Proteomic analysis of stimulated saliva in gastroesophageal reflux disease patients with and without erosive tooth wear: Observational study.

OBJECTIVE: To evaluate the difference in the proteomic profile of stimulated saliva in patients with gastroesophageal reflux disease (GERD) with (GE) and without (GNE) erosive tooth wear (ETW), regarding both human and bacterial proteins. METHODS: Stimulated saliva (SS) was collected from 16 patients (8/group). Samples were centrifuged at 4.500 g for 15 min under refrigeration to remove all debris. The supernatant from each saliva sample was taken and frozen at -80 °C. After extracting the proteins, they were submitted to reverse phase liquid chromatography and mass spectrometry (nLC-ESI-MS/MS). Label-free proteomic quantification was performed using Protein Lynx Global Service (PLGS) software (p < 0.05) for human and bacterial proteins. RESULTS: In total, 67 human proteins were common for GNE and GE groups. GNE group presented, compared to GE group, increase in proteins that confer antimicrobial and acid resistant properties, such as cystatins, histatin and immunoglobulins. However, GNE group had a marked decrease in subunits of hemoglobin (α, ß and delta). Regarding bacterial proteins, for SS, 7 and 10 unique proteins were identified in the GE and GNE groups, respectively. They are related to protein synthesis and energy metabolism and interact with human proteins typically found in saliva and supramolecular complexes of the acquired pellicle. CONCLUSIONS: Our data indicate that the stimulation of the salivary flow increases acid resistant and antimicrobial proteins in saliva, which might protect against ETW. CLINICAL SIGNIFICANCE: This pioneer study showed important differences in the human and bacterial proteome of SS in patients with GERD with or without ETW.

Acquired enamel pellicle protects gastroesophageal reflux disease patients against erosive tooth wear.

The objective of this study was to compare the protein profile of the acquired enamel pellicle (AEP) formed in vivo in patients with or without gastroesophageal reflux disease (GERD), and with or without erosive tooth wear (ETW). Twenty-four volunteers were divided into 3 groups: 1) GERD and ETW; 2) GERD without ETW; and 3) control (without GERD). The AEP formed 120 min after prophylaxis was collected from the lingual/palatal surfaces. The samples were subjected to mass spectrometry (nLC-ESI-MS/MS) and label-free quantification by Protein Lynx Global Service software. A total of 213 proteins were identified, or 119, 92 and 106 from each group, respectively. Group 2 showed a high number of phosphorylated and calcium-binding proteins. Twenty-three proteins were found in all the groups, including 14-3-3 protein zeta/delta and 1-phosphatidylinositol. Several intracellular proteins that join saliva after the exfoliation of oral mucosa cells might have the potential to bind hydroxyapatite, or participate in forming supramolecular aggregates that bind to precursor proteins in the AEP. Proteins might play a central role in protecting the dental surface against acid dissolution.

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

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

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

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

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

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

Acquired enamel pellicle protects gastroesophageal reflux disease patients against erosive tooth wear

Abstract The objective of this study was to compare the protein profile of the acquired enamel pellicle (AEP) formed in vivo in patients with or without gastroesophageal reflux disease (GERD), and with or without erosive tooth wear (ETW). Twenty-four volunteers were divided into 3 groups: 1) GERD and ETW; 2) GERD without ETW; and 3) control (without GERD). The AEP formed 120 min after prophylaxis was collected from the lingual/palatal surfaces. The samples were subjected to mass spectrometry (nLC-ESI-MS/MS) and label-free quantification by Protein Lynx Global Service software. A total of 213 proteins were identified, or 119, 92 and 106 from each group, respectively. Group 2 showed a high number of phosphorylated and calcium-binding proteins. Twenty-three proteins were found in all the groups, including 14-3-3 protein zeta/delta and 1-phosphatidylinositol. Several intracellular proteins that join saliva after the exfoliation of oral mucosa cells might have the potential to bind hydroxyapatite, or participate in forming supramolecular aggregates that bind to precursor proteins in the AEP. Proteins might play a central role in protecting the dental surface against acid dissolution.

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.

Radiotherapy changes the salivary proteome in head and neck cancer patients: evaluation before, during, and after treatment.

