Acquired pellicle and biofilm engineering with CaneCPI-5: Insights from proteomic and microbiomics analysis.

OBJECTIVE: In this in vivo proof-of-concept study, acquired pellicle engineering was implemented to promote alterations in the protein composition of the acquired enamel pellicle (AEP) and the bacterial composition of the dental biofilm after treatment with Sugarcane cystatin (CaneCPI-5). DESIGN: After prophylaxis, 10 volunteers rinsed (10 mL, 1 min) with the following solutions: 1) deionized water (H2O- negative control or 2) 0.1 mg/mL CaneCPI-5. The AEP and biofilm were formed along 2 or 3 h, respectively. The AEP was collected with electrode filter papers soaked in 3 % citric acid. After protein extraction, samples were analyzed by quantitative shotgun label-free proteomics. The biofilm microbiome was collected with a dental curette. The DNA was extracted, amplified, and analyzed by 16S-rRNA Next Generation Sequencing (NGS). RESULTS: Treatment with CaneCPI-5 increased several proteins with antimicrobial, acid-resistance, affinity for hydroxyapatite, structural and calcium binding properties, such as Cysteine-rich-3 (6-fold-p = 0.03), Cystatin-B (5.5-fold-p < 0.01), Neutrophil-defensin 1 (4.7-fold-p < 0.01), Mucin (3.9-fold-p < 0.01), Immunoglobulin-heavy-constant (3.8-fold-p < 0.01) and Lactotransferrin (2.8-fold-p < 0.01). Microbiome revealed that several commensal bacteria had their abundance increased after rinsing with CaneCPI-5, such as Corynebacterium and Neisseria, while Streptococcus and Prevotella nigrescens were decreased. The results indicate the efficiency of CaneCPI-5 in promoting beneficial changes in the AEP and biofilm, making this phytocystatin a potential target for incorporation into dental products. CONCLUSION: Cane demonstrated the capability to alter the protein composition of the acquired enamel pellicle (AEP) and the initial colonizers of the biofilm, enhancing the presence of proteins and bacteria crucial for dental protection.

The protective effect of the experimental TiF4 and chitosan toothpaste on erosive tooth wear in vitro.

This study evaluated the protective effect of TiF4 and chitosan toothpaste on erosive tooth wear (ETW) in vitro. Enamel and dentin samples were randomly assigned to toothpastes (n = 12): (G1) TiF4 (1400 ppm F-), (G2) 0.5% chitosan (75% deacetylation, 500 mPas), (G3) TiF4 (1400 ppm F-) plus 0.5% chitosan (75% deacetylation, 500 mPas), (G4) Placebo, (G5) Erosion Protection (Elmex-GABA, 1400 ppm F-). Twelve samples were only eroded. All samples were submitted to erosive pH cycles and G1 to G5 to abrasive challenges using toothpastes' slurries plus 45 s of treatment, for 7 days. The final profile was overlaid to the baseline one for the ETW calculation (µm). The data were subjected to Kruskal-Wallis/Dunn tests. TiF4 toothpastes, regardless of the presence of chitosan, were able to significantly reduce ETW compared to placebo, while chitosan alone was similar to placebo for both tissues. The toothpastes containing TiF4 were even superior to the commercial Elmex toothpaste on enamel, while they were similar on dentin; both were also significantly different from placebo for both tissues. TiF4 and Elmex toothpastes minimized the impact of brushing on eroded surface. In conclusion, TiF4 toothpastes, regardless the presence of chitosan, showed to be effective in minimizing ETW in vitro.

Effect of sweetener containing Stevia on the development of dental caries in enamel and dentin under a microcosm biofilm model.

