Hemoglobin Protects Enamel against Intrinsic Enamel Erosive Demineralization.

INTRODUCTION: This study investigated the changes in the acquired enamel pellicle (AEP) proteome when this integument is formed in vivo after treatment with sugarcane-derived cystatin (CaneCPI-5), hemoglobin (HB), and a statherin-derived peptide (StN15), or their combination and then exposed to an intrinsic acid challenge. The effectiveness of these treatments in preventing intrinsic erosion was also evaluated. METHODS: Ten volunteers, after prophylaxis, in 5 crossover phases, rinsed with the following solutions (10 mL, 1 min): control (deionized water-H2O) - group 1, 0.1 mg/mL CaneCPI-5 - group 2, 1.0 mg/mL HB - group 3, 1.88 × 10-5M StN15 - group 4, or a blend of these - group 5. Following this, AEP formation occurred (2 h) and an enamel biopsy (10 µL, 0.01 m HCl, pH 2.0, 10 s) was conducted on one incisor. The biopsy acid was then analyzed for calcium (Arsenazo method). The vestibular surfaces of the other teeth were treated with the same acid. Acid-resistant proteins in the residual AEP were then collected and analyzed quantitatively via proteomics. RESULTS: Compared to control, treatment with the proteins/peptide, mixed or isolated, markedly enhanced acid-resistant proteins in the AEP. Notable increases occurred in pyruvate kinase PKM (11-fold, CaneCPI-5), immunoglobulins and submaxillary gland androgen-regulated protein 3B (4-fold, StN15), Hb, and lysozyme C (2-fold, StN15). Additionally, a range of proteins not commonly identified in the AEP but known to bind calcium or other proteins were identified in groups treated with the tested proteins/peptide either in isolation or as a mixture. The mean (SD, mM) calcium concentrations released from enamel were 3.67 ± 1.48a, 3.11 ± 0.72a, 1.94 ± 0.57b, 2.37 ± 0.90a, and 2.38 ± 0.45a for groups 1-5, respectively (RM-ANOVA/Tukey, p < 0.05). CONCLUSIONS: Our findings demonstrate that all treatments, whether using a combination of proteins/peptides or in isolation, enhanced acid-resistant proteins in the AEP. However, only HB showed effectiveness in protecting against intrinsic erosive demineralization. These results pave the way for innovative preventive methods against intrinsic erosion, using "acquired pellicle engineering" techniques.

Acquired Pellicle and Biofilm Engineering by Rinsing with Hemoglobin Solution.

INTRODUCTION: The identification of acid-resistant proteins, including hemoglobin (Hb), within the acquired enamel pellicle (AEP) led to the proposition of the "acquired pellicle engineering" concept, which involves the modification of the AEP by incorporating specific proteins, presenting a novel strategy to prevent dental demineralization. OBJECTIVE: Combining in vivo and in vitro proof-of-concept protocols, we sought to reveal the impact of AEP engineering with Hb protein on the biofilm microbiome and enamel demineralization. METHODS: In the in vivo studies, 10 volunteers, in 2 independent experiments, rinsed (10 mL,1 min) with deionized water-negative control or 1.0 mg/mL Hb. The AEP and biofilm formed along 2 or 3 h, respectively, were collected. AEP was analyzed by quantitative shotgun-label-free proteomics and biofilm by 16S-rRNA next-generation sequencing (NGS). In in vitro study, a microcosm biofilm protocol was employed. Seventy-two bovine enamel specimens were treated with (1) phosphate-buffered solution (PBS), (2) 0.12% chlorhexidine, (3) 500 ppm NaF, (4) 1.0 mg/mL Hb, (5) 2.0 mg/mL Hb, and (6) 4.0 mg/mL Hb. The biofilm was cultivated for 5 days. Resazurin, colony forming units (CFU), and transversal microradiography were performed. RESULTS: Proteomics and NGS analysis revealed that Hb increased proteins with antioxidant, antimicrobial, acid-resistance, hydroxyapatite-affinity, calcium-binding properties and showed a reduction in oral pathogenic bacteria. In vitro experiments demonstrated that the lowest Hb concentration was the most effective in reducing bacterial activity, CFU, and enamel demineralization compared to PBS. CONCLUSION: These findings suggest that Hb could be incorporated into anticaries dental products to modify the oral microbiome and control caries, highlighting its potential for AEP and biofilm microbiome engineering.

Acquired enamel pellicle and biofilm engineering with a combination of acid-resistant proteins (CaneCPI-5, StN15, and Hemoglobin) for enhanced protection against dental caries - in vivo and in vitro investigations.

