Effect of nonionic and amphoteric surfactants on salivary pellicles reconstituted in vitro.

Surfactants are important components of oral care products. Sodium dodecyl sulfate (SDS) is the most common because of its foaming properties, taste and low cost. However, the use of ionic surfactants, especially SDS, is related to several oral mucosa conditions. Thus, there is a high interest in using non-ionic and amphoteric surfactants as they are less irritant. To better understand the performance of these surfactants in oral care products, we investigated their interaction with salivary pellicles i.e., the proteinaceous films that cover surfaces exposed to saliva. Specifically, we focused on pentaethylene glycol monododecyl ether (C12E5) and cocamidopropyl betaine (CAPB) as model nonionic and amphoteric surfactants respectively, and investigated their interaction with reconstituted salivary pellicles with various surface techniques: Quartz Crystal Microbalance with Dissipation, Ellipsometry, Force Spectroscopy and Neutron Reflectometry. Both C12E5 and CAPB were gentler on pellicles than SDS, removing a lower amount. However, their interaction with pellicles differed. Our work indicates that CAPB would mainly interact with the mucin components of pellicles, leading to collapse and dehydration. In contrast, exposure to C12E5 had a minimal effect on the pellicles, mainly resulting in the replacement/solubilisation of some of the components anchoring pellicles to their substrate.

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.

Physical-chemical interactions between dental materials surface, salivary pellicle and Streptococcus gordonii.

Dental materials are susceptible to dental plaque formation, which increases the risk of biofilm-associated oral diseases. Physical-chemical properties of dental material surfaces can affect salivary pellicle formation and bacteria attachment, but relationships between these properties have been understudied. We aimed to assess the effects of surface properties and adsorbed salivary pellicle on Streptococcus gordonii adhesion to traditional dental materials. Adsorption of salivary pellicle from one donor on gold, stainless steel, alumina and zirconia was monitored with a quartz crystal microbalance with dissipation monitoring (QCM-D). Surfaces were characterized by X-ray photoelectron spectroscopy, atomic force microscopy and water contact angles measurement before and after pellicle adsorption. Visualization and quantification of Live/Dead stained bacteria and scanning electron microscopy were used to study S. gordonii attachment to materials with and without pellicle. The work of adhesion between surfaces and bacteria was also determined. Adsorption kinetics and the final thickness of pellicle formed on the four materials were similar. Pellicle deposition on all materials increased surface hydrophilicity, surface energy and work of adhesion with bacteria. Surfaces with pellicle had significantly more attached bacteria than surfaces without pellicle, but the physical-chemical properties of the dental material did not significantly alter bacteria attachment. Our findings suggested that the critical factor increasing S. gordonii attachment was the salivary pellicle formed on dental materials. This is attributed to increased work of adhesion between bacteria and substrates with pellicle. New dental materials should be designed for controlling bacteria attachment by tuning thickness, composition and structure of the adsorbed salivary pellicle.

Characterization of the in situ pellicle ultrastructure formed under the influence of bovine milk and milk protein isolates.

OBJECTIVES: The present study aimed to investigate if bovine milk or milk protein isolates, respectively, alter the ultrastructure of thein situ pellicle and might therefore have an influence on oral health. METHODS: In situ pellicle samples were formed on bovine enamel slabs exposed in the oral cavity of three subjects for 6, 30, 60 or 120 min. After 3 min of pellicle formation, mouthrinses were performed for 3 min with (non-)homogenized UHT- or fresh milk (0.3% or 3.8% fat), 30% UHT-treated cream or different types of casein- or milk protein isolates containing preparations. The specimens were removed after the exposure times and transmission electron microscopy (TEM) was performed. Native pellicle samples served as controls. RESULTS: Topical ultrastructural pellicle modifications were detected after mouthrinses with all types of homogenized UHT- or fresh milk and after the application of a 3% native casein micelles containing experimental solution. Atypical globular protein structures, identified as casein micelles, were temporarily adsorbed onto the pellicle. They were closely associated with lipid droplets. Furthermore, the mouthrinses occasionally affected the morphology of salivary bacteria. However, no notable ultrastructural alterations remained after 120 min of pellicle formation. CONCLUSION: For the first time, bovine milk- and micellar casein-induced pellicle modifications were revealed by TEM. The adsorption of micellar casein is possibly due to its molecular interactions. CLINICAL SIGNIFICANCE: Bovine milk or micellar caseins provide some potential for the development of preventive strategies against bacterial biofilm formation or erosive processes at the tooth surface.

Effect of antimicrobial nanocomposites on Vibrio cholerae lifestyles: Pellicle biofilm, planktonic and surface-attached biofilm.

