the effect of the hormonal influence on periodontal tissues of adult female mice in proestrus phase of the estrous cycle, with experimentally induced obesity, through the cafeteria diet, whether or not associated with periodontal disease. For this study 20 adult female mice cycling, in proestrus phase of the estrous cycle were divided into control group (CON), ligature group (LIG), cafeteria group (CAF) and cafeteria and ligature group (CAFLIG). At 75 days of life, the animals from groups LIG and CAFLIG received a ligature around the right mandibular first molar which acted as a gum irritating for 30 days, favoring the accumulation of bacterial plaque and the consequent development of periodontal disease. After euthanasia, at 105 days of life, it was held the blood collection from the right brain stem to determine the estradiol and progesterone concentrations by using specific kits; and the dissection of the right hemi mandible, which was submitted to histological analysis. The obtained data were analyzed and evaluated using the ANOVA and Tukey tests so that the results showed a decrease in the concentration of female sex hormones when obesity and periodontal disease were associated; in the same way it was observed an evident bone resorption and a change in the morphology of the bone tissue in the group CAF/LIG. Based on the results obtained the current study can conclude that the decrease in female sex hormones associated with obesity and periodontal disease negatively influenced on the mandibular bone tissue / A obesidade tem sido tema de diversas polêmicas e controvérsias, seja no meio científico ou na população em geral. Poucos assuntos chamam tanto a atenção da sociedade e de estudiosos, quer devido ao elevadíssimo número de pessoas obesas a nível mundial, quer devido às suas potenciais complicações para a saúde dos indivíduos. Diversas doenças crônicas têm sido relacionadas à obesidade, dentre elas as doenças periodontais, as quais podem estar associadas à reabsorção óssea e ter seus mecanismos intensificados na obesidade. Atualmente, demonstra-se que o tecido ósseo sofre influência da obesidade, sendo que os hormônios sexuais femininos parecem um importante elo entre a obesidade e o ganho de massa óssea em pacientes obesos. Dada a importância da obesidade e sua considerável relação com a doença periodontal e o tecido ósseo, o objetivo deste estudo foi avaliar o efeito da influência hormonal nos tecidos periodontais de ratas adultas na fase do proestro do ciclo estral, com obesidade induzida experimentalmente, através da dieta de cafeteria, associada ou não à doença periodontal. Para a realização da pesquisa, 20 ratas fêmeas adultas ciclando, na fase do proestro do ciclo estral, foram divididas em grupo controle (CON), grupo ligadura (LIG), grupo cafeteria (CAF) e grupo cafeteria e ligadura (CAFLIG). Aos 75 dias de vida, os animais dos grupos LIG e CAFLIG receberam uma ligadura ao redor do primeiro molar inferior direito, a qual atuou como irritante gengival por 30 dias, favorecendo o acúmulo de placa bacteriana e o consequente desenvolvimento da doença periodontal. Após a eutanásia, aos 105 dias de vida, realizou-se a coleta de sangue do tronco cerebral direito para a determinação das concentrações de estradiol e progesterona utilizando-se kits específicos; e a dissecação da hemi-mandíbula direita, que foi submetida à análise histológica. Os dados obtidos foram analisados e avaliados por meio dos testes ANOVA e Tukey, sendo que os resultados demonstraram uma diminuição na concentração dos hormônios sexuais femininos quando obesidade e doença periodontal foram associadas; da mesma forma, observou-se reabsorção óssea acentuada e mudança na morfologia do tecido ósseo no grupo CAFLIG. Com base nos resultados obtidos, pode-se concluir que a diminuição nos hormônios sexuais femininos associados à obesidade e à doença periodontal interferiu negativamente no tecido ósseo mandibular

O Banco de Tecidos do Instituto de Ortopedia e Traumatologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo está desenvolvendo uma linha de estudos experimentais com o intuito de apresentar novos métodos de armazenamento de aloenxertos. Este estudo tem como objetivo comparar o método da criopreservação (- 80° C) com o da conservação em glicerol a 98% em temperatura ambiente. Foram analisadas tanto a capacidade de inibição de crescimento bacteriano e fúngico quanto às eventuais alterações histológicas. Para isso, foram estudadas 30 amostras de tecido ósseo trabecular coletadas de 10 pacientes submetidos à artroplastia total do quadril. Estas amostras foram divididas em três grupos de 10 espécimes, a saber: grupo controle, grupo criopreservado e grupo conservado em glicerol a 98% à temperatura ambiente. O período de armazenamento das amostras foi de um ano. Os resultados foram analisados estatisticamente pelo método de McNemar, com