OBJECTIVE: This study was designed in two-legs. In the in vivo, we explored the potential of a rinse solution containing a combination (Comb) of 0.1 mg/mL CaneCPI-5 (sugarcane-derive cystatin), 1.88 × 10- 5M StN15 (statherin-derived peptide) and 1.0 mg/mL hemoglobin (Hb) to change the protein profile of the acquired enamel pellicle(AEP) and the microbiome of the enamel biofilm. The in vitro, was designed to reveal the effects of Comb on the viability and bacterial composition of the microcosm biofilm, as well as on enamel demineralization. MATERIALS AND METHODS: In vivo study, 10 participants rinsed (10mL,1 min) with either deionized water (H2O-control) or Comb. AEP and biofilm were collected after 2 and 3 h, respectively, after rinsing. AEP samples underwent proteomics analysis, while biofilm microbiome was assessed via 16 S-rRNA Next Generation Sequencing(NGS). In vitro study, a microcosm biofilm protocol was employed. Ninety-six enamel specimens were treated with: 1)Phosphate-Buffered Solution-PBS(negative-control), 2)0.12%Chlorhexidine, 3)500ppmNaF and 4)Comb. Resazurin, colony-forming-units(CFU) and Transversal Microradiography(TMR) were performed. RESULTS: The proteomic results revealed higher quantity of proteins in the Comb compared to control associated with immune system response and oral microbial adhesion. Microbiome showed a significant increase in bacteria linked to a healthy microbiota, in the Comb group. In the in vitro study, Comb group was only efficient in reducing mineral-loss and lesion-depth compared to the PBS. CONCLUSIONS: The AEP modification altered the subsequent layers, affecting the initial process of bacterial adhesion of pathogenic and commensal bacteria, as well as enamel demineralization. CLINICAL RELEVANCE: Comb group shows promise in shaping oral health by potentially introducing innovative approaches to prevent enamel demineralization and deter tooth decay.

Differences in the Natural Enamel Surface and Acquired Enamel Pellicle following Exposure to Citric or Hydrochloric Acid.

OBJECTIVES: The aim of this study was to investigate variations in the interaction between enamel, that is, the acquired enamel pellicle (AEP) and citric or hydrochloric acid. MATERIALS AND METHODS: A 24-h AEP was formed on natural enamel specimens (n = 40) from pooled whole mouth human saliva. Samples were randomly allocated to citric (0.3%, pH 3.2) or hydrochloric (HCl) acid (0.01 M, pH 2.38) exposure for 30 or 300 s. The total protein concentration (TPC), and phosphorous and calcium concentrations of the pellicle were determined before and after acid exposure, and again after re-immersion in saliva. Surface roughness and tandem scanning confocal microscopy imaging were used to assess enamel changes. RESULTS: After 300 s of citric acid exposure, the mean ± SD TPC reduced from 5.1 ± 1.1 to 3.5 ± 1.1 mg/mL (p < 0.05). In contrast, after 300 s of HCl exposure, the mean TPC did not reduce significantly from baseline (6.6 ± 1.1 to 5.7 ± 0.7 mg/mL) but was significantly reduced in the reformed pellicle to 4.9 ± 1.2 mg/mL (p < 0.001). This reduction occurred after significant release of calcium and phosphorous from the enamel surface (p < 0.001). Thirty seconds of exposure to either acid had no obvious effect on the AEP. The surface roughness of the enamel decreased after acid exposure but no differences between groups was observed. CONCLUSIONS: These findings indicate that citric acid interacted with proteins in the AEP upon contact, offering enamel protection. In contrast, HCl appeared to bypass the pellicle, and reduced protein was observed only after changes in the enamel chemical composition.

In situ effect of enamel salivary exposure time and type of intraoral appliance before an erosive challenge.

OBJECTIVES: This study tested the effect of enamel salivary exposure time prior to an acid challenge (30 min, 1, 2, or 12 h) and type of intraoral appliance (palatal or mandibular) on initial erosion. METHODS: After initial surface hardness evaluation, enamel blocks (n = 340) were randomly divided into groups and volunteers (n = 20). The control group was not exposed to saliva previously to the erosive challenge. The volunteers wore palatal and mandibular appliances simultaneously. After salivary exposure, the blocks were subjected to acid exposure by immersion in hydrochloric acid (0.01 M, pH 2.3) for 30 s. Then, the enamel surface hardness was evaluated. Data were analyzed using ANOVA, Kruskal-Wallis and Tukey's test (p < 0.05). RESULTS: No difference was observed on percent surface hardness change (% SHC) in the enamel blocks between the types of intraoral appliances. Exposure to saliva for 30 min and 1 h promoted similar enamel resistance to the erosive attack, which was similar to the control group for both appliances. Blocks exposed to saliva for 2 h showed less hardness loss when compared to 30 min. Keeping the blocks in saliva during 12-h overnight resulted in similar percentage of enamel hardness loss compared to 2 h. CONCLUSIONS: A 2-hour in situ exposure to saliva is adequate to promote partial protection against initial erosive lesions, independently of the type of intraoral appliance used. CLINICAL SIGNIFICANCE: This finding will help researchers in the development of erosion studies, which will provide information for dentists to offer a better treatment for erosion.

