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

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

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

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

Biological sealing aspects surrounding anterior crowns on dental implants: clinical and photographic cases report

The biological sealing (BS) around implants is a dominant factor to determine the long-term success of peri-implant health. There are several features of the BS around implants in common with the soft tissue attached to teeth, such as the presence of crevicular fluid, acquired pellicle, epithelium; otherwise, the quality of the BS around implants is weaker compared with the junctional epithelium of natural teeth. Then, this article aimed to describe three cases report showing the presence of a BS (cuticle-crevice fluid-acquired pellicle) around the fixed crowns on dental implants in the anterior zone, through photographic analysis. It was used a Nikon 8100 camera with a 105 mm macro lens and a Macro Ring circular flash. A photographic profile examination was made always showing the clinical case and, specifically, the focal point in the crown-gingival tissue (prosthesis boundary and peri-implant tissue), highlighting the anatomical gingiva on the ceramic prosthetic crown at an angle between 140 to 160 degrees. Although cases 1 and 2 had 1-year follow-up and case 3 around 4 years, the common findings for all treatments done were: (i) oral rehabilitation with crowns on dental implants; (ii) patients satisfied with the esthetic and functional result; (iii) stability of the soft tissue around the crowns; (iv) all the patients had a good oral hygiene; (v) presence of a thin membrane associated with the acquire pellicle, similar to an annular cuticle, which we named cuticle-acquired pellicle complex or tertiary cuticle or prosthetic-implant cuticle. This complex (cuticle-crevicular fluid-acquired pellicle) is suggested to be the responsible by the BS on dental implants. Moreover, the cuticle (epithelial part in the peri-implant sulcus), although similar to teeth, may be considered a tertiary pellicle due to be found on ceramic crowns on dental implants, differently of the primary and secondary pellicle. Whitin the limitation of these three cases reports, the BS was reported and can be introduced the new concept of the "cuticle-crevicular fluid-acquired pellicle complex" or "prosthetic-implant cuticle".

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

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

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

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

Solutions and Gels Containing a Sugarcane-Derived Cystatin (CaneCPI-5) Reduce Enamel and Dentin Erosion in vitro.

The effect of solutions and gels containing a sugarcane-derived cystatin (CaneCPI-5) on the protection against enamel and dentin erosion in vitro was evaluated. Bovine enamel and dentin specimens were divided into 2 groups (n = 135 and 153/group for enamel and dentin, respectively) that were treated with solutions or chitosan gels containing 0.1 or 0.25 mg/mL CaneCPI-5. The positive controls for solutions and gels were Elmex Erosion Protection™ solution and NaF gel (12,300 ppm F), respectively. Deionized water and chitosan gel served as controls, respectively. The solutions were first applied on the specimens for 1 min and the gels for 4 min. Stimulated saliva was collected from 3 donors and used to form a 2-h acquired pellicle on the specimens. Then, the specimens were submitted to an erosive pH cycling protocol 4 times/day for 7 days (0.1% citric acid pH 2.5/90 s, artificial saliva/2 h, and artificial saliva overnight). The solutions and gels were applied again during pH cycling, 2 times/day for 1 min and 4 min, respectively, after the first and last erosive challenges. Enamel and dentin losses (µm) were assessed by contact profilometry. Data were analyzed by 2-way ANOVA and Tukey's test (p < 0.05). All the treatments significantly reduced enamel and dentin loss in comparison with controls. Both CaneCPI-5 concentrations had a similar protective effect against enamel erosion, but only the higher concentration was as effective against dentin erosion as the positive control. Regarding the vehicles, only the 0.1 mg/mL gel performed worse than the positive control for dentin. CaneCPI-5 reduced enamel and dentin erosion to a similar extent as the fluoride-containing vehicles. However, dentin requires higher CaneCPI-5 concentrations, in the case of gels. Solutions or gels containing CaneCPI-5 might be a new approach to protect against dental erosion.

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

OBJECTIVES: To 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. METHODS: Nine 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. RESULTS: Statherin 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. CONCLUSIONS: HNC and radiotherapy remarkably altered the proteome of the AEP. Antibacterial and acid-resistant proteins were decreased during radiotherapy. CLINICAL SIGNIFICANCE: Our 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.

