Characterization of the acquired pellicle formed in vivo over human enamel and its modification after mechanical and chemical salivary flow stimulation: proteomic study

Author(s)

Flávia Zaidan Cardoso dos Santos

• Document type: Master's Dissertation
• Press: Bauru.
• Institution: Universidade de São Paulo (USP). Faculdade de Odontologia de Bauru (FOB/SDB)
• Defense date: 2013-08-26

Examining board members

Marilia Afonso Rabelo Buzalaf; Juliano Pelim Pessan; Walter Luiz Siqueira Junior

• Advisor: Marilia Afonso Rabelo Buzalaf

Abstract

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 separation and mass spectrometric analyses were carried out with a nano-HPLC Proxeon linked to mass spectrometer (LC-ESI-MS/MS). The obtained MS/MS spectra were searched against human protein databases (UniProt and TrEMBL, Swiss Institute of Bioinformatics) using SEQUEST algorithm in Proteome Discoverer 1.3 software (Thermo Scientific). The combination of all 3 experiments yielded a total of 115 proteins/peptides identified by at least two peptides. All proteins were identified in at least three runs at each Group fifty-one proteins were found in all three Groups. The protein profile of the AEP observed in each study group was similar, but all groups presented specific proteins. Using the SIEVE quantification technology, it was observed that the amount of proteins that compose the AEP was greater in the groups in which the salivary stimulation was performed (2 and 3). The data suggest that salivary flow stimulation, either mechanical or chemical/gustatory, promotes the formation of AEP with higher concentration of protective proteins, such as carbonic anhydrase, mucins, cystatins and immunoglobulins, which may result with a greater protective character for the tooth enamel. However, further studies are necessary to fully understand the complex process of formation (under various conditions) and the dynamic functions of this important integument. (AU)

• FAPESP's process: 11/03112-9 - Determination of the composition of the acquired pellicle formed on enamel and dentin and its modification after chemical and mechanic stimulation of salivary flow: proteomic study
• Grantee: Flávia Zaidan Cardoso dos Santos
• Support Opportunities: Scholarships in Brazil - Master