In vitro salivary pellicles from adults and children have different protective effects against erosion.

OBJECTIVES: We aimed at analyzing the protective effects of salivary pellicles, formed with saliva from adults or children, on enamel from permanent or deciduous teeth. MATERIALS AND METHODS: Ninety human enamel specimens (45 permanent premolars and 45 deciduous canines) were ground, and the outer 200 µm of enamel was removed. We divided the teeth into three further subgroups: no salivary pellicle (control), adult salivary pellicle (AP), and child salivary pellicle (CP). We collected stimulated saliva from adults and children and placed 160 µl of either saliva on enamel specimens from AP and CP, respectively. Control specimens received no saliva. Specimens were stored at 37 °C for 2 h and then submitted to an erosive challenge (10 mL; 1 % citric acid; pH 3.6; 25 °C, 1 min). Pellicle formation and erosion was repeated for a total of 4 cycles. After every cycle, relative surface reflection intensity (rSRI) and surface microhardness (rSMH) were calculated. RESULTS: On permanent enamel, AP presented significantly better protective effects, with less rSMH loss (p < 0.001) and less rSRI loss (p < 0.001). On deciduous enamel, CP presented significantly better protective effects than AP and control (p < 0.05), for both measured parameters. CONCLUSION: We conclude that pellicles from adults and children promote different erosion protective effects, where adult pellicle provides better protection for permanent enamel, and child pellicle promotes better protection on deciduous enamel. CLINICAL RELEVANCE: The present results provide a better understanding toward the protective effect of salivary pellicle against dental erosion and brings light to one more factor involved in the erosion of deciduous teeth.

In vivo modification of the enamel pellicle and saliva resveratrol levels after use of resveratrol-containing orodispersible capsules.

OBJECTIVES: To evaluate in vivo 1) the bioavailability of trans-resveratrol when administered through sublingual capsules; 2) the effect of resveratrol on the protein composition of the acquired enamel pellicle (AEP). DESIGN: Ten volunteers received a sublingual capsule containing 50 mg of trans-resveratrol. Unstimulated saliva was then collected after 0, 30, 60, and 120 min and AEP was collected after 120 min following administration of the capsule. In the next week, the volunteers received a placebo sublingual capsule, and saliva and AEP were collected again. Saliva samples were analyzed for free trans-resveratrol using high-performance liquid chromatopgraphy (HPLC), and AEP samples were subjected to proteomic analysis (nLC-ESI-MS/MS). RESULTS: Trans-resveratrol was detected in saliva at all the time points evaluated, with the peak at 30 min. A total of 242 proteins were identified in both groups. Ninety-six proteins were increased and 23 proteins were decreased in the Resveratrol group. Among the up-regulated proteins, isoforms of cystatins, PRPs, Mucin-7, Histatin-1, Lactotrasnferrin and Lysozyme-C were increased and the isoforms of Protein S100, Neutrophil defensins, Albumin, PRPs, and, Statherin were decreased in Resveratrol group. CONCLUSION: The sublingual capsule is effective at increasing the bioavailability of trans-resveratrol in saliva. Several proteins involved in important processes to maintain systemic and oral health homeostasis were identified. These proteins differently expressed due to the presence of trans-resveratrol deserve attention for future studies, since they have important functions, mainly related to antimicrobial action.

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.

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.

Modulation of Streptococcus mutans Adherence to Hydroxyapatite by Engineered Salivary Peptides.