Statherin-derived peptide protects against intrinsic erosion.

OBJECTIVES: In the present study, we used an in vitro initial intrinsic erosion model to evaluate: (experiment 1) the influence of the degree of serine (Ser) phosphorylation of peptides containing the 15 N-terminal residues of statherin and (experiment 2) the effect of different concentrations of the peptide with the best performance in experiment 1 on initial enamel erosion. DESIGN: Bovine enamel specimens were divided into 6 groups (n = 15/group) for each experiment. In experiment 1, the peptides evaluated (at 1.88 × 10-5 M) were: not phosphorylated (StatSS), phosphorylated in Ser2 (StatpSS), phosphorylated in Ser3 (StatSpS) phosphorylated in Ser2 and Ser3 (StatpSpS). Phosphate buffer and human recombinant statherin were used as negative and positive controls, respectively. In experiment 2, StatpSpS was evaluated at different concentrations: 0.94, 1.88, 3.76 and 7.52 × 10-5 M. Phosphate buffer and 0.1 mg/mL CaneCPI-5 were employed as negative and positive controls, respectively. In each experiment, the specimens were incubated with the solutions for 2 h, then the AEP was allowed to form (under human pooled saliva) for 2 h. The specimens were then challenged with 0.01 M HCl for 10 s. Demineralization was evaluated by percentage of surface hardness change (%SHC). Data were analyzed by ANOVA and Tukey's test (p < 0.05). RESULTS: In experiment 1, only StatpSpS significantly reduced the % SHC in comparison with control. In experiment 2, 1.88 × 10-5 M StatpSpS significantly reduced the %SHC in comparison with control. CONCLUSIONS: This is the first study showing that statherin-derived peptide might protect against intrinsic erosion.

Protein Profile of the Acquired Enamel Pellicle after Rinsing with Whole Milk, Fat-Free Milk, and Water: An in vivo Study.

This study detected changes in the protein profile of the acquired enamel pellicle (AEP) formed in vivo after rinsing with whole milk, fat-free milk, or water. Nine subjects in good oral condition took part in the study. The acquired pellicle was formed in the morning, for 120 min, after prophylaxis with pumice. Following this, the volunteers rinsed with 10 mL of whole milk, fat-free milk, or deionized water for 30 s, following a blinded crossover protocol. After 60 min, the pellicle was collected with filter paper soaked in 3% citric acid and processed for analysis by liquid chromatography-electrospray ionization tandem mass spectrometry. The obtained tandem mass spectrometry spectra were searched against a human protein database (Swiss-Prot). The proteomic data related to protein quantification were analysed using the PLGS software. A total of 260 proteins were successfully identified in the AEP samples collected from all groups. Forty-nine were common to all 3 groups, while 72, 62, and 49 were specific to the groups rinsing with whole milk, fat-free milk, and water, respectively. Some were typical components of the AEP, such as cystatin-B, cystatin-SN, isoforms of α-amylase, IgA and IgG, lysozyme C, protein S100 A78, histatin-1, proline-rich protein 27, statherin, and lactotransferrin. Other proteins are not commonly described as part of the AEP but could act in defence 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 on bacterial adhesion and tooth dissolution. The use of fat-free milk could favourably modulate the adhesion of bacteria to the AEP as well as biofilm formation when compared with whole milk.

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)

Detecção de alterações na composição da película adquirida em função da sua localização na cavidade bucal: estudo proteômico / Detecção de alterações na composição da película adquirida em função da sua localização na cavidade bucal: estudo proteômicox

