Uma potencial alternativa para o transplante de tecidos é a engenharia de tecidos. Células-tronco mesenquimais e scaffolds eletrofiados são comumente utilizados nesta área devido à capacidade multipotente de diferenciação destas células e à rede de poros interconectados destas estruturas fibrosas. Além disso, bioreatores de perfusão podem ser utilizados para melhorar o transporte de nutrientes e reduzir o acúmulo de metabolitos tóxicos. Neste contexto, uma maneira de estudar e otimizar o sistema de cultivo é utilizar técnicas de modelagem para descrever interações ou processos individuais envolvidos no crescimento celular. Deste modo, o objetivo geral deste estudo é realizar o cultivo de células-tronco mesenquimais da polpa de dente decíduo (DPSCs) utilizando scaffolds tridimensionais eletrofiados de policaprolactona (PCL), biorreatores e técnicas de modelagem. Inicialmente foram testadas diferentes misturas de solventes (clorofórmio e metanol), a fim de produzir scaffolds com poros adequados ao cultivo tridimensional. Os diâmetros de fibra e de poro foram determinados por microscopia eletrônica de varredura (MEV). O crescimento e o metabolismo das células foram avaliados através da determinação da atividade metabólica e das concentrações de glicose e lactato do meio de cultivo, e a infiltração celular foi observada com a marcação do núcleo celular. Depois de estabelecidos os parâmetros de eletrofiação, o efeito da perfusão direta no desprendimento de DPSCs de scaffolds eletrofiados de PCL foi estudado. A atividade metabólica das células foi determinada para diferentes tempos de adesão, vazões e densidades de semeadura, e a tensão de cisalhamento na parede do poro foi calculada para cada vazão. A morfologia das células foi avaliada através de imagens de microscopia confocal e MEV. Paralelamente, foram realizadas simulações utilizando o software OpenFOAM para estudar como os parâmetros e variáveis de entrada (concentração inicial de glicose, porosidade e espessura do scaffold) afetam as saídas (fração volumétrica de células e concentração de substrato) de um modelo de proliferação celular que considera a difusão e o consumo de glicose. As contribuições do teor de oxigêno na cinética de crescimento de Contois e da variação da porosidade com o tempo devido à degradação do polímero também foram avaliadas. Inicialmente, foi observado que apenas um tamanho de poro maior que o diâmetro da célula permitiu a infiltração das células no scaffold. Então, observou-se que o aumento do tempo de adesão acarretou em maior espalhamento das células e, assim como a diminuição da densidade de semeadura e da tensão de cisalhamento, resultou em uma redução do desprendimento das células sob perfusão. Quanto ao modelo fenomenológico, observou-se maior sensibilidade à concentração inicial de glicose e à porosidade do scaffold, e aos parâmetros adimensionais relacionados à proliferação e morte celular e ao consumo de nutrientes. Além disso, o número inicial de células apresentou maior impacto no transporte de massa do que no crescimento celular. Neste estudo, foi possível obter scaffolds eletrofiados e conduções de cultivo dinâmico adequadas ao cultivo tridimensional de DPSCs, e elucidar os efeitos da limitação do transporte de massa e do oxigênio no crescimento celular, e da degradação do polímero no transporte de massa. / A potential alternative to tissue transplant is tissue engineering. Mesenchymal stem cells and electrospun scaffolds are commonly used in this field due to the multipotent differentiation capacity of these cells and the interconnected pore network of these fibrous structures. In addition, perfusion bioreactors can be used to enhance nutrient transport and reduce the accumulation of toxic metabolites. In this context, one way to study and optimize the culture system is to use modeling techniques to describe interactions or individual processes involved in cell growth. Thus, the objective of this study is to perform the three-dimensional culture of mesenchymal stem cells of dental pulp (DPSCs) using electrospun polycaprolactone (PCL) scaffolds, bioreactors and modeling techniques. Initially, different solvent mixtures (chloroform and methanol) were tested to produce scaffolds with pores suitable to three-dimensional culture. Fiber and pore diameter was determined using a scanning electron microscope. Cell growth and metabolism were evaluated through the metabolic activity and the culture medium concentration of glucose and lactate, and the cell infiltration was observed with cell nuclei staining. After the establishment of the elesctrospinning parameters, the effect of direct perfusion on DPSCs detachment from PCL electrospun scaffolds was investigated. The metabolic activity of the cells was determined for different adhesion times, flow rates and seeding densities and the pore wall shear stress was calculated for each flow rate. The cell morphology was evaluated through scanning electron and confocal microscopy imaging. In parallel, simulations with the software OpenFOAM were performed to study how parameters and inputs (initial glucose concentration, porosity and thickness of the scaffold) affect the outputs (cell volume fraction and substrate concentration) of a model of cell proliferation and glucose diffusion and consumption. The contribution of the oxygen in the Contois growth kinetics and the porosity variation with time due to polymer degradation was also evaluated. Initially, it was observed that only a pore size higher than the cell diameter allowed the infiltration of the cells through the scaffold. Then, it was observed that a higher adhesion time leaded to higher cell spreading in static conditions and, similar to smaller seeding densities and shear stresses, reduced cell detachment under perfusion. Regarding the phenomenological model, it was observed that the model is more responsive to the initial glucose concentration and scaffold porosity, and to the dimensionless parameters related to cell proliferation, death and nutrient uptake. Furthermore, the initial cell number had a more significant impact on mass transport than on cell growth. In this study, it was possible to obtain an electrospun scaffold and dynamic culture conditions suitable for the three-dimensional culture of DPSCs, and to elucidate the effects of transport limitations and of oxygen on cell growth, and of polymer degradation on mass transport were elucidated.

