Effect of arginine on dental biofilms and prevention of enamel demineralization

Abstract

This study will be divided in two distinct steps. The first step will aim to determine the effect of two different concentrations of arginine (2.5 and 8.0%), combined or not with 1,450 ppm sodium fluoride, on the composition and metabolism of salivary microcosm biofilms, as well as on the prevention of the demineralization of dental enamel. Saliva samples collected from 3 caries-free adults will be employed to produce a microbiological pool that will be used as a stock solution to start the biofilm growth on bovine enamel blocks. This study will be divided in two phases, according to distinct time of application of treatments: I) during 72 h of biofilm growth, and II) after 72 h of biofilm growth. In each experimental phase, the biofilms will be treated according to one of 6 experimental groups, as follows: 1) deionized water (DW, negative control), 2) 1,450 ppm sodium fluoride (NaF, positive control), 3) 2.5% arginine (ARG2.5%), 4) 8.0% arginine (ARG8.0%), 5) NaF + 2.5% arginine (NaFARG2.5%), and 6) NaF + 8.0% arginine (NaFARG8.0%). The second step will assess in situ effects of arginine-containing fluoride dentifrices on the microbial composition of dental biofilms and enamel demineralization. The tests will be performed on bovine enamel blocks fixed in palatal appliances of volunteers distributed in three groups, within a double-blind crossover design: control; 1,450 ppm NaF; and 1,450 ppm NaF + 8% arginine. The composition and viability of biofilms will be assessed by 3 distinct techniques: a) colony-forming units counts of specific microorganism groups (1st step), b) Human Oral Microbiome Identification using Next Generation Sequencing (HOMINGS) (1st and 2nd steps), and c) vitality of microbial cells in different depths of intact biofilms by confocal laser scanning microscopy (1st step). The metabolism of biofilms will be determined by the quantification of amounts of insoluble extracellular polysaccharides (colorimetric method) (1st and 2nd steps) and concentration of L-lactate in culture media (enzymatic spectrophotometric method) (1st step). The prevention of demineralization will be evaluated by the measurement of pH of culture media, quantification of calcium in culture medium by atomic absorption (1st step), quantification of fluoride and calcium in total biofilm (2nd step), and surface microhardness loss, integrated mineral loss and lesion depth by the mineral analyses of the structure of enamel blocks by microhardness tests and transverse microradiography (1st and 2nd steps). Statistical analysis will be based on the normality and homogeneity of the data. P values <0.05 will be considered significant. (AU)