índice de significância de 0,10. No que diz respeito à preservação da matriz óssea, não houve variações significativas nos dois grupos estudados em relação ao grupo controle. Não ocorreu crescimento bacteriano ou de fungos nas amostras armazenadas durante um ano em glicerol a 98%. Por ser extremamente reduzido em relação à criopreservação, o custo do método da conservação em glicerol a 98% o indica para uso em Bancos de Tecidos de pequena monta. Contudo, são ainda necessários posteriores estudos sobre as propriedades biomecânicas, remoção do glicerol do tecido ósseo e o processo de integração biológica dos mesmos com o receptor. / The Tissue Bank of Instituto de Ortopedia e Traumatologia do Hospital das Clínicas da Universidade de São Paulo is developing a line of experimental studies with the intention to present new methods of allografts storage. This study has the objective to compare the method of cryopreservation (-80 ºC) with glycerol 98% preservation at room temperature. It even analyses, in such way, the inhibition capacity of bacterial and fungal growth and any eventual histological changes. For this, 30 samples of trabecular bone tissue have been collected from 10 patients submitted to total hip arthroplasty. These samples were separated in three groups of 10 specimens, as following: a control group, a cryopreserved group and a glycerol 98% preserved group at room temperature. They were stored during a period of one year. The results were analysed by the McNemar statistic method, with a significance of 0,10. Concerning to bone matrix, no significant changes occurred to the two studied groups compared to the control group. Bacterial and fungal growth do not occurred in the stored samples for one year in glycerol 98%. For being extremely reduced when compared with cryopreservation method, the preservation cost in glycerol 98% indicates its use on small sum tissue banks. However, posterior studies about biomechanical properties, glycerol removal of bone tissue and their biological process of integration with the receiver are necessary.

A deficiência óssea requer procedimentos restauradores como uso de enxertos e substitutos ósseos para a reabilitação estética e funcional. Com o desenvolvimento dos biomateriais, a biocerâmica à base de fosfato de cálcio tornou-se alternativa promissora para a recomposição de estruturas ósseas, entretanto não apresenta potencial de osteoindução. O material hidróxido de cálcio (Ca(OH)2) demonstra propriedades antibacterianas e capacidade de induzir a formação de tecido ósseo. O objetivo deste trabalho foi avaliar o processo de reparo ósseo proporcionado pelo BTCP e Ca(OH)2 , isolados e associados. Realizou-se experimento em tíbias posteriores de dezoito coelhos. Dois animais receberam marcadores ósseos para fluorescência (alizarina, calceína e tetraciclina), e para cada tíbia foi utilizado um tipo de material de preenchimento (BTCP, Ca(OH)2, BTCP com Ca(OH)2 e sangue como controle); sendo eutanasiados após 56 dias para a análise de fluorescência e histomorfometria. Os 16 coelhos restantes foram aleatoriamente selecionados para receber os quatro materiais de preenchimento. Estes foram eutanasiados nos tempos de 14, 28, 42 e 56 dias para a análise morfológica microscópica com coloração em hematoxilina e eosina. Observou-se formação óssea em todos os grupos, e os que utilizaram BTCP apresentaram atraso para o início da reparação. O sítio preenchido com sangue ocorreu apenas o reparo da estrutura lesada, consistindo na reconstituição da cortical óssea e tecido medular em 28 dias. Aos 56 dias, o grupo de BTCP com Ca(OH)2 apresentou maior formação de trabéculas no interior da tíbia. Na avaliação histomofométrica, o marcador calceína apresentou maiores valores de deposição óssea em relação à alizarina e tetraciclina. Conclui-se que os biomateriais BTCP e Ca(OH)2 estão diretamente envolvidos na formação de tecido ósseo no interior do defeito; a combinação do Ca(OH)2 com BTCP mostrou aumento do potencial de formação óssea; e houve maior deposição óssea no período entre quinta e sexta semana de reparação indicado pelo marcador calceína. / Insufficient bone volume requires restorative procedures such as use of grafts and bone substitutes for cosmetic and functional rehabilitation. The development of biomaterials made available bioceramic based on calcium phosphate that is showing to be promising alternatives for the restoration of bony structures, however it do not has osteoinductive potential. Calcium hydroxide (Ca(OH)2) shows antibacterial properties and induce bone tissue formation as well. The objective of this research was to evaluate the bone healing process promoted by BTCP and Ca(OH)2 isolated or conjugated. The experiment was conducted in posterior rabbit tibiae of 18 animais. Two animais received bone markers (alizarin, calcein, tetracycline) and each of their tibias received