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Influência da técnica cirúrgica guiada para implantes sobre o aquecimento ósseo, a viabilidade celular imediata e a deformação de fresas após osteotomia para implantesSantos, Pâmela Letícia dos [UNESP] 22 February 2011 (has links) (PDF)
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santos_pl_me_araca.pdf: 11429192 bytes, checksum: d6972580cfad1fbdb5b39c5833a3da0b (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O propósito deste trabalho foi avaliar o efeito da osteotomia para implantes, seguindo o protocolo de cirurgia guiada comparando-o com a clássica, sobre o aquecimento tecidual, bem como verificar o desgaste das fresas por microscopia eletrônica de varredura e a viabilidade celular imediata. Para isso foram utilizadas tíbias de 20 coelhos, divididos em 2 grupos – GRUPO CONTROLE (GC) e GRUPO GUIADA (GG) - os quais foram divididos em 5 subgrupos (G): G0, GI, G2, G3, G4, que corresponderam às fresas sem uso e as utilizadas 10, 20, 30 e 40 vezes, respectivamente. Cada animal recebeu 10 seqüências de osteotomias, sendo 5 de cada grupo, aleatoriamente em cada tíbia, na velocidade de 1600 r.p.m, e por meio de irrigação externa. A oscilação térmica tecidual durante cada fresagem foi quantificada por meio de termopares. As brocas foram analisadas por microscopia eletrônica de varredura (MEV) e a quantificação da deformação das fresas foi realizada por meio do software ImageLab 2000. As áreas osteotomizadas foram coletadas, processadas para inclusão em parafina, e submetidas ao processamento imunoistoquímico para a expressão das proteínas osteoprotegerina (OPG), Receptor Ativador do Fator Nuclear k (RANKL), osteocalcina (OC) e caspase 3(cas 3). Os dados obtidos por meio da análise térmica foram submetidos ao teste de Mann-Whitney (p=0,05), já os valores de deformação das brocas foram submetidos ao teste t. O aquecimento ósseo no GG foi estatisticamente maior do que no GC. Na análise por meio do MEV, não houve diferença estatística entre os grupos, mas verificou-se que a deformação foi progressiva de acordo com a maior utilização das fresas. Na análise da viabilidade celular, não houve diferença... / The purpose of this study was to evaluate the effect of osteotomy for implants, following the protocol guided surgery comparing it with the classical, tissue heating, check the wear of the drills by scanning electron microscopy, and cell viability. For such purpose, the tibia of 20 rabbits were divided into 2 groups - Control Group (CG) and GUIDED GROUP (GG) - which were divided into five subgroups (G): G0, GI, G2, G3, G4, which corresponded to the cutters without used and used 10, 20, 30 and 40 times respectively. Each animal received 10 sequences osteotomies, 5 in each group randomly in each tibia at a speed of 1600 rpm, and through external irrigation. The tissue thermal oscillation during each milling was measured using thermocouples. The drills were analyzed by scanning electron microscopy (SEM), and quantification of the deformation of the cutters was performed using the software ImageLab2000. Osteotomized areas were collected, processed for paraffin, and subjected to immunohistochemical processing for the protein expression of osteoprotegerin (OPG), Receptor Activator of Nuclear Factor k (RANKL), osteocalcin (OC) and caspase 3 (cas 3). The data obtained by thermal analysis were tested using the Mann-Whitney test (p = 0.05), the strain values of the drills were tested using parametric t-test. Results in the heating bone in the GG was statistically higher than in CG. In the analysis by means of SEM, it was found that deformation was progressive in accordance with the increased use of milling drills. In the analysis of cell viability, no statistical difference when comparing each protein, OPG, RANKL and CASPASE-3, between groups. In the comparison between the OC and GG GC, there was a statistically significant difference. According to the... (Complete abstract click electronic access below)
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Efeito da desmineralização óssea sobre parâmetros de superfície e sobre o comportamento de pré-osteoblastos em cultura: estudo em microscopia eletrônica de varredura e confocal / Effect of bone demineralization on surface parameters and on behavior of pre-osteoblasts in culture: study in scanning electron microscopy and confocal microscopySamira Salmeron 02 April 2015 (has links)
A desmineralização óssea superficial tem se demonstrado favorável à consolidação de enxertos e ao comportamento celular, entretanto os mecanismos envolvidos ainda não estão esclarecidos. Os subsídios para o embasamento biológico da desmineralização, proporcionado por publicações anteriores, sugeriram que modificações na superfície óssea teriam influenciado o comportamento de pré-osteoblastos em cultura. Assim, este estudo objetivou comparar o efeito de duas concentrações de ácido cítrico na desmineralização de superfícies ósseas onde foram cultivadas células pré-osteoblásticas (MC3T3-E1), e analisar parâmetros de superfície comparando superfícies desmineralizadas a não desmineralizadas. Setenta amostras ósseas bicorticais foram removidas das calvárias de 35 ratos e divididas em grupos para as análises: 1) Microscopia Eletrônica de Varredura (MEV) para avaliação da área de recobrimento e espessura da camada de células sobre as amostras (n = 15) durante 24, 48 e 72 horas: Grupo AC.10 amostras desmineralizadas por 30 segundos com ácido cítrico 10 %; Grupo AC.50 amostras desmineralizadas