O objetivo do estudo foi avaliar as características da dentina cariada remanescente de molares decíduos quanto à coloração, consistência e microdureza, após um período médio de 3 anos e 8 meses da realização do capeamento pulpar indireto. O estudo clínico constou de 27 dentes que apresentavam lesões cariosas ativas com expressão radiográfica em metade interna de dentina. Aleatoriamente, foi realizado o capeamento pulpar indireto e utilizado como material capeador o hidróxido de cálcio (HC) e o cimento de ionômero de vidro resinoso modificado (CIVRM). As crianças foram acompanhadas através de exames clínicos e radiográficos, e ao longo de um período médio de 3 anos e 8 meses, observou-se um índice de sucesso de 89% para o grupo do HC e de 93% para o CIVRM, não havendo diferença estatisticamente significante entre os dois grupos (p=0,62). Os dentes considerados como sucesso no estudo clínico foram agrupados em um único grupo (grupo teste), que constou de 13 dentes que sofreram esfoliação natural ou exodontia por motivos ortodônticos. Além disso, foram selecionados 15 molares decíduos hígidos (grupo controle positivo) e 15, portadores de lesões cariosas ativas em metade interna de dentina (grupo controle negativo). As amostras do grupo teste tiveram suas respectivas restaurações removidas, a profundidade medida e a dentina remanescente avaliada por um operador calibrado, seguindo critérios descritivos, quanto à consistência e a coloração. Nos dentes do grupo controle positivo, foram realizados preparos cavitários oclusais em até 4 mm de profundidade, enquanto que, no grupo controle negativo, o mesmo operador do estudo clínico realizou a remoção parcial de tecido cariado in vitro. A partir daí, todos os dentes foram preparados para análise de microdureza, que foi realizada por um examinador calibrado, que empregou o princípio de cegamento. Na análise da consistência, todos os dentes do grupo teste (n=13) apresentaram-se endurecidos, enquanto que 9, apresentaram coloração amarela-clara (8 do CIVRM e 1 do HC) e 4, castanho-escura (1 do CIVRM e 3 do HC). Para o teste de microdureza no grupo teste, obteve-se uma média KHN de 40,81 (±16,28) MPa, enquanto que nos grupos controles positivo e negativo, foram alcançados valores médios de 62,73 (±11,24) MPa e 19,15 (±6,99) MPa, respectivamente. A análise estatística mediante o teste ANOVA indicou que houve diferença significativa entre os 3 grupos. Foi constatada a remineralização da dentina de dentes decíduos em que foi realizada a técnica do capeamento pulpar indireto após um período médio de 3 anos e 8 meses, através de critérios clínico (consistência) e laboratorial (análise da microdureza). / The aim of this study was to evaluate the characteristics of the deciduous molars carious dentin after a period of 3 years and 8 months (mean) of the indirect pulp capping through the color, consistency and microhardness analysis. The clinical study consisted of 27 primary teeth with deep acute carious lesions that were submitted to incomplete dentin caries removal, application of calcium hydroxide (restored with composit resin) or modified resin glass ionomer cement (restored with GIC). The children had been followed through clinical and radiographics examinations, and throughout an average period of 3 years and 8 months, an index of success of 93% for the GIC and 89% for the group of the CH was observed, no statistical difference was observed between the two groups (p=0,62). The teeth considered as success in the clinical study had been grouped in one group (test) that consisted of 13 teeth that had natural esfoliation or extraction for orthodontic reasons. Fifteen sound deciduous molars (positive control group) and 15 molars with deep acute carious lesions (negative control group) were selected. The samples of the test group had its respective restorations removed, measured depth and the remaining dentine evaluated by a calibrated operator, following descriptive standards, such as consistency and color. In the positive control group, cavity preparations had been carried in up to 3-4 mm of depth, while that, in the negative control group, the same operator of the clinical study proceeded the incomplete dentin caries removal in vitro. From there, all the teeth had been prepared for microhardness analysis that was carried through by a blind and calibrated examiner. In the analysis of the consistency, all the teeth of the test group (n=13) was considered hard, while that, 9 had presented yellow-clear coloration (8 of GIC and 1 of the CH) and 4 chestnut-dark (1 of GIC and 3 of the CH). For the microhardness analysis, the test group got an average KHN of 40,81 (±16,28) MPa, while the positive and negative control group, had been reached average values of 62,73 (±11,24) MPa and 19,15 (±6,99) MPa, respectively. The analysis statistics by means of test ANOVA indicates that it had significant difference between the 3 groups. The dentin remineralization of the deciduous tooth was evidenced where the technique of indirect pulp capping was carried through after an average period of 3 years and 8 months, through clinical (consistency) and laboratorial (microhardness analysis) standards.

