A new role for resveratrol: Protection of enamel against erosion.

OBJECTIVE: The aim of this study was to determine the effect of different concentrations of resveratrol in protecting enamel against initial dental erosion in vitro. METHODS: Ninety bovine enamel samples (4 × 4 mm) were divided into six groups: Phosphate buffered saline (negative control; PBS), Commercial solution (Elmex Erosion Protection™; positive control) and resveratrol at 4 different concentrations (1, 10, 100 or 400 µg/mL). Initially, the samples were incubated in saliva for the formation of the acquired pellicle (250 µL, 1 h, 37 °C, 250 rpm). Afterward, the samples were incubated in the respective treatments (250 µL, 1 min, 37 °C, 250 rpm) and then reincubated in saliva (250 µL, 1 h, 37 °C, 250 rpm). Finally, the samples were subjected to an erosive challenge by incubating in 1 % citric acid (1 mL, pH 3.5, 1 min, 25 °C, 250 rpm). The percentage surface microhardness change (% SMC) was assessed using a microhardness tester. Data were analyzed by Kruskal-Wallis and Dunn's tests (p < 0.05). RESULTS: The treatments with Elmex™ and resveratrol (1, 10 and 100 µg/mL) significantly protected enamel compared to the negative control, without significant differences among them. However, the group treated with the highest resveratrol concentration (400 µg/mL) did not show a significant difference from the negative control. CONCLUSIONS: Resveratrol at concentrations ranging from 1 to 100 µg/ml was effective in preventing loss of enamel surface microhardness. CLINICAL SIGNIFICANCE: This result suggests a potential new direction for the development of dental products based on resveratrol for the prevention of dental erosion.

Acquired pellicle engineering with the association of cystatin and vitamin E against enamel erosion.

OBJECTIVE: Evaluate CaneCPI-5 associated with Vitamin E in acquired enamel pellicle (AEP) engineering to prevent dental erosion. METHODS: 180 human enamel specimens were divided into 12 groups and treated with the following solutions: Cane+VitT and Cane+VitS- CaneCPI-5 + Vit E; Vit+CaneT and Vit+CaneS- Vit E + CaneCPI-5; VitT and VitS- Vit E; CaneT and CaneS- CaneCPI-5; ControlT and ControlS - AmF/NaF/SnCl2; WaterT and WaterS- Deionized water. Groups' name followed by "T" were first treated (200 µl; 2 min) and then incubated in human saliva (200 µl; 1 h) to form the AEP. For groups followed by "S", the AEP was formed and then treatment was applied. The erosive challenge consisted of immersion in 1% citric acid (1 min, 1x/day, for 3 days). The percentage of superficial hardness loss (%SHL) and the relative surface reflection intensity (%SRI) were subjected to normality and homogeneity tests, Shapiro-Wilk and Levene tests, respectively. Subsequently, the data were analyzed using two-way ANOVA, Tukey's test and Pearson's correlation (p < 0.005). RESULTS: For%SHL and%SRI, water controls showed significantly lower protective capacity. Cane+VitT, Cane+VitS, and Vit+CaneS presented the lowest%SHL, and VitT and VitS did not differ from Vit+CaneT, but they were different from the other groups (p = 0.002). The greatest%SRI was found for the Cane+VitT, Vit+CaneT, VitT, Cane+VitS, Vit+CaneS, and VitS groups, which did not significantly differ. CaneT and ControlT, showed similar reflections compared to CaneS and ControlS. CONCLUSION: CaneCPI-5 and Vitamin E demonstrated a synergistic protective effect against initial erosion. CLINICAL SIGNIFICANCE: The results open up new possibilities for preventive approaches against erosion through the acquired pellicle engineering, with the combination of CaneCPI-5 and Vitamin E, which demonstrated to be more effective than commercial stannous mouthwash. Further research is warranted to explore the potential of this combination in diverse clinical settings.

Different vehicles containing CaneCPI-5 reduce erosive dentin wear in situ.

