Enamel Pellicle Research Articles

Influence of periodic polyphenol treatment on the anti-erosive potential of the acquired enamel pellicle—A qualitative exploratory study

Objective: The aim of the present study was to investigate the impact of periodic polyphenol treatment on the ultrastructure and anti-erosive potential of an in-situ formed pellicle. Methods: Subjects wore intraoral appliances with buccally and palatally fixed bovine enamel specimens. During 6 h of intraoral pellicle formation, 100 ml black tea or tannic acid was applied ex-vivo every 25 min for 5 min. Untreated pellicles served as control. After the trial, specimens were immersed in 0.1% or 1% citric acid for 60 s and analysed for calcium release with atomic adsorption spectrometry and ultrastructure with transmission electron microscopy. Results: Specimens covered by pellicles treated with black tea or tannic acid released less calcium than untreated pellicles. Ultrastructural analyses reveal an increase in pellicle's thickness and density after treatment with polyphenols. Conclusions: Periodic polyphenol treatment of the pellicle modify its ultrastructure and increase its anti-erosive potential. Clinical significance: Consumption of polyphenolic beverages can enhance the anti-erosive potential of the enamel pellicle.

A Mass Spectrometric Approach to the Proteomic Profiling of the Canis lupus familiaris Acquired Enamel Pellicle on Hydroxyapatite Discs.

The acquired enamel pellicle (AEP) is a multi-protein film attached to the surface of teeth, which functions to lubricate the dental surface, form an anti-erosive barrier and exhibits antimicrobial properties. The initiation of AEP formation occurs within seconds of exposure to saliva, a biofluid rich in protein species. While there have been many publications on the formation of human AEP there is little research on the composition of canine AEP during its acquisition. The aim of these studies was to explore the composition of canine AEP formation, utilising hydroxyapatite (HA) discs as a tooth substitute matrix, over time. Qualitative and quantitative proteomics techniques using tandem mass tag labelled peptides and LC-MS/MS were used to follow the formation of canine AEP on hydroxyapatite discs over the course of an hour. Proteins adsorbed to the HA surface included highly abundant proteins in canine saliva, antimicrobial proteins, protease inhibitors and the buffering agent carbonic anhydrase. Greater understanding of the canine AEP deepens fundamental knowledge of the early processes driving bacterial colonisation of the tooth surface and subsequent plaque accumulation.

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.

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

ObjectiveTo study the proteomic alterations in the initial AEP after rinsing with CaneCPI-5, StN15 or Hb or their combination. Materials and methodsIn 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. ResultsRinsing 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. ConclusionRinsing with proteins/peptide, alone or combined, increased protective proteins in the initial AEP. Clinical RelevanceOur 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.

Influence of periodic milk or cream treatment on the anti-erosive potential of the acquired enamel pellicle

ObjectiveThe purpose of the present study was to assess the anti-erosive potential of the acquired enamel pellicle formed in situ under the influence of periodic milk or cream treatment. MethodsThe pellicle was formed on bovine enamel specimens in the oral cavity at buccal and palatal sites of upper molars in 6 subjects, using removable acrylic splints. During 6-h of intraoral exposure, splints were removed from the oral cavity every 25 min, treated with milk or cream for 5 min, and subsequently re-inserted into the oral cavity. After 6 h, pellicle covered specimens were immersed in citric acid (0.1 or 1.0 %) for 1 min, and processed for measurement of surface microhardness, determination of calcium release by atomic absorption spectroscopy, scanning and transmission electron microscopy. Statistical analysis was performed with SAS. ResultsStatistical analysis did not indicate major differences between erosive surface alterations on enamel specimens covered by pellicles treated with cream or milk, and those covered by control pellicles. In addition, TEM analysis did not reveal any differences concerning the ultrastructure of the different pellicle treatments during acid exposure. All pellicles were dissolved in part after exposure to 0.1 % citric acid and were nearly completely removed after treatment with 1.0% citric acid. ConclusionsIt is concluded that periodic treatment with milk or cream during pellicle formation in situ does not improve the protective potential of the acquired enamel pellicle against erosion. Clinical significanceModification of the pellicle by consumption of milk or cream prior to an acidic challenge cannot sufficiently protect enamel from erosion.