Made available in DSpace on 2014-06-11T19:31:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-09-05Bitstream added on 2014-06-13T19:01:54Z : No. of bitstreams: 1 crisci_fs_dr_arafo.pdf: 2406575 bytes, checksum: 7ed8c4c8f95948faa1e0430e502e380a (MD5) / Universidade Estadual Paulista (UNESP) / O presente estudo avaliou o efeito do laser de baixa intensidade diodo semicondutor de arseneto de gálio e alumínio em exposições pulpares induzidas e nos tecidos apicais e periapicais após tratamento endodôntico em dentes de macacos. Nas exposições pulpares, foi avaliado efeito do laser infra-vermelho associado ao hidróxido de cálcio, variando o tempo de aplicação, onde utilizou-se quatro macacos, totalizando 24 dentes, distribuídos em quatro grupos experimentais: Grupo I: Laser 2,5 segundos (dentes incisivos), Grupo II: Laser 40 segundos (dentes incisivos), Grupo III: Laser 40 segundos (dentes prémolares) e Grupo IV: Controle sem Laser (dentes pré-molares), por um período de 55 dias. Quanto ao efeito do laser sobre a agressividade tecidual do cimento endodôntico óxido de zinco e eugenol (OZE), após tratamento endodôntico nos tecidos apicais e periapicais, foi comparando o laser vermelho com o infravermelho, utilizando quatro macacos, totalizando 24 dentes, distribuídos em três grupos experimentais: Grupo I (Laser Vermelho), Grupo II (laser Infra-Vermelho) e Grupo III (Controle: Sem Laser), por um período de 22 dias. Decorrido os períodos experimentais de cada estudo, os animais foram mortos, os dentes ou peças removidas e preparadas para análise histológica. De acordo com a proposta e as condições específicas deste trabalho, os resultados nos permitem concluir que nas exposições pulpares, a irradiação com laser infra-vermelho (40 segundos) diminuiu a reação inflamatória e induziu a organização tecidual, bem como na formação da barreira mineralizada, apresentando diferenças estatísticas significantes entre os grupos (p<0,05), tendo como melhor resultado a irradiação com laser infra-vermelho (40 segundos), já nos tecidos apicais e periapicais a irradiação... / The present study evaluated the effect of the laser of low intensity diode semiconductor of gallium aluminum arsenide in exposed pulp induced in apical and periapical tissues after endodontic treatment in teeth of monkeys. In the exposed pulp, the effect of the laser infra-red associated to the hydroxide of calcium was evaluated varying the time of application, being used 04 monkeys, totaling 24 teeth, distributed in four experimental groups: Group I: Laser 2,5 seconds (incisive teeth), Group II: Laser 40 seconds (incisive teeth), Group III: Laser 40 seconds (premolar teeth) and Group IV: Control without Laser (premolar teeth), for a period of 55 days. As for the effect of the laser on the tissue aggressiveness of the endodontic zinco oxide and eugenol (OZE) sealer, after endodontic treatment o in the apical and Periapical tissues, the red laser was compared with the laser infra-red, using 04 monkeys, totaling 24 teeth, distributed in 03 experimental groups: Group I (Red Laser), Group II (Infra-red laser) and Group III (it Controls: Without Laser), for a period of 22 days. After the experimental periods of each study, the animals were killed, the teeth or pieces were removed and prepared for histological analysis. In agreement with the proposal and the specific conditions of this study work, the results allow to conclude us that in exposed pulp infra-red laser irradiation (40 seconds.) reduced the inflammatory reaction and induced the tissue organization, as well as the mineralized barrier formation and in apical and periapical tissues infra-red laser irradiation stimulated the cells of the periodontal tissue inducing periapical repair.

O objetivo do estudo foi avaliar as características da dentina cariada remanescente de molares decíduos quanto à coloração, consistência e microdureza, após um período médio de 3 anos e 8 meses da realização do capeamento pulpar indireto. O estudo clínico constou de 27 dentes que apresentavam lesões cariosas ativas com expressão radiográfica em metade interna de dentina. Aleatoriamente, foi realizado o capeamento pulpar indireto e utilizado como material capeador o hidróxido de cálcio (HC) e o cimento de ionômero de vidro resinoso modificado (CIVRM). As crianças foram acompanhadas através de exames clínicos e radiográficos, e ao longo de um período médio de 3 anos e 8 meses, observou-se um índice de sucesso de 89% para o grupo do HC e de 93% para o CIVRM, não havendo diferença estatisticamente significante entre os dois grupos (p=0,62). Os dentes considerados como sucesso no estudo clínico foram agrupados em um único grupo (grupo teste), que constou de 13 dentes que sofreram esfoliação natural ou exodontia por motivos ortodônticos. Além disso, foram selecionados 15 molares decíduos hígidos (grupo controle positivo) e 15, portadores de lesões cariosas ativas em metade interna de dentina (grupo controle negativo). As amostras do grupo teste tiveram suas respectivas restaurações removidas, a profundidade medida e a dentina remanescente avaliada por um operador calibrado, seguindo critérios descritivos, quanto à consistência e a coloração. Nos dentes do grupo controle positivo, foram realizados preparos cavitários oclusais em até 4 mm de profundidade, enquanto que, no grupo controle negativo, o mesmo operador do estudo clínico realizou a remoção parcial de tecido cariado in vitro. A