Uma potencial alternativa para o transplante de tecidos é a engenharia de tecidos. Células-tronco mesenquimais e scaffolds eletrofiados são comumente utilizados nesta área devido à capacidade multipotente de diferenciação destas células e à rede de poros interconectados destas estruturas fibrosas. Além disso, bioreatores de perfusão podem ser utilizados para melhorar o transporte de nutrientes e reduzir o acúmulo de metabolitos tóxicos. Neste contexto, uma maneira de estudar e otimizar o sistema de cultivo é utilizar técnicas de modelagem para descrever interações ou processos individuais envolvidos no crescimento celular. Deste modo, o objetivo geral deste estudo é realizar o cultivo de células-tronco mesenquimais da polpa de dente decíduo (DPSCs) utilizando scaffolds tridimensionais eletrofiados de policaprolactona (PCL), biorreatores e técnicas de modelagem. Inicialmente foram testadas diferentes misturas de solventes (clorofórmio e metanol), a fim de produzir scaffolds com poros adequados ao cultivo tridimensional. Os diâmetros de fibra e de poro foram determinados por microscopia eletrônica de varredura (MEV). O crescimento e o metabolismo das células foram avaliados através da determinação da atividade metabólica e das concentrações de glicose e lactato do meio de cultivo, e a infiltração celular foi observada com a marcação do núcleo celular. Depois de estabelecidos os parâmetros de eletrofiação, o efeito da perfusão direta no desprendimento de DPSCs de scaffolds eletrofiados de PCL foi estudado. A atividade metabólica das células foi determinada para diferentes tempos de adesão, vazões e densidades de semeadura, e a tensão de cisalhamento na parede do poro foi calculada para cada vazão. A morfologia das células foi avaliada através de imagens de microscopia confocal e MEV. Paralelamente, foram realizadas simulações utilizando o software OpenFOAM para estudar como os parâmetros e variáveis de entrada (concentração inicial de glicose, porosidade e espessura do scaffold) afetam as saídas (fração volumétrica de células e concentração de substrato) de um modelo de proliferação celular que considera a difusão e o consumo de glicose. As contribuições do teor de oxigêno na cinética de crescimento de Contois e da variação da porosidade com o tempo devido à degradação do polímero também foram avaliadas. Inicialmente, foi observado que apenas um tamanho de poro maior que o diâmetro da célula permitiu a infiltração das células no scaffold. Então, observou-se que o aumento do tempo de adesão acarretou em maior espalhamento das células e, assim como a diminuição da densidade de semeadura e da tensão de cisalhamento, resultou em uma redução do desprendimento das células sob perfusão. Quanto ao modelo fenomenológico, observou-se maior sensibilidade à concentração inicial de glicose e à porosidade do scaffold, e aos parâmetros adimensionais relacionados à proliferação e morte celular e ao consumo de nutrientes. Além disso, o número inicial de células apresentou maior impacto no transporte de massa do que no crescimento celular. Neste estudo, foi possível obter scaffolds eletrofiados e conduções de cultivo dinâmico adequadas ao cultivo tridimensional de DPSCs, e elucidar os efeitos da limitação do transporte de massa e do oxigênio no crescimento celular, e da degradação do polímero no transporte de massa. / A potential alternative to tissue transplant is tissue engineering. Mesenchymal stem cells and electrospun scaffolds are commonly used in this field due to the multipotent differentiation capacity of these cells and the interconnected pore network of these fibrous structures. In addition, perfusion bioreactors can be used to enhance nutrient transport and reduce the accumulation of toxic metabolites. In this context, one way to study and optimize the culture system is to use modeling techniques to describe interactions or individual processes involved in cell growth. Thus, the objective of this study is to perform the three-dimensional culture of mesenchymal stem cells of dental pulp (DPSCs) using electrospun polycaprolactone (PCL) scaffolds, bioreactors and modeling techniques. Initially, different solvent mixtures (chloroform and methanol) were tested to produce scaffolds with pores suitable to three-dimensional culture. Fiber and pore diameter was determined using a scanning electron microscope. Cell growth and metabolism were evaluated through the metabolic activity and the culture medium concentration of glucose and lactate, and the cell infiltration was observed with cell nuclei staining. After the establishment of the elesctrospinning parameters, the effect of direct perfusion on DPSCs detachment from PCL electrospun scaffolds was investigated. The metabolic activity of the cells was determined for different adhesion times, flow rates and seeding densities and the pore wall shear stress was calculated for each flow rate. The cell morphology was evaluated through scanning electron and confocal microscopy imaging. In parallel, simulations with the software OpenFOAM were performed to study how parameters and inputs (initial glucose concentration, porosity and thickness of the scaffold) affect the outputs (cell volume fraction and substrate concentration) of a model of cell proliferation and glucose diffusion and consumption. The contribution of the oxygen in the Contois growth kinetics and the porosity variation with time due to polymer degradation was also evaluated. Initially, it was observed that only a pore size higher than the cell diameter allowed the infiltration of the cells through the scaffold. Then, it was observed that a higher adhesion time leaded to higher cell spreading in static conditions and, similar to smaller seeding densities and shear stresses, reduced cell detachment under perfusion. Regarding the phenomenological model, it was observed that the model is more responsive to the initial glucose concentration and scaffold porosity, and to the dimensionless parameters related to cell proliferation, death and nutrient uptake. Furthermore, the initial cell number had a more significant impact on mass transport than on cell growth. In this study, it was possible to obtain an electrospun scaffold and dynamic culture conditions suitable for the three-dimensional culture of DPSCs, and to elucidate the effects of transport limitations and of oxygen on cell growth, and of polymer degradation on mass transport were elucidated.