OBJECTIVE: This study evaluated the protective capacity of a sugarcane-derived cystatin (CaneCPI-5) in different vehicles (1-solution and 2-chitosan gel) against erosive dentin wear in situ. METHODS: In part-1, 15 volunteers participated in a crossover protocol (solutions): Water; Elmex™ and CaneCPI-5. The volunteers wore an appliance with 4 dentin samples for 5 days. These samples were treated with a drop of the solutions for 1 min (4X/d), then the acquired pellicle (AP) was formed and the samples were subjected to erosive challenges (EROSION: citric acid, for 90 s, 4X/day). 2X/day, half of the samples were also abraded for 15 s (ABRASION). In part-2, 16 volunteers participated in a crossover protocol (gel): No gel, Chitosan gel, Chitosan gel + NaF and Chitosan gel + CaneCPI-5. The volunteers also wore an appliance. The samples were treated once/day with the gel or not for 4 min, then the AP was formed and the samples were subjected to erosive and abrasive challenges, as reported in part-1. Dentin wear was measured by profilometry. Data were analyzed by two-way RM-ANOVA and Sidak's tests (p < 0.05). RESULTS: Part-1: Elmex™ and CaneCPI-5 significantly reduced dentin loss in comparison with Water for the EROSION/ABRASION conditions (p < 0.05). Part-2, all the treated groups significantly reduced the dentin loss in comparison to the No gel. The greatest reduction was found for the gel + CaneCPI-5 group for the EROSION/ABRASION (p < 0.05). CONCLUSION: The solution and chitosan gel containing CaneCPI-5 protected against erosive dentin wear in situ. CLINICAL RELEVANCE: These different vehicles are probably sufficient for protecting people with high risk of developing erosive dentin wear.

Maquiberry Cystatins: Recombinant Expression, Characterization, and Use to Protect Tooth Dentin and Enamel.

Phytocystatins are proteinaceous competitive inhibitors of cysteine peptidases involved in physiological and defensive roles in plants. Their application as potential therapeutics for human disorders has been suggested, and the hunt for novel cystatin variants in different plants, such as maqui (Aristotelia chilensis), is pertinent. Being an understudied species, the biotechnological potential of maqui proteins is little understood. In the present study, we constructed a transcriptome of maqui plantlets using next-generation sequencing, in which we found six cystatin sequences. Five of them were cloned and recombinantly expressed. Inhibition assays were performed against papain and human cathepsins B and L. Maquicystatins can inhibit the proteases in nanomolar order, except MaquiCPIs 4 and 5, which inhibit cathepsin B in micromolar order. This suggests maquicystatins' potential use for treating human diseases. In addition, since we previously demonstrated the efficacy of a sugarcane-derived cystatin to protect dental enamel, we tested the ability of MaquiCPI-3 to protect both dentin and enamel. Both were protected by this protein (by One-way ANOVA and Tukey's Multiple Comparisons Test, p < 0.05), suggesting its potential usage in dental products.

Use of Reflectometer Optipen to assess the preventive effect of a sugarcane cystatin on initial dental erosion in vivo.

The sugarcane cystatin 5 (CaneCPI-5) showed protection against erosion and erosive tooth wear (ETW) under several protocols. However, evaluating these conditions in vivo is hard due to the lack of a suitable device. The aim of this study was to use clinically the relative surface reflection intensity (%SRI) by the Reflectometer Optipen to assess the acquired pellicle engineering with CaneCPI-5 rinse for the prevention of initial erosion in vivo. Nine volunteers were distributed in three cross-over phases, according to the rinse used, as follows: 1) Deionized water (negative control); 2) Elmex® (800 ppm Sn2+, 500 ppm F-; positive control); 3) 0.1 mg/mL CaneCPI-5. The following experimental steps were performed: Initially, the volunteers received prophylaxis and the initial %SRI was performed. Subsequently, they rinsed with the solutions (10 mL; 1min), followed by the formation of the acquired enamel pellicle (AEP; 120min). After, the erosive challenge with citric acid 1%, pH 2.5 (10 µL; 10s) was performed (in isolation) on the buccal surface of the maxillary central incisors (right and left). The calcium present in the acid was analyzed by Arsenazo III method. Finally, the final %SRI was performed. Data were analyzed by Kruskal-Wallis/Dunn's tests and Spearman's correlation were used (p < 0.05). For both variables, the negative control led to significantly less protection (lower reflectivity and higher calcium release) in comparison with the other groups. The best protection (higher reflectivity and lower calcium release) was observed for the Elmex® and the CaneCPI-5 groups, with no significant differences between them (p < 0.05). There was a significant correlation between both analyzes. The Reflectometer Optipen demonstrated to be a good device to assess clinically. Moreover, CaneCPI-5 rinse proved effective through acquired pellicle engineering against initial erosion in vivo.