por 30 segundos com ácido cítrico 50 %; e Grupo C (controle) amostras não desmineralizadas; 2) Microscopia Confocal para análise da área de expressão e intensidade de fluorescência das BMP-2, -4 e -7: AC.10 seis amostras desmineralizadas conforme item 1); AC.50 seis amostras desmineralizadas conforme item 1); C três amostras não desmineralizadas; 3) Microscopia Confocal para análise da rugosidade superficial média (Ra e Sa): Grupos AC.10 e AC.50 com cinco amostras cada, desmineralizadas conforme o item 1), sendo cada amostra seu próprio controle (análises antes e depois da desmineralização). Também foram avaliadas as distâncias entre picos (P-P) e entre picos e vales (P-V) antes e depois da desmineralização; 4) Microscopia Eletrônica de Varredura / Espectroscopia de Energia Dispersiva (MEV / EDS) para análise da composição superficial: mesmas amostras do item 3) foram avaliadas antes e depois da desmineralização quanto à porcentagem atômica (%A) de carbono, oxigênio, magnésio, fósforo, enxofre e cálcio. Análises estatísticas foram feitas adotando nível de significância de 95 %. Amostras desmineralizadas apresentaram células morfologicamente em estágios mais avançados de diferenciação do que as não desmineralizadas. A área de recobrimento superficial foi significantemente maior após 24 horas de cultura nos grupos teste do que no controle e a espessura da camada de células também foi maior nos grupos teste às 48 e 72 horas. Houve significantemente maior expressão de BMP-2 e -7 nos grupos teste do que no controle e, apenas AC.10 demonstrou maior expressão de BMP-4 do que os demais grupos, sem significância em relação a AC.50. Os parâmetros de superfície Ra e Sa foram inconclusivos, mas P-P e P-V diminuíram consideravelmente após a desmineralização para distâncias compatíveis com superfícies favoráveis à adesão e diferenciação celular. A análise da composição química superficial revelou diminuição da %A de enxofre e magnésio nos grupos teste. A concentração do ácido, embora não tenha apresentado diferença significante para a maioria das análises, pareceu ter influência positiva nos resultados para o ácido cítrico 10 %. Concluiu-se que a desmineralização superficial parece promover a proliferação e diferenciação celular, proporcionando superfícies com características de composição e topografia que favorecem o comportamento celular verificado. / The superficial bone demineralization has proved to be a favorable procedure for bone grafts consolidation and cell behavior, however the underlying mechanisms have not been clarified yet. Therefore, this study aimed to compare the effect of two concentrations of citric acid on demineralization of bone surfaces where pre-osteoblastic cells (MC3T3-E1) were cultivated, and analyze surface parameters comparing demineralized bone surfaces with non-demineralized surfaces. Seventy bicortical bone samples were harvested from the calvaria of 35 rats and divided into groups as follows: 1) Scanning Electron Microscopy (SEM) to evaluate the coating area and thickness of cells layers cultured on the samples (n = 15) for 24, 48, and 72 hours: Group CA.10 samples demineralized for 30 seconds with 10 % citric acid; Group CA.50 samples demineralized for 30 seconds with 50 % citric acid, and Group C (control) non-demineralized samples; 2) Confocal Microscopy for analysis of expression area and intensity of fluorescence of BMP-2, -4, and -7: CA.10 six samples demineralized as item 1); CA.50 six samples demineralized as item 1); Group C three non-demineralized samples; 3) Confocal Microscopy for surface mean roughness analysis (Ra and Sa): Groups CA.10 and CA.50 made up of five samples each and demineralized according to item 1), each sample was its own control (analysis before and after demineralization). The distances between peaks (P-P) and between peaks and valleys (P-V) were also evaluated before and after demineralization; 4) Scanning Electron Microscopy / Energy dispersive Spectroscopy (SEM / EDS) to analyze the surface composition: the same samples of item 3) were evaluated before and after demineralization for atomic percentage (%A) of carbon, oxygen, magnesium, phosphorus, sulfur and calcium. Statistical test was made by adopting the 95 % significance level. Demineralized samples showed cells with morphology in the later stages of differentiation than non-demineralized ones. The coating surface area by cells was significantly higher after 24 hours of culture in the test groups than in the control and the thickness of the layers were also greater in the test groups at 48 and 72 hours of evaluation. There was significantly higher expression of BMP-2 and -7 in test groups than in the control group, and only the CA.10 group showed higher BMP-4 expression than the other groups, but the difference was not statistically significant compared to the CA.50 group. Ra and Sa surface parameters were inconclusive, however P-P and P-V decreased considerably after demineralization to distances compatible with surfaces favorable to cell adhesion and cell differentiation. The chemical composition analysis of the surfaces revealed a decrease in the %A for sulfur and magnesium in test groups. Although the acid concentration did not shown significant difference for most analysis, it seemed to have a positive influence for the results with citric acid 10 %. It was concluded that the surface demineralization seems to promote cell proliferation and differentiation, providing surfaces with composition and topography that can favor observed cell behavior.
