Desenvolvimento de produtos para clareamento dental contendo a enzima horseradish peroxidase como agente catalisador
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Data
2019-12-09
Autores
Duque, Carla Caroline de Oliveira [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
O objetivo geral deste estudo foi avaliar a eficácia clareadora e toxicidade sobre células pulpares humanas (HDPCs) de dois produtos experimentais recomendados para terapia clareadora de consultório. Para isso, dois sistemas clareadores foram desenvolvidos: um gel clareador contendo um espessante catalisador (EP) e um primer polimérico catalisador (PR), indicado para aplicação sobre o esmalte previamente ao uso de agentes clareadores. Em ambos os produtos foi incorporada a enzima horseradish peroxidase (HRP) como agente catalisador do peróxido de hidrogênio (H2O2). Na primeira etapa deste estudo, géis clareadores com 10% e 20% de H2O2 foram preparados a partir de uma solução estoque de 35% de H2O2 combinada com um espessante contendo ou não as concentrações de 0,5, 1,0 e 2,0 mg/mL de HRP (HRP). Estes produtos foram avaliados quanto ao pH, temperatura, estabilidade de reação, formação de radicais-livres (EROs, sonda HORAC) e radicais hidroxila (HO•, sonda H2DCFDA), bem como eficácia clareadora (E). Um protocolo in vitro de pigmentação intrínseca de discos de esmalte/dentina bovinos foi usado para avaliar a citotoxicidade e a difusão trans-amelodentinária H2O2. De modo geral, o pH e a temperatura dos géis mantiveram-se constantes durante todo período de análise, sendo que a adição da enzima HRP ao EP acelerou a catálise do H2O2, estimulando a produção de EROs e HO• (ANOVA/Tukey; p<0,05). Além disso, a presença da HRP no EP aumentou o E do gel e reduziu a difusão trans-amelodentinária de H2O2 residual, minimizando a citotoxicidade desta molécula, a qual foi concentração/dependente (ANOVA/Tukey; p<0,05). Dentre os parâmetros testados, o gel contendo 10% de H2O2+2 mg/mL de HRP foi o que mostrou os melhores resultados. Embora o gel com 20% de H2O2+2mg/mL de HRP tenha apresentado E similar àquela obtida com o gel contendo 35% de H2O2 aplicado por 45 minutos sobre o esmalte (protocolo de consultório), este último foi altamente tóxico para as HDPCs. Por essa razão, o gel com 10% de H2O2 (HRP) foi selecionado para ser avaliado nas etapas subsequentes deste estudo. Com o objetivo de aproximar o E deste produto àquela proporcionada pelo gel de consultório, as concentrações de HRP incorporadas ao espessante do gel com 10% de H2O2. foram aumentadas para 4, 6 e 10 mg/mL. A concentração de 10 mg/mL de HRP resultou em valores de E similares ao protocolo de consultório (Dunnett’s; =5%), desse modo, esta mesma concentração foi selecionada para as etapas seguintes deste estudo, bem como para ser incorporada ao PR. Como o comportamento do PR se equiparou ao do EP, ambos os produtos foram aplicados (1x45, 1x30, 1x10 e 1x5 minutos) sobre discos de esmalte/dentina, cujas espessuras simulavam incisivos inferiores (2,3 mm 0,2) e superiores (3,5 mm 0,2). Apenas o gel com 10% de H2O2 + 10mg/mL de HRP (1x45 minutos) apresentou E similar ao grupo 35% de H2O2 (45 minutos). A aplicação do PR antes do gel com 10% de H2O2 (-HRP) não interferiu na eficácia clareadora para ambas as espessuras de discos usadas (ANOVA/Tukey; p>0,05). Quanto à citotoxicidade, apenas o grupo 35% de H2O2 (clareamento de consultório) reduziu a viabilidade das HDPCs (ANOVA/Tukey; p<0,05). Assim, foi possível concluir que a adição de HRP na composição de um gel com 10% de H2O2 exerceu atividade catalítica sobre o H2O2, evidenciada pelo aumento na formação do radical HO•. Desde que este radical tem intensa ação oxidativa, sua elevada produção potencializou a eficácia clareadora do produto e reduziu a difusão trans-amelodentinária de H2O2 residual, o que minimizou os efeitos tóxicos do procedimento clareador sobre as células pulpares.