A película adquirida do esmalte (PAE) é um filme orgânico, livre de bactérias, formado in vivo como resultado da adsorção seletiva de proteínas salivares sobre a superfície do dente, contendo também glicoproteínas e lipídeos. A presença de proteínas na PAE forma uma interface protetora sobre a superfície do dente, participando em todos os eventos interfaciais que ocorrem na cavidade bucal. O objetivo deste trabalho foi detectar alterações no perfil proteico da PAE formada in vivo de acordo com a sua localização nos arcos dentários. Fizeram parte da pesquisa 9 voluntários, com idade entre 18 e 35 anos, não fumantes, com bom estado de saúde geral e bucal. A película adquirida foi formada no período da manhã, por 120 minutos, após profilaxia com pedra pomes. Películas formadas nas regiões anterior vestibular superior e inferior (ULALa; dentes 13-23 e 33-43), anterior palatina superior (UAPa; dentes 13-23), anterior lingual inferior (LALi; dentes 33-43), posterior vestibular superior e inferior (ULPLa; dentes 14-17, 24-27, 34-37 e 44-47), posterior palatina superior (UPPa; dentes 14-17 e 24-27) e posterior lingual inferior (LPLi; dentes 34-37 e 44-47) foram coletadas separadamente para análise. Após a sua formação, a película foi coletada em papel filtro embebido em ácido cítrico a 3% e processada para análise 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. Um total de 363 proteínas foi encontrado, sendo 252 proteínas únicas de cada grupo e 25 proteínas comuns entre eles (como Protein S100-A8, Lysozyme C, Lactoferrin, Sthatherin, Ig alpha-2 chain C, ALB protein, Myeloperoxidase and Submaxillary gland androgen-regulated protein 3B). Na análise quantitativa, nove comparações foram realizadas e muitas proteínas foram diferentemente expressas entre os grupos, demonstrando assim que a localização na cavidade bucal pode alterar a composição da película adquirida do esmalte. Foram encontradas tanto proteínas típicas da película quanto proteínas não anteriormente descritas na película, cuja função na película foi inferida com base na literatura. Em conclusão houve diferença na composição proteica da película adquirida de acordo com a localização dos arcos dentários. Esses dados devem ser levados em conta quando se pensa no potencial protetor da película adquirida contra a desmineralização dentária, uma vez que esses resultados fornecem informações importantes para a compreensão dos diferentes papeis protetores da AEP dependendo da sua localização nos arcos dentários.(AU)

The acquired enamel pellicle (AEP) is a bacteria-free organic film formed in vivo as a result of selective adsorption of salivary proteins on the surface of the tooth. It also contains glycoproteins and lipids. The presence of proteins in the AEP forms a protective interface on the tooth surface that participates in the interfacial events that occur in the oral cavity. The objective of this study was to detect changes in the protein profile of the AEP formed in vivo according to its location in the dental arches. Nine volunteers, aged 18 to 35 years, non-smokers, with good general and oral health participated in the study. The acquired pellicle was formed in the morning, for 120 minutes, after prophylaxis with pumice. Pellicle formed at upper and lower anterior labial (ULALa; teeth 13-23 and 33-43), upper anterior palatal (UAPa; teeth 13-23), lower anterior lingual (LALi; teeth 33-43), upper and lower posterior labial (ULPLa; teeth 14-17 24 to 27, 34 to 37 and 44 to 47), upper posterior palatal (UPPa; teeth 14 to 17 and 24 to 27) and lower posterior lingual (LPLi; teeth 34 to 37 and 44 to 47) regions was collected separately for analysis. After its formation, the pellicle was collected with filter paper soaked in 3% citric acid and processed for analysis by LC-ESI-MS/MS. The MS/MS spectra obtained were compared with human protein databases (SWISS PROT). Label-free quantification was done using the PLGs software. A total of 363 proteins were found, of which 252 are unique proteins for one of the regions, while 25 proteins care to all of them, including Protein S100-A8, Lysozyme C, Lactoferrin, Sthatherin, Ig alpha-2 chain C, ALB protein, Myeloperoxidase and Submaxillary gland androgen-regulated protein 3B. In the quantitative analysis, 9 comparisons were made and many proteins were differently expressed among the groups, thus demonstrating that the location in the dental arches can change the composition of the AEP. Some proteins not previously found in the AEP were identified and their function in the AEP was inferred from the literature. In conclusion, the composition of the AEP changes as a function of its location in the dental arches. These data should be taken into account when we think about the protective potential of the acquired pellicle against tooth demineralization and provide important insights for understanding the differential protective roles of the AEP as a function of its location in the dental arches.(AU)