partir daí, todos os dentes foram preparados para análise de microdureza, que foi realizada por um examinador calibrado, que empregou o princípio de cegamento. Na análise da consistência, todos os dentes do grupo teste (n=13) apresentaram-se endurecidos, enquanto que 9, apresentaram coloração amarela-clara (8 do CIVRM e 1 do HC) e 4, castanho-escura (1 do CIVRM e 3 do HC). Para o teste de microdureza no grupo teste, obteve-se uma média KHN de 40,81 (±16,28) MPa, enquanto que nos grupos controles positivo e negativo, foram alcançados valores médios de 62,73 (±11,24) MPa e 19,15 (±6,99) MPa, respectivamente. A análise estatística mediante o teste ANOVA indicou que houve diferença significativa entre os 3 grupos. Foi constatada a remineralização da dentina de dentes decíduos em que foi realizada a técnica do capeamento pulpar indireto após um período médio de 3 anos e 8 meses, através de critérios clínico (consistência) e laboratorial (análise da microdureza). / The aim of this study was to evaluate the characteristics of the deciduous molars carious dentin after a period of 3 years and 8 months (mean) of the indirect pulp capping through the color, consistency and microhardness analysis. The clinical study consisted of 27 primary teeth with deep acute carious lesions that were submitted to incomplete dentin caries removal, application of calcium hydroxide (restored with composit resin) or modified resin glass ionomer cement (restored with GIC). The children had been followed through clinical and radiographics examinations, and throughout an average period of 3 years and 8 months, an index of success of 93% for the GIC and 89% for the group of the CH was observed, no statistical difference was observed between the two groups (p=0,62). The teeth considered as success in the clinical study had been grouped in one group (test) that consisted of 13 teeth that had natural esfoliation or extraction for orthodontic reasons. Fifteen sound deciduous molars (positive control group) and 15 molars with deep acute carious lesions (negative control group) were selected. The samples of the test group had its respective restorations removed, measured depth and the remaining dentine evaluated by a calibrated operator, following descriptive standards, such as consistency and color. In the positive control group, cavity preparations had been carried in up to 3-4 mm of depth, while that, in the negative control group, the same operator of the clinical study proceeded the incomplete dentin caries removal in vitro. From there, all the teeth had been prepared for microhardness analysis that was carried through by a blind and calibrated examiner. In the analysis of the consistency, all the teeth of the test group (n=13) was considered hard, while that, 9 had presented yellow-clear coloration (8 of GIC and 1 of the CH) and 4 chestnut-dark (1 of GIC and 3 of the CH). For the microhardness analysis, the test group got an average KHN of 40,81 (±16,28) MPa, while the positive and negative control group, had been reached average values of 62,73 (±11,24) MPa and 19,15 (±6,99) MPa, respectively. The analysis statistics by means of test ANOVA indicates that it had significant difference between the 3 groups. The dentin remineralization of the deciduous tooth was evidenced where the technique of indirect pulp capping was carried through after an average period of 3 years and 8 months, through clinical (consistency) and laboratorial (microhardness analysis) standards.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / In addition to chemical mechanical preparation, pulp therapy for primary teeth requires a root canal filling that is able to maintain the antimicrobial activity to sanitize the root canal. Since the materials are in close contact with the dental tissues, it is essential to perform biocompatibility testing to certify the safety of these products for clinical application. The aim of this study was to evaluate the cytotoxicity, oxidative stress, and genotoxicity in vitro of four iodoform pastes, three calcium hydroxide pastes, and their main components. Peripheral blood mononuclear cells and DNA from calf thymus were exposed to extracts of these products. Cytotoxicity was assessed with an MTT assay. The generation of reactive oxygen species (ROS) was evaluated by a DCFH-DA assay, and lipid peroxidation was evaluated by a TBARS assay. The genotoxicity was evaluated using alkaline comet assay and GEMO assay. All tests were performed at 24 and 72 h, except for GEMO. After Kolmogorov Smirnov test, the results were analyzed by Kruskal Wallis and Dunn s test, ANOVA, and Dunnett s test (p<0.05). In the MTT assay, the chlorhexidine, Maxitrol®, neomycin sulfate + bacitracin pastes, and their components (chlorhexidine, propylene glycol, and camphorated parachlorophenol) decreased cell viability after 24 h. After 72 h, no change was observed in cell viability and lipid peroxidation for any of the groups. Lipid peroxidation was observed with exposure to calcium hydroxide pastes, calcium hydroxide, and iodoform after 24 h. Exposure to chlorhexidine, Guedes-Pinto, calcium hydroxide pastes, chlorhexidine, neomycin sulfate + bacitracin, camphorated parachlorophenol, iodoform, and calcium hydroxide resulted in an increase in ROS after 24 h, whereas propylene glycol, iodoform pastes, and their components (except for iodoform alone), increased the ROS after 72 h. In the comet assay, damaged DNA was