Vibrio cholerae is an important human pathogen causing intestinal disease with a high incidence in developing countries. V. cholerae can switch between planktonic and biofilm lifestyles. Biofilm formation is determinant for transmission, virulence and antibiotic resistance. Due to the enhanced antibiotic resistance observed by bacterial pathogens, antimicrobial nanomaterials have been used to combat infections by stopping bacterial growth and preventing biofilm formation. In this study, the effect of the nanocomposites zeolite-embedded silver (Ag), copper (Cu), or zinc (Zn) nanoparticles (NPs) was evaluated in V. cholerae planktonic cells, and in two biofilm states: pellicle biofilm (PB), formed between air-liquid interphase, and surface-attached biofilm (SB), formed at solid-liquid interfaces. Each nanocomposite type had a distinctive antimicrobial effect altering each V. cholerae lifestyles differently. The ZEO-AgNPs nanocomposite inhibited PB formation at 4 µg/ml, and prevented SB formation and eliminated planktonic cells at 8 µg/ml. In contrast, the nanocomposites ZEO-CuNPs and ZEO-ZnNPs affect V. cholerae viability but did not completely avoid bacterial growth. At transcriptional level, depending on the nanoparticles and biofilm type, nanocomposites modified the relative expression of the vpsL, rbmA and bap1, genes involved in biofilm formation. Furthermore, the relative abundance of the outer membrane proteins OmpT, OmpU, OmpA and OmpW also differs among treatments in PB and SB. This work provides a basis for further study of the nanomaterials effect at structural, genetic and proteomic levels to understand the response mechanisms of V. cholerae against metallic nanoparticles.

Impact of oral astringent stimuli on surface charge and morphology of the protein-rich pellicle at the tooth-saliva interphase.

The proteinaceous pellicle layer, which develops upon contact with saliva on the surface of teeth, is important for the formation of oral biofilms and for the protection of teeth from abrasion and chemically induced erosion. Astringent food ingredients comprising polyphenols, cationic macromolecules, and multivalent metal salts are known to interact with the pellicle. However, astringent-induced changes in the physicochemical properties of the tooth-saliva interphase are not yet completely understood. Here we provide comprehensive insights into interfacial charging, ultrastructure, thickness, and surface roughness of the pellicles formed on the model substrates silicon oxide (SiO2), Teflon® AF, and hydroxyapatite, as well as on bovine enamel before and after incubation with the astringents epigallocatechin gallate, tannic acid, iron(III) salt, lysozyme, and chitosan. Quartz crystal microbalance with dissipation monitoring demonstrated viscous behavior of untreated pellicles formed in vitro on the different materials. Electrokinetic (streaming current) measurements revealed that cationic astringents reverse the charge of native pellicles, whereas polyphenols did not change the charge under physiological pH condition. In addition, transmission electron microscopy and atomic force microscopy showed a concentration-dependent increase in average film thickness and pellicle surface roughness as induced by astringents. These multifaceted alterations of the salivary pellicle may come along with an increase in roughness perceived on the teeth, which is part of the complex sensations of oral astringency.

Mechanisms of astringency: Structural alteration of the oral mucosal pellicle by dietary tannins and protective effect of bPRPs.

The interaction of tannins with salivary proteins is involved in astringency. This paper focussed on saliva lining oral mucosae, the mucosal pellicle. Using a cell-based model, the impact of two dietary tannins (EgC and EgCG) on the mucosal pellicle structure and properties was investigated by microscopic techniques. The role of basic Proline-Rich-Proteins (bPRPs) in protecting the mucosal pellicle was also evaluated. At low (0.05 mM) tannin concentration, below the sensory detection threshold, the distribution of salivary mucins MUC5B on cells remained unaffected. At 0.5 and 1 mM, MUC5B-tannin aggregates were observed and their size increased with tannin concentration and with galloylation. In addition, 3 mM EgCG resulted in higher friction forces measured by AFM. In presence of bPRPs, the size distribution of aggregates was greatly modified and tended to resemble that of the "no tannin" condition, highlighting that bPRPs have a protective effect against the structural alteration induced by dietary tannins.

Changes in the Proteomic Profile of Acquired Enamel Pellicles as a Function of Their Time of Formation and Hydrochloric Acid Exposure.

OBJECTIVE: Changes in the protein profile of acquired enamel pellicles (AEP) formed in vivo over different time periods were evaluated after the application of hydrochloric acid (HCl). METHODS: Nine subjects were submitted to dental prophylaxis with pumice. After 3 or 120 min, the teeth were isolated with cotton rolls and 50 µL of 0.1 M HCl (pH 1.0), 0.01 M HCl (pH 2.0), or deionized water were applied on the buccal surface of the teeth for 10 s. The AEP was then collected using an electrode filter paper presoaked in 3% citric acid. After protein extraction, the samples were submitted to reverse-phase liquid chromatography coupled to mass spectrometry (nano LC-ESI-MS/MS). Label-free quantification was performed (Protein Lynx Global Service software). RESULTS: A total of 180 proteins were successfully identified in the AEP samples. The number of identified proteins increased with the time of pellicle formation. Only 4 proteins were present in all the groups (isoforms of IgA, serum albumin, and statherin). The greatest number of proteins identified uniquely in one of the groups was obtained for the groups treated with HCl after 2 h of pellicle formation (approx. 50 proteins). CONCLUSION: Proteins resistant to removal by HCl, such as serum albumin and statherin, were identified even in the short-term AEP. In addition, 120-min pellicles present many proteins that are resistant to removal by HCl. This suggests an increase in protection against intrinsic acids with the time of pellicle formation, which should be evaluated in future studies.

