Abstract Objective To evaluate the protective effect of an experimental solution containing TiF4/NaF on the development of radiation-induced dentin caries lesions. Methodology bovine root samples were irradiated (70Gy) and distributed as following (n=12/group): Commercial Saliva (BioXtra), NaF (500 ppm F-), TiF4 (500 ppm F), TiF4/NaF (TiF4: 300 ppm F-, NaF: 190 ppm F-), and Phosphate buffer solution (PBS, negative control). Biofilm was produced using biofilm from irradiated patients and McBain saliva (0.2% of sucrose, at 37oC and 5% CO2) for five days. The treatments were applied 1x/day. Colony-forming units (CFU) were counted and demineralization was quantified by transversal microradiography. The ANOVA/Tukey test was applied for all parameters. Results All treatments reduced CFU for total microorganisms. TiF4 reduced Lactobacillus sp. (7.04±0.26 log10 CFU/mL) and mutans streptococci (7.18±0.28) CFU the most, when compared to PBS (7.58±0.21 and 7.75±0.17) and followed by NaF (7.12±0.31 and 7.34±0.22) and TiF4/NaF (7.16±0.35 and 7.29± 0.29). TiF4 and Commercial saliva showed the lowest integrated mineral loss (ΔZ-vol%.mm) (1977±150 and 2062±243, respectively) when compared to PBS (4540±335), followed by NaF (2403±235) and TiF4/NaF (2340±200). Commercial saliva was the only to significantly reduce mineral loss (LD-µm) (111±25) compared to PBS (153±24).Mean mineral loss (R-vol%) decreased by 35.2% for TiF4 (18.2±3.3) when compared to PBS (28.1±2.9) Conclusion: TiF4/NaF has a comparable anti-cariogenic effect to TiF4 and Commercial saliva under the model in this study. TiF4NaF TiFNaF TiF radiationinduced radiation induced lesions 70Gy Gy (70Gy n=12/group n12group ngroup n 12 group (n=12/group) BioXtra, BioXtra , (BioXtra) 500 (50 F, F F-) F) (TiF4 30 F- 19 PBS, (PBS control. control . control) 0.2% 02 0 2 (0.2 sucrose oC 5 CO2 CO days 1xday xday 1x day x 1x/day Colonyforming Colony forming (CFU microradiography ANOVATukey ANOVA Tukey parameters microorganisms sp 7.04±0.26 704026 7 04 26 (7.04±0.2 log log1 CFU/mL CFUmL mL 7.18±0.28 718028 18 28 (7.18±0.28 most 7.58±0.21 758021 58 21 (7.58±0.2 7.75±0.17 775017 75 17 7.12±0.31 712031 31 (7.12±0.3 7.34±0.22 734022 34 22 7.16±0.35 716035 16 35 (7.16±0.3 729 29 7.29 0.29. 029 0.29 0.29) ΔZvol%.mm ΔZvolmm ΔZ vol%.mm vol mm (ΔZ-vol%.mm 1977±150 1977150 1977 150 (1977±15 2062243 2062 243 2062±243 respectively 4540±335, 4540335 4540±335 4540 335 (4540±335) 2403±235 2403235 2403 235 (2403±235 2340±200. 2340200 2340±200 2340 200 (2340±200) LDµm LD µm (LD-µm 111±25 11125 111 25 (111±25 153±24.Mean 15324Mean Mean 153±24 .Mean 153 24 Rvol% Rvol R vol% (R-vol% 352 35.2 18.2±3.3 18233 3 (18.2±3.3 28.1±2.9 28129 1 9 (28.1±2.9 Conclusion anticariogenic anti cariogenic study (n=12/group (BioXtra 50 (5 (TiF 0.2 (0. 7.04±0.2 70402 (7.04±0. 7.18±0.2 71802 (7.18±0.2 7.58±0.2 75802 (7.58±0. 7.75±0.1 77501 7.12±0.3 71203 (7.12±0. 7.34±0.2 73402 7.16±0.3 71603 (7.16±0. 72 7.2 ΔZvol volmm 1977±15 197715 197 15 (1977±1 206224 206 2062±24 454033 4540±33 454 33 (4540±335 2403±23 240323 240 23 (2403±23 234020 2340±20 234 20 (2340±200 111±2 1112 11 (111±2 15324 153±2 (R-vol 35. 18.2±3. 1823 (18.2±3. 28.1±2. 2812 (28.1±2. ( 0. (0 7.04±0. 7040 (7.04±0 7.18±0. 7180 (7.18±0. 7.58±0. 7580 (7.58±0 7.75±0. 7750 7.12±0. 7120 (7.12±0 7.34±0. 7340 7.16±0. 7160 (7.16±0 7. 1977±1 19771 (1977± 20622 2062±2 45403 4540±3 45 (4540±33 2403±2 24032 (2403±2 23402 2340±2 (2340±20 111± (111± 1532 153± 18.2±3 182 (18.2±3 28.1±2 281 (28.1±2 7.04±0 704 (7.04± 7.18±0 718 (7.18±0 7.58±0 758 (7.58± 7.75±0 775 7.12±0 712 (7.12± 7.34±0 734 7.16±0 716 (7.16± 1977± (1977 2062± 4540± 4 (4540±3 2403± (2403± 2340± (2340±2 (111 18.2± (18.2± 28.1± (28.1± 7.04± 70 (7.04 7.18± 71 (7.18± 7.58± (7.58 7.75± 77 7.12± (7.12 7.34± 73 7.16± (7.16 (197 (4540± (2403 (2340± (11 18.2 (18.2 28.1 (28.1 7.04 (7.0 7.18 (7.18 7.58 (7.5 7.75 7.12 (7.1 7.34 7.16 (19 (4540 (240 (2340 (1 18. (18. 28. (28. 7.0 (7. 7.1 7.5 7.7 7.3 (454 (24 (234 (18 (28 (7 (45 (2 (23 (4