Influência de tensoativos sobre o efeito protetor da película e interação com NaF no desenvolvimento da erosão dental / Influence of surfactant agents on acquired pellicle protector effect and sodium fluoride interaction during dental erosion development

A erosão dental é um processo multifatorial que envolve a desmineralização do esmalte/dentina pela ação química de ácidos extrínsecos ou intrínsecos. A película adquirida é um filme, livre de bactérias, que cobre os dentes e atua como barreira de difusão ou membrana permeável seletiva, prevenindo o contato direto de ácidos com a superfície dos dentes. Os dentifrícios, normalmente usados no controle do biofilme bucal, possuem agentes tensoativos, que podem influenciar na adsorção de proteínas salivares, e atuar diretamente na formação da película adquirida e na liberação de fluoretos para o meio bucal. Assim, verificou-se a ação destes agentes na formação e proteção da película adquirida, sua interação com fluoreto de sódio (NaF) no esmalte, e consequentemente sua interferência na proteção contra a erosão dental. Foram testados três tensoativos (Lauril Sulfato de Sódio - LSS, Tween 20 ­ T20 e Cocoamidopropil Betaína - CAPB), em duas concentrações (1,0% e 1,5%). A água foi utilizada como controle negativo. Amostras de esmalte bovino foram submetidas a um modelo de des/remineralização com ácido cítrico durante 5 dias, imersão em saliva humana para formação de película adquirida e em soluções com os tensoativos testados, associados ou não ao NaF (275 ppm). A solução de NaF foi utilizada como controle positivo. A análise da energia de superfície do esmalte foi determinada por goniometria e a formação de película adquirida quantificada por espectroscopia (FTIR). A erosão inicial foi determinada por microdureza no primeiro dia (mensurada após o primeiro ácido, após o tratamento e após o segundo ácido) e a perda de estrutura de esmalte foi definida por perfilometria ao final de cinco dias de ciclo. Ainda, foi quantificado o flúor solúvel em KOH adsorvido na superfície do esmalte com eletrodo específico. Os resultados de goniometria mostraram que apenas o LSS e o CAPB em ambas concentrações diminuíram o ângulo de contato entre a água e o esmalte. Quanto à quantificação da formação de película, não foi possível verificar diferença significante entre os grupos testados. Com relação à erosão, os dados de dureza mostraram que os tensoativos, independente da concentração, não interferiram no reendurecimento do esmalte, porém o LSS a 1% e 1,5% interferiu no potencial de proteção do NaF, e o T20 a 1% e 1,5% e o CAPB a 1,5% protegeram o esmalte, porém não foram superiores ao efeito do NaF. Já a análise perfilometria mostrou que o T20 a 1% resultou em menores valores de perda que a 1,5%, e ainda que o CAPB 1% e 1,5% foi capaz de proteger comparado ao controle negativo, no entanto nenhum agente associado ao NaF protegeu mais do que o controle positivo. Os dados da concentração de flúor KOH-solúvel indicaram que os tensoativos reduziram a adsorção do CaF2 ao esmalte. Conclui-se que os tensoativos testados reduziram o ângulo de contato da água com o esmalte (exceção do T20). O LSS reduziu o potencial protetor do NaF e da película na erosão inicial e nenhum agente testado interferiu na capacidade protetora do NaF contra a progressão do desgaste erosivo(AU)