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The Use of Dynamic Fluid Flow Strategies for Bone Tissue Engineering ApplicationsSharp, Lindsay Ann 21 October 2009 (has links)
Bone is the second most transplanted tissue in the body, with approximately 2.2 million bone graft procedures performed annually worldwide. Currently, autogenous bone is the gold standard for bone grafting due to its ability to achieve functional healing; however, it is limited in supply and results in secondary injury at the donor site. Tissue engineering has emerged as a promising means for the development of new bone graft substitutes in order to overcome the limitations of the current grafts. In this research project, the specific approach for bone tissue engineering involves seeding osteoprogenitor cells within a biomaterial scaffold then culturing this construct in a biodynamic bioreactor. The bioreactor imparts osteoinductive mechanical stimuli on the cells to stimulate the synthesis of an extracellular matrix rich in osteogenic and angiogenic factors that are envisioned to guide bone healing in vivo. Fluid flow, which exerts a hydrodynamic shear stress on adherent cells, has been identified as one of the strongest stimuli on bone cell behavior. It has been shown to enhance the deposition of osteoblastic matrix proteins in vitro, and is particularly important for the delivery of oxygen and nutrients to cells within large scaffolds suitable for bone tissue regeneration. In particular, dynamic flow profiles have been shown to be more efficient at initiating mechanotransductive signaling and enhancing gene expression of osteoblastic cells in vitro relative to steady flow. However, the molecular signaling mechanisms by which bone cells convert hydrodynamic shear stress into biochemical signals and express osteoblastic matrix proteins are not fully understood. Therefore, the overall goal of this research project was to determine the effect of dynamic fluid flow on mechanotransductive signaling and expression of bioactive factors and bone matrix proteins.
In the first study, an intermittent flow regimen, in which 5 min rest periods were inserted during fluid flow, was examined. Results showed that signaling molecules, mitogen activated protein kinases (MAPKs) and prostaglandin E2, were modulated with the flow regimen, but that expression of bone matrix proteins, collagen 1α1, osteopontin, bone sialoprotein (BSP), and osteocalcin (OC), were similar under continuous and intermittent flow. Thus, this study suggested that variation of the flow regimen modulates mechanotranductive signaling. In the second study, four flow conditions were examined: continuous flow, 0.074 Hz, 0.044 Hz, and 0.015 Hz pulsatile flow. This study demonstrated that pulsatile flow enhances expression of BSP and OC over steady flow. Similarly, bone morphogenetic protein (BMP)-2 and -7 were enhanced with pulsatile flow, while BMP-4 was suppressed with all flow conditions, suggesting that the mechanism by which fluid flow enhances bone matrix proteins may involve the induction of BMP-2 and -7, but not BMP-4. In the third study, the molecular mechanism by which fluid flow simulates expression of BMPs was examined. Results from this study suggest that this mechanism may involve activation of MAPKs, but BMP-2, -4, and -7 are regulated through multiple different signaling pathways.
Overall, the results from this research demonstrate that dynamic flow modulates mechanotransductive signaling and expression of osteoblastic matrix proteins by osteoblast cells. In particular, BMPs, important for formation in vivo, were shown to be induced by fluid flow. Therefore, this work may be beneficial in understanding and developing 3D perfusion culture systems for the creation of a clinically effective engineering bone tissue. / Ph. D.
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Regulating human mammary epithelial stem cells transformation : an interplay between extrinsic and intrinsic signals / La régulation des cellules souches épithéliales mammaires humaines : un jeu entre signaux extrinsèques et intrinsèquesClément, Flora 05 May 2017 (has links)
L'incidence, le coût et l'issue fatale dans un nombre encore trop élevé de cas font du cancer un problème majeur en santé publique. Malgré les progrès réalisés dans le développement de thérapies ciblées, la plupart des cancers rechutent, vraisemblablement à cause de l'échappement des cellules souches cancéreuses (CSC) qui survivent et régénèrent la tumeur. L'enjeu clinique en cancérologie aujourd'hui est d'éliminer les cellules souches cancéreuses en épargnant les cellules souches normales. Pour atteindre cet objectif, il est primordial de comprendre leurs mécanismes spécifiques de transformation. Nous évaluons dans mon équipe de recherche l'implication du microenvironnement dans la transformation et la résistance des CSC épithéliales, à travers les effets de facteurs solubles et de contacts cellulaires : l'enzyme CD10, et la voie des BMPs (Bone Morphogenetic Proteins).Notre équipe étudie le rôle du dialogue permanent entre la CS normale et son microenvironnement qui régule la prolifération, et la survie des CS. Nous utilisons la glande mammaire et la prostate comme systèmes modèles car ces deux types d'épithélium présentent des similitudes, ce qui nous permet d'aborder la question de l'apparition et la résistance des CSC dans deux modèles tumoraux correspondants. Des dérégulations de la voie des BMPs, comme de l'enzyme CD10 sont observées dans ces tumeurs. Enfin, nous cherchons à comprendre comment les dérégulations de la voie des BMPs apparaissent, en s'intéressant principalement aux facteurs pouvant modifier directement le microenvironnement, tels que les polluants présents dans l'environnement (bisphénols, benzoapyrène) / It has been shown for a number of cancers that a cell population characterized by stem cell (SC) properties and therapeutic resistance is likely responsible for relapse several years after treatment. Current therapies kill most of the tumor cells, but fail to eradicate the so-called cancer stem cells (CSC). Therefore a complete cure of the disease will require the eradication of the tumor-sustaining CSC. We propose to study these CSC in the context of breast cancer as the existence of CSC as already been highlighted in this epithelia.CD10 is a membrane enzyme able to cleave several peptide of the microenvironment (such as oxytocin, bombesin, enkephalin.. ) that