O hidróxido de cálcio (HCa), o agregado de trióxido mineral (MTA) e o óleo-resina de copaíba (COP) isoladamente apresentam características biológicas do material de capeamento pulpar direto mais apropriado. Com o pressuposto que associados poderiam originar materiais mais apropriados para serem aplicados em capeamento pulpar direto, este estudo objetivou analisar in vitro proliferação, diferenciação e migração de células-tronco de polpa de dente decíduo humano esfoliado (SHEDs) em resposta a substâncias liberadas pelo COP isolado ou associado ao HCa ou ao MTA. Proliferação, diferenciação e migração de SHEDs (Linhagem PDH3) foram analisadas através do ensaio de redução do MTT; da atividade de fosfatase alcalina (ALP), formação de nódulos mineralizados pelo ensaio de Vermelho de Alizarina e expressão dos genes (BGLAP, DSPP, DMP1 e HSP-27) pelo qRT-PCR; e, do ensaio do Scratch, respectivamente. As células foram submetidas à ação de meios condicionados pelos biomateriais, de acordo com os seguintes grupos experimentais: COP isolado (COP); HCa isolado (HCa); HCa associado ao COP (HCa+COP); MTA isolado (MTA) e MTA associado ao COP (MTA+COP). Células crescidas em meio de cultura fresco serviram de controle. Os dados foram comparados utilizando ANOVA complementado pelo teste de Tukey (p 0,05). O grupo HCa apresentou número de células viáveis significativamente menor que o dos demais grupos, inclusive o do grupo HCa+COP (p<0,01) em todos os tempos experimentais. A atividade de ALP em 14 dias foi similar em todos os grupos experimentais. Em 21 dias, o grupo COP apresentou quantidade maior de mineralização que os demais grupos (p<0,01) e o grupo HCa+COP maior que o grupo HCa (p<0,01). O gene DMP1 não foi expresso pelas células em nenhum grupo experimental. O grupo COP apresentou as menores expressões dos genes BGLAP, DSPP e HSP-27. As SHEDs do grupo MTA+COP apresentaram superexpressão dos genes BGLAP, DSPP e HSP-27, e as do grupo HCa+COP superexpressão dos genes BGLAP e HSP-27. O grupo HCa foi o único que não apresentou células em migração em todos os tempos experimentais. Os demais grupos apresentaram migração similar à do grupo Controle, exceto o grupo MTA em 12 e 24 horas. As SHEDs dos grupos HCa+COP e MTA+COP migraram significativamente mais que as dos grupos HCa e MTA, respectivamente. O COP, isolado ou em associação aos demais biomateriais, não interfere na proliferação de SHEDs e quando associado ao HCa é capaz de anular a citotoxicidade deste biomaterial isolado. O COP, isoladamente ou em associação, não interfere na atividade de fosfatase alcalina. Isoladamente, o COP induz a maior diferenciação funcional e quando associado ao HCa melhora substancialmente a diferenciação induzida por este biomaterial isolado. Os biomateriais isolados ou associados não são capazes de induzir expressão de DMP1. O COP associado aos biomateriais induz superexpressão de genes relacionados à formação de matriz extracelular e de diferenciação odontoblástica. O COP isoladamente não interfere na migração celular e, quando associado ao HCa, anula por completo a inibição de migração causada por esse biomaterial isolado. Quando associado ao MTA, o COP aumenta a velocidade de migração das células em relação ao MTA isolado. Óleo-resina de copaíba (COP) promove proliferação, diferenciação e migração de células-tronco de polpa dental sendo uma proposta de um biomaterial para capeamento pulpar. / The calcium hydroxide (CaH), the mineral trioxide aggregate (MTA) and the oil-resin copaiba (COP) by themselves have biological characteristics of the ideal direct pulp capping material. With the assumption that when associated they could originate materials more appropriated for direct pulp capping, this study aimed to analyze the in vitro proliferation, differentiation and migration of stem cells from human exfoliated deciduous teeth (SHEDs) in response to substances leached from COP alone or associated with CaH or MTA. Proliferation, differentiation and migration of SHEDs (PDH3 lineage) were analyzed through the MTT reduction assay; alkaline phosphatase (ALP) activity, mineralized nodules formation using the Alizarin Red assay and gene expression (BGLAP, DSPP, DMP1 e HSP-27) using qRT-PCR; and the Scratch assay, respectively. The cells were submitted to the culture medium conditioned by the biomaterials according to the following experimental groups: COP alone (COP); CaH alone (CaH); CaH associated to COP (CaH+COP); MTA alone (MTA) and MTA associated to COP (MTA+COP). Cells grown in fresh culture medium served as control. Data were compared by ANOVA complemented by the Tukey´s test (p 0.05). The CaH group presented number of viable cells significantly smaller (p<0.01) than those of all other experimental groups, including the CaH+COP, during whole experimental time. The ALP activity in 14 days was similar in all experimental groups. In 21 days, the COP group presented the amount of mineralization higher (p<0.01) than those of all other groups. The gene DMP1 was not expressed by the cells in all experimental groups. The COP group presented the smallest expression (p<0.01) of BGLAP, DSPP and HSP-27 genes. The SHEDs of the MTA+COP group presented superexpression of the BGLAP, DSPP and HSP-27 genes, and in