Since the modification of the proteinaceous components of the Acquired Enamel Pellicle (AEP) could influence the adhesion of Streptococcus mutans, the most cariogenic bacteria, to dental surfaces, we assessed if engineered salivary peptides would affect the adherence and modulate the bacterial proteome upon adherence. Single-component AEPs were formed onto hydroxyapatite (HAp) discs by incubating them with statherin, histatin-3, DR9, DR9-DR9, DR9-RR14, RR14, and parotid saliva. Then, the discs were inoculated with S. mutans UA159 and the bacteria were allowed to adhere for 2 h, 4 h, and 8 h (n = 12/treatment/time point). The number of bacteria adhered to the HAp discs was determined at each time point and analyzed by two-way ANOVA and Bonferroni tests. Cell-wall proteins were extracted from adhered, planktonic, and inoculum (baseline) bacteria and proteome profiles were obtained after a bottom-up proteomics approach. The number of adhered bacteria significantly increased over time, being the mean values obtained at 8 h, from highest to lowest, as follows: DR9-RR14 > statherin > RR14 = DR9-DR9 > DR9 = histatin3 > saliva (p < 0.05). Treatments modulated the bacterial proteome upon adherence. The findings suggested a potential use of our engineered peptide DR9-DR9 to control S. mutans biofilm development by reducing bacterial colonization.

Protein-based engineering of the initial acquired enamel pellicle in vivo: Proteomic evaluation.

OBJECTIVE: To study the proteomic alterations in the initial AEP after rinsing with CaneCPI-5, StN15 or Hb or their combination. MATERIALS AND METHODS: In five crossover phases, after prophylaxis, 10 volunteers in 5 consecutive days, rinsed (10 mL, 1 min) with the following solutions: deionized water (H2O- negative control- 1), 0.1 mg/mL CaneCPI-5 (2), 1.88×10-5 M StN15 (3), 1.0 mg/mL Hb (4) or their combination (5). The AEP formed after 3 min was collected with electrode filter papers soaked in 3% citric acid. After protein extraction, samples were analyzed by quantitative shotgun label-free proteomics. RESULTS: Rinsing with the proteins/peptide increased the amounts of proteins in the AEP. The total numbers of proteins identified after rinsing with CaneCPI-5, StN15, Hb or their combination versus water, were 131, 167, 148 and 142, respectively. The treatment with the proteins/peptide or their combination increased proteins that bind calcium, phosphate and interact with distinct proteins, as well as proteins with antimicrobial and acid-resistant properties, such as, Cornifin-B (7.7, 12.6, and 4.3-fold for CaneCPI-5, StN15 and Hb, respectively), isoforms of Cystatin (2.2-2.4-fold for CaneCPI-5 and StN15), Proline-rich-protein 4 (4.3-fold; StN15), Histatin-1 (2.8-fold; StN15) and Hemoglobin (7.7-25-fold for Hb and Combination). Immunoglobulin, Keratin and Histone were exclusively identified upon treatment with the proteins/peptide, alone or combined. CONCLUSION: Rinsing with proteins/peptide, alone or combined, increased protective proteins in the initial AEP. CLINICAL RELEVANCE: Our results suggest that rinsing with the proteins/peptide or their combination increases the proteins capable of enhancing the protective function of the basal layer of AEP.

Engineered Salivary Peptides Reduce Enamel Demineralization Provoked by Cariogenic S. mutans Biofilm.

Engineering of the acquired enamel pellicle using salivary peptides has been shown to be a promising anticaries strategy. However, the mechanisms by which these peptides protect teeth against tooth decay are not fully understood. In this study, we evaluated the effect of the engineered salivary peptides DR9-DR9 and DR9-RR14 on enamel demineralization in two experimental conditions: (1) adsorbed onto the enamel surface forming the AEP, and (2) forming the AEP combined with their use to treat the biofilms 2×/day, using a validated cariogenic Streptococcus mutans in vitro biofilm model. Biofilms were grown for 144 h on enamel slabs and then collected to determine the bacterial viability (CFU/biofilm) and biofilm mass (mg protein/biofilm), and to extract cellular/extracellular proteins, which were characterized by mass spectrometry. The culture medium was changed 2×/day to fresh medium, and pH (indicator of biofilm acidogenicity) and calcium concentration (indicator of demineralization) was determined in used medium. DR9-RR14 peptide significantly reduced enamel demineralization (p < 0.0001) in both experimental conditions. However, this peptide did not have a significant effect on biofilm biomass (p > 0.05) nor did it modulate the expression of cellular and extracellular bacterial proteins involved in biofilm cariogenicity. These findings suggest that DR9-RR14 may control caries development mainly by a physicochemical mechanism.

Caries Management-The Role of Surface Interactions in De- and Remineralization-Processes.