can also interact with intracellular signalling pathway through its direct interaction with PTEN. Our results, and those of the literature, indicate that CD10 enzyme controls the fate of SC and is deregulated in normal breast and cancerous tissues. We showed that CD10 membrane expression allows the maintenance of immature cells partly through its enzymatic function that inhibits mammary stem cells differentiation. As CD10 has been described in breast cancer initiation, progression and resistance, we then decided to test the role of CD10 in tumor context. Our strategy consists in flow cytometry cell sorting for CD10+/CD10- cells to compare the functional properties of both sub-population. Only CD10+ cells are able to regenerate both CD10+ and CD10- subpopulations, and CD10+ cells exhibit higher expression of immature genes. Interestingly, modulating CD10 using stable expression of CD10 in our models and Sh strategies do not mimick the normal functions of CD10, indicating that CD10 could be more a marker of a certain population with immature properties prone to transformation rather than a driver. To better characterize the role of CD10 in luminal breast transformation, we developed a new human mammary model, initiated from immature cells to obtain transformed luminal epithelial cells and their resistant counterpart. We observed a higher level of CD10 expression during mammary epithelial cell transformation process. We then performed a microarray on CD10+ and CD10- subpopulations. Preliminary analysis seems to confirm that CD10 is a potential marker for a stem cell population prone to transformation rather than a direct driver of the cell transformation
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Rôle du microenvironnement dans le maintien et la résistance des cellules souches leucémiques de la Leucémie Myéloïde Chronique. voie BMP et contraintes mécaniques / Role of the microenvironment in maintenance and resistance of leukemic stem cells in Chronic Myelogenous Leukemia. BMP pathway and mechanical forcesLaperrousaz, Bastien 30 March 2015 (has links)
Une des principales causes d’échec dans le traitement des cancers est le développement de résistances aux drogues par les cellules tumorales. Les cellules souches cancéreuses (CSC) sont suspectées d’être responsables de ces rechutes, conduisant à la récurrence de la maladie et bien souvent au décès des patients. En clinique, il est donc nécessaire de développer des stratégies thérapeutiques capables de cibler ces CSC résistantes et aboutir à la guérison des patients. Les CSC sont régulées par un ensemble de signaux aussi bien biologiques que physiques au sein de la niche tumorale. Mon projet a pour objectif de déterminer l’implication du microenvironnement tumoral (voie de signalisation BMP et contraintes mécaniques) dans le maintien et la résistance des cellules souches leucémiques (CSLs) de la leucémie myéloïde chronique (LMC). Pour cela, nous avons combiné tests fonctionnels et moléculaires ainsi que l’analyse de la niche tumorale sur plus de 200 échantillons de patients atteints de LMC. Nous avons ainsi démontré que l’altération de la voie BMP intrinsèque aux cellules immatures de la LMC corrompt et amplifie la réponse à BMP2 et BMP4, présents en quantités anormalement abondantes au sein de la niche tumorale. Ces résultats récemment publiés dans Blood nous ont amenés à évaluer le rôle de la voie BMP dans le maintien des CSLs sous traitement par les ITK. La microscopie à force atomique nous a permis de démontrer que l’expression de BCR-ABL est suffisante pour induire une augmentation de la rigidité des cellules immatures de LMC par rapport à des cellules saines. Enfin, l’utilisation d’un système de confinement cellulaire nous a permis de démontrer que le stress mécanique contrôle la prolifération des cellules leucémiques immatures en régulant l’expression de gènes mécano-sensibles comme Twist-1. Ces résultats pourraient expliquer comment des CSLs tirent profit des contraintes mécaniques issues de leur microenvironnement afin d’acquérir un avantage prolifératif par rapport aux cellules saines. Ultimement, nous espérons que cette approche transdisciplinaire permettra d’identifier les molécules clés de la transduction de signaux mécaniques potentiellement impliqués dans le maintien et la résistance des CSC et ainsi proposer de nouvelles cibles pour contrer ces effets. / One of the main causes of treatment failure in cancers is the development of drug resistance by cancer cells. The persistence of cancer stem cells (CSCs) might explain cancer relapses as they could allow reactivation of cancer cells proliferation following therapy, leading to disease persistence and ultimately to patients’ death. Clinically, it is crucial to develop therapeutic strategies able to target resistant CSCs in order to cure the patients. CSCs are controlled by a variety of biochemical and biomechanical signals from the leukemic niche. My project aims to determine the involvement of the tumor microenvironment (BMP signaling pathway and mechanical stress) in the maintenance and resistance of Leukemic Stem Cells (LSCs) in Chronic Myelogenous Leukemia (CML). For this, we combined functional and molecular assays to the analysis of tumor microenvironment on more than 200 CML patients’ samples. We demonstrated that alterations of intracellular BMP signaling pathway in CP-CML primary samples corrupt and amplify the response to exogenous BMP2 and BMP4, which are abnormally abundant in the tumor microenvironment. These results, recently published in Blood led us to evaluate the role of the BMP pathway in LSC maintenance under TKI treatment. Atomic force microscopy allowed us to demonstrate that BCR-ABL expression alone is sufficient to increases the rigidity of immature CML cells compared to healthy ones. Finally, using a unique cell confining system, we were able to demonstrate that mechanical stress controls the proliferation of immature leukemic cells by regulating the expression of mechano-sensitive genes such as Twist-1. These results could explain how LSCs can benefit from a mechanical stress exerted by their microenvironment to acquire a proliferative advantage over normal cells. Ultimately, we hope that this transdisciplinary approach will help to identify key molecules in the transduction of mechanical signals potentially involved in maintenance and resistance of CSCs and thus offer new targets to counter these effects.