Dental erosion can be defined as a multifactorial process that induces tooth dissolution by intrinsic or extrinsic acids. Acquired pellicle is a film, free from bacteria, that covers all tooth tissues, and acts as a selective membrane that prevents direct contact of the acids with enamel/dentin surface. Dentifrices, frequently used in the biofilm control, have some constituents, such as surfactant agents, which influence on the adsorption of salivary proteins, and may directly affect the formation of salivary pellicle and the fluoride release on oral environment. Thus, it was verified the influence of surfactants over the protective effect of the acquired pellicle, and on the interaction of fluoride with enamel. Three different surfactants were tested (Sodium Lauryl Sulphate - SLS, Tween 20 ­ T20 and Cocoamidopropyl Betaine - CAPB), in 2 different concentrations (1.0% and 1.5%). Water was used as negative control. Bovine enamel samples were selected and submitted to an in vitro des/remineralization model with citric acid during 5 days, immersion in human saliva for acquired pellicle formation and immersion in the surfactant solutions, associated or not with sodium fluoride (NaF ­ 275ppm). A NaF solution was used as positive control. The surface wettability was determined by contact angle between water and the enamel using a tensiometer, and the acquired enamel pellicle formation was assessed using a spectrophotometer (FTIR). Initial erosion was defined by microhardness at the first cycle day (measured after the first acid, after treatment and after the second acid), and the structure loss was determined by profilometry. The KOH-soluble fluoride was also quantified after the end of the cycle. The surface energy analysis showed that only SLS and CAPB in both concentrations decreased the contact angle between enamel and water. Regarding the proteins quantification, no differences were found between the groups. Concerning initial erosion, microhardness data showed that all surfactants, in both concentrations, did not interfered with enamel remineralization, but 1% and 1,5% SLS interfered on NaF protective effect. 1% and 1,5% T20 and 1,5%, CAPB despite presenting some protective effect against new acid challenge, did not promote the same protection as NaF. Profilometry results showed that the 1% T20 promoted lower surface loss than at 1.5%, while 1% and 1.5% CAPB protected enamel compared to negative control group. However, no agent associated with NaF showed higher protection than the positive control. KOH-soluble fluoride analysis showed that all surfactants reduced the CaF2 adsorption over enamel surface. It can be concluded that the surfactants tested reduced the enamel contact angle (except for T20). The SLS decreased the protective potential of NaF associated with the pellicle in initial erosion and no agent tested interfered with the protective effect of NaF on enamel erosive wear(AU)

Exposure to acids changes the proteomic of acquired dentine pellicle.

OBJECTIVES: For the first time, this study characterized the proteome of the acquired pellicle formed on human dentine. The changes in this proteome after exposure to lactic or citric acid were also evaluated. METHODS: Volunteers (n=9) wore a mandibular device containing 6 specimens of human root dentine. After the device remained in the volunteers' oral cavities for 10min or 2h to allow the formation of the acquired pellicle in situ, the specimens were immersed in citric acid (1%, pH 2.5) or lactic acid (0.1M, pH 4.8) or deionized water for 20s. 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. After harvest, 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, 223 distinct proteins were identified in the dentine acquired pellicle in each of the different conditions. Exposure to citric acid dramatically reduced the number of identified proteins. This did not occur for lactic acid. Acid-resistant proteins, such as mucins, were identified after pellicle was exposed to lactic or citric acid. CONCLUSIONS: These proteins could be related to protective effect of tooth homeostasis. Moreover, in the future, they could be candidates to the development of a supplemental therapy for the prevention and treatment of dental caries and dental erosion. CLINICAL SIGNIFICANCE: This study indicates some acid-resistant proteins that could be used in dental products to prevent dental caries and erosion.

Inhibition of initial adhesion of oral bacteria through a lectin from Bauhinia variegata L. var. variegata expressed in Escherichia coli.

AIMS: The aim of the present work was to study the in vitro effect of native and recombinant Bauhinia variegata var. variegata lectins in inhibiting early adhesion of Streptococcus mutans, Streptococcus sanguis and Streptococcus sobrinus to experimentally acquired pellicle. METHODS AND RESULTS: Native lectin from B. variegata (BVL) was purified by affinity chromatography of extract of seeds. The recombinant lectin (rBVL-I) was expressed in E. coli strain BL21 (DE3) from a genomic clone encoding the mature B. variegata lectin gene using the vector pAE-bvlI. Recombinant protein deposited in inclusion bodies was solubilized and subsequently purified by affinity chromatography. The rBVL-I was compared to BVL for agglutination of erythrocytes and initial adherence of oral bacteria on a saliva-coated surface. The results revealed that rBVL-I acts similarly to BVL for agglutination of erythrocytes. Both lectins showed adhesion inhibition effect on Step. sanguis, Step. mutans and Step. sobrinus. CONCLUSION: We report, for the first time, the inhibition of early adhesion of oral bacteria by a recombinant lectin. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results support the proposed biotechnological application of lectins in a strategy to reduce development of dental caries by inhibiting the initial adhesion and biofilm formation.