Bioinspired caries preventive strategy via customizable pellicles of saliva-derived protein/peptide constructs

Dental caries has been the most widespread chronic disease globally associated with significant health and financial burdens. Caries typically starts in the enamel, which is a unique tissue that cannot be healed or regrown; nonetheless, new preventive approaches have limitations and no effective care has developed yet. Since enamel is a non-renewable tissue, we believe that the intimate overlaying layer, the acquired enamel pellicle (AEP), plays a crucial lifetime protective role and could be employed to control bacterial adhesion and dental plaque succession. Based on our identified AEP whole proteome/peptidome, we investigated the bioinhibitory capacities of the native abundant proteins/peptides adsorbed in pellicle-mimicking conditions. Further, we designed novel hybrid constructs comprising antifouling and antimicrobial functional domains derived from statherin and histatin families, respectively, to attain synergistic preventive effects. Three novel constructs demonstrated significant multifaceted bio-inhibition compared to either the whole saliva and/or its native proteins/peptides via reducing biomass fouling and inducing biofilm dispersion beside triggering bacterial cell death. These data are valuable to bioengineer precision-guided enamel pellicles as an efficient and versatile prevention remedy. In conclusion, integrating complementary acting functional domains of salivary proteins/peptides is a novel translational approach to design multifunctional customizable enamel pellicles for caries prevention.

Vitamin E: A potential preventive approach against dental erosion-an in vitro short-term erosive study

ObjectivesThis study evaluated the in vitro effect of different components of palm oil on enamel in a short-term erosive challenge. MethodsThe acquired enamel pellicle (AEP) was previously formed in situ for 2 h. Subsequently, the bovine enamel blocks were treated in vitro according to following solutions: G1–palm oil; G2–85% tocotrienol solution; G3–oily vitamin E; G4–oily vitamin A; G5–deionized water (negative control); G6-stannous-containing solution (Elmex® Erosion Protection Dental Rinse) (positive control). After application of the treatment solutions (500 µl, 30 s), the blocks were immersed in 0.5% citric acid (pH 2.4) during 30 s (initial erosion). The response variable was the percentage of surface hardness loss. Data were analyzed by one-way ANOVA and Fisher's Test (p < 0.05). ResultsThe positive control (G6), palm oil (G1) and oily vitamin E (G3) groups presented the lowest percentage of surface hardness loss, and were statistically different from the negative group (G5) (p < 0.05), and no differences were found between these three groups. The 85% tocotrienol solution (G2) and oily vitamin A groups (G4) were not different to the negative control group. ConclusionsStannous-containing positive control (Elmex® Erosion Protection), palm oil and oily Vitamin E were able to protect enamel against the erosive challenge performed in this in vitro study. In addition, vitamin E is probably the key ingredient of palm oil responsible for preventing enamel erosion. Clinical significanceVitamin E presented similar preventive effect to a commercial mouthwash stannous-containing solution (Elmex® Erosion Protection) against initial erosion and, it can be considered as a promising natural alternative for the formulations of solutions aiming to prevent erosive tooth wear.

Rinsing with Statherin-Derived Peptide Alters the Proteome of the Acquired Enamel Pellicle

Changes in the proteomic profile of the acquired enamel pellicle (AEP) formed for 3 min or 2 h after rinsing with a peptide containing the 15 N-terminal residues of statherin, with serines 2 and 3 phosphorylated (StatpSpS), were evaluated. Nine volunteers participated in 2 consecutive days. Each day, after professional tooth cleaning, they rinsed for 1 min with 10 mL of phosphate buffer containing 1.88 × 10⁻⁵ M StatpSpS or phosphate buffer only (control). The acquired pellicle formed on enamel after 3 min or 2 h was collected with electrode filter papers soaked in 3% citric acid. After protein extraction, samples were analyzed by quantitative shotgun label-free proteomics. In the 3-min AEP, 19 and 131 proteins were uniquely identified in the StatpSpS and control groups, respectively. Proteins typically found in the AEP were only found in the latter. Only 2 proteins (neutrophil defensins) were increased upon treatment with StatpSpS, while 65 proteins (among which are several typical AEP proteins) were decreased. In the 2-h AEP, 50 and 108 proteins were uniquely found in StatpSpS and control groups, respectively. Hemoglobin subunits and isoforms of keratin were only found in the StatpSpS group, while cystatin-C, cathepsin D, and cathepsin G, isoforms of heat shock 70 and protocadherin were exclusively found in the control group. In addition, 23 proteins were increased upon treatment with StatpSpS, among which are histatin-1, serum albumin, and isoforms of neutrophil defensin and keratin, while 77 were decreased, most of them were typical AEP proteins. In both evaluated periods, rinsing with StatpSpS profoundly changed the proteomic profile of the AEP, which might impact the protective role of this integument against carious or erosive demineralization. This study provides important insights on the dynamics of the protein composition of the AEP along time, after rinsing with a solution containing StatpSpS.