not present with exposure to iodoform pastes for both times. However, in the GEMO assay, the chlorhexidine paste showed genotoxic effects. Calcium hydroxide paste and calcium hydroxide caused DNA damage in both tests. Thus, the pastes and components varied in their ability to induce cytotoxicity, genotoxicity, and oxidative stress. In general, the Guedes-Pinto, Maxitrol®, and neomycin sulfate + bacitracin pastes exhibited better biocompatibility in vitro. / A terapia pulpar em dentes decíduos requer como complemento ao preparo químico-mecânico, pastas obturadoras capazes de manter o poder antimicrobiano, ajudando na sanificação do canal radicular. Por estabelecerem íntimo contato com os tecidos dentários, é imprescindível a realização de testes de biocompatibilidade que atestem a segurança desses produtos para aplicação clínica. O objetivo desse estudo foi avaliar a capacidade de quatro pastas iodoformadas, três pastas de hidróxido de cálcio, e seus principais componentes, em causar citotoxicidade, estresse oxidativo e genotoxicidade in vitro. Células mononucleares de sangue periférico e DNA purificado de timo de bezerro (teste GEMO) foram expostos a extratos desses produtos. O ensaio MTT avaliou a citotoxicidade. A geração de espécies reativas de oxigênio foi avaliada pelo teste DCFH-DA, e a peroxidação lipídica pelo ensaio TBARS. A genotoxicidade foi avaliada pelo Ensaio Cometa Alcalino e GEMO. Todos os testes foram realizados em 24 e 72 horas, exceto o GEMO. Após teste de normalidade Kolmogorov-Smirnov, os resultados foram analisados por Kruskal-Wallis e Dunn, ANOVA e Dunnet, com p<0,05. No MTT, as pastas Clorexidina, Maxitrol®, Sulfato de Neomicina+Bacitracina, os componentes clorexidina, PMCC e o propilenoglicol diminuiram a viabilidade celular em 24h. Em 72h, nenhum produto provocou diminuição da viabilidade celular e lipoperoxidação. A lipoperoxidação foi causada por pastas de hidróxido de cálcio, o hidróxido de cálcio e o iodofórmio em 24h. O aumento das ERO foi provocado pelas pastas Clorexidina, Guedes-Pinto, e pastas de hidróxido de cálcio,além dos produtos clorexidina, Sulfato de Neomicina+Bacitracina, PMCC, iodofórmio e hidróxido de cálcio em 24h, e pelo propilenoglicol, pastas iodoformadas e seus componentes, com exceção do iodofórmio, em 72h. No ensaio cometa, nenhuma pasta iodoformada danificou o DNA em ambos os tempos experimentais. Enquanto no GEMO, a pasta clorexidina mostrou efeito genotóxico. As pastas à base de hidróxido de cálcio e o hidróxido de cálcio causaram danos ao DNA nos dois ensaios. Concluiu-se que as pastas e componentes avaliados variaram quanto à capacidade de indução de citotoxicidade, extresse oxidativo e genotoxicidade. As pastas Guedes-Pinto, Maxitrol® e Sulfato de Neomicina+Bacitracina apresentaram melhor desempenho quanto à biocompatibilidade in vitro.

Resumo: O presente estudo avaliou a reação histológica do efeito do laser de baixa intensidade diodo semicondutor de arseneto de gálio e alumínio (GaA1As, com comprimento de onda de 785 nm, infravermelho, com potência de emissão de 50 mW, densidade de energia de 3 J/cm2, em emissão contínua, por meio de ponta especial de fibra óptica, por um período de 2 segundos) em exposições pulpares induzidas em dentes de ratos. Foram utilizados 60 ratos, dos quais cada animal ofereceu dois dentes (totalizando 120), que foram distribuídos em 5 grupos experimentais: grupo I (controle), exposição capeada com hidróxido de cálcio; grupo II, laser imediato + hidróxido de cálcio; grupo III, laser imediato + hidróxido de cálcio + laser após 24 horas; grupo IV, laser imediato + hidróxido de cálcio + laser após 24 e 48 horas; e grupo V, somente a aplicação do laser. Decorridos os períodos experimentais de sete e 30 dias, os animais foram mortos, as peças removidas e preparadas para análise histológica. De acordo com a proposta e as condições específicas deste trabalho, os resultados nos permitem concluir que as associações laser imediato + hidróxido de cálcio e laser imediato + hidróxido de cálcio + laser 24 horas, foram os grupos que apresentaram os melhores resultados tanto aos sete dias (efeito antiinflamatório), quanto aos trinta dias (efeito no reparo tecidual). / Abstract: The present study evaluated the histological reaction of the low poweer laser effect, diode laser semiconductor of gallium aluminum arsenide (GaA1As, with wavelenght of 785 nm, infra-red, potency of emission of 50 mW, energy density of 3 J/cm2, with continuous emission, through special tip of optic fiber, for a period of two seconds) in exposed pulp induced in teeth of mice. Sixty mice were used, of which each animal offered two teeth (totaling 120), that were distributed in 5 experimental groups: group I(control group) exposed capping with calcium hydroxide; group II, immediate laser + calcium hydroxide; group III, immediate laser + calcium hydroxide + laser after 24 hours; group IV, immediate laser + calcium hydroxide + laser after 24 and 48 hours; and group V, only laser application. After the experimental periods of seven and thirty days, the animals were killed, the pieces were removed and prepared for histological analysis. In agreement with the proposal and the specific conditions of this study, the results allow us conclude that the associations of immediate laser + calcium hydroxide and immediate laser + calcium hydroxide + laser after 24 hours, were the groups that presented the best results in the periods of seven days (anti-inflammatory effect), and thirty days (effect in the tissue repair). / Orientador: Fábio Luiz Camargo Villela Berbert / Coorientador: Lizeti Toledo de Oliveira Ramalho / Banca: Renato de Toledo Leonardo / Banca: Jesus Djalma Pécora / Mestre