Effect of vegetable oils applied over acquired enamel pellicle on initial erosion.

OBJECTIVE: The prevalence of dental erosion has been recently increasing, requiring new preventive and therapeutic approaches. Vegetable oils have been studied in preventive dentistry because they come from a natural, edible, low-cost, and worldwide accessible source. This study aimed to evaluate the protective effect of different vegetable oils, applied in two concentrations, on initial enamel erosion. MATERIAL AND METHODS: Initially, the acquired pellicle was formed in situ for 2 hours. Subsequently, the enamel blocks were treated in vitro according to the study group (n=12/per group): GP5 and GP100 - 5% and pure palm oil, respectively; GC5 and GC100 - 5% and pure coconut oil; GSa5 and GSa100 - 5% and pure safflower oil; GSu5 and GSu100 - 5% and pure sunflower oil; GO5 and GO100 - 5% and pure olive oil; CON- - Deionized Water (negative control) and CON+ - Commercial Mouthwash (Elmex® Erosion Protection Dental Rinse, GABA/positive control). Then, the enamel blocks were immersed in artificial saliva for 2 minutes and subjected to short-term acid exposure in 0.5% citric acid, pH 2.4, for 30 seconds, to promote enamel surface softening. The response variable was the percentage of surface hardness loss [((SHi - SHf) / SHf )×100]. Data were analyzed by one-way ANOVA and Tukey's test (p<0.05). RESULTS: Enamel blocks of GP100 presented similar hardness loss to GSu100 (p>0.05) and less than the other groups (p<0.05). There was no difference between GP5, GC5, GC100, GSa5, GSu100, GSa100, GSu5, GO5, GO100, CON- and CON+. CONCLUSION: Palm oil seems to be a promising alternative for preventing enamel erosion. However, further studies are necessary to evaluate a long-term erosive cycling.

Effect of vegetable oils applied over acquired enamel pellicle on initial erosion

Abstract Objective The prevalence of dental erosion has been recently increasing, requiring new preventive and therapeutic approaches. Vegetable oils have been studied in preventive dentistry because they come from a natural, edible, low-cost, and worldwide accessible source. This study aimed to evaluate the protective effect of different vegetable oils, applied in two concentrations, on initial enamel erosion. Material and Methods Initially, the acquired pellicle was formed in situ for 2 hours. Subsequently, the enamel blocks were treated in vitro according to the study group (n=12/per group): GP5 and GP100 - 5% and pure palm oil, respectively; GC5 and GC100 - 5% and pure coconut oil; GSa5 and GSa100 - 5% and pure safflower oil; GSu5 and GSu100 - 5% and pure sunflower oil; GO5 and GO100 - 5% and pure olive oil; CON− - Deionized Water (negative control) and CON+ - Commercial Mouthwash (Elmex® Erosion Protection Dental Rinse, GABA/positive control). Then, the enamel blocks were immersed in artificial saliva for 2 minutes and subjected to short-term acid exposure in 0.5% citric acid, pH 2.4, for 30 seconds, to promote enamel surface softening. The response variable was the percentage of surface hardness loss [((SHi - SHf) / SHf )×100]. Data were analyzed by one-way ANOVA and Tukey's test (p<0.05). Results Enamel blocks of GP100 presented similar hardness loss to GSu100 (p>0.05) and less than the other groups (p<0.05). There was no difference between GP5, GC5, GC100, GSa5, GSu100, GSa100, GSu5, GO5, GO100, CON− and CON+. Conclusion Palm oil seems to be a promising alternative for preventing enamel erosion. However, further studies are necessary to evaluate a long-term erosive cycling.

Oral astringent stimuli alter the enamel pellicle's ultrastructure as revealed by electron microscopy.

