Analysis of the antimicrobial and anti-caries effects of TiF4 varnish under microcosm biofilm formed on enamel

Abstract Titanium tetrafluoride (TiF4) is known for interacting with enamel reducing demineralization. However, no information is available about its potential antimicrobial effect. Objectives This study evaluated the antimicrobial and anti-caries potential of TiF4 varnish compared to NaF varnish, chlorhexidine gel (positive control), placebo varnish and untreated (negative controls) using a dental microcosm biofilm model. Material and Methods A microcosm biofilm was produced on bovine enamel previously treated with the varnishes, using inoculum from human saliva mixed with McBain saliva, under 0.2% sucrose exposure, for 14 days. All experiments were performed in biological triplicate (n=4/group in each experiment). Factors evaluated were: bacterial viability (% dead and live bacteria); CFU counting (log10 CFU/mL); and enamel demineralization (transverse microradiography – TMR). Data were analysed using ANOVA/Tukey's test or Kruskal-Wallis/Dunn's test (p<0.05). Results Only chlorhexidine significantly increased the number of dead bacteria (68.8±13.1% dead bacteria) compared to untreated control (48.9±16.1% dead bacteria). No treatment reduced the CFU counting (total microorganism and total streptococci) compared to the negative controls. Only TiF4 was able to reduce enamel demineralization (ΔZ 1110.7±803.2 vol% μm) compared to both negative controls (untreated: ΔZ 4455.3±1176.4 vol% μm). Conclusions TiF4 varnish has no relevant antimicrobial effect. Nevertheless, TiF4 varnish was effective in reducing enamel demineralization under this model.

The antimicrobial effect of fluoride depends on its concentration 20,21 . Varnish is the highest fluoride concentrated vehicle, with the advantage of having resinous base, which allows a long contact time with the tooth surface 16 . Most varnishes contain NaF as active agent, which has shown to be able to protect the teeth against dental caries when applied twice a year (46% of preventive fraction in permanent dentition) 16 .
On the other hand, our research group has tested the anticariogenic effect of an experimental 4% TiF 4 varnish compared to 5.42% NaF varnish under abiotic environment 15 . Our results have shown greater effect of TiF 4 varnish compared to NaF varnish due to its chemical reaction with enamel surface, promoting deposition of Ti compounds with high acid resistance.
However, none of our studies have tested its potential as antimicrobial agent, another possible mechanism of action related to fluorides. We expected that the glaze layer produced by TiF 4 varnish could alter the microorganism adhesion and, consequently, the biofilm growth and viability.
The use of microcosm biofilm, produced from microorganisms in human saliva, can benefit studies with monospecies or multispecies biofilms, allowing the presence of high number of microorganisms and interactions between them in the presence of fluoride or antimicrobial agents. Considering that 1) most studies on antimicrobial action of fluoride have been done using monospecies or dual-species 4,12,[20][21][22] and 2) the lack of knowledge on the antimicrobial effect of TiF 4 , the aim of this study was to compare the antimicrobial and anticariogenic effects of TiF 4 varnish with NaF varnish, chlorhexidine gel (positive control), placebo varnish (without any active agent) and untreated specimens (negative controls) using a microcosm biofilm model on bovine enamel.
This research tested the following null hypotheses: 1) There is no significant difference between the fluoride varnishes and positive control on the microbial viability; 2) There is no significant difference between the fluoride varnishes and positive control on CFU counting for total microorganisms and total streptococci; 3) There is no significant difference between the fluoride varnishes and positive control in reducing enamel demineralization.

Saliva collection
This study was firstly approved by the local Ethical Committee (CEEA 38143714.7.0000.5417). Saliva was collected from 2 healthy donors, who fit the following inclusion criteria: 1) normal salivary flow (stimulated saliva flow >1 mL/min and non-stimulated saliva flow

Tooth sample preparation and treatment
One hundred twenty (60 for viability assay and 60 for CFU counting) enamel samples (4 mm x 4 mm) were prepared from bovine teeth, using a semi-precision cutting machine (Buehler; Lake Bluff,

Microcosm biofilm formation
The human saliva was defrosted and mixed with

Results
In respect to the biofilm viability, only chlorhexidine was able to significantly increase the number of dead bacteria compared to untreated control (p<0.002), but it did not significantly differ from placebo. No significant differences were found between fluoride treatments or among fluoride treatments and negative or positive control. Figure 1 shows a representative confocal picture of the biofilm from each treatment group and Figure 2 shows the viability data. CFU  counting for total microorganism and total streptococci showed no significant differences among treatments (p>0.05) (Table 1). TiF 4 and NaF varnishes were similarly able to significantly reduce the integrated mineral loss and the average mineral loss compared to the untreated group and chlorhexidine, while only TiF 4 was significantly different from placebo varnish.
Both TiF 4 and NaF reduced lesion depth compared to control, but only TiF 4 was significantly different from placebo. Treatment with chlorhexidine did not reduce enamel demineralization (Table 2). Figure 3     we applied a commercial chlorhexidine gel that was able to reduce the microorganism viability in our study.
However, chlorhexidine had no effect on CFU counting.
We believe that chlorhexidine affects the viability of microorganisms not directly involved with dental caries, which are in lower quantity in our microcosm biofilm and, therefore, it did not have significant influence on the total microorganisms CFU counting.
Other possible explanation is that the bacteria affected one in situ study that tested the effect of AmF/NaF mouthrinse on the adhesion of bacteria to enamel and dentin, which showed some inhibition only for dentin, but not for enamel 10 , in agreement with our study.
Our study is the first one dealing with microcosm biofilm to test the antimicrobial effect of NaF and