Development and analyses of experimental bleaching products based on the use of a hemic peroxidase as buster agent lopment and analyses of experimental bleaching products based on the use of a hemic peroxidase as buster agent

Author(s)

Carla Caroline de Oliveira Duque

• Document type: Doctoral Thesis
• Press: Araraquara. 2020-01-30.
• Institution: Universidade Estadual Paulista (Unesp). Faculdade de Odontologia. Araraquara
• Defense date: 2019-12-09
• Advisor: Carlos Alberto de Souza Costa; Diana Gabriela Soares dos Passos

Abstract

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. (AU)

• FAPESP's process: 16/10928-9 - Development and analysis of innovative bleaching products using a hemic-peroxidase as buster agent
• Grantee: Carla Caroline de Oliveira Duque
• Support Opportunities: Scholarships in Brazil - Doctorate