New insights into the protective effect of statherin-derived peptide for different acquired enamel pellicle formation times on the native human enamel surfaces.

OBJECTIVES: This study evaluated the protective impact of acquired enamel pellicle (AEP) engineering with statherin-derived peptide (StatpSpS), considering different AEP formation times. DESIGN: A total of 120 native human enamel specimens were divided into 2 main groups: 1) No AEP engineering and 2) AEP engineering with StatpSpS (pretreatment for 1 min; 37 °C, under agitation). Each group was further divided into 4 subgroups: No pellicle, or 1, 60-and-120 min AEP formation times (human saliva; 37 °C). The specimens were then subjected to an erosive challenge (1% citric acid; pH 3.6; 1 min; 25 °C). This procedure was repeated for 5 cycles. Relative surface reflection intensity (%SRI) was measured and scanning electron microscopy (SEM) of the enamel surface was done. RESULTS: All AEP engineering groups protected against initial dental erosion in comparison with No pellicle (p < 0.001), likewise all groups with AEP, independent of engineering or formation times (p 0.001). Furthermore, engineering with StatpSpS even without the presence of AEP protected the enamel when compared to the No engineering/No pellicle group (p < 0.0001). No difference was observed regarding the protection from the different AEP formation times (p > 0.05). Regarding the SEM analysis, in the "No AEP engineering & No AEP" group, a more severe effect of citric acid was observed, with more enamel prism heads and scratches on the surface when compared with the other groups. CONCLUSIONS: AEP provides almost instant protection at formation times even as short as 1 min, protecting the native enamel against erosion. Treatment with StatpSpS by itself provides similar protection as the AEP.

Solutions Containing a Statherin-Derived Peptide Reduce Enamel Erosion in vitro.

The effect of solutions containing a statherin-derived peptide (Stn15pSpS) on the protection against enamel erosion in vitro was evaluated. Bovine enamel specimens were divided into 4 groups (n = 15/group): (1) deionized water (negative control), (2) Elmex Erosion Protection™ (positive control), (3) 1.88 × 10-5 M Stn15pSpS, and (4) 3.76 × 10-5 M Stn15pSpS. The solutions were applied on the specimens for 1 min. Stimulated saliva was collected from 3 donors and used to form a 2-h acquired pellicle on the specimens. Then, the specimens were submitted to an erosive pH-cycling protocol 4 times/day, for 7 days (0.01 M HCl pH 2.0/45 s, artificial saliva/2 h, and artificial saliva overnight). The solutions were applied again during pH-cycling, 2 times/day for 1 min after the first and last erosive challenges. Enamel loss (µm) was assessed by contact profilometry. Data were analyzed by Kruskal-Wallis and Dunn's test (p < 0.05). The best protection against erosion was conferred by Elmex Erosion Protection that significantly differed from all the other treatments, followed by the solutions containing Stn15pSpS, regardless of the concentration. However, 3.76 × 10-5 M Stn15pSpS did not differ from the negative control. The solution containing the lower concentration of Stn15pSpS protected against erosion in vitro, which should be confirmed using protocols that more closely resemble the clinical condition.

Teeth with acute apical abscess vs. teeth with chronic apical periodontitis: a quantitative and qualitative proteomic analysis.