the CaH+COP group the cells superexpressed the BGLAP and HSP-27 genes. The CaH group was the only one where there was no cell migration during whole experiment. Migration of all other groups was similar to that of the control group, except by the MTA group in 12 and 24 hours. The CaH+COP and MTA+COP migrated significantly more than the CaH and MTA groups, respectively. COP, alone or in association to the other biomaterials, does not interfere with the proliferation of the SHEDs, and when associated to CaH is able to annul the cytotoxicity of the CaH alone. The COP, alone or in association to the other biomaterials, does not interfere with the ALP activity. The COP alone induces the highest functional differentiation of the SHEDs and when associated to the CaH substantively improves this differentiation. The biomaterials, alone or in association, are not able to induce the expression of the DMP1 gene. The COP associated to the biomaterials induces superexpression of the genes related to the extracellular matrix formation and odontoblastic differentiation. The COP alone does not interfere with the cell migration and, when associate to CaH, completely annuls the inhibition of migration caused by this material alone. When associated to MTA the COP increases the cell migration speed compared to the effect of the MTA alone. Oil-resin copaiba (COP) promotes proliferation, differentiation and migration of stem cells from dental pulp with a proposal for a biomaterial for pulp capping.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-08-21T18:31:15Z No. of bitstreams: 1 patriciayannedeoliveira.pdf: 1047015 bytes, checksum: 1a92b1ec7fc7cdc1e6d9f8551e6c4790 (MD5) / Rejected by Adriana Oliveira (adriana.oliveira@ufjf.edu.br), reason: Favor verificar metadata.dc.contributor.advisor2: Resende on 2017-08-24T12:05:18Z (GMT) / Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-08-24T12:09:35Z No. of bitstreams: 1 patriciayannedeoliveira.pdf: 1047015 bytes, checksum: 1a92b1ec7fc7cdc1e6d9f8551e6c4790 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-08-25T12:00:33Z (GMT) No. of bitstreams: 1 patriciayannedeoliveira.pdf: 1047015 bytes, checksum: 1a92b1ec7fc7cdc1e6d9f8551e6c4790 (MD5) / Made available in DSpace on 2017-08-25T12:00:33Z (GMT). No. of bitstreams: 1 patriciayannedeoliveira.pdf: 1047015 bytes, checksum: 1a92b1ec7fc7cdc1e6d9f8551e6c4790 (MD5) Previous issue date: 2017-07-27 / Objetivo: Neste estudo in vitro, avaliou-se a proliferação de células-tronco de polpa dentária (DPSCs) após a aplicação de laser de baixa intensidade. Métodos: A análise da proliferação de DPSCs cultivadas com DMEM e SFB a 10% foi realizada pelo ensaio de redução de MTT. Estas células foram irradiadas a cada 12 horas durante 72 horas ou de 24 em 24 horas durante 72 horas, com um laser Vermelho-InGaAlP (660nm, 30mW e 0,5 ou 1J/cm2) durante 16 ou 33 segundos. O melhor parâmetro dado pelo ensaio do MTT foi utilizado para analisar a diferenciação osteogênica e a viabilidade celular através do teste Trypan Blue. Para a análise estatística foi utilizado o teste ANOVA com nível de significância de 5% (p <0,05). Resultados: Através do MTT, foi possível observar que a menor dose de laser (0,5J /cm2) em aplicações as 0 e 48 horas obteve as melhores taxas de proliferação comparada a todos os outros grupos. Além disso, o laser de baixa intensidade não influenciou estatísticamente na diferenciação osteogênica e na viabilidade celular após a coloração com o vermelho de alizarina e o teste Trypan Blue no melhor parâmetro encontrato pelo MTT (0,5J/cm2). Conclusões: Ao analisar os resultados e considerando os parâmetros utilizados, podemos observar que o laser de baixa intensidade é uma ferramenta que favorece a proliferação de DPSCs. Finalmente, outros estudos devem ser realizados a fim de melhor definir os parâmetros para as aplicações de células-tronco. / Purpose: In this in vitro study, the proliferation of dental pulp stem cells (DPSCs) was evaluated after the application of low intensity laser. Methods: Analysis of the proliferation of DPSCs cultured with DMEM and 10% FBS was performed by the MTT reduction assay. These cells were irradiated every 12 hours for 72 hours or every 24 hours for 72 hours, with a RedInGaAlP laser (660nm, 30mW and 0.5 or 1J/cm2) for 16 or 33 seconds. The best parameter recorded by MTT was used to analyze the osteogenic differentiation and viability using the Trypan Blue test. For the statistical analysis the ANOVA test was used with significance level of 5% (p <0.05). Results: Through the MTT, it was possible to observe that the lowest dose of the laser (0.5J/cm2) in applications at 0 and 48 hours obtained the best proliferation rates then all the other groups. In addition, the low level laser did not appear to influence on the osteogenic differentiation nor the viability of the cells by the Trypan Blue test in the best parameter found by MTT (0.5J /cm2). Conclusions: When analyzing the results and considering the parameters used, we can observe that the low intensity laser is a tool that favors the proliferation of dental pulp stem cells. Finally, further studies should be carried out in order to better define parameters for stem cell applications.

No description available.