Determinação da mudança na composição da película adquirida formada sobre esmalte e dentina após exposição ácida: estudo proteômico / Determination of change in the composition of the acquired pellicle formed on enamel and dentin after acid exposure: proteomic study

O objetivo deste trabalho foi analisar as mudanças no perfil protéico em películas adquiridas formadas in situ, em diferentes tempos, sobre o esmalte e sobre a dentina humana após a exposição a dois tipos de ácido: lático e cítrico. Foram utilizados 162 blocos de esmalte e 162 blocos de dentina humana (3X3 mm). Os experimentos foram realizados em três dias consecutivos. Em cada dia, os voluntários (n=9) utilizavam um dispositivo mandibular contendo 6 blocos de esmalte e 6 blocos de dentina. Na sequência, os voluntários permaneceram com o dispositivo na cavidade bucal por 10 ou 120 minutos para formação de película adquirida. Os blocos foram então imersos em ácido cítrico (1%, pH 2,5) ou ácido lático (0,1 M pH 4,8) ou água deionizada por 20 segundos. Na sequência, a película foi removida com um papel de filtro umedecido em ácido cítrico a 3%. Este procedimento foi repetido por mais dois dias adicionais e foi confeccionado um pool com os papeis de filtro obtidos dos 9 voluntários, para cada tipo de substrato, tempo de coleta e meio de imersão. Após extração das proteínas, as mesmas foram submetidas à cromatografia líquida de fase reversa interligada a um espectrômetro de massas (nLC-ESI-MS/MS). Os dados obtidos de MS/MS foram processados e submetidos conjuntamente ao programa SEQUEST [Proteome Discoverer 1.3 (Thermo Scientific)]. As buscas foram feitas utilizando-se os bancos de dados SWISS-PROT e TrEMBL. Para o esmalte, a taxa de identificação de proteínas foi baixa (13 proteínas no total). Já para a dentina, a taxa de identificação de proteínas foi maior (223 proteínas no total), sendo que a exposição ao ácido cítrico reduziu dramaticamente o número de proteínas identificado, o que não aconteceu para o ácido lático. Proteínas ácido-resistentes foram identificadas tanto para o esmalte quanto para a dentina, destacando-se as queratinas e as mucinas, respectivamente. Estas proteínas, ou os peptídeos oriundos delas responsáveis pelo efeito protetor...

The purpose of this work was to analyze changes in protein profile in the acquired pellicle formed on enamel and dentine at different times in situ after exposure to lactic and citric acids. Enamel (n=162) and dentin (n=162) blocks (3X3 mm) were be used. The experiments were conducted on three consecutive days. Each day, volunteers (n = 9) used a mandibular device containing 6 blocks of enamel and 6 of human dentin. After the volunteers remained with the device in the oral cavity for 10 or 120 minutes in order to allow the formation of the acquired pellicle, the blocks were immersed in citric acid (1%, pH 2.5) or lactic acid (0.1 M, pH 4,8) or deionized water for 20 seconds. Following, the pellicle was collected with an electrode filter paper soaked in 3% citric acid. This procedure was repeated for two additional days and 'pools' with the filter papers obtained from the 9 volunteers for each type of substrate, sampling time and type of acid were made. After extraction, proteins were subjected to reverse phase liquid chromatography coupled to mass spectrometry (nLC-ESI-MS/MS). MS/MS data were processed and submitted to SEQUEST software [Proteome Discoverer 1.3 (Thermo Scientific)]. Searches were done using SWISS-PROT and TrEMBL databases. The rate of protein identification was low for enamel (13 proteins in total). As for dentin, the rte of protein identification was higher (223 proteins in total). Exposure to citric acid dramatically reduced the number of identified proteins, what did not occur for lactic acid. Acid-resistant proteins, especially keratins and mucins were identified for enamel and dentin, respectively. These proteins, or the peptides originated from them that are responsible for the protective effect, are candidates to be used for the enrichment of dental products, aiming to prevent dental caries and erosion.