O objetivo do estudo foi avaliar as características da dentina cariada remanescente de molares decíduos quanto à coloração, consistência e microdureza, após um período médio de 3 anos e 8 meses da realização do capeamento pulpar indireto. O estudo clínico constou de 27 dentes que apresentavam lesões cariosas ativas com expressão radiográfica em metade interna de dentina. Aleatoriamente, foi realizado o capeamento pulpar indireto e utilizado como material capeador o hidróxido de cálcio (HC) e o cimento de ionômero de vidro resinoso modificado (CIVRM). As crianças foram acompanhadas através de exames clínicos e radiográficos, e ao longo de um período médio de 3 anos e 8 meses, observou-se um índice de sucesso de 89% para o grupo do HC e de 93% para o CIVRM, não havendo diferença estatisticamente significante entre os dois grupos (p=0,62). Os dentes considerados como sucesso no estudo clínico foram agrupados em um único grupo (grupo teste), que constou de 13 dentes que sofreram esfoliação natural ou exodontia por motivos ortodônticos. Além disso, foram selecionados 15 molares decíduos hígidos (grupo controle positivo) e 15, portadores de lesões cariosas ativas em metade interna de dentina (grupo controle negativo). As amostras do grupo teste tiveram suas respectivas restaurações removidas, a profundidade medida e a dentina remanescente avaliada por um operador calibrado, seguindo critérios descritivos, quanto à consistência e a coloração. Nos dentes do grupo controle positivo, foram realizados preparos cavitários oclusais em até 4 mm de profundidade, enquanto que, no grupo controle negativo, o mesmo operador do estudo clínico realizou a remoção parcial de tecido cariado in vitro. A partir daí, todos os dentes foram preparados para análise de microdureza, que foi realizada por um examinador calibrado, que empregou o princípio de cegamento. Na análise da consistência, todos os dentes do grupo teste (n=13) apresentaram-se endurecidos, enquanto que 9, apresentaram coloração amarela-clara (8 do CIVRM e 1 do HC) e 4, castanho-escura (1 do CIVRM e 3 do HC). Para o teste de microdureza no grupo teste, obteve-se uma média KHN de 40,81 (±16,28) MPa, enquanto que nos grupos controles positivo e negativo, foram alcançados valores médios de 62,73 (±11,24) MPa e 19,15 (±6,99) MPa, respectivamente. A análise estatística mediante o teste ANOVA indicou que houve diferença significativa entre os 3 grupos. Foi constatada a remineralização da dentina de dentes decíduos em que foi realizada a técnica do capeamento pulpar indireto após um período médio de 3 anos e 8 meses, através de critérios clínico (consistência) e laboratorial (análise da microdureza). / The aim of this study was to evaluate the characteristics of the deciduous molars carious dentin after a period of 3 years and 8 months (mean) of the indirect pulp capping through the color, consistency and microhardness analysis. The clinical study consisted of 27 primary teeth with deep acute carious lesions that were submitted to incomplete dentin caries removal, application of calcium hydroxide (restored with composit resin) or modified resin glass ionomer cement (restored with GIC). The children had been followed through clinical and radiographics examinations, and throughout an average period of 3 years and 8 months, an index of success of 93% for the GIC and 89% for the group of the CH was observed, no statistical difference was observed between the two groups (p=0,62). The teeth considered as success in the clinical study had been grouped in one group (test) that consisted of 13 teeth that had natural esfoliation or extraction for orthodontic reasons. Fifteen sound deciduous molars (positive control group) and 15 molars with deep acute carious lesions (negative control group) were selected. The samples of the test group had its respective restorations removed, measured depth and the remaining dentine evaluated by a calibrated operator, following descriptive standards, such as consistency and color. In the positive control group, cavity preparations had been carried in up to 3-4 mm of depth, while that, in the negative control group, the same operator of the clinical study proceeded the incomplete dentin caries removal in vitro. From there, all the teeth had been prepared for microhardness analysis that was carried through by a blind and calibrated examiner. In the analysis of the consistency, all the teeth of the test group (n=13) was considered hard, while that, 9 had presented yellow-clear coloration (8 of GIC and 1 of the CH) and 4 chestnut-dark (1 of GIC and 3 of the CH). For the microhardness analysis, the test group got an average KHN of 40,81 (±16,28) MPa, while the positive and negative control group, had been reached average values of 62,73 (±11,24) MPa and 19,15 (±6,99) MPa, respectively. The analysis statistics by means of test ANOVA indicates that it had significant difference between the 3 groups. The dentin remineralization of the deciduous tooth was evidenced where the technique of indirect pulp capping was carried through after an average period of 3 years and 8 months, through clinical (consistency) and laboratorial (microhardness analysis) standards.