Cette thèse concerne la fonctionnalisation des biomatériaux, le titane et l'alliage de titane Ti6Al4V ainsi que le PEEK (Poly-Éther-Éther-Ketone) pour l'application de ces biomatériaux dans les domaines de l'implantologie dentaire et de la reconstruction maxillo-faciale. Au cours de la première partie de ce travail nous avons synthétisé quatre peptides bi-fonctionnels avec une grande affinité pour la surface implantaire de Ti et de Ti6Al4V et également pour les kératinocytes oraux. La spectroscopie de force de la cellule unique (Single Cell Force Spectroscopy - AFM) a été utilisée pour étudier l'adhésion d'une cellule sur les surfaces fonctionnalisées par les quatre peptides bi-fonctionnels. A l'aide du test colorimétrique de para-nitrophenyl phosphate (pNPP) on a démontré l'adhésion des kératynocytes après 4 heures d'incubation sur les surfaces fonctionnalisées avec les peptides bi-fonctionnels. Les résultats de ces études ont démontré la présence d'un peptide bi-fonctionnel qui augmente l'adhésion cellulaire instantanée et l'adhésion 4h après incubation des kératinocytes oraux sur les métaux. Ce peptide résiste à l'influence de facteurs externes tels que l'adsorption d'albumine et représente une proposition prometteuse pour la fonctionnalisation de l'implant de Ti et de Ti6Al4V. La deuxième partie de notre mémoire de thèse décrit une méthode de dépôt par spray des films des multicouches de PLL/PGA (poly-l-lysine/acide polyglutamique), des protéines et des cellules souches pulpaires. Les outils de spray d'usage unique ont été adaptés aux exigences de la bonne pratique de fabrication (GMP - good manufacturing practice). Les premiers tests de prolifération cellulaire ont démontré qu'elle n'a pas été affectée par le spray. L'étude en AFM a démontré que la rugosité et l'épaisseur du film augmentaient exponentiellement avec l'augmentation du nombre des couches déposées. Le traitement physique par UV rayonnement ainsi que le traitement par la déshydratation et la réhydratation des films ont provoqué des changements dans l'épaisseur, l'élasticité et la dureté des films. Le dépôt des protéines sur les MPEs a aussi augmenté l'épaisseur et a influencé la dureté de la surface. Les changements chimiques de la structure des MPEs après le traitement physique et après le dépôt des protéines naturelles ont été étudiés par la spectroscopie Raman. La prolifération cellulaire au 1er, 3e et 8e jours après leur spray sur des films de MPEs et des protéines a été ensuite évaluée. Les MPEs traitées par UV combiné avec déshydratation/réhydratation ainsi que les PEMs couvertes par protéine - CaP ont entrainé une meilleure adhésion et prolifération cellulaires que les MPEs non-traitées. Finalement la méthode de dépôt par spray des MPEs, protéines et aussi des cellules souches pulpaires a été appliquée sur la surface de PEEK. La meilleure prolifération cellulaire au 8e jour après le spray était sur la surface couverte par le film de (PLL-PGA)5-protéine/CaP. Une étude in vitro sur le degré de minéralisation au 21e jour après l'incubation des cellules pulpaire dans un milieu ostéconductuer sur des différentes (MPEs couvertes-PEEK) surfaces a été effectuée. Les résultats de la microscopie électronique à balayage (MEB), microscopie électronique à transmission (MET), combiné avec des études histologiques ont confirmé la formation d'une matrice extracellulaire minéralisée des autour des cellules pulpaires sur la surface de PEEK. Conclusions: Dans ce travail nous avons décrit une nouvelle approche de fonctionnalisation des surfaces de Ti and Ti6Al4V par des peptides bi-fonctionnels en proposant une séquence prometteuse qui augmente l'adhésion des kératinocytes oraux. Ensuite nous avons développé une méthode de fonctionnalisation de la surface de PEEK par des multicouches de polyeléctrolytes, des protéines naturelles et des cellules souches de la pulpe dentaire. La différentiation ostéoblastique in vitro a été finalement évaluée. / The objective of this thesis was to develop new techniques of surface functionalization of titanium; titanium alloy (Ti6Al4V) and PEEK (poly-ether-ether ketone) surfaces for their application on dental implantology and maxillofacial surgery. During the first part of the thesis we have synthesized four metal binding-cell specific peptides (MCSPs) with high affinity to titanium surface and to oral keratinocytes cells. Single Cell Force Spectroscopy (AFM) was used to study the instantaneous cell adhesion force of keratinicyte cell on MCSPs functionalized surfaces. The colorimetric para-nitrophenyl phosphate (pNPP) essay demonstrates the surface cell adhesion four hours after incubation. The results demonstrate the presence of one bi-functional peptide (MCSP-2) who increases both: the instantaneous and the 4 h cell adhesions. MCSP-2 resist to the influence of external factors like BSA adsorption and could be an interesting candidate for implant surface functionalisation. In the second part of the thesis we have developed a spray deposition method of polyelectrolyte multilayer (PEM) films build. PLL/PGA (poly-l-lysine/poly-glutamic acid) and proteins were used for surface coating. Consequently dental pulp stem cells (DPSC) are sprayed on the PEM films. The aims were to improve a spray -method for cells and the polyelectrolytes deposition on the PEEK implant. The entire spray device was designed for single use, which correspond to good manufacturing practice (GMP) conditions. Cell proliferation in 24 hours after spraying was not disturbed by the spray. Physicochemical properties of PEMs deposited by spray on glass surfaces were performed by Atomic Force Microscopy (AFM) and by contact angle measurements. The results have demonstrated the changes in films thickness and films roughness with increasing numbers of layers corresponding to exponential growth of the films. Physical treatment by UV irradiation and drying-wetting process affects the film thickness and the film elasticity