a type offiller material (BTCP, Ca(OH)2, BTCP plus Ca(OH)2 and blood as control group), being euthanized after 56 days for the histomorphometry and fluorescence analysis. The remaining 16 rabbits were randomly selected to receive one of the 4 filling materiais. These rabbits were euthanized on 14,28,42 and 56 days for the micromorphological analysis in hematoxylin and eosin slides. The bone formation was observed in ali groups, and who used BTCP had delay to the start of repair. The site filled with blood occurred only repair of the damaged structure, consisting of the reconstitution of the bone cortical and medullary tissue in 28 days. After 56 days, the BTCP group with Ca(OH)2 showed higher trabecular formation inside the tibia. In histomorfometric evaluation, the marker calcein had higher bone deposition values in relation to alizarin and tetracycline. It was concluded that the biomaterials BTCP and Ca(OH)2 are directly involved in the formation of bone tissue; the conjugation of Ca(OH)2 with BTCP increased bone formation potential; and there was greater bone deposition in the period between the fifth and sixth week of repairs indicated by calcein marker.

Orientador: Willian Fernando Zambuzzi / Resumo: Com o aumento progressivo da expectativa de vida da população, defeitos ósseos se tornou um problema de saúde pública. Por sua vez, o sistema esquelético abriga um conjunto de células que, de maneira hierárquica, sustentam a formação do osso ao longo da vida, sendo sua capacidade regenerativa comprometida durante o processo de envelhecimento. Com o aumento da expectativa de vida, a população idosa vem crescendo nos últimos anos e com ela o aumento eminente de fraturas ósseas. Sabe-se que o desenvolvimento e regeneração ósseos são eventos complexos e controlados por mecanismos parácrinos de sinalização intercelulares, destacando que a osteogênese está acoplada, principalmente, à angiogênese. Embora relatado, este mecanismo de comunicação entre células endoteliais e células osteoprogenitoras não está bem elucidado, sobretudo considerando o repertório de moléculas tróficas envolvidas. A fim de compreender melhor estes mecanismos, o objetivo deste trabalho foi desenvolver metodologias capazes de mimetizar o microambiente endotelial-ósseo, bem como desvendar eventos acoplados entre os diferentes tipos celulares envolvidos no processo, sobretudo gerando tecido endotelial in vitro, equivalente ao original. Para isso, fizemos uso de células humanas primárias, as quais foram submetidas a diferentes protocolos experimentais, onde a mesma densidade de células endoteliais arterial (HCAEC) e venosa (HUVEC) e de musculatura lisa (AoSMC) (cultivo misto) foi plaqueada em tubos cônicos sem tr... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: With the progressive increase in the life expectancy of the population, bone defects became a public health problem. In turn, the skeletal system houses a set of cells that, in a hierarchical way, support the formation of the bone throughout life, and its regenerative capacity is compromised during the aging process. With the increase in life expectancy, the elderly population has been increasing in recent years and with it the eminent increase of bone fractures. It is known that bone development and regeneration are complex events and controlled by paracrine mechanisms of intercellular signaling, emphasizing that osteogenesis is mainly coupled with angiogenesis. Although reported, this mechanism of crosstalk between endothelial cells and osteoprogenitor cells is not well elucidated, especially considering the repertoire of involved trophic molecules. In order to better understand these mechanisms, the objective of this work was to develop methodologies capable of mimicking the endothelial-bony microenvironment, as well as unveiling the coupled events between the different cell types involved in the process, especially generating endothelial tissue in vitro, equivalent to the original one. For this, we used primary human cells, which were submitted to different experimental protocols, where the same density of arterial (HCAEC) and venous (HUVEC) endothelial cells and smooth muscle cell (AoSMC) (mixed culture) were plated in tubes (s), and after 72 hours the generated spheroid... (Complete abstract click electronic access below) / Mestre