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 Edineia Teixeira (edineia.teixeira@unioeste.br) on 2018-04-20T13:11:28Z No. of bitstreams: 2 Giovani _Hartmann2018.pdf: 1083042 bytes, checksum: 4c6c31040314a6b8b1914db7eec998ac (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-04-20T13:11:28Z (GMT). No. of bitstreams: 2 Giovani _Hartmann2018.pdf: 1083042 bytes, checksum: 4c6c31040314a6b8b1914db7eec998ac (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-01-18 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / This study analyzed sericin as a potential biomaterial in contact with dentin pulp, histologically comparing its response to direct pulp capping with calcium hydroxide. These are the first morphological and functional data derived from application of this protein directly on pulp exposure. 20 maxillary first molars from Wistar male mice were used, with 60 days of age, between 200 and 300 grams, which were divided in 4 groups (n=5): G1 and G3, controls, capped with calcium hydroxide in 7 and 30 days, respectively; G2 and G4, capped with sericin in 7 and 30 days, respectively. Circular cavities were prepared for pulp exposure, where capping materials were applied, being posteriorly restored with glass ionomer cement. At the end of the groups period, animals were euthanized and molars were histologically processed for analysis in light microscopy to evaluate presence of necrosis in pulp tissue, inflammatory cells infiltration and tertiary dentin formation. After 7 days, there was less necrosis and inflammatory cells infiltration in G1 when compared to G2 (p=0.007 and p=0.008, respectively). After 30 days, a sample of G3 induced tertiary dentin formation and G4 showed decrease in inflammation (p=0.041) compared to G2. Among the determined experiment conditions, it was concluded that sericin did not present to be viable to treatment as it did not induce tertiary dentin formation, although, its contact with pulp tissue has shown improve in inflammatory response during the treatment and new cells proliferation. / Este estudo analisou a sericina como potencial biomaterial em contato com a polpa dental comparando histologicamente sua resposta ao capeamento pulpar direto com hidróxido de cálcio. Estes são os primeiros dados morfológicos e funcionais procedentes da aplicação desta proteína diretamente sobre a exposição da polpa. Foram utilizados 20 primeiros molares superiores de ratos Wistar machos, com 60 dias de idade entre 200 e 300 gramas, os quais foram divididos em 4 grupos (n=5): G1 e G3, controles, capeados com hidróxido de cálcio em 7 e 30 dias, respectivamente; G2 e G4, capeados com sericina em 7 e 30 dias, respectivamente. Cavidades circulares foram preparadas para exposição pulpar, onde foram aplicados os materiais capeadores, sendo posteriormente restauradas com cimento de ionômero de vidro. Transcorridos os tempos dos grupos, os animais foram eutanasiados e os molares foram processados histologicamente para análise em microscopia de luz para avaliar presença de necrose no tecido pulpar, infiltração de células inflamatórias e formação de dentina terciária. Aos 7 dias, a necrose e a infiltração de células inflamatórias foram menores em G1 quando comparado ao G2 (p=0,007 e p=0,008, respectivamente). Aos 30 dias, uma amostra do G3 induziu formação de dentina terciária e G4 apresentou diminuição de inflamação (p=0,041) em relação ao G2. Dentro das determinadas condições experimentais, concluiu-se que a sericina não se mostrou viável ao tratamento por não induzir formação de dentina terciária, entretanto, seu contato com o tecido pulpar demonstrou melhora na resposta inflamatória ao longo do tratamento e proliferação de novas células.

>Magister Scientiae - MSc / Tuberculosis (TB) is a disease that results from infection by Mycobacterium tuberculosis, which is regarded the most common infecting organism. TB has killed countless numbers of people particularly in underdeveloped countries. TB bacteria can remain inactive or in dormant state for years without causing symptoms or spreading to other subjects, but as soon as the immune system of the host becomes weakened, the bacteria become active and infect mainly the lungs along with other parts of body. TB cases are further aggravated by other illnesses that affect the immune system, such as human immune virus (HIV), which is very prevalent in resource-poor countries. Interferon-gamma (IFN-γ) is a TB biomarker that has found to have all the qualities that are needed to help and cure Tuberculosis disease. Early diagnosis and treatment are essential measures for effectively controlling the disease. Traditional microbial culture-based tests are the most common methodologies currently used. Usually, these methods involve cell culture, cell counts, and cell enrichment, but this process is time-consuming and laborious, especially for the slow-growing bacteria like M. tuberculosis. Sputum smear is one of the methods currently used to detect acid fast bacilli (AFB) in clinical specimens or fluorescent staining. It is a cost-effective tool for diagnosing patients with TB and to monitor the progress of treatment especially in developing countries. The traditional method of inoculating solid medium such as Lowerstein-Jensen (L-J) or 7H10/7H11 media is also used currently it is slow and takes 6-8 weeks of incubation to diagnose the infection and further more time to determine the susceptibility patterns. The microscopic observation drug susceptibility (MODS) assay they are also used currently they rely on light microscopy to visualize the characteristic cording morphology of M. tuberculosis in liquid culture. MODS has shorter time to culture positivity (average 8 days) compared with