OBJECTIVE: This study compared the effect of commercial and pure sweetener containing stevia to that of aspartame, to sucrose and xylitol on the development of dental caries. METHODS: 228 bovine enamel and root dentin were exposed to microcosm biofilm model using human saliva. From the 2nd to the 5th day, the samples were exposed daily to McBain saliva supplemented with 0.2% of the respective sweeteners/sugar, under 5% CO2 and 37 °C. The lactic acid and the colony-forming units (CFU) were quantified. The demineralization was analyzed by TMR. The data were compared statistically (Kruskal-Wallis/ Dunn, p<0.05). RESULTS: Pure stevia, pure aspartame, xylitol and control were able to significantly reduce 92% of lactate production compared to sucrose. Stevia finn, aspartame finn and sucrose showed similar production of lactic acid (around 0.45±0.12 g/L and 0.67±0.18 g/L, for enamel and dentin, p<0.0001). With respect to total lactobacilli and S. mutans/S. sobrinus CFU, xylitol and control did not show growth on enamel, while CFU numbers were found in stevia finn, aspartame finn and sucrose groups for both tissues. Enamel and dentin demineralization was significantly reduced for xylitol, control, pure stevia and pure aspartame (85% and 83% reduction, respectively) compared to stevia finn, aspartame finn and sucrose, which in turn did not differ from each other (sucrose ΔZ: 2913.7 ± 646.7 vol%.µm for enamel and 3543.3 ± 432.5 vol%.µm for dentin). CONCLUSIONS: Commercial sweeteners containing stevia and aspartame proved to be as cariogenic as sucrose, which may be due to the other components, since the pure forms were not cariogenic. CLINICAL RELEVANCE: Our study showed that some commercial sweeteners (aspartame and stevia) are as cariogenic as sucrose, which may be due to the presence of lactose. The population should be advice about the presence of lactose in such brand names, to avoid their consume.

Effect of TiF4/NaF and chitosan solutions on biofilm formation and prevention of dentin demineralization.

OBJECTIVE: This study evaluated the effect of experimental solutions containing TiF4/NaF and chitosan on bacterial species of microcosm biofilm and on dentin demineralization. DESIGN: Microcosm biofilm was produced from human saliva mixed with McBain medium (0.2% sucrose) on bovine dentin for 5 days, under 5% CO2 and 37 °C. From the 2nd day to 5th day, the treatments were applied (1×60s/day) as following: (1) NaF (500 ppm F-, positive control); (2) TiF4 and NaF (TiF4: 190 ppm Ti4+ and 300 ppm F-; NaF: 190 ppm F-); (3) similar to 2 plus 0.5% chitosan (Ch 500 mPa.s, 75% deacetylation); (4) phosphate buffer solution (negative control); and (5) 0.5% chitosan (Ch 500 mPa.s, 75% deacetylation). CFU counting was performed for total microorganism, total streptococci, total lactobacilli and mutans streptococci. Dentin demineralization was measured by transverse microradiography-TMR. The data were compared using ANOVA/Tukey or Kruskal-Wallis/Dunn tests (p < 0.05). RESULTS: No differences were found between the treatments with respect to CFU counting (p > 0.05). Dentin treated with TiF4/NaF plus chitosan solution presented the lowest demineralization compared to the negative control and pure chitosan solution. On the other hand, this experimental solution did not significantly differ from TiF4/NaF solution, being both able to significantly reduce mineral loss. CONCLUSION: TiF4/NaF plus chitosan solution, at suitable pH to be clinically applicable, had no antimicrobial effect, but it was able to reduce dentin caries development under this model.

Effect of TiF4/NaF and chitosan solutions on the development of enamel caries under a microcosm biofilm model.