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Efeito da desmineralização ácida da interface enxerto-leito na consolidação de enxertos ósseos autógenos em bloco / Influence of acid demineralization of contacting osseous surfaces on the consolidation of autogenous onlay bone graftsDomingues, Roberta Santos 24 May 2013 (has links)
Para testar a hipótese de que a desmineralização in situ das superfícies de contato enxerto-leito, e a forma como o enxerto é estabilizado ao leito, podem influenciar os mecanismos envolvidos na consolidação do enxerto, fragmentos ósseos de 10 mm de diâmetro foram removidos das metáfises proximais tibiais de 36 coelhos (Oryctolagus Cuniculus) e transplantados para uma área adjacente. Na tíbia esquerda dos animais, as superfícies de contato do enxerto e do leito hospedeiro foram desmineralizadas com ácido cítrico pH 1,0 por 3 minutos antes dos enxertos serem fixados ao leito. Na tíbia direita, o transplante do bloco ósseo não foi precedido de desmineralização. Metade dos enxertos foi imobilizada sobre o leito pela superposição de uma membrana não reabsorvível de politetrafluoretileno colada com cianoacrilato ao leito à distância da interface enxerto-leito. A outra metade dos enxertos foi fixada por um parafuso de titânio no centro do enxerto. Assim, foram formados 4 grupos de estudo: membrana (M), membrana + ácido (MA), parafuso (P) e parafuso + ácido (PA). Três animais de cada grupo forneceram espécimes para análise microscópica quantitativa e qualitativa aos 15, 30 e 45 dias de pós-operatório. A análise qualitativa demonstrou que não houve formação óssea na interface em nenhum espécime aos 15 dias e que nos demais períodos, em todos os grupos, a quantidade de tecido ósseo neoformado na interface e seu estágio de maturação aumentaram com o tempo. Ambos os métodos de fixação empregados foram eficientes em manter os enxertos em posição, porém a membrana promoveu menor reabsorção da estrutura do enxerto. A análise quantitativa computadorizada revelou que, aos 30 dias, os grupos MA e PA apresentaram maior área de formação óssea na interface (71,34 ± 12,03%; 56,74 ± 2,15% respectivamente) em relação aos grupos M e P (51,75 ± 11,02%; 43,95 ± 4,05% respectivamente) e superfícies de consolidação óssea mais extensas (93,41 ± 5,95%; 93,73 ± 4,96% respectivamente) do que os grupos sem tratamento ácido (73,49 ± 7,7%; 73,77 ± 11,77% respectivamente para M e P), sendo essas diferenças estatisticamente significantes (p<0,05). Aos 45 dias de pós-operatório, os grupos MA e PA (71,18 ± 8,9%; 58,97 ± 3,97% respectivamente) apresentaram resultados superiores aos grupos M e P (59,78 ± 11,28%; 46,08 ± 3,53% respectivamente) em relação à área de neoformação óssea na interface, porém essa diferença não foi significativa. Concluiu-se que a desmineralização ácida das superfícies contactantes nos enxertos ósseos autógenos em bloco na tíbia de coelhos promoveu a osteogênese na interface enxerto-leito e acelerou a consolidação dos enxertos. Além disso, quando o tratamento ácido foi associado ao uso de membrana como método de fixação, a consolidação e grau de reabsorção óssea foram otimizados. / In order to test the hypothesis that the demineralization \"in situ\" of contacting surfaces of bone graft/bone bed and the fixation method used for graft stabilization can influence the mechanisms involved in the consolidation of the graft, bone fragments of 10 mm in diameter were removed from the proximal tibial metaphysis of thirty-six male rabbits (Oryctolagus Cuniculus) and transplanted to an adjacent area. In the left tibia of the animals, the contacting surfaces of the graft and host bed were demineralized with citric acid pH 1.0 for 3 minutes before the grafts were fixed to the receptor bed. In the right tibia, the bone block transplantation was not preceded by demineralization. Half of the grafts were immobilized on the bone bed by a nonresorbable polytetrafluoroethylene membrane glued with cyanoacrylate adhesive to the host bed distant from the bone graft-bone bed interface. The other half of the grafts were fixed by a titanium screw in the center of the graft. Thus, four groups were formed: membrane (M), membrane + acid (MA), screw (P) and screw + acid (PA). Three animals from each group provided specimens for quantitative and qualitative microscopic analysis at 15, 30 and 45 days postoperatively. Qualitative analysis showed no significant bone formation at the interface in any specimen of the groups at 15 days and on the other periods in all groups, the amount of newly formed bone at the interface as well as the stage of bone maturation increased with time. Both fixation methods were effective in maintaining the graft in position, but the membrane resulted in less resorption of the graft. Quantitative analysis, performed by means of a computer program for image analysis, showed that at day 30, groups MA and PA, showed greater area of bone formation at the interface (71.34 ± 12.03%; 56.74 ± 2 15%) than groups M and P (51.75 ± 11.02%, 43.95 ± 4.05%) and more osseointegrated bone surfaces (93.41 ± 5.95%, 93.73 ± 4.96%) than those without acid treatment (73.49 ± 7.7%, 73.77 ± 11.77%), and these differences were statistically significant. At 45 days postoperatively MA and PA groups (71.18 ± 8.9%, 58.97 ± 3.97%) showed better results than the M and P groups (59.78 ± 11.28%, 46 , 08 ± 3.53%) compared to the area of new bone formation at the interface and osseointegrated surfaces, but these differences were not significant. It was concluded that the acid demineralization of contacting surfaces in autogenous onlay bone grafts in the tibia of rabbits promotes osteogenesis in bone graft-host bed interface and accelerates the consolidation of the grafts. Furthermore, the association of this surface treatment to the use of membrane/cyanoacrylate fixation method, optimizes the results regarding consolidation and degree of bone graft resorption.