LJ medium (average ~26 days), they are very expensive. The Gen-Probe assay specific for M. tuberculosis complex is a rapid detection that is also used, nucleic acid amplification (NAA) test results can be obtained as fast as in two hours (provided if a positive culture is present); it also has a high sensitivity of 99% and specificity of 99.2%. It holds the disadvantage of needing of positive culture that can take several days. Enzyme-linked immunosorbent assay (ELISA), is a test that uses antibodies and colour change to identify a substance. ELISA is an assay that uses a solid-phase enzyme immunoassay (EIA) to detect the presence of a substance, usually an antigen, in a liquid sample or wet sample. It can be used to detection of Mycobacterium antibodies in tuberculosis. The Amplified Mycobacterium Tuberculosis Direct Test (AMTDT) is used for the detection of M. tuberculosis it enables the amplification and detection of M. tuberculosis rRNA directly from respiratory specimens. The diagnostic methods employing genetechnology based on the amplification of DNA or RNA are expected to improve the speed, sensitivity, and specificity of Mycobacterium tuberculosis detection. TB rapid cultivation detection technique, such as MB/BacT system, BactecMGIT 960 system and flow cytometry. The BACTEC MGIT960 system (Becton Dickinson, Sparks, MD) performs incubation and reading of the tubes continuously inside the machine using a predefined algorithm to interpret the fluorescent signal and giving the results as positive or negative. When performing DST, the BACTEC MGIT960 interprets the results as susceptible or resistant to the antibiotic under study. Results are available within 8 days. A recent meta-analysis of the published studies found high accuracy and high predictive values associated with the use of BACTEC MGIT960. These methods are more sensitive and rapid than the traditional microbial culture-based methods. However, they cannot provide the detection results in real-time and most of these methods are centralized in large stationary laboratories because complex instrumentation and highly qualified technical staff are required. Recently, Food and Drug Administration (FDA) approved two new assays that were introduced. These two assays detect in vitro a specific immune response to M. tuberculosis. These tests are the QuantiFERON-TB Gold In-Tube (Cellestis/Qiagen, Carnegie, Australia) and the T-SPOT.TB assay (Oxford Immunotec, Abingdon, United Kingdom). Both assays use whole blood from the patient and measure the production of interferon gamma after the whole blood is exposed to specific antigens from M. tuberculosis. These tests are based on the knowledge that IFN-γ is a product of an active cell-mediated immune response induced by M. tuberculosis. However, TB detection remains a major obstacle due to several drawbacks of these methods. To date, the number of diagnosis approaches for TB has increased as the disease continues to be a major public health problem worldwide and most conventional detection technologies present difficulties in recognizing the presence of M. tuberculosis, since they are time consuming, do not provide clinically reliable results and significantly lack of sensitivity. This thesis focusedon developing two binary and one ternary-electrochemically quantum dots, all synthesised at room temperature in aqueous media for detecting (IFN-γ). Copper telluride (CuTe) and Zinc telluride (ZnTe) was prepared to check how does the two quantum dot behave individual and also to check on how they behave when they are combined and formed ternary quantum dots (CuZnTe). The electrochemical studies of the binary CuTe quantum dots, ZnTe quantum dots and the ternary CuZnTe core-shell quantum dots reveal that ternary quantum dots were stable and showed a significant enhancement in the conductivity of CuZnTe core-shell solution compared to that of CuTe and ZnTe, all studied in solution. The three different quantum dots were capped with three different capping reagents which are tetraethyl orthosilicate (TEOS), thioglycolic acid (TGA), (3-mercaptopropyl) trimethoxysilane (MPS). In the study, a label-free electrochemical immunosensor for the detection of interferon gamma (IFN-γ) was prepared for the first time using ternary quantum dots. The biosensor consists of water-soluble silica coated Copper Zinc telluride (CuZnTe core-shell) quantum dots conjugated to a gold electrode. The antibody-antigen were then conjugated on the CuZnTe core-shell QD modified gold electrode. Results from synthesis of two different binary quantum dots are also presented in the study and compared to the results of the CuZnTe core-shell QDs. The CuTe quantum dots had a small average size which was confirmed through HRTEM, SAXS and XRD analysis

La division cellulaire asymétrique est un processus crucial dans le développement des organismes multicellulaires puisqu’elle permet la génération de la diversité cellulaire. Les cellules qui se divisent de façon asymétrique doivent tout d’abord se polariser et correctement orienter leur fuseau mitotique pour ségréger des déterminants cellulaires en deux entités distinctes. L’embryon du nématode C. elegans est un modèle robuste et largement utilisé pour étudier la division cellulaire asymétrique. Dans cet embryon, le point d'entrée du spermatozoïde détermine l'axe de polarité antéro-postérieur. Suite à la fécondation, le cortex embryonnaire est uniformément contractile et un complexe conservé formé des protéines PAR-3, PAR-6 et PKC-3 (nommé complexe PAR-3 ci-dessous) est localisé sur l'ensemble du cortex. La complétion de la méiose maternelle induit une relaxation corticale au postétieur et un flux cortical vers l’antérieur de l’embryon. Ces