OBJECTIVES: This electron microscopic study aimed at investigating effects of oral astringent stimuli on the enamel pellicle's morphology. METHODS: Pellicles were formed in situ within 30min on bovine enamel slabs, fixed to individuals' upper jaw splints. The pellicle-coated specimens were immersed in vitro in seven diverse astringent solutions and subsequently analyzed by scanning electron microscopy (SEM), energy dispersive X-ray (EDX) spectroscopy, as well as transmission electron microscopy (TEM). Four biocompatible astringents, namely the polyphenol epigallocatechin gallate, the metal salt iron(III) sulfate, the basic protein lysozyme, and the aminopolysaccharide chitosan, were additionally applied in situ. After rinsing the oral cavity with these compounds, the pellicle's ultrastructure was imaged by SEM and TEM, respectively. Untreated pellicle samples served as controls. RESULTS: Exposure to polyphenols and lysozyme induced particularly thicker and electron-denser pellicles in comparison to the control pellicle with similar characteristics in vitro and in situ. In contrast, acidic chitosan and metal salt solutions, respectively, revealed minor pellicle alterations. The incorporation of Fe and Al into the pellicles treated with the corresponding inorganic salts was verified by EDX analysis. CONCLUSIONS: Astringent-induced pellicle modifications were for the first time visualized by TEM. The ultrastructural alterations of the dental pellicle may partly explain the tooth-roughening effect caused by oral astringent stimuli. CLINICAL SIGNIFICANCE: Astringents might modify the pellicle's protective properties against dental erosion, attrition, as well as bacterial adhesion, and by this means may influence tooth health. The findings may thus be particularly relevant for preventive dentistry.

Effect of gels containing chlorhexidine or epigallocatechin-3-gallate on the protein composition of the acquired enamel pellicle.

OBJECTIVE: This study evaluated changes in protein profile of the acquired enamel pellicle (AEP) formed in vivo, after application of gels containing chlorhexidine or EGCG and further challenge with citric acid. DESIGN: AEP was formed in 9 volunteers for 2h and then treated with one of the following gels: placebo, 400µM EGCG or 0.012% chlorhexidine. A thin layer of gel was applied and after 1min the excess was removed. One hour after gel application, the AEP was collected from the buccal surface (upper and lower jaw) of one of the sides with filter paper dipped in 3% citric acid. On the other side, erosive challenge was performed through gentle application of 1% citric acid (pH 2.5) for 20s (using a pipette) followed by washing with deionized water. The AEP was collected as mentioned before. Proteomic analysis was performed through liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The MS/MS spectra obtained were compared with human protein databases (SWISS-PROT). Label-free quantitation was done using the PLGS software. RESULTS: In total, 223 proteins were identified. After treatment with EGCG and CHX gels, proteins with potential functions to protect against caries and erosion such as PRPs, calcium-bind proteins and Statherin were increased. When EGCG and CHX-treated AEPs were challenged with citric acid, there was increase in cystatins and Profilin-1. CONCLUSION: CHX- and EGCG-treated AEPs, submitted to challenge with citric acid or not, had remarkable changes in their proteomic profiles.

In situ evaluation of fluoride-, stannous- and polyphosphate-containing solutions against enamel erosion.

OBJECTIVE: To evaluate the anti-erosive effect of solutions containing sodium fluoride (F: 225ppm of fluoride), sodium fluoride+stannous chloride (F+Sn: 225ppm of fluoride+800ppm of stannous), sodium fluoride+stannous chloride+sodium linear polyphosphate (F+Sn+LPP: 225ppm of fluoride+800ppm of stannous+2% of sodium linear polyphosphate), and deionized water (C: control), using a four-phase, single-blind, crossover in situ clinical trial. METHODS: In each phase, 12 volunteers wore appliances containing 4 enamel specimens, which were submitted to a 5-day erosion-remineralization phase that consisted of 2h of salivary pellicle formation with the appliance in situ, followed by 2min extra-oral immersion in 1% citric acid (pH 2.4), 6x/day, with 90min of exposure to saliva in situ between the challenges. Treatment with the test solutions was performed extra-orally for 2 min, 2x/day. At the end of the experiment, surface loss (SL, in µm) was evaluated by optical profilometry. Data were analyzed using ANOVA and Tukey tests (α=0.05). The surface of additional specimens was evaluated by x-ray diffraction after treatments (n=3). RESULTS: C (mean SL±standard-deviation: 5.97±1.70) and F (5.36±1.59) showed the highest SL, with no significant difference between them (p>0.05). F+Sn (2.68±1.62) and F+Sn+LPP (2.10±0.95) did not differ from each other (p>0.05), but presented lower SL than the other groups (P<0.05). Apatite and stannous deposits on specimen surfaces were identified in the x-ray analysis for F+Sn and F+Sn+LPP. CONCLUSIONS: Sodium fluoride solution exhibited no significant anti-erosive effect. The combination between sodium fluoride and stannous chloride reduced enamel erosion, irrespective of the presence of linear sodium polyphosphate. CLINICAL SIGNIFICANCE: Under highly erosive conditions, sodium fluoride rinse may not be a suitable alternative to prevent enamel erosion. A rinse containing sodium fluoride and stannous chloride was shown to be a better treatment option, which was not further improved by addition of the sodium linear polyphosphate.