OBJECTIVE: To quantitatively and qualitatively analyze the proteomic profile of teeth with acute apical abscesses (AAA) compared with teeth with chronic apical periodontitis (CAP) and to correlate the expression of detected human proteins with their main biological functions. MATERIALS AND METHODS: Samples were obtained from root canals of 9 patients diagnosed with AAA and 9 with CAP. Samples were analyzed by reversed-phase liquid chromatography coupled to mass spectrometry. Label-free quantitative proteomic analysis was performed by Protein Lynx Global Service software. Differences in protein expression were calculated using the t-test (p < 0.05). RESULTS: In total, 246 human proteins were identified from all samples. Proteins exclusively found in the AAA group were mainly associated with the immunoinflammatory response and oxidative stress response. In the quantitative analysis, 17 proteins were upregulated (p < 0.05) in the AAA group, including alpha-1-acid glycoprotein, hemopexin, fibrinogen gamma chain, and immunoglobulin. Additionally, 61 proteins were downregulated (p < 0.05), comprising cathepsin G, moesin, gelsolin, and transketolase. Most of the proteins were from the extracellular matrix, cytoplasm, and nucleus. CONCLUSIONS: The common proteins between the groups were mainly associated with the immune response at both expression levels. Upregulated proteins mostly belonged to the acute-phase proteins, while the downregulated proteins were associated with DNA/RNA regulation and repair, and structural function. CLINICAL RELEVANCE: The host response is directly related to the development of apical abscesses. Thus, understanding the behavior of human proteins against the endodontic pathogens involved in this condition might contribute to the study of new approaches related to the treatment of this disease.

Gels containing statherin-derived peptide protect against enamel and dentin erosive tooth wear in vitro.

The effect of gels containing a statherin-derived peptide (Stn) on the protection against enamel and dentin erosive tooth wear (ETW) in vitro was evaluated. Bovine enamel and dentin specimens were divided into 2 groups (n = 15 and 18/group for enamel and dentin, respectively) that were treated with Chitosan or Carboxymethylcellulose (CMC) gels containing Stn15pSpS at 1.88 × 10-5 M or 3.76 × 10-5 M. Chitosan or CMC gels without active ingredients served as negative controls, while chitosan gel containing 1.23% F (as NaF) and acidulated phosphate fluoride gel (1.23% F) served as positive controls. The gels were applied on the specimens for 4 min. Stimulated saliva was collected from 3 donors and used to form a 2-h acquired pellicle on the specimens. Then, the specimens were submitted to an erosive pH cycling protocol 4 times/day for 7 days (0.01 M HCl pH 2.0/45 s, artificial saliva/2 h, and artificial saliva overnight). The gels were applied again during pH cycling, 2 times/day for 4 min after the first and last erosive challenges. Enamel and dentin loss (µm) were assessed by contact profilometry. Scanning electron microscopy (SEM) was analyzed using a cold field emission. Data were analyzed by two-way ANOVA (for chitosan and CMC gels, separately) and Tukey's multiple comparison test. SEM images showed changes to enamel topography after application oft the gels containing Stn or F. Regarding CMC-based gels, for enamel, none of the treatments significantly reduced ETW in comparison with placebo; for dentin, however, gels containing Stn, regardless the concentration, significantly reduced the ETW. Moreover, Chitosan-based gels, regardless the Stn concentration, were able to protect enamel and dentin against ETW. Gels containing Stn might be a new approach to protect against ETW.

New insights into the anti-erosive property of a sugarcane-derived cystatin: different vehicle of application and potential mechanism of action.

