A randomized, duple-blind crossover clinical trial study was performed to evaluate the bioavailability of intraoral fluoride in biomarkers of exposure (biofilm and saliva) after the use of dentifrice with experimental fluoride for one week, with wash-out periods between them. The biofilm was collected 1 hour (h) and 12h after toothbrushing, and saliva was collected 1h (T1, T15, T30, T45, T60 min) and 12h. The data was obtained by potentiometry (ion selective electrode) for fluoride in these samples.
Study population The sample size was determined by sample calculation \[25\], according to previous studies \[26\], α = 5%, β = 10%, and a dropout ratio of 10%. Eighteen individuals with an average of 26 years old, including both males and females, participated in this study. The eligibility criteria were people over 18 years old, without systemic commitment and living in the city of João Pessoa-Paraíba, without water fluoridation (\< 0.1 μg/mL F). Individuals were excluded if they used drugs that interfere with biofilm formation and salivary flow or fluoride products (\> 5000 ppm) in the last 4 weeks, used orthodontic appliances, or had cavity lesions, periodontal disease, and/or tooth sensitivity.
Experimental Dentifrices The participants were allocated randomly to use the following experimental dentifrices according to table 1. They were coded by an independent investigator not involved in the trial. A simple randomization was performed taking into account a cross design. The unit of randomization was "dentifrice", so all dentifrices had the same chance of being selected. The design of the study was created for each participant and for each new one another draw was made.
Clinical Stage First, the participants were submitted to dental prophylaxis and scraping to remove all biofilm and dental calculus. They used a placebo, dentifrice without fluoride, for 7 days. Then, the Whitford protocol was followed \[16\]. Briefly, the subjects were instructed to brush their teeth three times/day for 1 min (transversal technique) and also to rinse their mouths after brushing with 10 mL of water. On the seventh day, the subjects were instructed to brush only the occlusal surfaces and do not use dental floss, to allow biofilm accumulation. After going to bed, they abstained from eating or drinking anything except water and did not brush their teeth. The next morning, after 12 hours of last brushing, and fasting, the first samples of saliva and biofilm (upper and lower right hemi-arch) were collected. Then, the volunteers brushed the occlusal surfaces for 1 min. Soon after, saliva samples were collected in the following times: t1, t15, t30, t45, and t60 minutes. Biofilme samples (left side) were collected later (1 hour after brushing). Resting saliva was collected for 5 minutes. The biofilm was collected from all tooth surfaces, in both buccal and lingual areas, using a 3S hollenback spatula, from which the samples were immediately transferred to an eppendorff tube, centrifuged and subsequently dried at 90°C for two hours and then weighed. The saliva samples were centrifuged at 6,000 rpm for 10 minutes in order to separate the saliva debris.
Determination of the concentration of fluoride The samples were analyzed by the hexamethyldisiloxane (HMDS) facilitated diffusion method of Taves \[20\] modified by Whitford \[21\]. The analyzes were performed with a fluoride specific electrode (model 9409; Orion Research) and a potentiometer (model EA 940; Orion Research, Cambridge, MA, USA) (Model 720 A Orion). Fluoride standards (1-100 nM) were used to prepare calibration curves.
Statistical analysis The data were analyzed using descriptive and inferential statistics, using the statistical program SPSS - v. 21.0 and software Graphpad. The Shapiro-Wilk test was applied to test the normality of the in vivo study data (n \<50). Nonparametric tests were performed. The area under the curve (AUC) was calculated by taking baseline values (12 h after the last brush) up to 60 min after the last brush to indicate the effectiveness of F retention in saliva over time. For the purpose of comparing the concentration of F in saliva and biofilm between the groups, the ANOVA test of repeated measures was performed followed by the Bonferroni post-test. The relationship between the concentrations of biofilm and salivary fluoride was determined using Spearman's correlation coefficient. This study assumed a significance level of 5% (p \<0.05).