Effect of Tannic Acid on the Protective Properties of the in situ Formed Pellicle.

OBJECTIVES: In the present in situ/ex vivo study the impact of tannic acid on the erosion-protective properties of the enamel pellicle was tested. Additionally, the antiadherent and antibacterial effects of tannic acid were evaluated. METHODS: The pellicle was formed in situ on bovine enamel samples fixed on individual splints worn by 6 subjects. Following 1 min of pellicle formation the volunteers rinsed for 10 min with tannic acid. After further oral exposure for 19 min, 109 min, and 8 h overnight, respectively, slabs were incubated in HCl ex vivo (pH 2.0, 2.3, 3.0) over 120 s. Subsequently, kinetics of calcium and phosphate release were measured photometrically. Samples after a 1-min fluoride mouth rinse as well as enamel samples with and without a 30-min in situ pellicle served as controls. Antiadherent effects were evaluated after a 1-min rinse with tannic acid and oral exposure of the slabs overnight. DAPI (4',6-diamidino-2-phenylindole) combined with concanavalin A staining and live/dead staining was used for fluorescence microscopic visualization and quantification of adherent bacteria and glucans. Modification of the pellicle's ultrastructure by tannic acid was evaluated by transmission electron microscopy (TEM). RESULTS: Tannic acid significantly improved the erosion-protective properties of the pellicle in a pH-dependent manner. Bacterial adherence and glucan formation on enamel were significantly reduced after rinses with tannic acid as investigated by fluorescence microscopy. TEM imaging indicated that rinsing with tannic acid yielded a sustainable modification of the pellicle; it was distinctly more electron dense. CONCLUSION: Tannic acid offers an effective and sustainable approach for the prevention of caries and erosion.

Análise proteômica da película adquirida formada sobre resinas compostas experimentais contendo ou não carga e/ou inibidores de proteases: estudo in situ / Proteomic analysis of acquired pellicle formed on experimental composite resins containing or not filler and/or protease inhibitors: study in situ

Na cavidade oral, qualquer superfície exposta é propensa à formação da película adquirida (PA), sendo considerada um filme orgânico, livre de bactérias que se forma in vivo como resultado da adsorção seletiva de proteínas e glicoproteínas salivares às superfícies solidas que estão expostas ao meio bucal. O objetivo deste trabalho será avaliar a influência da adição ou não de carga (vidro de bário alumina silicato e sílica) e/ou de inibidores de proteases (EGCG ou CHX) a resinas compostas experimentais no perfil proteico da PA formada sobre estes espécimes, utilizando estratégias proteômicas quantitativas livres de marcadores. Foram preparadas 324 amostras de esmalte bovino (6x6x2mm), foi feita uma cavidade no centro de 4x4mm, a qual foi preenchida com resinas experimentais. As amostras foram divididas em 6 grupos de 54 espécimes cada, de acordo com os grupos experimentais: Sem carga, sem inibidor (NF-NI); carga, sem inibidor (F-NI); sem carga e CHX (NF-CHX); carga e CHX (F-CHX); sem carga e EGCG (NF-EGCG); carga e EGCG (F-EGCG). Nove adultos jovens de ambos os gêneros paticiparam, usando um aparelho mandibular removível (BISPM - Bauru in situpellicle model) com duas amostras de cada grupo. O experimento foi conduzido por 9 dias consecutivos, durante a manhã por 120 minutos. A PA foi obtida através da ajuda do papel filtro de eletrotodo, umidecido em 3% de ácido cítrico. A película coletada, foi processada por LC-ESI-MS/MS. Os espectros MS/MS obtidos foram confrontados com bases de dados de proteínas humanas (SWISS-PROT). A quantificação livre de marcadores foi feita utilizando o software PLGS. A diferença de expressão entre os grupos foi expressa como p<0.05 para as proteínas down-regulated e 1-p>0.95 para as proteínas up-regulated. Um total de 140 proteínas foram identificadas na PA. Destas, 16 foram encontradas em comum em todos os grupos, dentre elas muitas proteínas típicas da PA, tais como, duas isoformas de Basic salivary proline-rich protein, Cystatin-S, Cystatin-AS, Cystatin-SN, Histatin-1, Ig alpha-1 chain C region, Lysozyme C, Mucin-7, Proline-rich protein 4, Protein S100- A9, Salivary acidic proline-rich phosphoprotein ½, Statherin e Submaxillary gland androgen-regulated protein 3B. O número total de proteínas identificadas em cada grupo foi 31, 51, 18, 38, 106 and 54 para NF-NI, F-NI, NF-CHX, F-CHX, NF-EGCG e F-EGCG, respectivamente. A respectiva quantidade de proteínas exclusivas de cada grupo foi 6, 14, 1, 6, 51 e 5. A maioria das proteínas que não são comumente descritas na PA e que tem funções distintas no organismos, estando envolvidas no metabolismo, sinalização celular, adesão celular, divisão celular, transporte, síntese proteica e degradação foram encontradas no grupo NF-EGCG. Estes resultados demonstram que houve uma diferença no perfil preteico da PA, devido à composição das resinas experimentais, além de oferecer informações importantes sobre o desenvolvimento de materiais restauradores com componentes que podem aumentar a proteção na cavidade oral.(AU)