Salivary Pellicle Modification with Grape-seed Extract: In Vitro Study on the Effect on Bacterial Adhesion and Biofilm Formation.

Grape-seed extract (GSE) contains polyphenols that readily adhere to proteins and modify the acquired enamel pellicle (AEP). The first step in biofilm formation is bacterial adhesion to the AEP-covered enamel. The aim of this in vitro study was to test whether AEP modification with GSE, fluoride (F-), or their combination (GSE+F-) modulates bacterial adhesion, biofilm metabolism and composition, or cariogenic demineralisation of the enamel. The study comprised 3 parts: 1) single-strain Streptococcus gordonii species, 2) a five-species biofilm model, or 3) biofilm (re-)formation using the five-species biofilm model after removal of initial biofilm with toothbrushing. Human whole-mouth stimulated saliva was used to form an AEP on human enamel specimens. The AEP was incubated in water (control), or modified with GSE, F-, or GSE+F-. Bacterial adhesion, biofilm diversity, metabolic activity, biofilm mass, and cariogenic demineralisation (surface hardness) of enamel were assessed after incubation in bacterial broths after 4 h or 22 h. Differences between groups were analysed with one-way ANOVA and post-hoc Bonferroni tests. GSE and GSE+F- statistically significantly decreased single-strain S. gordonii adhesion, but had no relevant influence when the five-species biofilm model was used. In the biofilm (re-)formation model, GSE reduced bacterial adhesion compared to GSE+F-, while F- caused less cariogenic demineralisation than was found in the control group. AEP modified with GSE retards S. gordonii adhesion, but it does not influence the formation, metabolism and composition of a cariogenic multi-species biofilm.

Preventive Applications of Polyphenols in Dentistry-A Review.

Polyphenols are natural substances that have been shown to provide various health benefits. Antioxidant, anti-inflammatory, and anti-carcinogenic effects have been described. At the same time, they inhibit the actions of bacteria, viruses, and fungi. Thus, studies have also examined their effects within the oral cavity. This review provides an overview on the different polyphenols, and their structure and interactions with the tooth surface and the pellicle. In particular, the effects of various tea polyphenols on bioadhesion and erosion have been reviewed. The current research confirms that polyphenols can reduce the growth of cariogenic bacteria. Furthermore, they can decrease the adherence of bacteria to the tooth surface and improve the erosion-protective properties of the acquired enamel pellicle. Tea polyphenols, especially, have the potential to contribute to an oral health-related diet. However, in vitro studies have mainly been conducted. In situ studies and clinical studies need to be extended and supplemented in order to significantly contribute to additive prevention measures in caries prophylaxis.

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.

Radiotherapy changes acquired enamel pellicle proteome in head and neck cancer patients