BACKGROUND: Bioadhesion and surface interactions on enamel are of essential relevance for initiation, progression and prevention of caries and erosions. Salivary proteins on and within initial carious and erosive lesions can facilitate or aggravate de- and remineralization. This applies for the pellicle layer, the subsurface pellicle and for proteins within initial carious lesions. Little is known about these proteinaceous structures related to initial caries and erosion. Accordingly, there is a considerable demand for an understanding of the underlying processes occurring at the interface between the tooth surface and the oral cavity in order to develop novel agents that limit and modulate caries and erosion. Objectives and findings: The present paper depicts the current knowledge of the processes occurring at the interface of the tooth surface and the oral fluids. Proteinaceous layers on dental hard tissues can prevent or aggravate demineralization processes, whereas proteins within initial erosive or carious lesions might hinder remineralization considerably and restrict the entry of ions into lesions. CONCLUSIONS: Despite the fact that organic-inorganic surface interactions are of essential relevance for de- and remineralization processes at the tooth surface, there is limited knowledge on these clinically relevant phenomena. Accordingly, intensive research is necessary to develop new approaches in preventive dentistry.

A systematic review of the proteomic profile of in vivo acquired enamel pellicle on permanent teeth.

OBJECTIVES: To provide a comprehensive review on the proteomic profile of in vivo acquired enamel pellicle (AEP) formed on permanent teeth. DATA/SOURCES: PubMed, EMBASE (Ovid) and Web of Science databases were searched for eligible studies (up to December 2020). Studies reporting mass spectrometry-based proteomic analysis of in vivo AEP were included. Risk of bias assessment was performed. Qualitative and quantitative proteomic data were extracted, integrated, then followed by bioinformatic analysis. STUDY SELECTION: Eleven studies were included, involving 122 systemically and dentally healthy adults from four different research groups. Pooled AEP samples from study participants were the normal practice for all included studies. A total of 257/93/108/870 non-redundant proteins were detectable from the in vivo ≤ 5 min/10-min/60-min/2-h AEP samples, respectively. Fifteen "core in vivo 2-h AEP proteins", generally associated with immune and/or inflammatory responses, were consistently identifiable from all four research groups. Eight included studies conducted relative quantitative proteomic analysis, while no statistical analysis could be undertaken due to the inherent limitation of the relative quantification in the proteomics analyses of these studies. CONCLUSIONS: A systematic review on adult in vivo AEP proteomic profile was undertaken. The results provide a comprehensive appreciation of the AEP proteome in healthy individuals from in vivo sampling. Further studies are warranted to clarify the biological role of AEP on oral health, particularly at an individual level. CLINICAL SIGNIFICANCE: A comprehensive appreciation of the proteomic profile of in vivo AEP in healthy individuals is essential to further understand its functions in oral health and disease. The information generated also provides insights for future studies.

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.

Comparative proteomic analysis on acquired enamel pellicle at two time points in caries-susceptible and caries-free subjects.

OBJECTIVES: The aim of this study was to analyse the protein profiles of acquired enamel pellicle (AEP) at two time points (5 min and 2 h) between caries-susceptible and caries-free subjects using label-free and parallel reaction monitoring (PRM) proteomic approaches, and to discover potential biomarkers for dental caries. METHODS: Sixty participants (30 caries-susceptible (DMFT>5) and 30 caries-free subjects (DMFT = 0)) were included. Their AEP at 5 min and 2 h was separately sampled, and the AEP materials were quantitatively analysed using label-free proteomics. Bioinformatics analysis of differentially expressed proteins was subsequently conducted and target proteins were verified using PRM analysis. RESULTS: A total of 82 and 39 differentially expressed proteins were identified at the 5-minute and 2 -h points, respectively, between the caries-susceptible and caries-free groups. Some proteins related to immune response and antibacterial activity were identified, and the characteristic enriched biological processes of up- and down-regulated proteins were presented using bioinformatics analysis. Subsequently, five intriguing target proteins of each group were chosen for PRM analysis and their presence was successfully verified in the 5-minute and 2 -h AEPs, respectively. CONCLUSIONS: The proteins in the AEPs of caries-susceptible and caries-free subjects at both time points presented unique protein profiles related to immune response and antibacterial activity. Mucin-7 can be regarded as an important potential AEP biomarker for dental caries. CLINICAL SIGNIFICANCE: The results of the study identified potential biomarkers, which can facilitate the design of new bio-functional agents for the diagnosis and treatment of dental caries in future.

Interaction between the Oral Microbiome and Dental Composite Biomaterials: Where We Are and Where We Should Go.