OBJECTIVE: A new sugarcane-derived cystatin (CaneCPI-5) showed anti-erosive properties when included in solutions and strong binding force to enamel, but the performance of this protein when added to gel formulations and its effect on surface free energy (SFE) requires further studies. 1) to evaluate the protective effect of gels containing different concentrations of CaneCPI-5 against initial enamel erosion (Experiment 1); and 2) to analyze the SFE (γS) after treating the enamel surface with CaneCPI-5 solution (Experiment 2). METHODOLOGY: In Experiment 1, 75 bovine enamel specimens were divided into five groups according to the gel treatments: placebo (negative control); 0.27%mucin+0.5%casein (positive control); 0.1 mg/mL CaneCPI-5; 1.0 mg/mL CaneCPI-5; or 2.0 mg/mL CaneCPI-5. Specimens were treated with the gels for 1 min, the AP was formed (human saliva) for 2 h and the specimens were incubated in 0.65% citric acid (pH=3.4) for 1 min. The percentage of surface hardness change (%SHC) was estimated. In Experiment 2, measurements were performed by an automatic goniometer using three probing liquids: diiodomethane, water and ethylene glycol. Specimens (n=10/group) remained untreated (control) or were treated with solution containing 0.1 mg/mL CaneCPI-5, air-dried for 45 min, and 0.5 µL of each liquid was dispensed on the surface to measure contact angles. RESULTS: Gels containing 0.1 and 1.0 mg/mL CaneCPI-5 significantly reduced %SHC compared to the other treatments (p<0.05). Treated enamel showed significantly lower γS than control, without changes in the apolar component (γSLW), but the polar component (γSAB=Lewis acid-base) became more negative (p<0.01). Moreover, CaneCPI-5 treatment showed higher γS - (electron-donor) values compared to control (p<0.01). CONCLUSIONS: Gels containing 0.1 mg/mL or 1.0 mg/mL CaneCPI-5 protected enamel against initial dental erosion. CaneCPI-5 increased the number of electron donor sites on the enamel surface, which may affect AP formation and could be a potential mechanism of action to protect from erosion.

Preventive effect of chitosan gel containing CaneCPI-5 against enamel erosive wear in situ.

OBJECTIVE: This study evaluated the preventive effect of a chitosan gel containing CaneCPI-5 against enamel erosion and erosion + abrasion in situ. METHODS: Sixteen volunteers participated in a crossover, double-blind protocol, comprising 4 phases: (1) no treatment (Nt); (2) chitosan gel (Cg); (3) chitosan gel + 12,300 ppm NaF (Cg + NaF); and (4) chitosan gel + 0.1 mg/mL CaneCPI-5 (Cg + Cane). Volunteers wore an appliance containing 4 specimens. Once/day, they applied the gel (except for Nt) (4 min/specimen). Erosive challenges were performed extra-orally (0.1% citric acid, 90 s, 4 × /day; ERO). Specimens were also abraded (toothbrush, 15 s/specimen, 2 × /day; ERO + ABR). Enamel wear was assessed by profilometry and relative surface reflection intensity (%SRI). Two-way RM-ANOVA/Sidak's tests and Spearman's correlation were used (p < 0.05). RESULTS: For profilometry, ERO + ABR promoted significantly greater wear when compared with ERO. There was a significant difference among all treatments. The lowest enamel loss occurred for Cg + Cane, followed by Cg + NaF, Cg, and Nt (p < 0.05). The %SRI was significantly lower for ERO + ABR when compared to ERO, only for the Nt group. The greatest %SRI was found for the Cg + NaF and Cg + Cane groups, which did not differ significantly, regardless of the conditions. The lowest %SRI was found for the Nt and Cg groups, which did not differ from each other, regardless of the conditions. The Nt group did not differ significantly from the Cg + NaF (ERO). There was a significant correlation between both analyses. CONCLUSION: The incorporation of CaneCPI-5 in the chitosan gel prevented erosive wear in situ. CLINICAL RELEVANCE: These results open a new perspective for the use of CaneCPI-5 in other application vehicles, such as chitosan gel.

Proteomic analysis of infected root canals with apical periodontitis in patients with type 2 diabetes mellitus: A cross-sectional study.