and increases the stiffness of the film. The deposition of protein-CaP and collagen coatings on PEM films increased the layer thickness and influenced the hardness of the surface. Chemical changes in the polyelectrolyte structure during the physical treatment and after proteins deposition were studied by Raman spectroscopy. Cell proliferation of the pulp cells at 1st, 3th and 8th days after pulp cells deposition on PEMs coated glass surfaces, was then evaluated. The results demonstrated that 10 UV/ dray/wetted films and the natural proteins coated films enhanced cell adhesion and cell proliferation. The protein-CaP PEMs covered surface creates the best microenvironment to ensure the cell behavior. Finally PEM films coatings are applied on PEEK implant surface. The AFM study shows the changes of homogeneity and roughness of this surface after PEM film deposition. Contact angle measurement demonstrates decreases of surface hydrophobicity. Cell proliferation of dental pulp stem cells (DPSC) on (PLL-PGA)5-protein/CaP coated PEEK surface was highest compared to other functionalized surfaces. In vitro study of DPSCs osteogenic differentiation was evaluated by the degree of mineralization of the extracellular matrix on the PEEK surface at 21st day after cell incubation. Scanning electron microscopy (SEM), transmission electron microscopy (TEM) combined with histological studies improved the formation of mineralized extracellular matrix and confirmed the osteogenic differentiation of DPSCs on the PEMs coated PEEK surface. Conclusions: In this work we validated the method of Ti and Ti6Al4V functionalization with bi-functional peptides. One peptide that could increase the epithelial cell adhesion to the surface was proposed. Spray deposition technics of PEMs, protein and pulp stem cells were applied for PEEK implant functionalization. Finally we evaluated the differentiation of dental pulp stem cells on the PEEK surface in vitro.

A Dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree Of Master of Science in Medicine, 2016. / Dental stem cells (DSCs) have been identified in teeth and their supporting tissues. They represent an exclusive source of adult stem cells, easily isolated and manipulated for tissue repair and regeneration. This research project evaluated the neurogenic potential of the dental pulp stem cells (DPSCs) and stem cells from the pulp of human exfoliated deciduous teeth (SHEDs) in a South African cohort. Sixty non-carious permanent and deciduous teeth were extracted from healthy patients aged between 18 and 30 years and 5 and 10 years, at the University of the Witwatersrand's Oral Health Clinic in Johannesburg Charlotte Maxeke Academic Hospital, South Africa. The cells, isolated from the extracted pulp tissue were cultured, counted and then phenotyped by flow cytometry analysis. The cells were further expanded in a neural induction medium and immunocytochemistry analysis for Ki-67, doublecortin (DCX) and nestin were performed. Large colonies of both DPSCs and SHEDS were harvested from the extracted pulp tissues and positively cultured. Flow cytometry analysis confirmed the presence of CD44+ and CD29+ cells as well as the known mesenchymal stem cell markers CD90 and CD105. Both DPSCs and SHEDs demonstrated successful proliferation and neural differentiation. This study confirmed that DPSCs and SHEDs are highly proliferative human adult stem cells that exhibit a neurogenic potential that may contribute in the treatment of neurological disorders. / AC2017

Mais de 200 mutações patogênicas já foram descritas no gene que codifica a Cx26 (GJB2) que levam à surdez hereditária. A mais frequente destas mutações é a c.35delG. Ela é a principal causa de surdez não sindrômica com padrão de herança autossômico recessivo na população brasileira e em diversas populações do mundo. O genoma humano contém 21 diferentes genes de proteínas da família das conexinas que são expressos em diversos tecidos. Os canais comunicantes e hemicanais formados por conexinas facilitam a passagem de pequenos metabólitos entre as células adjacentes e entre a célula e o meio extracelular, promovendo a homeostasia celular. Este estudo teve o objetivo de avaliar os efeitos da mutação c.35delG em homozigose no gene GJB2 em SHEDs sobre a diferenciação celular e sobre a expressão das Cx26 (GJB2), Cx30 (GJB6), Cx31 (GJB3), Cx43 (GJA1) e Cx50 (GJA8) em SHEDs (Stem cells from human exfoliated deciduous teeth). Para isso, obtivemos linhagens de SHEDs a partir de 3 indivíduos portadores da mutação c.35delG em homozigose e de 3 indivíduos controle, sem a mutação. Nossos resultados indicaram que SHEDs portadoras da mutação apresentam maiores taxas de diferenciação em adipócitos e em osteócitos do que SHEDs de indivíduos controle. Por meio de RT-PCR, RT-PCR quantitativa e citometria de fluxo identificamos a expressão dos genes GJB2 (Cx26), GJB6 (Cx30) e GJA1 (Cx43) e suas respectivas proteínas, tanto em SHEDs dos indivíduos com a mutação como em SHEDs de indivíduos controle. Não há expressão dos genes GJA8 (Cx50) e GJB3 (Cx31) e suas respectivas proteínas em SHEDs. Por meio de RT-PCR quantitativa observamos aumento dos níveis de expressão do RNAm de GJA1 e redução de RNAm de GJB2 em SHEDs oriundas de pacientes com c.35delG, quando comparadas às de amostras controle. Resultados obtidos por meio de Western Blotting mostraram aumento de aproximadamente 50% na expressão da Cx43 corroborando os resultados obtidos por meio de RT-PCR quantitativo. Detectamos que a expressão da Cx26 em células de indivíduos com mutação é 50% menor do que em células de indivíduos controle. Os aumentos observados das taxas de diferenciação em adipócitos e em osteócitos podem estar relacionados ao aumento da expressão da Cx43. Sabe-se que a Cx43 possui importante papel na manutenção de pré-adipócitos durante a fase de expansão clonal na adipogênese. A Cx43 também está relacionada à sinalização celular nas vias de proliferação