Determinação da composição da película adquirida formada sobre o esmalte humano in vivo e da sua modificação após estimulação química e mecânica do fluxo salivar: estudo proteômico / Characterization of the acquired pellicle formed in vivo over human enamel and its modification after mechanical and chemical salivary flow stimulation: proteomic study

Determinação da composição da Película Adquirida formada sobre o esmalte humano in vivo e da sua modificação após estimulação química e mecânica do fluxo salivar: estudo proteômico. Todas as superfícies sólidas expostas na cavidade bucal são cobertas por uma camada proteinácea denominada película adquirida do esmalte. Trata-se de um filme orgânico, livre de bactérias, que recobre os tecidos dentários e é composto basicamente por proteínas. Diversos trabalhos têm se concentrado na caracterização e no impacto protetor da película adquirida formada sobre a superfície do esmalte. Porém tanto para a película adquirida formada sobre o esmalte in situ quanto in vivo, as considerações sobre o tipo de saliva que contribuiu para sua formação são bastante limitadas, senão inexistentes. Com base nisto, o objetivo deste estudo foi comparar o perfil proteico em películas adquiridas formadas in vivo sobre o esmalte humano em condições de repouso, bem como analisar as películas formadas após estimulação salivar mecânica e química, através de espectrometria de massa. Os experimentos foram realizados por três dias consecutivos, seguindo um delineamento cruzado. Em cada dia, os voluntários (n=9) receberam uma meticulosa profilaxia dentária. Em seguida, aguardaram por duas horas para que a película adquirida fosse formada naturalmente sobre o esmalte (saliva não estimulada Grupo 1, controle). Após o período em questão, cada quadrante da boca foi enxaguado, seco e a película adquirida foi então coletada com auxílio de um papel filtro de eletrodos (electrode wick filter paper, Bio-Rad) embebido em ácido cítrico 3%. Os procedimentos descritos acima foramrepetidos em outros dois dias consecutivos para cada voluntário, sendo que em um dos dias os voluntários estimularam o fluxo salivar mecanicamente (saliva com estimulação mecânica Grupo 2), através da mastigação de Parafilm®, durante o mesmo período de tempo. Em outro dia, o fluxo salivar foi estimulado através de...

All solid surfaces exposed in the oral cavity are covered by a closely adherent protein film termed the acquired enamel pellicle (AEP). It is an organic, bacteria-free film that overlies the hard dental tissues and is basically composed by glycoproteins and proteins. Recently, many studies have focused on the characterization and the protector impact of the acquired pellicle formed over the enamel surface. However either for the in situ or in vivo formed AEP, information regarding the type of saliva which has contributed for its formation is quite limited, or even inexistent. Based on this, the aim of the present study was to compare the protein profile of acquired pellicles formed in vivo over the human enamel surfaces under resting condition and also to analyze the protein profile of the pellicles formed after mechanical and chemical salivary flow stimulation using mass spectrometry. The experiments were performed on three consecutive days, following a crossover design. On each day, the volunteers (n=9) received a meticulous dental prophylaxis and then waited for two hours to allow the AEP formation (non stimulated saliva - group 1). After the time span, each quadrant of the mouth was rinsed with water, dried by air spray and then the collection of the AEP was carried out using a filter paper (electrode wick filter paper, Bio-Rad) presoaked in 3% citric acid The procedures described above were repeated on two other consecutive days for each volunteer. However, in one day their salivary flow was mechanically stimulated (mechanical stimulation Group 2) by chewing on Parafilm® during the same period of time. In another day, the salivary flow was chemically stimulated (Chemical stimulation Group 3) by 2% citric acid drops on each side of the tongue, once per minute, for the same period of time. For each group, a pool with the wick filters from all 9 volunteers was made. After the extraction and digestion of the proteins from the paper strips, peptide...