Dental composites are routinely placed as part of tooth restoration procedures. The integrity of the restoration is constantly challenged by the metabolic activities of the oral microbiome. This activity directly contributes to a less-than-desirable half-life for the dental composite formulations currently in use. Therefore, many new antimicrobial dental composites are being developed to counteract the microbial challenge. To ensure that these materials will resist microbiome-derived degradation, the model systems used for testing antimicrobial activities should be relevant to the in vivo environment. Here, we summarize the key steps in oral microbial colonization that should be considered in clinically relevant model systems. Oral microbial colonization is a clearly defined developmental process that starts with the formation of the acquired salivary pellicle on the tooth surface, a conditioned film that provides the critical attachment sites for the initial colonizers. Further development includes the integration of additional species and the formation of a diverse, polymicrobial mature biofilm. Biofilm development is discussed in the context of dental composites, and recent research is highlighted regarding the effect of antimicrobial composites on the composition of the oral microbiome. Future challenges are addressed, including the potential of antimicrobial resistance development and how this could be counteracted by detailed studies of microbiome composition and gene expression on dental composites. Ultimately, progress in this area will require interdisciplinary approaches to effectively mitigate the inevitable challenges that arise as new experimental bioactive composites are evaluated for potential clinical efficacy. Success in this area could have the added benefit of inspiring other fields in medically relevant materials research, since microbial colonization of medical implants and devices is a ubiquitous problem in the field.

Proteomics of acquired pellicle in gastroesophageal reflux disease patients with or without erosive tooth wear.

OBJECTIVES: This in vivo study compared the protein profile of the acquired enamel pellicle (AEP) in volunteers 1) with gastroesophageal reflux disease (GERD) and erosive tooth wear (ETW) (BEWE ≥ 9; GE group); 2) with GERD without ETW (BEWE = 0; GNE group) and 3) control (without GERD and BEWE = 0; C group). MATERIALS AND METHODS: Twenty-four subjects (8/group) participated. AEP was formed during 120 min and collected. After protein extraction, the samples were submitted to reverse phase liquid chromatography coupled to mass spectrometry. Label-free proteomic quantification was performed using Protein Lynx Global Service software. RESULTS: In total, 458 proteins were identified. Seventy-six proteins were common to all the groups. The proteomic profile of the AEP was quite different among the distinct groups. The numbers of proteins exclusively found in the C, GE and GNE groups were 113, 110 and 81, respectively. Most of the proteins exclusively identified in the C and GNE groups bind metals, while those in the GE group are mainly membrane proteins. Many proteins were found exclusively in the reflux groups. In the quantitative analyses, when the GNE group was compared with the GE group, the proteins with the highest decreases were Lysozyme C, Antileukoproteinase, Cathepsin G, Neutrophil defensins and Basic salivary proline-rich proteins, while those with the highest increases were subunits of Hemoglobin, Albumin and isoforms of Cystatin. CONCLUSION: Profound alterations in the proteomic profile of the AEP were seen in GNE compared with GE volunteers, which might play a role in the resistance to ETW seen in the first. CLINICAL SIGNIFICANCE: This pioneer study compared the proteomic profile of the AEP of patients with GERD with or without ETW. Increased proteins in those without ETW might be protective and are good candidates to be added to dental products to protect against erosion caused by intrinsic acids.

Protective effect of milk against dental demineralization: understanding the mechanisms / Efeito protetor do leite contra a desmineralização dentária: compreensão dos mecanismos

This study aimed to answer the following questions: 1) does whole fluoridated milk protect more against enamel and dentin erosion than fat-free fluoridated milk? 2) does the protective effect of fluoridated milk against erosion follow a dose-response relationship? 3) is the treatment with whole or fat-free fluoridated milk before the first erosive challenge more protective against enamel and dentin erosion? 4) does the fat content of milk change the proteomic profile of the acquired enamel pellicle (AEP)? This study was divided into 2 parts. The first part analyzed in vitro the effect of milk against dental erosion, considering three factors: type of bovine milk (whole/fat-free), presence of different fluoride concentrations (0- 10.0 ppm) and time of application (before/after erosive challenge). Bovine enamel (n=15/group) and root dentin (n=12/group) specimens were submitted to the following treatments: 0.9% NaCl solution (negative control)( after first erosive challenge); whole milk with 0, 2.5, 5.0, 10.0 ppm F; fat-free milk with 0, 2.5, 5.0, 10.0 ppm F; 0.05% NaF solution (positive control) (before or after first erosive challenge). Specimens were submitted to demineralization - remineralization regimes, 4 times/ day, for 5 days. The response variables were enamel and dentin loss, evaluated by profilometry (µm). Data were analyzed using Kruskal­Wallis/Dunn's test (p<0.05). The presence of fluoride, especially at 10 ppm, was the most important factor in reducing dental erosion. The second part detected changes in protein profile of AEP formed in vivo after rinsing with whole milk, fat-free milk or water. Nine subjects with good oral conditions participated. The AEP was formed in the morning, for 120 min, after prophylaxis with pumice. In sequence, the volunteers rinsed with 10 mL of whole milk, fat-free milk or deionized water for 30 s, following a blind, crossover protocol. After 60 min, the AEP was collected with filter paper soaked in 3% citric acid and processed for analysis by liquid chromatography-electrospray ionization tandem mass spectrometry (LCESI- MS/MS). The obtained MS/MS spectra were searched against human protein database (SWISS­PROT). The proteomic data related to protein quantification were analyzed using the PLGS software. A total of 260 proteins were successfully identified in the AEP samples collected in all groups. Forty-nine were common to the 3 groups, while 72, 62 and 49 were specific for groups treated with whole milk, fat-free milk and water, respectively. Some were typical components of the AEP, such as Cystatin-B, Lysozyme C, Histatin-1, Statherin and Lactotransferrin. Other proteins are not commonly described as part of the AEP but could act in the defense of the organism against pathogens. Distinct proteomic profiles were found in the AEP after rinsing with whole or fat-free milk, which could have an impact in bacterial adhesion and tooth dissolution. The use of fat-free milk could favorably modulate the adhesion of bacteria in the AEP and the biofilm formation in comparison to whole milk.(AU)