In the oral cavity, any exposed surface is prone to the formation of the acquired pellicle (AP), an organic film, free of bacteria, which is formed in vivo as a result of the selective adsorption of salivary proteins and glycoproteins to the solid surfaces exposed to the oral environment. The objective of this work was to evaluate the influence of the addition or not of filler (Barium glass alumina silicate and silica) and/or protease inhibitors [epigallocatechin-3-gallate (EGCG) or chlorhexidine (CHX)] to experimental composite resins in the protein profile of the AP formed on these specimens, using quantitative label-free proteomic analysis. Three-hundred and twenty-four samples of bovine enamel (6x6x2mm) were prepared. A cavity (4x4mm) was made, filled with experimental resins and divided into 6 groups of 54 specimens each, according to the experimental groups: no filler, no inhibitor (NF-NI); filler, no inhibitor (F-NI); no filler plus CHX (NF-CHX); filler plus CHX (F-CHX); no filler plus EGCG (NF-EGCG); filler plus EGCG (F-EGCG). Nine young adults of both genders participated using a removable jaw appliance (BISPM - Bauru in situ pellicle model)) with 2 slabs of each group. The experiment was carried out in 9 consecutive days, during the morning for 120 minutes. The pellicle was obtained through the aid of electrodes filter paper moistened in 3% citric acid. The pellicles collected were processed for analysis by LC-ESI-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. Difference in expression among the groups was expressed as p<0.05 for down-regulated proteins and 1-p>0.95 for up-regulated proteins. A total of 140 proteins were identified in the AP. From these, 16 were found in all the groups, among which are many proteins typically found in the AP, such as two isoforms of Basic salivary proline-rich protein, Cystatin-S, Cystatin-AS, Cystatin-SN, Histatin-1, Ig alpha-1 chain C region, Lysozyme C, Mucin-7, Proline-rich protein 4, Protein S100-A9, Salivary acidic proline-rich phosphoprotein ½, Statherin and Submaxillary gland androgen-regulated protein 3B. The total number of proteins identified in each group was 31, 51, 18, 38, 106 and 54 for NF-NI, F-NI, NF-CHX, F-CHX, NF-EGCG and F-EGCG, respectively. The respective amount of proteins exclusively in each group was 6, 14, 1, 6, 51 and 5. Most of the proteins that are not commonly described in the AP that have distinct functions in the organism, being involved in metabolism, cell signaling, cell adhesion, cell division, transport, protein synthesis and degradation were found most prominently in the NF-EGCG group. These results demonstrate that there was a difference in the protein profile of the AP due to the composition of the experimental resins, beyond offering important information on the development of restorative materials with components that can increase the protection in the oral cavity.(AU)

Alterações no perfil proteômico da película adquirida formada in vivo e em diferentes tempos, após exposição ao ácido clorídrico / Changes in the proteomic profile of acquired enamel pellicles formed in vivo for different times, after exposure to hydrochloric acid

A saliva é um importante meio de proteção contra danos ao esmalte e dentina, e é quando ela entra em contato com a superfície dentária, que ocorre uma adsorção seletiva de proteínas salivares, glicoproteínas e lipídeos. Esta adsorção forma um filme orgânico, que é isenta de bactérias, que quando formada sobre esmalte dentário é denominada de película adquirida do esmalte (PAE). A presença destas proteínas recobrindo os tecidos dentários auxilia na lubrificação, tem capacidades de tamponamento e de remineralização, tornando-se um importante fator de proteção contra erosão dentária. O objetivo deste trabalho foi detectar as alterações no perfil protéico na película adquirida do esmalte (PAE) formada in vivo, após a exposição ao ácido clorídrico. Os experimentos foram realizados em 12 dias consecutivos. Em cada dia, os voluntários (n=9), com idade entre 18 a 35 anos, não fumantes, e com um bom estado de saúde geral e bucal, eram submetidos a uma profilaxia dentária com pedra pomes. Depois de 3 min ou 120 min e após a formação PAE, os dentes eram isolados com rolos de algodão e submetidos a 3 procedimentos distintos, sendo um deles realizado a cada dia: aplicação de 50µL de ácido clorídrico (0,1 M, pH 1), ácido clorídrico (0,01 M, pH 2) ou água deionizada por 10 segundos. A aplicação foi feita, em todos os dentes dos arcos superiores e inferiores na face vestibular. Na sequência, a película foi removida com um papel de filtro umedecido em ácido cítrico a 3%. Este procedimento foi repetido por mais uma vez e foi feito um "pool' com os papeis de filtro obtidos dos 9 voluntários, para cada procedimento e tempo de formação (Água-3min, Água-2h, pH2-3min, pH2-2h, pH1-3min e pH1-2h). Após extração das proteínas, as mesmas foram submetidas à cromatografia líquida de fase reversa interligada a um espectrômetro de massas (nLC-ESI-MS/MS). Quantificação proteômica livre de marcadores foi feita utilizando o software (Protein Lynx Global Service software). Um total de 180 proteínas foram encontradas nas amostras de PAE. E o número de proteínas identificadas crescia conforme aumentou-se o seu tempo de formação da película. Somente 4 proteínas foram presentes em todos os grupos sendo estas isoforms de IgA, Serum albumin e Statherin. Um grande número proteínas foram identificadas como sendo únicas dos grupos tratados com HCl, depois de 2h de formação de película (~ 50 proteínas). Em conclusão as proteínas são resistentes a remoção por HCl, e tanto que Serum Albumin e Statherin, foram identificadas em películas formadas em tempos precoces. Para películas formadas no tempo de 120-min foram encontradas muitas proteínas que são resistentes a remoção por HCl. Este fato sugere um aumento da proteção contra ácidos intrínsecos conforme o tempo de formação de película, o que deverá ser avaliada em estudos futuros.(AU)