Determinação da composição da película adquirida formada in situ sobre o esmalte e dentina humanos através de análise proteômica / Determination of the composition of the acquired pellicle formed in situ on human enamel and dentin: proteomic study

A película adquirida (PA) é um filme formado pela adsorção seletiva de proteínas, glicoproteínas e lipídeos à superfície dentária. A presença de proteínas na PA forma uma interface protetora sobre a superfície do dente, participando em todos os eventos interfaciais que ocorrem na cavidade bucal, tais como des- e remineralização, lubrificação das superfícies dos dentes, e aderência bacteriana. Com o advento da proteômica, tem havido um aumento considerável no conhecimento acerca do perfil proteico de PAs adquiridas formadas sobre o esmalte dentário, em diferentes situações, mas nenhum trabalho até o momento descreveu o perfil proteômico de PAs formadas sobre a dentina. Este estudo foi pioneiro em comparar o perfil proteico de PAs formadas in situ sobre o esmalte e a dentina, nos tempos de 10 minutos e 2 horas, utilizando análise proteômica quantitativa livre de marcadores. Os experimentos foram realizados por três dias consecutivos. Em cada dia, os 9 voluntários receberam profilaxia dentária e em seguida utilizaram um aparelho vestibular com 6 blocos de esmalte e 6 de dentina humanos por 10 minutos ou 2 horas. Após esses períodos, a PA formada era coletada com auxílio de um papel filtro de eletrodos embebido em ácido cítrico 3%. Para as análises foi realizado um pool com os papéis dos 9 voluntários de todos os dias, para cada substrato e tempo de formação. Após a extração e digestão das proteínas, a separação dos peptídeos foi realizada por nano-HPLC (nano-Cromatografia Líquida de Alta Performace), interligada a um espectrômetro de massa (nLC-ESI-MS/MS). Os dados MS/MS obtidos foram processados e pesquisados em bancos de dados de proteínas humanas (UniProt e TrEMBL), utilizando o algoritmo SEQUEST no software Proteome Discoverer 1.3. Para a PA formada sobre o esmalte, foram identificadas 160 e 64 proteínas, nos tempos de formação de 10 minutos e 2 horas, respectivamente. Os respectivos números de proteínas identificadas para a dentina foram 86 e 52...

The acquired pellicle (AP) is a film that results from selective adsorption of proteins, glicoproteins and lipids on the tooth surface. The presence of proteins in the AP forms a protective interface on the tooth surface that participates in all the surface events occurring in the oral cavity, such as de- and remineralization, lubrification of the tooth surfaces and bacterial adherence. With the advent of Proteomics, considerable increase in the knowledge of the protein profile of the AP formed on tooth enamel, under different circunstances, has been observed. However, so far the proteomic profile of the AP formed on dentin has not been described. This is the first study to compare the proteomic profile of APs formed in situ for 10 minutes and 2 hours, on enamel and dentin, using quantitative label-free proteomics. The experiments were conducted for 3 consecutive days. Each day, 9 volunteers were submitted to dental prophylaxis and in sequence wore a vestibular device containing 6 human enamel and 6 human dentin blocks for 10 minutes or 2 hours. After these periods, the PA formed was collected with an electrode filter paper soaked in 3% citric acid. The papers from the 9 volunteers, for each substrate and time of pellicle formation were pooled and used for analysis. After protein extraction and digestion, peptides were separated by nano-HPLC (High-performance liquid chromatography) coupled to a mass spectrometer (nLC-ESI- MS/MS). The obtained MS/MS spectra were searched against human protein databases (UniProt and TrEMBL) using SEQUEST algorithm in Proteome Discoverer 1.3 software. For the AP formed on enamel, 160 and 64 proteins were identified for the times of pellicle formation of 10 minutes and 2 hours, respectively. The respective numbers of identified proteins for dentin were 86 and 52, respectively. For the times of 10 minutes and 2 hours, respectively, 25 and 11 proteins were common to both substrates. They were submitted to label-free...