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Aspectos celulares, teciduais e moleculares da osteogênese ectópica e ortotópica induzida pela matriz alogênica óssea e dentinária / Cellular, tissue and molecular aspects of the ectopic and orthotopic osteogenese induced by bone and dentine allogenic matrixCestari, Tania Mary 08 April 2009 (has links)
O objetivo do atual trabalho, foi correlacionar os eventos celulares e teciduais com a expressão das proteínas VEGF, BMP-7, RANKL e OPG durante a osteogênese ectópica e ortotópica, induzida pela matriz óssea (MO) e dentinária (MD) alogênica. Matrizes alogênicas desmineralizada em HCl a 0,6N, obtidas de fêmur e incisivo de ratos, fori implantada entre as fáscias musculares da coxa e em defeito trans-ósseo de 8mm de diâmetro nos ossos parietais. As análises radiográfica e histomorfométrica da neoformação óssea e, a imunohistoquímica e o western blotting para as proteínas VEGF, BMP, RANKL e OPG, mostraram que: a) o volume da região do enxerto nos sítios ortotópicos reduziu 19% em 42 dias; b) em ambos tipos de enxerto e locais de implantação, ocorreu formação de tecido cartilaginoso e ósseo; c) nos sítios intramusculares, a reabsorção da matriz alogênica e a remodelação do tecido cartilaginoso, ósseo e medular foi mais acelerado, em relação a implantação ortotópico; d) o aumento na densidade de volume dos vasos sanguíneos e no número de osteoblastos/osteócitos e osteoclastos ocorreu simultaneamente e estava associado à maior reabsorção da matriz alogênica e à formação do tecido medular (hematopoiético); e) as proteínas VEGF, BMP-7, RANKL, OPG foram expressas em condrócitos, osteoblastos ativos, osteócitos recém aprisionados na matriz e em células estromais próximas aos osteoblastos ou às áreas da matriz alogênica reabsorvida; e f) a expressão das proteínas VEGF, BMP-7, RANKL e OPG foi maior no grupo MO. O pico de expressão dessas proteínas ocorreu nos períodos de 14 aos 21 dias no grupo da MO e 21 e 28 dias no grupo da MD. Concluímos que, a capacidade osteoindutora da matriz alogênica desmineralizada está relacionado a origem da matriz e ao sítio de implantação e que, as proteínas VEGF, BMP-7, RANKL e OPG estão associadas a maior reabsorção da matriz implantada, promovendo uma rápida e contínua liberação dos morfógenos contidos em seu interior que, induzem temporal e espacialmente a formação óssea/medular. / The aim of the present work was to correlate the cellular and tissue events with the expression of VEGF, BMP-7, RANKL and OPG during ectopic and orthotopic osteogenesis, induced by bone and dentin allogeneic matrix. Allogenic matrices obtained from femur and incisor of rats and demineralized in 0.6 N HCl were implanted into a intramuscular pocket and a 8mm-diameter bone defect in the skull. The radiographic and histomorphometric analysis of new bone formation, and immunohistochemistry and western blotting for VEGF, BMP, RANKL and OPG proteins, showed that: a) the total volume of the graft region in orthotopic site decreased 19% at 42 days b) in both graft types and implantation sites occurred formation of cartilaginous and bone tissues, c) in intramuscular sites, the resorption of allogenic matrix and remodeling of the new formed cartilage and bone were faster, in relation to orthotopic implantation sites; d) the increase in the volume density of blood vessels and in the number of osteoblasts/osteocytes and osteoclasts occurred simultaneously and was associated with greater reabsorption of the allogenic matrix and hematopoietic bone marrow formation; e) VEGF, BMP-7, RANKL, OPG proteins were expressed in chondrocytes, active osteoblasts, newly osteocytes confined and stromal cells located near the osteoblasts or in the surface of the reabsorbed matrix; and f) the VEGF, BMP-7, RANKL and OPG expression was higher in MO grafts than in the MD. The peak of expression of these proteins each occurred at 14 and 21 days in MO and 21 and 28 days in MD. We concluded that, the osteoinductive capacity of allogeneic demineralized matrix is related to matrix origin and implantation site and that the VEGF, BMP-7, RANKL and OPG proteins are associated with greater reabsorption of the implanted matrix, promoting rapid and continuous matrix-release morphogens that induces spatially and temporally the bone and bone marrow formation.