Saliva it is an important factor against enamel and dentin damages. When the saliva enter in contact with the dental surface, results in a selective adsorption of salivary proteins, glycoproteins and lipids. This adsorption formed an organic free-bacterial film, which when formed in the enamel, is denominated acquired enamel pellicle. The presence of this proteins covering the enamel tissues, has the function of lubrication, buffering and remineralization capabilities, making it an important factor against dental erosion. The objective of this study was detected changes in the protein profile of acquired enamel pellicles (AEP) formed in vivo for different times, after application of hydrochloric acid (HCl). The experimental was realized in 12 consecutive days. On each day, nine subjects, (aged 18 to 35 years, non-smokers, with good general and oral health) were submitted to dental prophylaxis with pumice. After 3 or 120 min, time of formation of the acquired pellicle, the teeth were isolated with cotton rolls and, submitted for a 3 different procedures, one procedure of each day, 50 µL of 0.1 M HCl (pH = 1.0), 0.01 M HCl (pH = 2.0) or deionized water were applied on the buccal surface of the teeth for 10 s. The application of HCl was in all teethes from the superior and lower arch, in vestibular surface. In sequence the AEP was collected using an electrode filter paper pre-soaked in 3% citric acid. This procedures was repeted for one more day. After protein extraction, the samples were submitted to reverse phase liquid chromatography coupled to mass spectrometry (nLC-ESI-MS/MS). Label-free quantification was performed (Protein Lynx Global Service software). A total of 180 proteins were successfully identified in the AEP samples. The number of identified proteins increased with the time of pellicle formation. Only 4 proteins were present in all the groups (isoforms of IgA, Serum albumin and Statherin). The greatest number of proteins identified uniquely in one of the groups was obtained for the groups treated with HCl after 2 h of pellicle formation (~ 50 proteins). Conclusion: Proteins resistant to removal by HCl, such as Serum Albumin and Statherin, were identified even in the short-term AEP. In addition, 120-min pellicle present many proteins that are resistant to removal by HCl. This suggests an increase in the protection against intrinsic acids along the time of pellicle formation, which should be evaluated in future studies.(AU)

The Interactions of CPP-ACP with Saliva.

The repair of early dental caries lesions has been demonstrated by the application of the remineralisation technology based on casein phosphopeptide-stabilised amorphous calcium phosphate complexes (CPP-ACP). These complexes consist of an amorphous calcium phosphate mineral phase stabilised and encapsulated by the self-assembly of milk-derived phosphopeptides. During topical application of CPP-ACP complexes in the oral cavity, the CPP encounters the enamel pellicle consisting of salivary proteins and peptides. However the interactions of the CPP with the enamel salivary pellicle are not known. The studies presented here reveal that the predominant peptides of CPP-ACP complexes do interact with specific salivary proteins and peptides of the enamel pellicle, and provide a mechanism by which the CPP-ACP complexes are localised at the tooth surface to promote remineralisation.

Structural modifications of the salivary conditioning film upon exposure to sodium bicarbonate: implications for oral lubrication and mouthfeel.