OBJECTIVES: To evaluate the effect of experimental solutions containing TiF4/NaF and chitosan on bacterial species and on enamel caries prevention. METHODS: Microcosm biofilm was produced from human saliva mixed with McBain saliva (0.2% sucrose) on bovine enamel for five days, under 5% CO2 and 37 °C. From the second day until the end, the treatments were applied (1 × 60 s/day): (1) NaF (500 ppm F-, positive control); (2) TiF4 and NaF (TiF4: 190 ppm Ti4+ and 300 ppm F-; NaF: 190 ppm F-); (3) similar to 2 plus 0.5% chitosan (Ch 500 mPas, 75% deacetylation); (4) phosphate buffer solution (negative control); and (5) 0.5% chitosan (Ch 500 mPas, 75% deacetylation). CFU counting was performed for total microorganism, total streptococcus, total lactobacillus and Streptococcus mutans. Enamel demineralization was measured by transverse microradiography-TMR. The data were compared using ANOVA/Tukey or Kruskal-Wallis/Dunn tests (p < .050). RESULTS: No differences were found between the treatments with respect to CFU counting (ANOVA, p > .050). Enamel treated with TiF4/NaF plus chitosan solution presented the lowest demineralization compared to the negative control and pure chitosan solution. On the other hand, this experimental solution did not significantly differ from TiF4/NaF and NaF solutions, being all of them able to significantly reduce mineral loss (50-74%), but only TiF4/NaF plus chitosan reduced lesion depth (55%) compared to the negative control (p = .001). CONCLUSION: TiF4/NaF plus chitosan solution had no antimicrobial effect, but it was able to reduce enamel caries development in 79% compared to control under this model. CLINICAL SIGNIFICANCE: This study showed that TiF4/NaF plus chitosan solution had no antimicrobial effect, but it was able to reduce enamel caries development under a microcosm biofilm model.

Protective effect of titanium tetrafluoride and silver diamine fluoride on radiation-induced dentin caries in vitro.

This in vitro study evaluated the protective effect of titanium tetrafluoride (TiF4) varnish and silver diamine fluoride (SDF) solution on the radiation-induced dentin caries. Bovine root dentin samples were irradiated (70 Gy) and treated as follows: (6 h): 4% TiF4 varnish; 5.42% NaF varnish; 30% SDF solution; placebo varnish; or untreated (negative control). Microcosm biofilm was produced from human dental biofilm (from patients with head-neck cancer) mixed with McBain saliva for the first 8 h. After 16 h and from day 2 to day 5, McBain saliva (0.2% sucrose) was replaced daily (37 °C, 5% CO2) (biological triplicate). Demineralization was quantified by transverse microradiography (TMR), while biofilm was analyzed by using viability, colony-forming units (CFU) counting and lactic acid production assays. The data were statistically analyzed by ANOVA (p < 0.05). TiF4 and SDF were able to reduce mineral loss compared to placebo and the negative control. TiF4 and SDF significantly reduced the biofilm viability compared to negative control. TiF4 significantly reduced the CFU count of total microorganism, while only SDF affected total streptococci and mutans streptococci counts. The varnishes induced a reduction in lactic acid production compared to the negative control. TiF4 and SDF may be good alternatives to control the development of radiation-induced dentin caries.

Effect of titanium tetrafluoride/sodium fluoride solutions containing chitosan at different viscosities on the protection of enamel erosion in vitro.

OBJECTIVE: The aim of this in vitro study was to evaluate the protective effect against enamel erosion of experimental solutions containing TiF4/NaF and Chitosan compared to a commercial SnCl2/NaF/AmF solution. DESIGN: Bovine enamel samples were divided (n = 15/group) into: (1) commercial solution SnCl2/NaF/AmF (500 ppm F-, positive control); (2) NaF/TiF4 (490 ppm F-); (3) similar to 2 plus 0.5 % chitosan (Ch) (500 mPas), (4) similar to 2 plus 0.5 % chitosan (2000 mPas), (5) negative control (water), (6) 0.5 % chitosan (500 mPas) and (7) 0.5 % chitosan (2000 mPas). The samples were submitted to a pH cycling (0.1 % citric acid, 4 × 90 s/day, interposed by artificial saliva) and daily treatment application (after the last erosive challenge, 1 × 30 s/day) for seven days. After the first day, the surface reflection intensity changes (% rSRI) were measured. After 7 days, the erosive enamel loss was quantified by contact profilometer. The % rSRI and the enamel loss (µm) were compared using ANOVA/Tukey and Kruskal-Wallis/Dunn, respectively (p < 0.05). RESULTS: The solution containing TiF4/NaF plus Ch 500 mPas was the only able to reduce the early erosive demineralization compared to negative control (p = 0.003). However, it did not differ from the other solutions. Enamel samples treated with SnCl2/NaF/AmF presented the lowest median loss value [0.72 (0.18) µm] followed by both TiF4 + Ch [1.24 (0.49) and 1.28 (0.25)]; which significantly differed from the negative control [1.70 (0.27)]. CONCLUSION: The experimental solution containing TiF4/NaF plus chitosan (2000 mPas) has comparable effect to SnCl2/NaF/AmF on the protection against enamel erosion.