Este estudo objetivou responder as seguintes questões: 1) o leite integral fluoretado protege mais contra a erosão do esmalte e dentina do que o leite fluoretado desnatado? 2) o efeito protetor do leite fluoretado segue um padrão dose-resposta? 3) o tratamento com leite integral ou leite desnatado fluoretado antes do primeiro desafio erosivo protege mais contra a erosão do esmalte e dentina? 4) o leite contendo gordura altera o perfil proteico da película adquirida do esmalte (PAE)? O estudo foi dividido em 2 partes. Na primeira parte foi realizado um estudo in vitro, considerando três fatores: tipo de leite bovino (integral/ desnatado), diferentes concentrações de fluoreto e tempo de aplicação (antes/após desafio erosivo). Os espécimes de esmalte bovino (n=15 /grupo) e dentina radicular (n=12 /grupo) foram submetidos aos seguintes tratamentos: solução de NaCl a 0,9% (controle negativo)(após o desafio erosivo); Leite integral com 0, 2,5, 5,0, 10,0 ppm F Leite desnatado com 0, 2,5, 5,0, 10,0 ppm F 0,05% de solução de NaF (controle positivo) (antes ou após o primeiro desafio erosivo). Os espécimes foram submetidos a regimes de desmineralização e remineralização, 4 vezes/dia, durante 5 dias. As variáveis de resposta foram perda de esmalte e dentina, avaliadas por perfilometria (µm). Os dados foram analisados usando o teste de Kruskal-Wallis / Dunn (p <0,05). A presença de fluoreto, especialmente na concentração de 10 ppm, demonstrou ser o fator mais importante na redução da erosão dentária. A parte II do estudo detectou alterações no perfil proteico da PAE formada in vivo após bochecho com leite integral, leite desnatado ou água. Nove indivíduos com boas condições de saúde bucal participaram. A PAE foi formada pela manhã, durante 120 minutos, após profilaxia com pedra-pomes. Em seguida, os voluntários bochecharam com 10 mL de leite integral, leite desnatado ou água deionizada durante 30 s, seguindo um protocolo cego e cruzado. Após 60 min, a película foi coletada com papel de filtro embebido em ácido cítrico a 3% e processada para análise por cromatografia líquida acoplada à espectrometria de massas com ionização por eletrospray (LC-ESI-MS / MS). Os espectros MS/MS obtidos foram confrontados com bases de dados de proteínas humanas (SWISSPROT). Os dados proteômicos relacionados à quantificação de proteínas foram analisados usando o software PLGS. Um total de 260 proteínas foi identificado nas amostras de PAE coletadas em todos os grupos. Quarenta e nove eram comuns aos 3 grupos, enquanto 72, 62 e 49 eram específicas para grupos tratados com leite integral, leite desnatado e água, respectivamente. Algumas proteínas encontradas são típicas da PAE, como Cistatina-B, Lisozima C, Histatina-1, Estaterina e Lactotransferrina. Outras proteínas não são comumente descritas como parte da PAE, mas podem atuar na defesa do organismo contra patógenos. Perfis proteômicos distintos foram encontrados na PAE após o bochecho com leite integral ou desnatado, o que poderia ter um impacto na adesão bacteriana e na dissolução dentária. O uso de leite desnatado pode modular favoravelmente a adesão de bactérias na PAE e a formação do biofilme em comparação com o leite integral.(AU)