e diferenciação durante a osteogênese. Indivíduos portadores da mutação c.35delG apresentam surdez sem outros fenótipos associados sugerem que ocorra redundância funcional entre as conexinas. Além disso, o aumento da expressão de GJA1 (Cx43) nas SHEDs oriundas dos indivíduos surdos com a mutação c.35delG falam a favor da existência de um mecanismo de regulação compensatório a ser esclarecido, que aumenta a síntese de Cx43 na redução ou ausência da Cx26 funcional / More than 200 patogenic mutations in the connexin 26 gene (GJB2) were associated to deafness. The most frequent is the c.35delG mutation, which is the most common cause of nonsydromic recessive hearing loss in the Brazilian population, and also in several populations in the world. The human genome contains 21 genes that comprise the gene family of the connexins, expressed in many different tissues. Connexins are components of gap junctions and hemichannels, which facilitate the transference of small metabolites between cells and between cells and the extracelular medium. The aim of this study was to investigate the effects of the c.35delG mutation on adipogenesis, chondrogenesis and osteogenesis, and also its effects on mRNA and protein expression related to Cx26 (GJB2), Cx30(GJB6), Cx31 (GJB3), Cx43 (GJA1) and Cx50 (GJA8) in SHEDs (stem cells from human exfoliated deciduous teeth). For this purpose, 3 SHED lines from patients with c.35delG mutation in homozygosis and 3 lines from individuals without mutation were established. We observed that the rates of induced differentiation into adipocytes and osteocytes were higher in SHEDs from individuals with the c.35delG mutation than in SHEDs from control individuals. By RT-PCR, real time RT-PCR and flow cytometry were detected the expression of GJB2, GJB6 and GJA1 genes and their respective proteins Cx26, Cx30 and Cx43 in SHEDs from control samples and in SHEDs with the mutation. The expression of GJA8 (Cx50) and of GJB3 (Cx31) were not detected in SHEDS from both groups. Quantitative gene expression analysis using real-time RT-PCR revealed significantly elevated levels of GJA1 mRNA expression and decreased GJB2 mRNA expression in cells from patients with c.35delG, when compared to cells from normal controls. Western Blotting analysis showed an increase of about of 50% of the protein Cx43 in cells with c.35delG mutation, in comparison to cells from normal controls, in accordance with the real-time RT-PCR results. We detected that Cx26 protein expression in samples with c.35delG mutation is approximately 50% lower than in SHEDs from normal controls. The observed increase in differentiation rates related to adipogenesis and osteogenesis may be explained by the higher levels of Cx43 expression. This is supported by the fact that Cx43 was reported to play an important role in the maintenance of pre-adipocytes during clonal expansion and it was also related to cell signaling pathways in proliferation and differentiation during osteogenesis. Individuals with c.35delG in homozygosis present only deafness, without other symptoms, which suggests that functional redundancy between connexins may exist. The increase of Cx43 expression in SHEDs from deaf patients with c.35delG mutation found by us may be related to a compensation mechanism to be clarified, which results in increase of the production of Cx43 when functional connexin Cx26 is reduced or absent

Síndrome de Angelman (AS - MIM 105830) é causada pela ausência de função do gene UBE3A que codifica uma proteína ubiquitina - ligase (E6-AP). Esse gene é expresso bialelicamente em vários tecidos exceto no cérebro, onde a expressão é preferencialmente materna. O RNA anti-senso de UBE3A é considerado o regulador dessa expressão diferencial entre os alelos, e faz parte de um transcrito grande que só o alelo paterno é expresso devido ao imprinting genômico; no cérebro, esse transcrito se entende até a região anti-senso de UBE3A, mas nos demais tecidos o transcrito é menor e não engloba a região anti-senso. Este trabalho visa obter um modelo para estudo da AS. Células-tronco da polpa do dente (SHEDs) de pacientes com deleção do segmento 15q11-q13 ou mutação no gene UBE3A foram caracterizadas e submetidas à diferenciação neuronal. A diferenciação foi analisada através do estudo de RNA e proteínas para marcadores neuronais e, também, por testes funcionais. As SHEDs são células-tronco mesenquimais e constituem uma população heterogênea. Essas células ou algumas dessas células já expressam algumas proteínas neuronais ou de células excitáveis como nestina, &beta;-tubulina III, MAP2 e proteína de canais dependentes de voltagem de sódio e potássio. Um ponto interessante é que as SHEDs apresentam baixa expressão do UBE3A anti-senso e a expressão do UBE3A nas células de pacientes é menor que 50% da expressão encontrada nas células de controles, que pode indicar a ocorrência de expressão preferencial materna desse gene em outros tipos celulares além de neurônios maduros. Quando induzidas à diferenciação neurogênica, a maioria das linhagens controles apresentou aumento da expressão de MAP2 e, principalmente, &beta;-tubulina III; e a maioria das linhagens de pacientes com AS não apresentou aumento notável na expressão dessas proteínas, exceto uma linhagem de paciente que aumentou a expressão de &beta;-tubulina III. As células induzidas à diferenciação apresentaram aumento estatisticamente significativo da condutância de sódio através de canais de sódio dependentes de voltagem. Com a análise de expressão de UBE3A e do UBE3A anti-senso é possível afirmar que a expressão deles não alterou com a diferenciação neuronal. Assim, é possível concluir que as células-tronco da polpa do dente, com o protocolo de diferenciação neurogênica, progrediram na via de diferenciação, mas a maioria das células não atingiu o estágio de maturação necessário para que ocorresse o imprinting do UBE3A ou a via de diferenciação não ia em direção a neurônios