Efeito da ativação enzimática de um gel clareador sobre a cinética de degradação do peróxido de hidrogênio, eficácia clareadora, difusão e citotoxicidade trans-amelodentinária
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Data
2017-08-07
Autores
Ortecho Zuta, Uxua [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
No presente estudo, a enzima horseradish peroxidase (HRP) foi empregada como agente catalizador de um gel contendo 35% de peróxido de hidrogênio (H2O2), objetivando a aceleração da sua taxa de decomposição em radicais livres, aumento sobre a eficácia clareadora e minimização da citotoxicidade indireta sobre células pulpares. Três grupos experimentais foram estabelecidos: CN: sem tratamento; PH35%: 35% de H2O2 e PH35%+HRP: 35% de H2O2 com adição de HRP (10 mg/mL). A cinética de degradação do H2O2 e liberação de radicais livres foram avaliados nos períodos de 0, 5, 10 e 15 min, por meio de sondas fluorescentes específicas. A eficácia clareadora foi avaliada em espectrofotômetro UV-vis (E) durante 6 sessões (3x15 min), em discos de esmalte e dentina simulando incisivos inferiores (2,3 mm de espessura). Para análise biológica, o clareamento foi realizado sobre discos adaptados em câmaras pulpares artificiais, sendo o meio de cultura em contato com a dentina (extrato) coletado e aplicado por 1 h sobre células MDPC-23 previamente semeadas (80% confluência). A viabilidade celular (teste de MTT), morfologia (MEV), lesão à membrana citoplasmática (ensaio de live/dead) e estresse oxidativo (carboxy-H2DCFDA) foram avaliados. Discos não submetidos ao clareamento foram empregados como controle negativo. Observou-se que na presença de HRP houve aceleração da degradação do H2O2 com concomitante aumento na formação de radicais livres em comparação ao gel sem adição da enzima. Estes resultados foram correlacionados com o aumento significante nos valores de E em todas as sessões para o gel com HRP em relação ao grupo PH35%, sendo detectada intensa aceleração do efeito clareador. A citotoxicidade trans-amelodentinária foi significativamente minimizada na presença de HRP, para todos os parâmetros testados. Este efeito foi associado a redução significativa na difusão de H2O2 residual e radicais livres pela estrutura dental no grupo contendo HRP em relação ao grupo PH35%. Concluiu-se que a adição da HRP em géis clareadores a base de H2O2 acelera e otimiza o efeito clareador, bem como minimiza a difusão de subprodutos pela estrutura dental, reduzindo o efeito tóxico do produto sobre células odontoblastóides in vitro. Este efeito foi associado a intensa aceleração da decomposição do H2O2 no gel clareador, mediado pela HRP.
In the present study, the horseradish peroxidase (HRP) enzyme was used as a catalytic agent of a 35% hydrogen peroxide (H2O2) bleaching gel aiming to accelerate its degradation, increase the bleaching effectiveness and reduce the toxic effects on pulp cells. Three experimental groups were stablished: CN: no treatment; PH35%: 35% H2O2 and PH35%+HRP: 35% H2O2 in contact with HRP (10 mg/mL). H2O2 degradation rate and free radicals release was assessed after 0, 5, 10 and 15 min, with specific fluorescence probes. Bleaching effectiveness in the presence or absence of HRP was assessed with a UV-vis spectrophotometer (E) on enamel/dentin discs simulating mandibular incisors (2.3 mm thickness) throughout 6 bleaching sessions (3x15 min). For the biological assays, bleaching protocol was performed onto discs adapted to artificial pulp chambers, and the culture medium in contact with dentin surface (extract) was collected and applied for 1 h on odontoblast-like MDPC-23 cells previously seeded (80% confluence). The cell viability (MTT assay), morphology (SEM) cell membrane damage (live/dead assay) and oxidative stress (carboxyH2DCFDA) were evaluated. The amount of residual H2O2 and free radicals capable to diffuse through the discs was quantified. Discs not subjected to bleaching were used as negative control. According to the results, in the presence of HRP there was acceleration of H2O2 degradation associated with increased generation of free radicals in comparison to plain H2O2 solution. These results were correlated with increased E values at each bleaching session on HRP-containing gel relative to positive control, resulting in significant acceleration of bleaching effect. The trans-enamel and transdentinal cytotoxicity was significantly minimized in the presence of HRP, for all tested parameters. This effect was associated with reduced H2O2 and free radicals diffusion through dental structure for the group containing HRP in comparison to positive control. It was concluded that adding HRP to bleaching gels promotes an optimization and acceleration of tooth bleaching, associated with minimization of residual H2O2 diffusion and odontoblast-like cells cytotoxicity, mediated by increased release of highly oxidative molecules on tooth surface.
In the present study, the horseradish peroxidase (HRP) enzyme was used as a catalytic agent of a 35% hydrogen peroxide (H2O2) bleaching gel aiming to accelerate its degradation, increase the bleaching effectiveness and reduce the toxic effects on pulp cells. Three experimental groups were stablished: CN: no treatment; PH35%: 35% H2O2 and PH35%+HRP: 35% H2O2 in contact with HRP (10 mg/mL). H2O2 degradation rate and free radicals release was assessed after 0, 5, 10 and 15 min, with specific fluorescence probes. Bleaching effectiveness in the presence or absence of HRP was assessed with a UV-vis spectrophotometer (E) on enamel/dentin discs simulating mandibular incisors (2.3 mm thickness) throughout 6 bleaching sessions (3x15 min). For the biological assays, bleaching protocol was performed onto discs adapted to artificial pulp chambers, and the culture medium in contact with dentin surface (extract) was collected and applied for 1 h on odontoblast-like MDPC-23 cells previously seeded (80% confluence). The cell viability (MTT assay), morphology (SEM) cell membrane damage (live/dead assay) and oxidative stress (carboxyH2DCFDA) were evaluated. The amount of residual H2O2 and free radicals capable to diffuse through the discs was quantified. Discs not subjected to bleaching were used as negative control. According to the results, in the presence of HRP there was acceleration of H2O2 degradation associated with increased generation of free radicals in comparison to plain H2O2 solution. These results were correlated with increased E values at each bleaching session on HRP-containing gel relative to positive control, resulting in significant acceleration of bleaching effect. The trans-enamel and transdentinal cytotoxicity was significantly minimized in the presence of HRP, for all tested parameters. This effect was associated with reduced H2O2 and free radicals diffusion through dental structure for the group containing HRP in comparison to positive control. It was concluded that adding HRP to bleaching gels promotes an optimization and acceleration of tooth bleaching, associated with minimization of residual H2O2 diffusion and odontoblast-like cells cytotoxicity, mediated by increased release of highly oxidative molecules on tooth surface.
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Palavras-chave
Clareamento dental, Peróxido de hidrogênio – Toxicidade, Odontoblastos, Tooth bleaching, Hydrogen peroxide – Toxicity, Odontoblasts