ObjectivesTo evaluate in vivo the proteomic profile of the acquired enamel pellicle (AEP) in patients with head and neck cancer (HNC) before, during and after radiotherapy. MethodsNine patients, after prophylaxis, had their AEPs collected before (BRT), during (DRT; 2–5 weeks) and after (ART; 3–4 months) radiotherapy. AEP was also collected from nine healthy patients (Control). The proteins were extracted in biological triplicate and processed by label-free proteomics. ResultsStatherin was increased more than 9-fold and several hemoglobin subunits were increased more than 5-fold DRT compared to BRT, while lactotransferrin, proline-rich proteins, cystatins, neutrophil defensins 1 and 3 and histatin-1 were decreased. ART, there was an increase in lactotransferrin and several isoforms of histones, while statherin and alpha-amylase proteins were decreased. MOAP-1 was exclusively found ART in comparison to BRT. When compared to Control, AEP of patients BRT showed an increase in proteins related to the perception of bitter taste, mucin-7 and alpha-amylases, while cystatin-S was decreased. ConclusionsHNC and radiotherapy remarkably altered the proteome of the AEP. Antibacterial and acid-resistant proteins were decreased during radiotherapy. Clinical significanceOur results provide important information for designing more effective dental products for these patients, in addition to contributing to a better understanding of the differential protective roles of the AEP proteins during radiotherapy. Moreover, some proteins identified in the AEP after radiotherapy may serve as prognostic markers for survival of HNC patients.

A sugarcane cystatin (CaneCPI-5) alters microcosm biofilm formation and reduces dental caries

The antimicrobial and anticaries effects of CaneCPI-5 were evaluated. Ninety bovine enamel samples were treated for 60 s with either phosphate-buffered-saline (PBS), 0.12% chlorhexidine (CHX), 0.05 mg ml−1 CaneCPI-5, 0.1 mg ml−1 CaneCPI-5 or 0.5 mg ml−1 CaneCPI-5. They were incubated with inoculum (human saliva + McBain's saliva) for the first 8 h. From then until the end of the experiment, the enamel was exposed to McBain saliva with sucrose and, once a day, for 5 days, they were treated with the solutions. At the end of the experimental period, resazurin and viable plate count assays were performed. Enamel demineralization was also measured. All concentrations of CaneCPI-5 and CHX significantly reduced the activity of biofilms compared with PBS. For viable plate counts, all treatments similarly reduced the lactobacilli and total streptococci; for the mutans streptococci, 0.05 mg ml−1 CaneCPI-5 performed better than CHX. All CaneCPI-5 concentrations significantly reduced the integrated mineral loss. This study represents the first step regarding the use of CaneCPI-5 within the concept of acquired enamel pellicle and biofilm engineering to prevent dental caries.

The effect of red wine in modifying the salivary pellicle and modulating dental erosion kinetics.

This study investigated the potential of red wine in modulating dental erosion kinetics in the presence or absence of salivary pellicle. Polished human enamel specimens were used in two conditions; presence or absence of acquired enamel pellicle; and subdivided according to exposure: red wine, orange juice, apple juice, or citric acid. The specimens were incubated in clarified whole human saliva (presence of acquired enamel pellicle) or in a humid chamber (absence of acquired enamel pellicle) for 2h at 37°C, then in the test substances for 1min, at 25°C, under shaking. This was repeated four times. Surface hardness was measured initially and after each cycle and surface reflection intensity was measured initially and after all cycles. In the presence of acquired enamel pellicle, red wine caused the least surface hardness loss, followed by orange juice, apple juice, and citric acid. Statistically significantly less surface reflection intensity loss was observed for red wine and orange juice than for apple juice and citric acid. In the absence of acquired enamel pellicle, red wine and orange juice caused less surface hardness loss than apple juice and citric acid. Orange juice showed the least surface reflection intensity loss, followed by red wine, citric acid, and apple juice. The polyphenol composition of these drinks can notably modulate the erosion kinetics.

Proteomic profiles of the acquired enamel pellicle formed in vitro, in situ, or in vivo.

This study compared the protein profile of the acquired enamel pellicle (AEP) formed under three conditions: in vitro, in situ, and in vivo. Nine volunteers participated in all procedures. In the in vitro condition, the volunteers donated saliva, in which specimens were incubated to form the AEP. In the in situ condition, the volunteers used an oral device containing specimens where the AEP was formed. In the in vivo condition, the AEP was collected from the volunteers own teeth. All AEPs were formed for 120min, collected and processed by mass spectrometry. Overall, a total of 321 proteins were identified, among which 37 proteins are commonly considered typical in the AEP. For each of the in vitro, in situ, and in vivo conditions, respectively, 66, 174, and 170 proteins were identified. For the in vitro condition, 17 pellicle-typical proteins were not identified. Furthermore, several proteins with important functions within the AEP presented differences in expression in the three conditions. The qualitative profile of the proteins, especially the typical ones, is different in the in vitro condition. In addition, there are important quantitative differences that may interfere when attempting to extrapolate in vitro results to an in situ and in vivo condition.