Proteomics of acquired enamel pellicle in volunteers with gastroesophageal reflux with dental erosion or not / Proteoma da película adquirida em voluntários com refluxo gastresofágico com erosão dentária ou não

This study compared the protein profile of the acquired enamel pellicle (PAE) in 1) volunteers with gastroesophageal reflux disease (GERD) and dental erosion (BEWE ≥ 9 or grade 3 in the upper anterior sextant, all incisors affected; GE group); 2) volunteers with GERD without dental erosion (BEWE=0; GNE group) and 3) control volunteers (without GERD and dental erosion; BEWE = 0; C group). Twenty four subjects (8 in each group) participated. After dental prophylaxis, the AEP was allowed to form during 120 min and was then collected from the vestibular surface of the upper and lower teeth, with filter paper pre-soaked in 3% citric acid. After protein extraction, the samples were submitted to reverse phase liquid chromatography coupled to mass spectrometry (nLC-ESI-MS/MS). Label-free proteomic quantification was performed using Protein Lynx Global Service (PLGS) software. In total, 458 proteins were identified. Seventy-six proteins were common to all the groups. The proteomic profile of the AEP was quite different among the distinct groups. The numbers of proteins exclusively found in the C, GE and GNE groups were 113, 110 and 81, respectively. Most of the proteins exclusively identified in the C and GNE groups bind metals, while those in the GE group are mainly membrane proteins. Many proteins were found exclusively in the reflux groups. Heat-shock proteins were not found in GE. Histatins and Histones were not found in GNE, while Serine/threonine-protein kinases were only identified in GNE. In the quantitative analysis, when the GNE group was compared with the GE group, the proteins with the highest decreases were Lysozyme C, Antileukoproteinase, Cathepsin G, Neutrophil defensins and Basic salivary proline-rich proteins, while those with the highest increases were subunits of Hemoglobin, Albumin and isoforms of Cystatin. Profound alterations in the proteomic profile of the AEP were seen in GNE compared with GE volunteers, which might play a role in the resistance to dental erosion seen in the first. (AU)

Este estudo comparou o perfil proteico da película adquirida do esmalte adquirida (PAE) em 1) voluntários com doença de refluxo gastroesofágico (DRGE) e erosão dentária (BEWE ≥ 9 ou grau 3 no sextante anterior superior, todos os incisivos afetados; 2) voluntários com DRGE sem erosão dentária (BEWE = 0; grupo GNE) e 3) voluntários de controle (sem DRGE e erosão dentária, BEWE = 0; grupo C). Participaram vinte e quatro indivíduos (8 em cada grupo). Após a profilaxia dentária, permitiu-se que a PAE se formasse durante 120 minutos e foi então coletada a partir da superfície vestibular dos dentes superiores e inferiores, com papel de filtro previamente embebido em ácido cítrico a 3%. Após a extração da proteína, as amostras foram submetidas a cromatografia líquida de fase reversa acoplada a espectrometria de massa (nLC-ESI-MS / MS). A quantificação proteômica livre de marcadores foi realizada utilizando o software de Protein Lynx Global Service (PLGS). No total, foram identificadas 458 proteínas. Setenta e seis proteínas foram comuns a todos os grupos. O perfil proteômico da AEP foi bastante diferente entre os grupos distintos. O número de proteínas encontradas exclusivamente nos grupos C, GERD com erosão e GERD sem erosão foi de 113, 110 e 81, respectivamente. A maioria das proteínas exclusivamente identificadas nos grupos C e GERD sem erosão se liga a metais, enquanto que as do grupo GERD com erosão são principalmente proteínas de membrana. Muitas proteínas foram encontradas exclusivamente nos grupos de refluxo. As proteínas Heat-shock não foram encontradas no GERD com erosão. Histatins e Histones não foram encontradas no GERD com erosão, enquanto Serine/threonine-protein kinases foram identificadas apenas no GERD sem erosão. Na análise quantitativa, quando o grupo GERD sem erosão foi comparado com o grupo GERD com erosão, as proteínas com as maiores diminuições foram Lysozyme C, Antileukoproteinase, Cathepsin G, Neutrophil defensins and Basic salivary prolinerich proteins enquanto aquelas com os maiores aumentos foram subunidades de Hemoglobin, Albumin e isoformas de Cystatin. Maiores alterações no perfil proteômico da PAE foram observadas no GERD sem erosão em comparação com os voluntários GERD com erosão, o que pode ter um papel na resistência à erosão dentária observada anteriormente. (AU)