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PROTEOMICS OF ACQUIRED ENAMEL PELLICLE AND SALIVA IN VOLUNTEERS WITH GASTROESOPHAGEAL REFLUX WITH DENTAL EROSION OR NOT AND IN VITRO EVALUATION OF PROTECTIVE POTENTIAL OF HEMOGLOBIN AGAINST DENTAL EROSION
This study will compare the protein profile of the acquired enamel pellicle (AEP) formed in vivo on the lingua/palatal surfaces, as well as of saliva, in patients with gastroesophageal reflux disease (GERD) with dental erosion, patients with GERD without dental erosion and control patients (without GERD and dental erosion). The potential of the modification of the AEP with different concentrations of hemoglobin (Hb) to protect against initial enamel erosion will also be evaluated in vitro, since in preliminary experiments we observed increased expression of different subunits of Hb in GERD patients without dental erosion, when compared to GERD patients with dental erosion. The in vivo phase will consist of 3 parallel arms, in which 24 volunteers will be divided into 3 groups, namely: a) patients with GERD and dental erosion (BEWE e 9 or grade 3 in the upper anterior sextant (with all incisors affected), b) patients with GERD and without dental erosion (BEWE = 0); c) control patients (GERD and without BEWE = 0). Volunteers (n=8/group) will be submitted to dental prophylaxis and AEP will be allowed to form for 120 min. The AEP will be collected from the palatal/lingual surfaces (from second molar to second molar) with wick filters pre-soaked in 3% citric acid. A pool with the filters obtained from 8 volunteers from the same group will be done. Non-stimulated saliva will be collected before prophylaxis, while stimulated saliva will be collected after collection of AEP. After protein extraction, samples will be submitted to reverse phase liquid chromatography coupled to mass spectrometry (nLC-ESI-MS/MS). Label-free proteomic quantification will be performed using Protein Lynx Global Service (PLGS) software. In the in vitro phase, 75 bovine enamel specimens (4 X 4 mm) will be prepared and divided into 5 groups: a control group, consisting of deionized water and 4 experimental groups consisting of solutions containing proteins): 4 mg / mL Hb, 2 mg / mL Hb, 1 mg / mL Hb, 0.1 mg / mL Hb or 0.01 mg/mL CaneCPI-5 (positive control). The specimens will be exposed to protein solutions under agitation at 30 ° C for 2 h. Once per day for 3 days the specimens will be incubated in these solutions or deionized water for 2 h at 37ºC under agitation and then in pool of human saliva for another 2 h at 37°C for the of the AEP. They will then be washed in deionized water and challenged with HCl 0.01 M (pH 2.3) for 10 s under agitation. Analysis of surface hardness (SMH) will be performed and alterations in SMH (SMH baseline - SMH post erosion) will be calculated daily. Data will be checked for normality and homogeneity for the selection of the appropriate statistic test (p<0.05).
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