AIM: This study aimed to quantitatively and qualitatively determine the proteomic profile of apical periodontitis (AP) in type 2 diabetes mellitus (T2DM) patients in comparison with systemically noncompromised patients and to correlate the protein expression of both groups with their biological functions. METHODOLOGY: The sample consisted of 18 patients with asymptomatic AP divided into two groups according to the presence of T2DM: diabetic group-patients with T2DM (n = 9) and control group-systemically healthy patients (n = 9). After sample collection, the root canal samples were prepared for proteomic analysis using reverse-phase liquid chromatography-mass spectrometry. Label-free quantitative proteomic analysis was performed by Protein Lynx Global Service software. Differences in protein expression between groups were calculated using t-test (p < .05). Biological functions were analysed using the Homo sapiens UniProt database. RESULTS: A total of 727 human proteins were identified in all samples. Among them, 124 proteins common to both groups were quantified, out of which 65 proteins from the diabetic group showed significant differences compared with the control: 43 upregulated (p < .05) and 22 downregulated (p < .05) proteins. No significant differences in protein expression were seen for the remaining 59 proteins (p > .05). Most proteins with differences in expression were related to immune/inflammatory response. Neutrophil gelatinase-associated lipocalin, Plastin-2, Lactotransferrin and 13 isoforms of immunoglobulins were upregulated. In contrast, Protein S100-A8, Protein S100-A9, Histone H2B, Neutrophil defensin 1, Neutrophil defensin 3 and Prolactin-inducible protein were downregulated. CONCLUSIONS: Quantitative differences were demonstrated in the expression of proteins common to diabetic and control groups, mainly related to immune response, oxidative stress, apoptosis and proteolysis. These findings revealed biological pathways that provide the basis to support clinical findings on the relationship between AP and T2DM.

Characterization of white spot lesions formed on human enamel under microcosm biofilm for different experimental periods.

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.

Acquired Pellicle Engineering Using a Combination of Organic (Sugarcane Cystatin) and Inorganic (Sodium Fluoride) Components against Dental Erosion.

This study evaluated the combination of a sugarcane cystatin (CaneCPI-5) and sodium fluoride (NaF) in acquired pellicle engineering for the prevention of dental erosion in vitro. Seventy-five human enamel specimens were prepared and divided into 5 treatment groups (n = 15/group): Deionized water (Control); Elmex™ (SnCl2/NaF/AmF); 0.1 mg/mL CaneCPI-5; 500 ppm NaF; and CaneCPI-5+NaF (Combination). The specimens were individually treated (200 µL; 2 min; 37°C), then incubated in human saliva (200 µL; 1 h, at 37°C) for acquired pellicle formation. Afterward, the specimens were submitted to an erosive challenge (1% citric acid [CR], pH 3.6, 10 mL, 2 min, 25 °C). This sequence was conducted 5 times. Percentage of surface microhardness change (%SMC), relative surface reflection intensity (rSRI), and calcium released to the CR were measured and analyzed by one-way ANOVA followed by Tukey's test (p < 0.05). In general, all the treatments (SnCl2/NaF/AmF, CaneCPI-5, NaF, and Combination) significantly protected the enamel when compared the control group. Regarding %SMC and rSRI, the Combination was the most effective treatment, reducing the %SMC significantly (p < 0.01) when compared to all the other treatments, although this difference was not significant in the CR analysis. All treatments demonstrated a protective effect on enamel against dental erosion; however, the combination of CaneCPI-5 with NaF showed a greater protection.

New insights into the anti-erosive property of a sugarcane-derived cystatin: different vehicle of application and potential mechanism of action

Abstract A new sugarcane-derived cystatin (CaneCPI-5) showed anti-erosive properties when included in solutions and strong binding force to enamel, but the performance of this protein when added to gel formulations and its effect on surface free energy (SFE) requires further studies. Objective 1) to evaluate the protective effect of gels containing different concentrations of CaneCPI-5 against initial enamel erosion (Experiment 1); and 2) to analyze the SFE (γS) after treating the enamel surface with CaneCPI-5 solution (Experiment 2). Methodology In Experiment 1, 75 bovine enamel specimens were divided into five groups according to the gel treatments: placebo (negative control); 0.27%mucin+0.5%casein (positive control); 0.1 mg/mL CaneCPI-5; 1.0 mg/mL CaneCPI-5; or 2.0 mg/mL CaneCPI-5. Specimens were treated with the gels for 1 min, the AP was formed (human saliva) for 2 h and the specimens were incubated in 0.65% citric acid (pH=3.4) for 1 min. The percentage of surface hardness change (%SHC) was estimated. In Experiment 2, measurements were performed by an automatic goniometer using three probing liquids: diiodomethane, water and ethylene glycol. Specimens (n=10/group) remained untreated (control) or were treated with solution containing 0.1 mg/mL CaneCPI-5, air-dried for 45 min, and 0.5 µL of each liquid was dispensed on the surface to measure contact angles. Results Gels containing 0.1 and 1.0 mg/mL CaneCPI-5 significantly reduced %SHC compared to the other treatments (p<0.05). Treated enamel showed significantly lower γS than control, without changes in the apolar component (γSLW), but the polar component (γSAB=Lewis acid-base) became more negative (p<0.01). Moreover, CaneCPI-5 treatment showed higher γS - (electron-donor) values compared to control (p<0.01). Conclusions Gels containing 0.1 mg/mL or 1.0 mg/mL CaneCPI-5 protected enamel against initial dental erosion. CaneCPI-5 increased the number of electron donor sites on the enamel surface, which may affect AP formation and could be a potential mechanism of action to protect from erosion.