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Biomaterial integration within 3D stem cell aggregates for directed differentiationBratt-Leal, Andrés Miguel 14 November 2011 (has links)
The derivation of embryonic stem cells (ESCs) has created an invaluable resource for scientific study and discovery. Further improvement in differentiation protocols is necessary to generate the large number of cells needed for clinical relevance. The goal of this work was to develop a method to incorporate biomaterial microparticles (MPs) within stem cell aggregates and to evaluate their use for local control of the cellular microenvironment for directed differentiation.
The effects of unloaded MPs on ESC differentiation were first determined by controlled incorporation of poly(lactic-co-glycolic acid) (PLGA), agarose and gelatin MPs. Embryoid body (EB) formation, cell viability, and gross morphology were not affected by the presence of the MPs. Further analysis of gene expression and patterns of phenotypic marker expression revealed alterations in the differentiation profile in response to material incorporation. The ability of MPs to direct ESC differentiation was investigated by incorporation of growth factor loaded MPs within EBs. MPs were loaded with bone morphogenetic protein-4 (BMP-4). BMP-4 loaded MPs incorporated within EBs induced mesoderm gene expression while inhibiting expression of an ectoderm marker compared to untreated EBs.
Finally, magnetic MPs (magMPs) were incorporated within EBs to induce magnetic sensitivity. The responsiveness of EBs to applied magnetic fields was controlled by the number of magMPs incorporated within the aggregates. Magnetic guidance was then used to control the precise location of single EBs or populations of EBs for bioreactor culture and for construction of heterogeneous cell constructs. Overall, the results indicated that PSC differentiation within spheroids is sensitive to various types of biomaterials. Incorporation of MPs within EBs can be used to direct ESC differentiation by control of the cellular environment from microscale interactions, by delivery of soluble factors, to macroscale interactions, by control of EB position in static and suspension cultures.
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Sequential Growth Factor Delivery From Complexed Microspheres For Bone Tissue EngineeringBasmanav, Fitnat Buket 01 September 2007 (has links) (PDF)
Complexed microspheres of poly(4-vinyl pyridine) (P4VN) and alginic acid were prepared by internal gelation method and subsequent freeze drying.
The 4% and 10% microspheres were loaded with Bone Morphogenetic Protein-2 (BMP-2) and Bone Morphogenetic Protein-7 (BMP-7), respectively for in vitro studies and were entrapped in PLGA foams. Foams containing only 4%, BMP-2 microspheres, only 10%, BMP-7 microspheres and both populations were prepared. Control samples of each group were prepared with drug free microspheres. Bone marrow derived stem cells from rat femur and tibia isolated by a surgical operation, were seeded onto foams.
Proliferation of cells on foams containing both microsphere populations was higher at all time points regardless of the presence of BMPs. This was attributed to different porosity characteristics. Proliferation was higher at all times in control samples in comparison to their positive samples for all groups, suggesting proliferation attenuation related enhancement in osteogenic activity due to BMP supply.
Alkaline phosphatase (ALP) activities were lower at all time points for foams containing both microsphere populations regardless of BMP presence. This was attributed to different physical characteristics of foams confirmed by the inverse correlation between proliferation and osteogenic differentiation. Total and specific ALP activity results demonstrated the significant positive influence of all BMP containing types in enhancing osteogenic differentiation. Best results were obtained with co-administration of sequential delivery performing 4% and 10% microspheres loaded with BMP-2 and BMP-7, respectively.
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The role of bone morphogenic proteins in human aortic valvular endothelial cellsAnkeny, Randall Francis 01 April 2010 (has links)
In the United States alone, there are nearly 49,000 aortic valvular repairs or replacements each year, and this number is expected to rise. Unlike atherosclerosis, the molecular mechanisms contributing to this side-dependent disease development are limited, which contributes to the lack of therapeutic treatments. Once clinically manifested, options for treatment are limited to valvular replacement or repair. Therefore understanding the mechanobiology and cellular responses in aortic valves may provide important information for disease development and possible biomarkers or therapeutic treatments.
Aortic valve disease occurs on one side of the valvular leaflet. The fibrosa side, which faces the aorta, is prone to disease development, while the ventricularis remains relatively unaffected. The hemodynamics is hypothesized to play a role in side dependent disease formation. The fibrosa endothelium is exposed to oscillatory flow while the ventricularis endothelium is exposed to a pulsatile unidirectional flow. Previous work by our group has shown that bone morphogenic protein-4 is a mechanosensitve inflammatory cytokine in the vasculature. In the following study, we proposed that mechanosensitive bone morphogenic proteins play a role in side specific aortic valve disease.