Abstract Microcosm biofilms can reproduce the complexity of a dental biofilm. However, different forms of cultivation have been used. The impact of the culture atmosphere on the development of microcosm biofilms and their potential to cause tooth demineralization has not yet been deeply studied. Objective This study analyzes the effects of three experimental cultivation models (microaerophile vs. anaerobiosis vs. experimental mixed) on the colony-forming units (CFU) of the cariogenic microorganisms and tooth demineralization. Methodology 90 bovine enamel and 90 dentin specimens were distributed into different atmospheres: 1) microaerophilia (5 days, 5% CO2); 2) anaerobiosis (5 days, jar); 3) mixed (2 days microaerophilia and 3 days anaerobiosis), which were treated with 0.12% chlorhexidine (positive control – CHX) or Phosphate-Buffered Saline (negative control – PBS) (n=15). Human saliva and McBain’s saliva containing 0.2% sucrose were used for microcosm biofilm formation, for 5 days. From the second day to the end of the experiment, the specimens were treated with CHX or PBS (1x1 min/day). Colony-forming units (CFU) were counted, and tooth demineralization was analyzed using transverse microradiography (TMR). Data were subjected to two-way ANOVA and Tukey’s or Sidak’s test (p<0.05). Results CHX was able to reduce total microorganism’s CFU compared to PBS (differences of 0.3–1.48 log10 CFU/mL), except for anaerobiosis and microaerophilia in enamel and dentin biofilm, respectively. In the case of dentin, no effect of CHX on Lactobacillus spp. was observed. CHX significantly reduced enamel demineralization compared to PBS (78% and 22% reductions for enamel and dentin, respectively). Enamel mineral loss did not differ when compared with the other atmospheres; however, the enamel lesion depth was greater under anaerobiosis. Dentin mineral loss was lower under anaerobiosis when compared with the other atmospheres. Conclusion The type of atmosphere has, in general, little influence on the cariogenic ability of the microcosm biofilm. However studied microaerophile vs colonyforming colony forming (CFU 9 atmospheres 1 ( CO2 CO CO2) 2 jar jar) anaerobiosis, , anaerobiosis) 012 0 12 0.12 positive PhosphateBuffered Phosphate Buffered negative n=15. n15 n n=15 . 15 (n=15) McBains McBain s 02 0.2 formation experiment 1x1 x (1x min/day. minday min/day min min/day) Colonyforming Colony counted TMR. TMR (TMR) twoway two way Tukeys Tukey Sidaks Sidak p<0.05. p005 p p<0.05 05 (p<0.05) microorganism differences 03148 48 0.3–1.4 log log1 CFU/mL, CFUmL CFU/mL mL CFU/mL) respectively spp observed 78% 78 (78 22 respectively) however general 01 0.1 n1 n=1 (n=15 0. 1x (TMR p00 p<0.0 (p<0.05 0314 4 0.3–1. 7 (7 n= (n=1 p0 p<0. (p<0.0 031 0.3–1 (n= p<0 (p<0. 03 0.3– (n p< (p<0 0.3 (p< (p