OBJECTIVES: Salivary glands are affected during radiotherapy in the head and neck region, leading to a reduction in salivary flow and changes its composition. Besides negatively affecting the oral soft tissues, this can also lead to dental impairment. Thus, we evaluated the effect of radiotherapy in the proteomic profile of the saliva in patients with head and neck cancer (HNC). MATERIALS AND METHODS: HNC patients had their saliva collected before (BRT), during (2-5 weeks; DRT), and after (3-4 months; ART) radiotherapy. Saliva was also collected from healthy volunteers (control; C). Samples were processed for proteomic analysis. RESULTS: In total, 1055 proteins were identified, among which 46 were common to all groups, while 86, 86, 286, and 395 were exclusively found in C, BRT, DRT, and ART, respectively. Remarkably, alpha-enolase was increased 35-fold DRT compared with BRT, while proline-rich proteins were decreased. ART there was a 16-fold increase in scaffold attachment factor-B1 and a 3-fold decrease in alpha-enolase and several cystatins. When compared with C, salivary proteins of BRT patients showed increases cystatin-C, lysozyme C, histatin-1, and proline-rich proteins CONCLUSION/CLINICAL REVELANCE: Both HNC and radiotherapy remarkably change the salivary protein composition. Altogether, our results, for the first time, suggest investigating alpha-enolase levels in saliva DRT in future studies as a possible biomarker and strategy to predict the efficiency of the treatment. Moreover, our data provide important insights for designing dental products that are more effective for these patients and contribute to a better understanding of the progressive changes in salivary proteins induced by radiotherapy. Graphical abstract.

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

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

Is there difference in the comparative and quantitative salivary proteome between stimulated and unstimulated saliva in head and neck cancer patients treated by radiotherapy?

Stimulation of saliva production is an alternative to improve the quality of life of patients treated by radiotherapy. However, there is no information about changes in the salivary proteome of stimulated and unstimulated saliva in these patients. OBJECTIVES: Thus, we evaluated the difference in the proteomic profile of stimulated and unstimulated saliva in patients with head and neck cancer (HNC) treated by radiotherapy. METHODS: Stimulated and unstimulated saliva were collected from 9 patients with HNC before (BRT), during (DRT; 2-5 weeks) and after (ART; 3-4 months) treatment. Healthy patients paired by age and gender also had their saliva collected (C; control group). The stimulated and unstimulated salivary flow were evaluated (p < 0.05). Salivary proteins were extracted and processed for shotgun proteomic analysis. RESULTS: Significant differences were observed between stimulated and unstimulated salivary flows for C and BRT (p greater than 0.001), but not for DRT and ART. Proteins involved with apoptosis, antibacterial and acid-resistance were decreased in stimulated saliva in comparison to unstimulated saliva DRT and ART. Isoforms of keratins were not identified in control and BRT. CONCLUSION: there is a marked difference in the protein profile of stimulated and unstimulated salivary flows in HNC patients treated by radiotherapy. In addition, saliva stimulation in patients with HNC decreases important proteins involved with dental protection. The unstimulated salivary flow seems to be the best alternative to search for biomarkers. Our results contribute in an unprecedented way to understand the changes in the salivary proteome of different flows in HNC patients undergoing radiotherapy treatment.

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.

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.

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

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

The proteomic profile of the acquired enamel pellicle according to its location in the dental arches.

OBJECTIVE: This study evaluated the variation in the protein profile of the acquired enamel pellicle (AEP) formed in vivo according to its location in the dental arches. DESIGN: The AEP was formed for 120min in 9 volunteers. Pellicle formed at upper+lower anterior facial (ULAFa; teeth 13-23 and 33-43), upper anterior palatal (UAPa; teeth 13-23), lower anterior lingual (LALi; teeth 33-43), upper+lower posterior facial (ULPFa; teeth 14-17 24-27, 34-37 and 44-47), upper posterior palatal (UPPa; teeth 14-17 and 24-27) and lower posterior lingual (LPLi; teeth 34-37 and 44-47) regions were collected separately and processed for analysis by label-free LC-ESI-MS/MS. RESULTS: Three-hundred sixty three proteins were identified in total, twenty-five being common to all the locations, such as Protein S100-A8, Lysozyme C, Lactoferrin, Statherin, Ig alpha-2, ALB protein, Myeloperoxidase and SMR3B. Many proteins were found exclusively in the AEP collected from one of the regions (46-UAPa, 33-LALi, 59-ULAFa, 31-ULPFa, 44-LPLi and 39-UPPa). CONCLUSIONS: The protein composition of the AEP varied according to its location in the dental arches. These results provide important insights for understanding the differential protective roles of the AEP as a function of its location in the dental arches.

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)