The salivary conditioning film (SCF) that forms on all surfaces in the mouth plays a key role in lubricating the oral cavity. As this film acts as an interface between tongue, enamel and oral mucosa, it is likely that any perturbations to its structure could potentially lead to a change in mouthfeel perception. This is often experienced after exposure to oral hygiene products. For example, consumers that use dentifrice that contain a high concentration of sodium bicarbonate (SB) often report a clean mouth feel after use; an attribute that is clearly desirable for oral hygiene products. However, the mechanisms by which SB interacts with the SCF to alter lubrication in the mouth is unknown. Therefore, saliva and the SCF was exposed to high ionic strength and alkaline solutions to elucidate whether the interactions observed were a direct result of SB, its high alkalinity or its ionic strength. Characteristics including bulk viscosity of saliva and the viscoelasticity of the interfacial salivary films that form at both the air/saliva and hydroxyapatite/saliva interfaces were tested. It was hypothesised that SB interacts with the SCF in two ways. Firstly, the ionic strength of SB shields electrostatic charges of salivary proteins, thus preventing protein crosslinking within the film and secondly; the alkaline pH (≈8.3) of SB reduces the gel-like structure of mucins present in the pellicle by disrupting disulphide bridging of the mucins via the ionization of their cysteine's thiol group, which has an isoelectric point of ≈8.3.

Sequence of stannous and sodium fluoride solutions to prevent enamel erosion.

OBJECTIVES: Investigate the timing of stannous (SnF2) and sodium fluoride (NaF) application with and without salivary pellicle to prevent enamel erosion. METHODS: Human buccal molar enamel samples (n=120, REC ref 12/LO/1836) were randomly assigned to three groups testing SnF2 and NaF basic fluoride formulation and commercial mouthrinses with and without the presence of human saliva. Samples were randomly allocated to 2 subgroups: immersion in either fluoride for 1 min either before or after citric acid immersion (0.3%, pH 3.2, 10 min), and the cycle repeated 5 times. For human saliva group, samples were immersed in 80 ml of natural saliva for 24 h prior to the experiment. Analysis was done using non-contacting profilometry and microhardness change. Data were not normal and were log transformed. A linear model tested statistical differences between the groups. RESULTS: SnF2 application before erosion statistically reduced step height compared to application after erosion for all groups (solutions: 6.5 µm (±1.2), 7.5 µm (±0.8); p=0.01, mouthrinses: 3.2 µm (±0.6), 4.2 µm (±0.7); p<0.0001, mouthrinses with saliva: 2.5 µm (±0.4), 3.1 µm (±0.6); p=0.002, before and after respectively). In contrast, application of NaF before erosion increased step height compared to application after, but this was only statistically significant for the saliva group (before: 5.6 µm (±0.3) and after: 4.9 µm (±0.3); p=0.023). Presence of saliva increased microhardness change (p<0.0001). Within this group, greatest microhardness change was observed when SnF2 was applied before erosion and when NaF was applied after erosion (SnF2: 156.6KHN (±32.8), 123KHN (±20.1); p=0.02. NaF: 119.5KHN (±33.5), 218KHN (±24.9), before, and after respectively). CONCLUSION: SnF2 reduced step height formation overall when compared to NaF, but particularly when applied before citric acid immersion. In contrast, NaF reduced step height when applied after citric acid immersion, but only in the presence of saliva. CLINICAL SIGNIFICANCE: Stannous fluoride can be recommended over sodium fluoride to patients at risk of dental erosion and is optimally applied before erosion occurs. If sodium fluoride is to be used in the presence of saliva it is optimally applied after erosion has occurred.

Identification of acid-resistant proteins in acquired enamel pellicle.

OBJECTIVES: This study characterized the proteome profile of the acquired pellicle formed in vivo on enamel. Changes in this proteome profile after exposure to lactic or citric acid were also evaluated. METHODS: Volunteers (n=8) were subjected to dental prophylaxis. After 2 h to allow the formation of the acquired pellicle, the teeth were isolated with cotton rolls and 1 mL of citric acid (1%, pH 2.5) or lactic acid (0.1 M pH 4.8) or deionized water was gently applied with a pipette on the anterior teeth (both maxillary and mandibular) for 10 s. In sequence, the pellicle was collected with an electrode filter paper soaked in 3% citric acid. This procedure was repeated for two additional days following a crossover protocol. Proteins were subjected to reverse phase liquid chromatography coupled to mass spectrometry (nLC-ESI-MS/MS). MS/MS data were processed and submitted to Proteome Discoverer software. Searches were done using SWISS-PROT and TrEMBL databases for human proteins. RESULTS: In total, seventy-two proteins were present in all groups and were submitted to quantitative analysis (SIEVE). Some of these proteins were increased more than two-fold after exposure to the acids. Among them, cystatin-B was increased 20- and 13-fold after exposure to citric and lactic acids, respectively. Additionally, some proteins were identified in only one of the groups (18, 5, and 11 proteins for deionized water, citric and lactic acids, respectively). CONCLUSIONS: Our results open new insights regarding potentially acid-resistant proteins that could be added to dental products to prevent acidic dissolution of the teeth.