Influência do peróxido de hidrogênio sobre a diferenciação celular e a mineralização do tecido pulpar após procedimento clareador. Estudo histológico e imunoistoquímico
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Data
2018-04-13
Autores
Benetti, Francine [UNESP]
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Universidade Estadual Paulista (Unesp)
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Introdução: Através de modelo experimental caracterizado por nosso grupo de pesquisa, e protocolo clareador adaptado, verificamos que o peróxido de hidrogênio (H2O2) contido no gel clareador pode gerar efeitos ao tecido pulpar, que ainda não estão completamente compreendidos. Outros estudos mostram uma indução à mineralização, levando à posterior calcificação de grande parte do tecido pulpar e à formação de nódulos. Objetivos: Os objetivos deste trabalho foram divididos em duas etapas: 1 – Verificar os efeitos do H2O2 na expressão de marcadores da mineralização no tecido pulpar, por meio da imunomarcação de osteocalcina (OCN) e osteopontina (OPN); e a presença de resposta celular específica ao estresse oxidativo, por meio de imunomarcação com anticorpo para espécies reativas de oxigênio (EROs); 2 – Determinar a capacidade de resposta ao estresse oxidativo gerado pelo H2O2 no tecido pulpar, por meio da imunomarcação de Heme-oxigenase-1 (HO-1); investigar os efeitos do gel clareador sobre a diferenciação odontoblástica, por meio da imunomarcação do fator de transcrição Jun-D; e a influência do estresse oxidativo gerado pelo H2O2 na identificação de células-tronco mesenquimais (CTMs) do tecido pulpar, por meio da técnica de imunofluorescência, com identificação concomitante de positividade celular para CD90, CD73, CD105 e negatividade para CD45. Materiais e métodos: Foram utilizados 60 ratos Wistar que tiveram os molares superiores direitos ou esquerdos clareados com 0,01 mL de H2O2 35%, em uma aplicação de 30 minutos, de forma randomizada. Os molares do lado não clareado serviram de controle. Após 0 horas, 2, 3, 7, 15 e 30 dias (n=10), os animais foram mortos e as maxilas processadas para avaliação histológica, imunoistoquímica (OCN, OPN, EROs, HO-1 e Jun-D) e de imunofluorescência (CD90, CD73, CD105, CD45). Os resultados foram submetidos a testes estatísticos específicos (p<0,05). Resultados: No tempo de 0 horas, houve necrose em toda a polpa coronária (p<0,05), e aos 2 e 3 dias, no terço oclusal (p<0,05); aos 7, 15 e 30 dias, não houve inflamação, assim como no controle (p>0,05). Dentina terciária estava presente aos 7 dias, aumentando em 15 e 30 dias (p<0,05). Em relação aos marcadores de mineralização, OCN foi ausente imediatamente após procedimento clareador, aumentando ao longo dos períodos, se tornando significativa aos 15 e 30 dias (p<0,05); OPN apresentou maior imunomarcação aos 7 e 15 dias no grupo clareado (p<0,05). A imunomarcação de EROs foi significativa em todos os terços da polpa coronária no grupo clareado aos 7 e 15 dias, e no terço cervical aos 2 e 30 dias, comparada ao controle (p<0,05). HO-1 revelou maior imunomarcação no grupo clareado nos terços médio e cervical da polpa coronária aos 2 e 3 dias, em todos os terços aos 7 dias, e no terço oclusal aos 15 dias, quando comparado ao grupo controle (p<0,05). Imunomarcação nuclear para Jun-D foi significativa no grupo clareado no terço cervical da polpa coronária aos 2 e 3 dias, e nos terços oclusal e médio aos 7 dias, quando comparado ao grupo controle (p<0,05), reduzindo nos demais períodos (p>0,05). Poucas células CD90+/CD73+/CD105+/CD45- foram observadas no tecido pulpar do grupo controle e do grupo clareado em todos os períodos de análise (p>0,05). Conclusões: Pode-se concluir que: 1 – A redução da inflamação e o processo de reparo pulpar após procedimento clareador está associado com o aumento de OCN, e OPN participa durante o processo de reparo; EROs está presente no processo de defesa celular contra o estresse oxidativo decorrente do H2O2. 2 – As células pulpares apresentam capacidade de resposta ao estresse oxidativo expressando HO-1 nos períodos onde há inflamação, até o início do reparo; Jun-D é presente no tecido pulpar durante a redução do processo inflamatório e início da produção de dentina terciária; a presença de estresse oxidativo não influencia o número de células CD90+/CD73+/CD105+/CD45- identificadas in vivo no tecido pulpar.