Abstract Quantification of collagen degradation is an important parameter to evaluate dentin caries for preventive aid. Objectives: Evaluate preventive methods against root collagen degradation by the hydroxyproline assay (HYP) and microradiography technique (MRT). Methodology: Five bovine root dentin blocks were obtained and subjected to an artificial demineralization process by acetate buffer (pH 5) to induce carious lesion formation. Samples were subjected to the following therapeutic treatments: 1) 0.12% chlorhexidine for 1 min, 2) 2% fluoride for 1 min, 3) Nd:YAG Laser (400 μm diameter optical fiber, 10 Hz frequency, 60 mJ/pulse energy, 48 J/cm2 energy density, in noncontact mode for 10 s), 4) deionized water (control) for 1 min, 5) MRT control group (without treatment and removal of collagen). Samples were exposed to degradation by a collagenase enzyme for five days. The enzyme solution was collected, by colorimetry in a spectrophotometer, from the collagen matrix for the hydroxyproline release analysis. The same samples were subjected to an additional two days of demineralization to induce the progression of mineral loss. Samples were analyzed by MRT for the visualization of their degraded areas (estimation of lesion depth and mineral loss). ANOVA was applied to compare hydroxyproline release rates. MRT data were subjected to the Kruskal-Wallis test, followed by the Dunn’s test. Comparisons between the initial five-day and the subsequent two-day demineralization processes were performed by repeated t-test or Wilcoxon (p<0.05) measurements. Results: The amount of HYP released from the dentin samples failed to show significant differences among the groups (p=0.09). Fluoride and chlorhexidine were able to interact with the samples, reducing the progression of dentin caries after removal of the demineralized organic matrix. CHX was the only treatment able to show significant lower lesion depth than the negative control. Conclusion: Chlorhexidine and fluoride were effective in reducing root caries progression.

Abstract The initial characteristics of white spot lesion (WSLs), such as the degree of integrated mineral loss (ΔZ), depth and pattern of mineral distribution, have an impact on further demineralization and remineralization. However, these lesion parameters have not been evaluated in WSLs produced from microcosm biofilms. Objective: This study characterized artificial white spot lesions produced on human enamel under microcosm biofilm for different experimental periods. Methodology: In total, 100 human enamel specimens (4x4mm) were assigned to 5 distinct groups (n=20/group) differing according to the period of biofilm formation (2, 4, 6, 8 or 10 days). Microcosm biofilm was produced on the specimens from a mixture of human and McBain saliva at the first 8h. Enamel samples were then exposed to McBain saliva containing 0.2% sucrose. WSLs formed were characterized by quantitative light-induced fluorescence (QLF) and transverse microradiography (TMR). Data were analyzed by ANOVA/Tukey or Kruskal-Wallis/Dunn tests (p<0.05). Results: A clear time-response pattern was observed for both analyses, but TMR was able to better discriminate among the lesions. Regarding QLF analysis, median (95%CI; %) changes in fluorescence ∆Z were -7.74(-7.74:-6.45)a, -8.52(-8.75:-8.00)ab, -9.17(-10.00:-8.71)bc, -9.58(-10.53:-8.99)bc and -10.01(-11.44:-9.72)c for 2, 4, 6, 8, and 10 days, respectively. For TMR, median (95%CI; vol%.µm) ∆Z were 1410(1299-1479)a, 2420(2327-2604)ab, 2775(2573-2899)bc, 3305(3192-3406)cd and 4330(3972-4465)d, whereas mean (SD; µm) lesion depth were 53.7(12.3)a, 71.4(12.0)a, 103.8(24.8)b, 130.5(27.2)bc, 167.2(39.3)c for 2, 4, 6, 8 and 10 days, respectively. Conclusion: The progression of WSLs formed on human enamel under microcosm biofilm can be characterized over 2-10 days, both by QLF and TMR analyses, although the latter provides better discrimination among the lesions.