Acquired pellicle protein-based engineering protects against erosive demineralization

ObjectivesTo 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 methodsIn 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. ResultsTreatment 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). ConclusionsTreatment with CaneCPI-5, HB or StN15 remarkably increases acid-resistant proteins in the AEP, protecting against erosion. Clinical significanceOur 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.

Deep Proteomic Insights into the Individual Short-Term Pellicle Formation on Enamel-An In Situ Pilot Study.

Dental pellicle formation starts instantaneously after oral hygiene due to the adsorption of salivary proteins to all orally exposed surfaces. The pellicle acts as a physiological mediator, protects the tooth surface from mechanical damages and reduces acid-induced enamel demineralization. The aim of this pilot study is to identify and characterize individual proteomic profiles of the initial pellicle formed on dental enamel and to compare the profiles with the corresponding saliva to analyze specific adsorption patterns occurring during pellicle formation. The 3-min pellicle of five subjects formed in situ on bovine enamel is eluted chemically and analyzed separately by nano-mass spectrometry. The analysis of the corresponding saliva is conducted in parallel. Up to 498 pellicle proteins and up to 1032 salivary proteins are identified on an individual level. Comparison of the salivary and pellicle protein profiles demonstrates the pellicle formation to be highly individual. Nineteen proteins are significantly enriched in the 3-min pellicle of all subjects and 22 proteins are significantly depleted indicating that pellicle formation relies on selective adsorption. The short-term enamel pellicle is composed of several hundreds of adsorbed salivary proteins and reveals a highly individual proteomic profile.

Proteins from whole mouth saliva mediate greater protection against severe erosive tooth wear than proteins from parotid saliva using an in vitro model

Objectivesto investigate how the composition of the acquired enamel pellicle (AEP) affected a laboratory model of erosive tooth wear (ETW) on human enamel by comparing whole mouth saliva (WMS) to parotid saliva (PS). Methods60 enamel specimens were prepared from extracted human teeth and were randomly assigned to 4 experimental groups: WMS (n = 20), PS (n = 20), artificial saliva (AS, n = 10) and deionised water (DW, n = 10). Following incubation, a subset of WMS (n = 5) and PS (n = 5) groups were used to collect the AEP before the erosive challenge. The rest of the blocks, had their AEP collected after five cycles of acid, wash and saliva and were then assessed for mean step height changes using a non-contacting profilometer (n = 10 each). AEP samples were collected from the enamel specimens by rubbing with filter papers soaked in sodium dodecyl sulfate. Total protein in AEP was quantified using BCA assay, individual protein components of AEP were separated and analysed using SDS-PAGE and western blot for [mucin 5b, albumin, carbonic anhydrase VI (CA VI), statherin]. Specific antibody binding was quantified using purified protein standards of known concentration. Samples of AEP were also analysed by LC/MS/MS sequencing. ResultsWMS group had significantly (p < 0.0001) less acid-induced erosion (step height [4.16 (0.9) μm]) than PS group [6.41 (0.3) μm]. The amount of total protein, mucin 5b and albumin were more dominant in WMS pellicles than PS (p < 0.0001) whereas CA VI and statherin were dominant in PS pellicles (p < 0.0001). ConclusionThe composition of the acquired enamel pellicle influences the degree of protection from acid attack, possibly by altering the mechanism of protection. The in-vitro model used in this study was severe enough to cause tissue loss as opposed to just softening of the surface structure. AEP from WMS was more protective than that of PS, and its likely mechanisms of protection seem to be as a physical barrier rather than stabilising the crystal structure. SignificanceThe protective salivary proteins against in-vitro erosion models differ from in-vivo studies. Therfore, it can be recommended that in-vitro laboratory models of ETW need to be assessed carefully to represent the clinical environment more closely.

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

ObjectivesThe 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. MethodsSixty 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. ResultsA 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. ConclusionsThe 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 significanceThe 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.