Introduction Through an experimental model characterized by our research group, and adapted bleaching protocol, we verified that the hydrogen peroxide (H 2 O 2 ) of bleaching gel can generate effects on the pulp tissue, which are not yet completely understood. Studies show an induction to mineralization, leading to subsequent calcification of a large part of the pulp tissu e and to the formation of nodules. Objectives The objective s of this study were divided into two stages: 1 – To verify the effects of H 2 O 2 on the expression of mineralization markers in pulp tissue, through of immunolabelling of the osteocalcin (OCN) and o steopontin (OPN); and the presence of specific cellular response to oxidative stress, by immunolabeling with antibody to reactive oxygen species (ROS); 2 – To determine the capacity of response to oxidative stress generated by H 2 O 2 in pulp tissue , through of immunolabelling of Heme - oxigenase - 1 (HO - 1); to investigate the effects of the bleaching gel on the odontoblastic differentiation, through the immunolabelling of the transcription factor Jun - D; and the influence of the oxidative stress generated by H 2 O 2 on the identification of mesenchymal stem cells ( MSC s) of the pulp tissue by the immunofluorescence technique, with the concomitant identification of cellular positivity for CD90, CD73, CD105 and negativity for CD45. Materials and methods Sixty Wistar rats were used, and the right or left upper molars were bleached with 0.01 mL of 35% H 2 O 2 , in a direct application of 30 minutes , r andomly . T he molars on the unbleached side served as controls. After 0 hours, 2, 3 , 7, 15 and 30 days (n=10), the animals were ki lled and the jaws processed for histological, immunohistochemical (OCN, OPN, ROS, HO - 1 and Jun - D) and immunofluorescence ( CD90, CD73, CD105, CD45 ) analysis. The results were submitted to specific statistical tests ( P <0.05). Results At 0 hours, there was necrosis throughout the coronary pulp ( P <0.05), and at 2 and 3 days, in the occlusal third ( P <0.05); at 7, 15 and 30 days, there was no inflammation, as well as in the control ( P >0.05). Tertiary dentin was present at 7 days, increasing in 15 and 30 days ( P <0.05). In relation to mineralization markers, OCN was absent immediately after bleaching procedure, increasing over the periods, becoming significant at 15 and 30 days ( P <0.05); OPN has higher immunolabelling at 7 and 15 days in the bleached group ( P <0.05 ). Immuno labelling of ROS was significant in all thirds of the coronary pulp in the bleached group at 7 and 15 days, and in the cervical third at 2 and 30 days, compared to the control group ( P <0.05). HO - 1 showed higher immunolabelling in the bleached grou p in the middle and cervical thirds of the coronary pulp at 2 and 3 days, in all thirds at 7 days, and in the occlusal third at 15 days, when compared to the control group ( P <0.05). Nuclear immuno labelling for Jun - D was significant in the bleached group in the cervical third of the coronary pulp at 2 and 3 days, and in the occlusal and middle thirds at 7 days, when compared to the control group ( P <0.05), reducing in the other periods ( P >0.05). Low number of CD90+/CD73+/CD105+/CD45 - cells were observed in the pulp tissue of the control group and the bleached group in all periods of analysis ( P >0.05). Conclusions It is concluded that : 1 – The reduction of inflammation and the pulp repair process after bleach ing is associated with increased of OCN, and OPN participates during the repair process; ROS are present in the cellular defence process against oxidative stress by H 2 O 2 . 2 – The pulp cells had capacity to respond to oxidative stress expressing HO - 1 in the periods where there is inflammation, until the beginning of the repair; Jun - D is present in the pulp tissue during the reduction of the inflammatory process and the beginning of the production of tertiary dentin; the presence of oxidative stress does not influence the number of CD90+/CD73+/CD105+/CD45 - cells identified in vivo in the pulp tissue.