Les forces musculaires appliquées à l’os entraînent des contraintes produisant un effet ostéogénique. Cet aspect est à ce jour très mal connu. L’électromyostimulation est une alternative aux contractions musculaires volontaires et permet de mieux de contrôler les paramètres de l’effort. Après un rappel sur le tissu osseux et l’ostéoporose, la revue de littérature effectuée dans le cadre de cette thèse permet de cerner les aspects fondamentaux liés à l’application de la stimulation électrique. Deux axes de recherche sont proposés :Pour le Modèle animal 4 thématiques sont étudiées : 1-Identification des points moteursdu membre pelvien du rat, et mise en place d’un protocole de stimulation électrique.2-Evaluation in vivo des contraintes appliquées à l’os par stimulation électrique. 3-Evaluation de l’effet d’un mois de stimulation électrique sur la force et l’endurance musculaire.4-Détermination de l’effet d’un mois de stimulation électrique sur les caractéristiques mécaniques de l’os in vitro. L’application à l’homme concerne deux aspects de la mise en oeuvre d’une stimulation électrique : 1 l’évaluation de l’Influence de la fréquence de stimulation électrique sur la force et la fatigue musculaire.2- la caractérisation d’un protocole de stimulation électrique pour le renforcement musculaire et la prévention de l’ostéoporose.Les résultats obtenus montrent que la stimulation électrique musculaire sous les conditions d’application définies peut avoir un effet ostéogénique. La transposition de ces résultats à l’homme reste à faire cependant les protocoles de stimulation pouvant être utilisés ont été précisés. Cette technique thérapeutique préventive, non invasive pouvant être appliquée à des sujets vieillissant ou à mobilité réduite devrait permettre de minimiser l’ostéoporose. / The muscular strengths applied to the bone pull(entail) constraints producing an ostéogénique effect. This aspect is this day very badly known. The électromyostimulation is an alternative in the voluntary muscular contractions and allows of better to check(control) the parameters of the effort. After a reminder(abseiling) on the bone tissue and the osteoporosis, the magazine(review) of literature made within the framework of this thesis allows to encircle the fundamental aspects bound(connected) with care of the electric stimulation. Two areas of research are proposed:pour the animal model 4 themes are studied: 1 - identification of the driving points of the pelvien member(limb) of the raz, and organized by a protocol of stimulation in vivo électrique.2-evaluations of the constraints applied to the bone by electric stimulation. 3 - evaluations of the effect of a month of electric stimulation on the strength and the stamina musculaire.4-Détermination of the effect of a month of electric stimulation on the mechanical characteristics of the in vitro bone. The application to the man concerns two aspects of the implementation of an electric stimulation: 1 the evaluation of the Influence of the frequency of electric stimulation on the strength and the muscular 2-fatigue the characterization of a protocol of electric stimulation for the muscular intensification and the prevention of the osteoporosis. The obtained results(profits) show that the muscular electric stimulation under the defined conditions of application can have an ostéogénique effect. The transposition of these results(profits) to the man remains to make however the protocols of stimulation being able to be used were specified. This preventive, not invasive therapeutic technique which can be applied to aging subjects or to reduced mobility should allow to minimize the osteoporosis.

Tissue engineering is a multidisciplinary field that is rapidly emerging as a promising new approach in the restoration and reconstruction of tissues. In this approach, three dimensional (3D) scaffolds are of great importance. Scaffolds function both as supports for cell growth and depot for sustained release of required active agents (e.g. enzymes, genes, antibiotics, growth factors). Scaffolds should possess certain properties in accordance with usage conditions. Wet-spinning is a simple technique that has been widely used for the fabrication of porous scaffolds for tissue engineering applications. Natural polymers can effectively be used in scaffold fabrication due to their biocharacteristics. Among natural polymers, chitosan and alginate are two of the most studied ones in tissue engineering and drug delivery fields because of being biologically renewable, biodegradable, biocompatible, non-antigenic, non-toxic and biofunctional. In this study, two kinds of porous scaffolds were produced as chitosan and alginate coated chitosan fibrous scaffolds by wet-spinning technique In order to investigate the delivery characteristics of the scaffolds, loading of gentamicin as a model antibiotic and bovine serum albumin (BSA) as a model protein was carried out in different loading models. Resultant scaffolds were characterized in terms of their structural formation, biodegradation, biomineralization, water uptake and retention ability and mechanical properties. Additionally, release kinetics of gentamicin and BSA were examined. Efficiency of gentamicin on Escherichia coli (E.coli) was examined. Characterization of scaffolds revealed their adequacy to be used in bone tissue engineering applications and capability to be employed as bioactive agent delivery systems.