contractions corticales asymétriques mènent à la formation d'un domaine antérieur contenant le complexe PAR-3, tandis que le cortex postérieur, dont le complexe PAR-3 s’est délocalisé, est enrichi avec les protéines PAR-2 et PAR-1. Par conséquent, les domaines formés par les protéines PAR définissent un pôle antérieur et un pôle postérieur dans l'embryon suite au remodelage du cytosquelette. Les protéines PAR-4 et PAR-5 restent localisées de façon uniforme dans l'embryon. Curieusement, les protéines PAR exercent une régulation par rétroaction sur la contractilité corticale. Il a été montré qu’une des protéines PAR récemment identifiée, PAR-5, est orthologue à la protéine adaptatrice 14-3-3 et joue un rôle important dans la contractilité corticale. En dépit de son rôle central dans la contractilité corticale et le processus de polarisation cellulaire, le mécanisme par lequel PAR-5 régule la contractilité corticale n’est pas bien compris. Le but de ce projet est de mieux comprendre comment PAR-5 et ses interacteurs contrôlent la régulation des contractions corticales et, de ce fait, la polarité cellulaire. Dans un essai de capture de la protéine GST (GST pull-down), nous avons identifié plusieurs nouveaux interacteurs de PAR-5. Parmi ceux-ci, nous avons trouvé CAP-2 (protéine de coiffage de l'actine), qui a été identifiée dans des éxpériences de capture de 14-3-3 dans trois systèmes modèles différents. CAP-2 est un hétérodimère des protéines CAP, qui sont impliquées dans la régulation de l'actine. Nous avons trouvé que la déplétion des protéines CAP par interférence à l’ARN dans des vers de type sauvage mène à une augmentation létalité embryonnaire, ce qui suggère que ces protéines jouent un rôle important dans le développement embryonnaire. L'imagerie en temps réel d'embryons déplétés pour les protéines CAP montre qu’ils ont une diminution des contractions corticales avec un sillon de pseudoclivage mois stable, suggérant un défaut dans la régulation du cytosquelette d'actine-myosine. Ceci a également été confirmé par la diminution de la vitesse et du nombre de foci de NMY-2::GFP. En outre, ces embryons montrent une légère diminution de la taille du croissant cortical de PAR-2 lors de la phase d’établissement de la polarité. Les embryons déplétés en CAP-2 montrent également un retard dans la progression du cycle cellulaire, mais le lien entre ce phénotype et la régulation des contractions corticales reste à être précisé. La caractérisation des protéines CAP, des régulateurs du remodelage du cytosquelette, permettra d'améliorer notre compréhension des mécanismes qui sous-tendent l'établissement et le maintien de la polarité cellulaire, et donc la division cellulaire asymétrique. / Asymmetric cell division is a crucial step in organism development, as it allows the generation of cellular diversity. In order to achieve asymmetric division cells need to polarize their cell fate determinants and properly orient their mitotic spindle before division. The C. elegans embryo is a powerful and widely used model to study asymmetric cell division. In the embryo the sperm entry site determines the anterior-posterior axis of polarity. In the newly fertilized embryo, shortly after meiosis, the cortex is uniformly contractile and the conserved PAR-3/PAR-6/PKC-3 complex (hereafter referred to as the PAR-3 complex) is localised on the entire cortex. Entry of the sperm triggers posterior smoothening and anterior-directed cortical flows. Asymmetric cortical contractions result in the formation of an anterior domain containing the PAR-3 complex, while the posterior-pole cortex, depleted of the PAR-3 complex, is enriched in PAR-2 and PAR-1 proteins. Therefore PAR domains define an anterior and a posterior pole of the embryo in response to cytoskeleton remodelling. The PAR-4 and PAR-5 proteins remain localized uniformly throughout the embryo. Intriguingly, the PAR proteins exert a feedback regulation on cortical contractility. PAR-5, one of the lately identified PAR proteins, was shown to be an ortholog of the adaptor protein 14-3-3 and to play an important role in cortical contractility. Despite its central role in cortical contractility and henceforth the polarization process, little is known on how PAR-5 regulates cortical contractility. The aim of this project is to better understand the regulation of cortical contractions via the PAR-5 protein and its interactors, and how they control cell polarity. In a GST pull down assay we identified several new interactors of PAR-5. Among these we found CAP-2 (actin capping protein), which was also pulled down with 14-3-3 in three different model systems. CAP-2 has been implicated in actin regulation. Interestingly we found that depletion of CAP proteins by RNA interference in wild type worms results in increased embryonic lethality, suggesting an important role in embryonic development. Live imaging of embryos depleted of CAP proteins shows that these embryos have decreased cortical contractions with a less stable pseudo cleavage furrow, indicating a defect in the regulation of the actin-myosin cytoskeleton. This was further confirmed by the decreased velocity and the number of NMY-2::GFP foci in CAP depleted embryos. Furthermore, these embryos show mild decrease in PAR-2 domain size during the polarity establishment phase. cap-2(RNAi) embryos also show a delay in cell cycle progression, however the role of the cell cycle delay in the regulation of cortical contractions has to be determined. The characterization of CAP proteins, which are cytoskeleton-remodeling regulators, will improve our understanding of the mechanisms underling the establishment and maintenance of cell polarity, and thereby asymmetric cell division.