Characterization of white spot lesions formed on human enamel under microcosm biofilm for different experimental periods

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.

Proteomic profile of the acquired enamel pellicle of professional wine tasters with erosive tooth wear.

The aim of this study was to compare the acquired enamel pellicle protein profile of professional wine tasters with mild and moderate erosive tooth wear. Twelve professional wine tasters participated (3 from a low tooth wear group; 9 from a high tooth wear group). Acquired enamel pellicle samples were collected and processed for proteomic analysis (nLC-ESI-MS/MS). The acquired enamel pellicle proteomic profile was different between the groups. The proteins found exclusively in the low tooth wear group were histatins 1 and 3 and mucins 7 and 21. When comparing the wear groups, proteins with higher levels in the low tooth wear group included neutrophil defensins (1 and 3), lysozyme C, lysozyme, myeloperoxidase, and squalene monooxygenase. In conclusion, the findings indicate that the proteins found at higher levels in the low tooth wear group and proteins exclusively found in the low tooth wear group might be protective and, therefore, could be good candidates for further studies regarding their potential to be added to dental products to protect professional wine tasters from extrinsic erosive tooth wear.

Acquired pellicle engineering with proteins/peptides: Mechanism of action on native human enamel surface.

OBJECTIVE: This study investigated the mechanism of action of different proteins/peptides (separately or in combination), focusing on how they act directly on the native enamel surface and on modifying the salivary pellicle. METHODS: A total of 170 native human enamel specimens were prepared and submitted to different treatments (2 h; 37 °C): with deionized water, CaneCPI-5, Hemoglobin, Statherin, or a combination of all three proteins/peptides. The groups were subdivided into treatment acting on the enamel surface (NoP - absence of salivary pellicle), and treatment modifying the salivary pellicle (P). Treatment was made (2 h; 37 °C) in all specimens, and later, for P, the specimens were incubated in human saliva (2 h; 37 °C). In both cases, the specimens were immersed in 1% citric acid (pH 3.6; 2 min; 25 °C). Calcium released from enamel (CaR) and its relative surface reflection intensity (%SRI) was measured after 5 cycles. Between-group differences were verified with two-way ANOVA, with "presence of pellicle" and "treatment" as factors (α = 0.05). RESULTS: The presence of pellicle provided better protection regarding %SRI (p < 0.01), but not regarding CaR (p = 0.201). In relation to treatment, when compared to the control group, all proteins/peptides provided significantly better protection (p < 0.01 for %SRI and Car). The combination of all three proteins/peptides demonstrated the best protective effect (p < 0.01 for %SRI). CONCLUSION: Depending on the protein or peptide, its erosion-inhibiting effect derives from their interaction with the enamel surface or from modifying the pellicle, so a combination of proteins and peptides provides the best protection. CLINICAL SIGNIFICANCE: The present study opens a new direction for a possible treatment with a combination of proteins for native human enamel, which can act directly on the enamel surface as well on the modification of the salivary pellicle, for the prevention of dental erosion.

Antimicrobial and anti-caries effects of a novel cystatin from sugarcane on saliva-derived multi-species biofilms.