Abstract: This randomized three-armed controlled clinical trial compared the effect of titanium tetrafluoride (TiF4) and sodium fluoride (NaF) varnishes on caries control in smooth surfaces of permanent dentition and children's acceptability. Sixty children (6-8 y/o) were randomly divided into TiF4 (2.45% F–), NaF (2.26% F–) or placebo (control) groups. Varnishes were applied on permanent teeth once a week for the first 4 weeks and after the 6th and 12th months of the study. The variables were as follows: International Caries Detection and Assessment System (ICDAS) scores, quantitative fluorescence changes, visual plaque index (VPI) and degree of acceptability. Two-way RM-ANOVA, ANOVA/Tukey and χ2 tests were performed (p < 0.05). No differences were found between the treatments with respect to ICDAS scores (p = 0.32). Only TiF4 reduced the mean fluorescence loss significantly at 18 months compared to the baseline (p = 0.003). TiF4 showed a lower percentage of new caries lesions by tooth surface than the placebo, while NaF did not induce such a change (p < 0.014). Regardless of the treatment, more than 95% of the participants reported being satisfied. For all groups, the VPI decreased significantly at 3 months compared to the baseline value (p < 0.001), with no differences between the treatments (p = 0.17). TiF4 had a similar ability to control caries lesions as NaF; however, only TiF4 differed from the placebo (p = 0.004). The acceptability of TiF4 varnish was similar to that of NaF varnish.

Abstract Objectives: The aim of this study was to assess the effect of Myracrodruon urundeuva All. and Qualea grandiflora Mart. leaves hydroalcoholic extracts on viability and metabolism of a microcosm biofilm and on enamel demineralization prevention. Methodology: Microcosm biofilm was produced on bovine enamel using inoculum from pooled human saliva mixed with McBain saliva, under 0.2% sucrose exposure, for 14 days. The biofilm was daily-treated with the extracts for 1 min. At the end, it was analyzed with respect to viability by fluorescence, CFU counting and extracellular polysaccharides (phenol-sulphuric acid colorimetric assay) and lactic acid (enzymatic assay) production. The demineralization was measured by TMR. The data were compared using ANOVA or Kruskal-Wallis (p<0.05). Results: M. urundeuva All. at 100, 10 and 0.1 μg/mL and Q. grandiflora Mart. at 100 and 0.1 μg/mL reduced biofilm viability similarly to positive control (chlorhexidine) and significantly more than the negative-vehicle control (35% ethanol). M. urundeuva at 1000, 100 and 0.1 μg/mL were able to reduce both lactobacilli and mutans streptococci CFU counting, while Q. grandiflora (1000 and 1.0 μg/mL) significantly reduced mutans streptococci CFU counting. On the other hand, the natural extracts were unable to significantly reduce extracellular polysaccharides and lactic acid productions neither the development of enamel carious lesions. Conclusions: The extracts showed antimicrobial properties on microcosm biofilm, however, they had no effect on biofilm metabolism and caries protection.

Abstract Microcosm biofilm has been applied to induce carious lesions in dentin. However, no study has been done to compare the impact of the type of model for providing nutrients to microcosm biofilm formation on dentin. Objective This study compared the performance of two kinds of models (static and semi-dynamic) on the biofilm formation and the development of dentin carious lesions. Material and Methods In both models, biofilm was produced using inoculum from pooled human saliva mixed with McBain saliva for the first 8 h (5% CO2 and 37°C). Afterwards, for the static model, the samples were placed in 24-wells microplate containing McBain saliva with 0.2% sucrose, which was replaced at 24 h. In the semi-dynamic model, the samples were submitted to artificial mouth system with continuous flow of McBain saliva with 0.2% sucrose (0.15 ml/min, 37°C) for 10 h a day (for the other 14 h, no flow was applied, similarly to the static model). After 5 days, biofilm viability was measured by fluorescence and dentin demineralization by transverse microradiography. Results Biofilm viability was significantly lower for the static compared with semi-dynamic model, while dentin demineralization was significantly higher for the first one (p<0.05). The static model was able to produce a higher number of typical subsurface lesions compared with the semi-dynamic model (p<0.05). Conclusions The type of model (static and semi-dynamic) applied in the microcosm biofilm may have influence on it's viability and the severity/profile of dentin carious lesions.