Complexo de fluoreto de sódio/quitosana: síntese, caracterização, viabilidade celular e efeitos no esmalte desmineralizado e na formação de biofilme de S. mutans

Abstract

The aim of this study was to synthesize a chitosan gel with sodium fluoride (2.8% NaF/Chit), without crosslinking agent; characterize it by rheological tests and electrical impedance spectroscopy (EIS); test cell viability (MTT assay); and evaluate the antibacterial effect of the gel on the formation of S. mutans biofilm and the demineralization of bovine enamel using the microhardness test. The NaF/Chit gel was synthesized at a concentration of 2.8% by adding Chit to the NaF solution in 1% acetic acid (AAc) solution. The viscosity (η) of the NaF/Chit gel was evaluated by oscillatory and stationary rheological titration and the EIS evaluated the electrical response of the complex. The MTT assay was performed on RAW 264.7 macrophage culture. The agar diffusion assay was performed against S. mutans in 5 groups: 2.8% Chit gel, 2.8% NaF solution, 2.8% NaF/Chit gel, 1.23% FFA gel (acidulated fluorine phosphate - positive control) and 1% AAc solution (negative control). Enamel blocks (4 x 4mm) were divided into 5 groups (n = 5 samples/group) according to the substances tested above. The formation of the S. mutans biofilm on the enamel surface occurred over 48 hours, with daily application of the substances for 90 seconds, and a bacteria count was performed (CFU/mL). Knoop surface microhardness (KHN) values were obtained before and after biofilm. The Kruskal-Wallis test was used to compare CFU/mL between groups and the 2-way repeated measures ANOVA and Tukey's post-hoc tests were used to evaluate microhardness (α= 0.05). The rheological test showed thixotropy of the NaF/Chit gel and the EIS indicated a NaF solution with greater conduction charge, higher ion concentration and low viscosity in relation to other gels. The conduction charge of the Chit gel was lower and had high viscosity. NaF/Chit presented ions from the dissociation of NaF, greater viscosity and electrophoretic effects of interaction with the medium. The MTT assay indicated a cell viability percentage of less than 50% for all groups: 2.8% NaF (0.047 mg/mL), 2.8% Chitosan (1.69 mg/mL), 2.8% NaF/Chit (0.043 mg/mL), AAc (0.059 mg/mL) and FFA (0.068 mg/mL). The 2.8% Chit gel showed the highest inhibition halo formation (14.1 mm  0.7) and AAc (5.7 mm  0.6) the lowest. There was no significant difference for the S. mutans count between the gels: 2.8% Chit (3.9 x 102 CFU/mL), 2.8% NaF/Chit (3.8 x 102 CFU/mL) and FFA (3.6 x 102 CFU/mL). However, the 2.8% NaF/Chit gel presented a higher post-biofilm KHN value (308.0  4.30 KHN) compared to the other groups (p < 0.05). It can be concluded that the 2.8% NaF/Chit gel was effectively synthesized without cross-linking agent; demonstrated thixotropy; showed toxicity in RAW 264.7 cells; and inhibited the growth of S. mutans after 48h without significant difference from the FFA gel. Furthermore, the 2.8% NaF/Chit gel provided a protective effect on the surface, strengthening the enamel structure and reducing demineralization after the formation of the S. mutans biofilm.