The main objective of this study was to assess the bleaching effectiveness and cytotoxicity on human dental pulp cells (HDPCs) of two experimental products recommended in-office tooth bleaching therapy. For this, two bleaching systems were developed: a gel containing a catalyst thickener (CT) and a polymeric catalyst primer (CP) indicated for application on the enamel prior to the use of bleaching agents, were prepared. The horseradish peroxidase enzyme (HRP) was incorporated in both products as a catalyst for hydrogen peroxide (H2O2) molecules. In the first step of this study, 10% and 20% H2O2 bleaching gels were prepared from a 35% H2O2 stock solution combined with a thickener with or without concentrations of 0.5, 1.0 and 2.0 mg/mL of HRP (HRP). These products were evaluated concerning the pH, temperature, reaction stability, free radicals (ROS, HORAC probe) and hydroxyl radical (OH•, H2DCFDA probe) formation, as well as bleaching efficacy (E). An in vitro protocol for intrinsic pigmentation of bovine enamel/dentin discs was used to assess the cytotoxicity as well as the trans-enamel and trans-dentinal diffusion of H2O2. Overall, the pH and temperature of the gels remained constant during all the analysis period. The addition of HRP enzyme to the CT accelerated the H2O2 catalysis, stimulating the production of ROS and OH• (ANOVA/Tukey; p<0,05). Furthermore, the HRP added to the CT enhanced E of the gel and reduced the trans-enamel and trans-dentinal diffusion of residual H2O2, minimizing the cytotoxicity of the bleaching gel, which was H2O2-concentration dependent (ANOVA/Tukey; p<0,05). Among the tested parameters, the gel containing 10% H2O2 + 2 mg/mL HRP determined the best results. Despite the 20% H2O2 + 2mg/mL HRP gel presented E similar to the conventional in-office protocol, this product caused high toxic effects to the HDPCs. For this reason, the gel with 10% H2O2 (HRP) was selected to be evaluated in the subsequent steps of this study. In order to approximate the E of this product to that obtained with the gel used for in-office bleaching (35% H2O2), higher concentrations of HRP (4, 6 and 10 mg/mL) were added to the 10% H2O2 gel thickener. Since the 10 mg/mL of HRP provided an E similar to the traditional protocol (Dunnett's; = 5%), this concentration incorporated into CP was selected for the following laboratorial tests. Since the general behavior of CP and CT was similar, both products were applied (1x45, 1x30, 1x10 and 1x5 minutes) on enamel/dentin discs with thickness of 2.3 mm 0.2 (mimic lower incisors) or 3.5 mm 0.2 (mimic upper incisors). Only the bleaching gel with 10% H2O2 + 10 mg/mL HRP (1x45 minutes) presented E similar to the 35% H2O2 group (45 minutes). For both enamel/dentin thickness no E difference was observed when PC was applied on enamel before the gel with 10% H2O2 (-HRP) (ANOVA/Tukey; p0,05). Only the 35% H2O2 group (conventional in-office bleaching) reduced the viability of HDPCs (ANOVA/Tukey; p<0,05). Therefore, one can conclude that the presence of HRP in bleaching gel with 10% H2O2 triggered the catalysis of H2O2 molecules, increasing the OH• generation. Since this free-radical has intense oxidative effects, its high concentration in the enamel/dentin discs enhanced the bleaching efficacy of the product and reduced the trans-enamel and trans-dentinal diffusion of residual H2O2. The low concentration of residual H2O2 associated with the fact that OH• has a very short life-time minimized the toxicity of the product to pulp cells.
The main objective of this study was to assess the bleaching effectiveness and cytotoxicity on human dental pulp cells (HDPCs) of two experimental products recommended in-office tooth bleaching therapy. For this, two bleaching systems were developed: a gel containing a catalyst thickener (CT) and a polymeric catalyst primer (CP) indicated for application on the enamel prior to the use of bleaching agents, were prepared. The horseradish peroxidase enzyme (HRP) was incorporated in both products as a catalyst for hydrogen peroxide (H2O2) molecules. In the first step of this study, 10% and 20% H2O2 bleaching gels were prepared from a 35% H2O2 stock solution combined with a thickener with or without concentrations of 0.5, 1.0 and 2.0 mg/mL of HRP (HRP). These products were evaluated concerning the pH, temperature, reaction stability, free radicals (ROS, HORAC probe) and hydroxyl radical (OH•, H2DCFDA probe) formation, as well as bleaching efficacy (E). An in vitro protocol for intrinsic pigmentation of bovine enamel/dentin discs was used to assess the cytotoxicity as well as the trans-enamel and trans-dentinal diffusion of H2O2. Overall, the pH and temperature of the gels remained constant during all the analysis period. The addition of HRP enzyme to the CT accelerated the H2O2 catalysis, stimulating the production of ROS and OH• (ANOVA/Tukey; p<0,05). Furthermore, the HRP added to the CT enhanced E of the gel and reduced the trans-enamel and trans-dentinal diffusion of residual H2O2, minimizing the cytotoxicity of the bleaching gel, which was H2O2-concentration dependent (ANOVA/Tukey; p<0,05). Among the tested parameters, the gel containing 10% H2O2 + 2 mg/mL HRP determined the best results. Despite the 20% H2O2 + 2mg/mL HRP gel presented E similar to the conventional in-office protocol, this product caused high toxic effects to the HDPCs. For this reason, the gel with 10% H2O2 (HRP) was selected to be evaluated in the subsequent steps of this study. In order to approximate the E of this product to that obtained with the gel used for in-office bleaching (35% H2O2), higher concentrations of HRP (4, 6 and 10 mg/mL) were added to the 10% H2O2 gel thickener. Since the 10 mg/mL of HRP provided an E similar to the traditional protocol (Dunnett's; = 5%), this concentration incorporated into CP was selected for the following laboratorial tests. Since the general behavior of CP and CT was similar, both products were applied (1x45, 1x30, 1x10 and 1x5 minutes) on enamel/dentin discs with thickness of 2.3 mm 0.2 (mimic lower incisors) or 3.5 mm 0.2 (mimic upper incisors). Only the bleaching gel with 10% H2O2 + 10 mg/mL HRP (1x45 minutes) presented E similar to the 35% H2O2 group (45 minutes). For both enamel/dentin thickness no E difference was observed when PC was applied on enamel before the gel with 10% H2O2 (-HRP) (ANOVA/Tukey; p0,05). Only the 35% H2O2 group (conventional in-office bleaching) reduced the viability of HDPCs (ANOVA/Tukey; p<0,05). Therefore, one can conclude that the presence of HRP in bleaching gel with 10% H2O2 triggered the catalysis of H2O2 molecules, increasing the OH• generation. Since this free-radical has intense oxidative effects, its high concentration in the enamel/dentin discs enhanced the bleaching efficacy of the product and reduced the trans-enamel and trans-dentinal diffusion of residual H2O2. The low concentration of residual H2O2 associated with the fact that OH• has a very short life-time minimized the toxicity of the product to pulp cells.
Descrição
Palavras-chave
Toxidade, Clareamento dental, Peroxidase, Toxicity, Tooth-Bleaching, Horseradish Peroxidase