que apresentam imprinting do UBE3A. / Angelman syndrome (AS - MIN 105830) is caused by the loss of function of the maternal UBE3A gene, which encodes an ubiquitin protein ligase (E6-AP). UBE3A displays biallelic expression in most of tissues, but maternal predominant expression is observed in the brain. A RNA antisense that is paternally expressed in some regions in the brain is considered to be responsible for this tissue-specific imprinting; UBE3A antisense is part of a large transcript that starts at SNURF-SNRPN gene and is paternally expressed, and in the brain this transcript includes UBE3A antisense region however in other tissues this region is not included. The aim of the present study is to develop a new model for studying AS. Dental pulp stem cells (SHEDs) were characterized and differentiated by an already described protocol. SHEDs intrinsically express some neuronal proteins as nestin, &beta;-tubulin III, MAP2 and voltage-gated sodium channels and potassium channels. Interestingly, SHEDs also present a low expression of UBE3A antisense, and UBE3A expression in cells from patients with AS is lower than 50% of the cells from normal control, so it is possible that preferential maternal expression of this gene might occur in some cells beyond mature neurons. After the neuronal differentiation, most control lineages and one lineage of AS patients had an increase of MAP2 and &beta;-tubulin III expression. Two control lineages and most lineages from AS patients did not have a notable increase of expression of these proteins. Neuronal differentiated cells displayed an increase in conductance through voltage-gated sodium channels. Analysis of UBE3A and UBE3A antisense expression in SHEDs and cells induced to differentiate into neurons indicated no changes in their expression. Thus, after neuronal differentiation induction, dental pulp stem cells progressed through neuronal differentiation pathway. However, most cells did not reach the stage which UBE3A imprinting occurs or the neuronal differentiation is resulting in a cell that do not present UBE3A imprinting.

Malgré le développement de biomatériaux de plus en plus nombreux dans le domaine des greffes osseuses et de la préservation alvéolaire, les résultats sont toujours insuffisants pour assurer des reconstructions ad integrum du tissu osseux. Les techniques d’ingénieries osseuses semblent être la piste à privilégier pour améliorer nos techniques chirurgicales. Le silicium poreux est un matériau prometteur pour l’ingénierie tissulaire et notamment pour la régénération osseuse. En effet, son état de surface permet une adhésion cellulaire importante et ses propriétés non toxique et résorbable en fond un matériau porteur de cellules souches intéressant. Les cellules souches de la pulpe dentaire (DPSC) sont des cellules facilement accessibles dans la cavité buccale. Leurs capacités de prolifération et de différenciation associées au silicium poreux semblent être un atout pour les applications thérapeutique pour la régénération osseuse. Des résultats d’études ultérieures in vitro ont montré leur fort intérêt à une application in vivo. Dans ce travail thèse, nous avons tester l’association silicium poreux et cellules souches de la pulpe dentaire, aux même caractéristiques énoncées dans l’étude de référence in vitro, chez l’animal. Pour cela, le matériau a été produit sous forme de particules de manière a être utilisé comme moyen de comblement osseux, associé ou non à des DPSC. Le modèle de queue de rat a été développé et tester pour diminuer le nombre d’animaux nécessaire à l’étude tout en conservant la puissance statistique des résultats. Les études ont montré la possibilité d’utiliser ce modèle pour la régénération de défauts osseux crées chirurgicalement. De plus, il semblerait que ce modèle puisse également être utile pour les études sur l’ostéointégration de système implantables et sur la régénération osseuse autour de ces implants. Le silicium poreux a ensuite été testé dans ces conditions, associé ou non aux DPSC, en comparaison avec un témoin positif et un témoin négatif. Cette association est apparue comme une piste prometteuse pour la régénération osseuse in vivo. / Despite the development of biomaterials in the field of bone grafts and alveolar preservation, the results are no sufficient to made reconstructions ad integrum of bone tissue. Bone engineering techniques seem to be the preferred way to improve our surgical techniques. Porous silicon is a promising material for tissue engineering and especially for bone regeneration. Indeed, its surface allows cell adhesion. And then, it’s a non-toxic and bioresorbable interesting material properties carrying stem cells. Dental pulp stem cells (DPSC) are easily accessible cells in the oral cavity. Their proliferation and differentiation capacities associated with porous silicon appear to be attractive for therapeutic applications in bone regeneration. The results of the in vitro studies have shown the interest for in vivo application. In this thesis, we have tested the combination of porous silicon and dental pulp stem cells in vivo experimentation, using the same characteristics of the in vitro reference study. For this, the material was produced in particle form to be used as bone filling material, associated or not with DPSC. The rat-tail model was developed and tested to reduce the number of animals needed for the study while maintaining the statistical power of the results. Studies have shown the possibility of using this model for bone regeneration defects surgically created. In addition, it seems that this model can also be useful for studies on osseointegration of implantable systems and bone regeneration around these implants. Then, the porous silicon was tested under these conditions, with or without DPSC, in comparison with a positive control and a negative control. This association has emerged as a promising approach for bone regeneration in vivo.