The effect of commercial whitening toothpastes on erosive dentin wear in vitro.

OBJECTIVES: This study evaluated the erosive tooth wear promoted by commercial whitening toothpastes on eroded dentin in vitro. DESIGN: Ninety bovine roots were embedded, polished and subjected to the baseline profile analysis. The samples were protected in 2/3 of the dentin surface and were randomly assigned to 6 groups (n = 15/group): Oral-B 3D White; Close-up Diamond Attraction Power White; Sorriso Xtreme White 4D; Colgate Luminous White; Crest and erosion only. All samples were submitted to erosive pH cycles (4 × 90 s in 0.1% citric acid, pH 2.5, per day) and abrasive challenges (2 × 15 s, per day) for 7 days. The samples were subjected to abrasion, using toothbrushing machine, soft toothbrushes and slurries of the tested toothpastes (1.5 N, 1:3 water). Between the challenges, the samples were immersed in artificial saliva. The final profile was overlaid to the baseline profile for the calculation of the erosive dentin wear (µm). The data were subjected to Kruskal-Wallis/Dunn tests (p < 0.05). RESULTS: Colgate Luminous White (4.7 µm) and Sorriso Xtreme White 4D (4.0 µm) promoted the highest wear, similarly to Oral-B 3D White (2.3 µm). Oral-B 3D White promoted similar wear compared to Crest (1.1 µm) and Close-up Diamond Attraction Power White (1.2 µm); however, it induced significant higher dentin wear compared to erosion only (1.0 µm). Close-up Diamond Attraction Power White and Crest did not increase the erosive wear compared to erosion only. CONCLUSION: Some whitening toothpastes increase the wear of eroded dentin, which should be considered by the dentist when prescribing them to patient with root exposure.

Effect of an experimental mouth rinse containing NaF and TiF4 on tooth erosion and abrasion in situ.

OBJECTIVE: This study compared the effect of an experimental NaF/TiF4 mouth rinse with a commercial tin/F mouth rinse on the prevention of tooth wear in situ. METHODS: Fifteen subjects took part in this crossover and double-blind study, in which they wore a palatal appliance with 8 bovine teeth samples (4 enamel and 4 root dentine) in each of 3 phases (5 days each). Half of the samples were subjected to erosive challenges, and the other half to erosive plus abrasive challenges. The phases corresponded to the use of 1) Experimental solution containing NaF/TiF4 (189 ppm Ti+4, 500 ppm F-, pH 4.4); 2) commercial solution containing SnCl2/NaF/AmF (800 ppm Sn+2, 500 ppm F-, pH 4.5, Elmex®/GABA, positive control); 3) distilled water (negative control). Erosive challenges were performed using 0.1% citric acid (pH 2.5) for 90 s 4 times per day. The abrasion was done using a toothbrush and slurry of fluoride toothpaste, for 15 s 2 times per day. Thereafter, the subjects rinsed with the tested mouth rinse for 60s. Tooth wear was measured using contact profilometry (µm) and submitted to a two-way RM ANOVA/Bonferroni test (p < 0.05). RESULTS: No significant differences were detected between the experimental and the commercial mouth rinses, regardless of the challenge. Both fluoride mouth rinses were able to significantly reduce tooth wear compared to the negative control (p < 0.0001). No significant differences were detected with respect to tooth wear between the challenges (erosion and erosion plus abrasion). CONCLUSION: The experimental NaF/TiF4 mouth rinse has a similar protective effect to the commercial one against tooth wear in situ. CLINICAL SIGNIFICANCE: The experimental NaF/TiF4 solution protected against tooth wear in situ, regardless of the challenge (erosion or erosion plus abrasion), for both enamel and dentine, similarly to a commercial solution (tin/F-Elmex®) applied for this proposal. This result supports the conduction of clinical trials and a possible application of this solution in the future.