This study evaluated the antimicrobial (anti-biofilm) and anti-caries (enamel demineralization prevention) effects of a new cystatin derived from sugarcane (CaneCPI-5). Microcosm biofilm was produced on bovine enamel specimens (4 x 4 mm; n=48) from a mixture of human saliva and McBain saliva at the first 8 h. From this moment until the end of the experiment, the enamel specimens were exposed to lsaMcBain saliva containing 0.2% sucrose and, once a day, they were treated with the test solutions for 1 min. This treatment was performed for 5 days. The solutions evaluated were: PBS (negative control), 0.12% chlorhexidine (positive control), 0.1 mg/ml CaneCPI-5 and 1.0 mg/ml CaneCPI-5. The biofilm viability was determined by fluorescence using confocal microscopy and the enamel demineralization was quantified using transverse microradiography (TMR). The data were analyzed by ANOVA/Tukey or Kruskal-Wallis/Dunn tests for biofilm and enamel, respectively (p<0.05). With respect to the antimicrobial effect, all treatment solutions significantly reduced the biofilm viability compared with PBS. The best antimicrobial effect was found for 1.0 mg/ml CaneCPI-5 (82.37±10.01% dead bacteria) that significantly differed from 0.12% chlorhexidine (73.13±15.07% dead bacteria). For the anti-caries effect, only 0.12% chlorhexidine (ΔZ: 2610, 1683-4343) performed significantly better than PBS (ΔZ: 8030, 7213-9115), but 0.12% chlorhexidine did not significantly differ from 0.1 mg/ml Cane-CPI-5. Under this experimental model, CaneCPI-5 significantly reduced the biofilm viability, but this effect was not reflected on its anti-caries potential.

Acquired pellicle protein-based engineering protects against erosive demineralization.

OBJECTIVES: To evaluate, in vivo: 1) proteomic alterations in the acquired enamel pellicle (AEP) after treatment with sugarcane-derived cystatin (CaneCPI-5), hemoglobin (HB), statherin-derived peptide (StN15) or their combination before the formation of the AEP and subsequent erosive challenge; 2) the protection of these treatments against erosive demnineralization. MATERIALS AND METHODS: In 5 crossover phases, after prophylaxis, 10 volunteers rinsed (10 mL, 1 min) with: deionized water-1, 0.1 mg/mL CaneCPI-5-2, 1.0 mg/mL HB-3, 1.88 × 10-5 M StN15-4 or their combination-5. AEP was formed (2 h) and enamel biopsy (10 µL, 1%citric acid, pH 2.5, 10 s) was performed on one incisor for calcium analysis. The same acid was applied on the vestibular surfaces of the remaining teeth. The acid-resistant proteins within the remaining AEP were collected. Samples were quantitatively analyzed by label-free proteomics. RESULTS: Treatment with the proteins/peptide, isolated or combined, increased several acid-resistant proteins in the AEP, compared with control. The highest increases were seen for PRPs (32-fold, StN15), profilin (15-fold, combination), alpha-amylase (9-fold; StN15), keratins (8-fold, CaneCPI-5 and HB), Histatin-1 (7-fold, StN15), immunoglobulins (6.5-fold, StN15), lactotransferrin (4-fold, CaneCPI-5), cystatins, lysozyme, protein S-100-A9 and actins (3.5-fold, StN15), serum albumin (3.5-fold, CaneCPI-5 and HB) and hemoglobin (3-fold, StN15). Annexin, calmodulin, keratin, tubulin and cystatins were identified exclusively upon treatment with the proteins/peptide, alone or combined. Groups 2, 3 and 4 had significantly lower Ca released from enamel compared to group 1 (Kruskal-Wallis/Dunn's, p < 0.05). CONCLUSIONS: Treatment with CaneCPI-5, HB or StN15 remarkably increases acid-resistant proteins in the AEP, protecting against erosion. CLINICAL SIGNIFICANCE: Our results show, for the first time, that treatment with proteins/peptide remarkably increases acid-resistant proteins in the AEP, protecting against erosive demineralization. These findings open an avenue for a new preventive approach for erosive demineralization, employing acquired pellicle engineering procedures that may in the future be incorporated into dental products.