Application of fluoride disturbs plaque microecology and promotes remineralization of enamel initial caries

Abstract

Background: The caries-preventive effect of topical fluoride application has been corroborated by a number of clinical studies. However, the effect of fluoride on oral microecology remains unclear.

Objective: To monitor the effect of fluoride on dental plaque microecology and demineralization/remineralization balance of enamel initial caries.

Methods: Three-year-old children were enrolled and treated with fluoride at baseline and 6 months. International Caries Detection and Assessment System II indices of 52 subjects were measured at baseline, 3, 6, and 12 months. Supragingival plaque samples of 12 subjects were collected at baseline, 3 and 14 days for 16S rRNA sequencing.

Results: Changes in microbial community structure were observed at 3 days after fluoridation. Significant changes in the relative abundance of microorganisms were observed after fluoride application, especially Capnocytophaga, unidentified Prevotellaceae and Rothia. Functional prediction revealed that cell movement, carbohydrate and energy metabolism were affected significantly after fluoride application. Fluoride significantly inhibited enamel demineralization and promoted remineralization of early demineralized caries enamel at 3 months.

Conclusion: Fluoride application significantly inhibited the progression of enamel initial caries and reversed the demineralization process, possibly by disturbing dental plaque microecology and modulating the physicochemical action of demineralization/remineralization. This deepened our understanding of caries-preventive effects and mechanisms of fluoride.

Keywords: 16S rRNA sequencing; Fluoride; ICDAS II; enamel initial caries; oral microecology.

Figure 1.

Detection teeth positions and sites using ICDASII. Representative positions and detected sites were selected, namely, the occlusal surface (O), gingival 1/3 of the buccal surface (GB), occlusal 2/3 of the buccal surface (OB) of the first deciduous molar teeth 54, 64, 74 and 84 and gingival 1/3 of the labial surface (GL), incisal 2/3 of the labial surface (IL) of the deciduous central incisor teeth 51, 61, 71 and 81.

Figure 2.

The CONSORT flow diagram of the progress through the phases of the trial (enrollment, allocation, follow-up, and analysis). N indicates the number of subjects. The number of lost subjects and the reasons for lost to follow-up are listed in the diagram.

Figure 3.

Alpha diversity analysis at different sampling time points. Alpha diversity indices of different samples at a 97% congruence threshold were statistically analyzed (a: Chao1; b: ACE; c: Shannon; d: Simpson), and the amount of data selected for homogenization was based on the cutoff = 32,438. H0, H3d and H2w represent the time points before, 3 days and 14 days after the first fluoride application, respectively.

Figure 4.

Beta diversity analysis at different sampling time points. Beta diversity were evaluated by PCoA analysis based on unweighted UniFrac distances (a) and weighted UniFrac distances (c). The data were analyzed by Wilcoxon rank sum tests (b, d). The asterisks indicate P values (one asterisk, P < 0.05; two asterisks, P < 0.01). H0, H3d and H2w represent the time points before, 3 days and 14 days after the first fluoride application, respectively.

Figure 5.

Bacterial abundance, distribution and differential microbiota compositions of supragingival plaque samples. Stacked bar charts showing the relative abundance of the supragingival plaque microbiota (the top 10) at the phylum level (a) and genus level (b) at three time points. The plot of PLS-DA analysis (c) indicates the difference between samples and groups, and the VIP scores plot of PLS-DA (d) reflects the key genera between groups at different time points. The abscissa represents the VIP scores and the ordinate represents the key genera with VIP scores >1. The histogram of LDA value distribution revealed the effect size of each differentially featured taxa (LDA > 2, P < 0.05) between baseline and 3 days (e) and between baseline and 14 days (f). Different colors suggest the enrichment of certain taxa in corresponding groups. H0, H3d and H2w represent the time points at baseline, 3 days and 14 days after the first fluoride application.

Figure 6.

Analysis of significant differences in predicted metabolic functions between groups. Paired Wilcoxon rank sum test revealed the differences in predicted metabolic functions between baseline and 3 days (a), between 3 days and 14 days (b), and between baseline and 14 days (c). The abscissa represents the P values and the ordinate represents subfunctions of predicted metabolic pathways. P < 0.05 means statistically significant. H0, H3d and H2w represent the time points at baseline, 3 days and 14 days after the first fluoride application.

Figure 7.

The proportions of different ICDAS II codes at different time points. a-e showed the change in the proportions of different ICDAS II codes at different time points at the sites of the gingival 1/3 of the buccal surface (GB, a), occlusal 2/3 of the buccal surface (OB, b), gingival 1/3 of the labial surface (GL, c), incisal 2/3 of the labial surface (IL, d) and occlusal surface (O, e). The abscissa represents the ICDAS II codes (0, 1, 2, 3–6) and the ordinate represents percentages of different codes at different time points. The asterisks indicate P values (one asterisk, P < 0.05),which represents the proportion of an ICDAS II code significantly different from the baseline.