Recently, the bone morphogenic proteins (BMPs) have been found in calcified human aortic valves. Furthermore, BMP-4 in vascular endothelial cells is increased by oscillatory shear stress. However, the role of the BMPs in aortic valve endothelial cells and their contribution to aortic valve calcification remains unstudied. Therefore, the overall objective of this dissertation was to investigate how disease and hemodynamics affects the BMP pathway and inflammation in human aortic valvular endothelial cells. By understanding how the bone morphogenic proteins are regulated and what roles they play in aortic valve disease, we will have better insight into endothelial cell regulation and contribution in aortic valve pathology. The central hypothesis of this project was that oscillatory flow conditions on the fibrosa side of the aortic valve stimulate endothelial cells to produce BMP-4, which then activates an inflammatory response leading to accumulation of inflammatory cells, calcification, and ultimately valve impairment. This hypothesis was tested through three specific aims using calcified human aortic valves, non-calcified human aortic valves, and side-specific human aortic valve endothelial cells.
We first worked to establish the importance of the BMPs in the aortic valvular endothelium by looking at two populations of aortic valves: 1) calcified human aortic valves were obtained from patients undergoing valve replacement, and 2) non-calcified valves were obtained from recipient hearts of patients undergoing heart transplantation. Using immunohistochemical techniques, we examined the BMPs, BMP antagonists, and SMADs. Surprisingly, we identified that the ventricularis endothelium had higher BMP expression in both calcified and non-calcified human aortic valves. Furthermore, no disease-dependent BMP expression was detected. Next, we examined the BMP antagonists and found that there was robust BMP antagonist expression in the ventricularis endothelium and very low expression in the fibrosa endothelium. Finally, to determine if the BMP pathway was activated, we stained for the canonical BMP signaling molecule phosphorylated-SMAD 1/5/8 and found increased staining in the endothelium of calcified human aortic valves. Furthermore, a significant increase in SMAD 1/5/8 phosphorylation was seen in the endothelium of calcified fibrosa when compared to the non-calcified fibrosa. Finally, inhibitory SMAD 6 was significantly increased in the ventricularis endothelium of non-calcified human aortic valves. These findings suggest that preferential activation of BMP pathways, controlled by the balance between the BMPs and their inhibitors, play an important role in side-dependent calcification of human AVs.
We next wanted to examine the role of shear stress in BMP regulation, but before doing so, we needed to examine the endothelial response to fluid shear stress to validate the phenotype of our isolated human aortic valve endothelial cells. KLF2 and eNOS expression in vascular endothelial cells has been shown to be increased by laminar flow and to have anti-inflammatory effects by decreasing VCAM-1 levels. Conversely, oscillatory shear stress has been shown to increase NF-kappa B translocation and increase ICAM-1 and E-selectin. We found laminar shear stress causes human aortic valve endothelial cells align parallel to flow and have robust increases of KLF2 and eNOS and decreases in VCAM-1 levels; however, laminar shear-treated cells had similar levels of NF-kappa B activation as oscillatory treated cells while ICAM-1 and E-selectin was not affected by shear stress. In contrast, oscillatory shear had higher levels of monocytes bound which may be due to eNOS's protective effects under laminar shear and robust VCAM-1 expression in oscillatory shear. These studies suggest differential regulation of human aortic valvular endothelial cells than published reports on human aortic endothelial cells which adds to the growing evidence that valvular endothelial cells are phenotypically different than vascular endothelial cells.
After verifying the shear response of our endothelial cells, we next determined the shear response of the BMPs and BMP antagonists and described BMPs' effect on inflammation. Previously, BMP-4 has been shown in vitro and in vivo to be increased in endothelial cells exposed to oscillatory flow, while the closely-related BMP-2 has not been shown to be shear sensitive. In this study we have found that BMPs -2 and -4 are shear sensitive while BMP-6 is not. Furthermore, we have found that follistatin is decreased by laminar flow only in the ventricularis, while MGP1 is decreased in the fibrosa valvular endothelial cells under both oscillatory and laminar flow. Finally, incubation with noggin did not affect monocyte adhesion after shear, suggesting differential regulation of inflammation in human aortic valvular endothelial cells.
By addressing the specific aims of this project, we have investigate disease- and side-dependent valvular endothelial BMP expression in vivo, shear regulation of valvular endothelial inflammation in vitro, and shear regulation of valvular endothelial BMP expression in vitro. Our results suggest that the BMP pathway is playing a role in side specific aortic valve disease development; however, regulation of the BMPs does not appear to be shear regulated in vivo. Other factors that may be affecting BMP production include including pulsatile pressures, bending stresses, cyclic stretch, and humeral stimuli present in the blood of the patients. However, in vitro we have found BMPs -2 and -4 to be shear-regulated in human aortic valvular endothelial cells. Shear-induced inflammation in human aortic valve endothelial cells seems to be VCAM-1-dependent, and BMP-independent. Finally, by identifying factors that are modulated in calcific- and shear-dependent processes, new targets for the early detection of aortic valve disease can be determined and new therapeutics to slow or stop the progression of aortic valve disease may be discovered.
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