Introduction Through an experimental model characterized by our research group, and adapted bleaching protocol, we verified that the hydrogen peroxide (H 2 O 2 ) of bleaching gel can generate effects on the pulp tissue, which are not yet completely understood. Studies show an induction to mineralization, leading to subsequent calcification of a large part of the pulp tissu e and to the formation of nodules. Objectives The objective s of this study were divided into two stages: 1 – To verify the effects of H 2 O 2 on the expression of mineralization markers in pulp tissue, through of immunolabelling of the osteocalcin (OCN) and o steopontin (OPN); and the presence of specific cellular response to oxidative stress, by immunolabeling with antibody to reactive oxygen species (ROS); 2 – To determine the capacity of response to oxidative stress generated by H 2 O 2 in pulp tissue , through of immunolabelling of Heme - oxigenase - 1 (HO - 1); to investigate the effects of the bleaching gel on the odontoblastic differentiation, through the immunolabelling of the transcription factor Jun - D; and the influence of the oxidative stress generated by H 2 O 2 on the identification of mesenchymal stem cells ( MSC s) of the pulp tissue by the immunofluorescence technique, with the concomitant identification of cellular positivity for CD90, CD73, CD105 and negativity for CD45. Materials and methods Sixty Wistar rats were used, and the right or left upper molars were bleached with 0.01 mL of 35% H 2 O 2 , in a direct application of 30 minutes , r andomly . T he molars on the unbleached side served as controls. After 0 hours, 2, 3 , 7, 15 and 30 days (n=10), the animals were ki lled and the jaws processed for histological, immunohistochemical (OCN, OPN, ROS, HO - 1 and Jun - D) and immunofluorescence ( CD90, CD73, CD105, CD45 ) analysis. The results were submitted to specific statistical tests ( P <0.05). Results At 0 hours, there was necrosis throughout the coronary pulp ( P <0.05), and at 2 and 3 days, in the occlusal third ( P <0.05); at 7, 15 and 30 days, there was no inflammation, as well as in the control ( P >0.05). Tertiary dentin was present at 7 days, increasing in 15 and 30 days ( P <0.05). In relation to mineralization markers, OCN was absent immediately after bleaching procedure, increasing over the periods, becoming significant at 15 and 30 days ( P <0.05); OPN has higher immunolabelling at 7 and 15 days in the bleached group ( P <0.05 ). Immuno labelling of ROS was significant in all thirds of the coronary pulp in the bleached group at 7 and 15 days, and in the cervical third at 2 and 30 days, compared to the control group ( P <0.05). HO - 1 showed higher immunolabelling in the bleached grou p in the middle and cervical thirds of the coronary pulp at 2 and 3 days, in all thirds at 7 days, and in the occlusal third at 15 days, when compared to the control group ( P <0.05). Nuclear immuno labelling for Jun - D was significant in the bleached group in the cervical third of the coronary pulp at 2 and 3 days, and in the occlusal and middle thirds at 7 days, when compared to the control group ( P <0.05), reducing in the other periods ( P >0.05). Low number of CD90+/CD73+/CD105+/CD45 - cells were observed in the pulp tissue of the control group and the bleached group in all periods of analysis ( P >0.05). Conclusions It is concluded that : 1 – The reduction of inflammation and the pulp repair process after bleach ing is associated with increased of OCN, and OPN participates during the repair process; ROS are present in the cellular defence process against oxidative stress by H 2 O 2 . 2 – The pulp cells had capacity to respond to oxidative stress expressing HO - 1 in the periods where there is inflammation, until the beginning of the repair; Jun - D is present in the pulp tissue during the reduction of the inflammatory process and the beginning of the production of tertiary dentin; the presence of oxidative stress does not influence the number of CD90+/CD73+/CD105+/CD45 - cells identified in vivo in the pulp tissue.
Descrição
Palavras-chave
Clareação dentária, Estresse oxidativo, Resposta pulpar, Peróxido de hidrogênio, Dentinogênese, Células-tronco, Dental bleaching