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Effect of saliva enrichment with CaneCPI-5, Stn15pSpS or hemoglobin, isolated or combined, associated or not with EGCG, in thickness, viscoelastic properties, interfacial load and morphology of the acquired enamel pellicle

Grant number: 21/00203-5
Support type:Scholarships in Brazil - Post-Doctorate
Effective date (Start): June 01, 2021
Effective date (End): May 31, 2023
Field of knowledge:Health Sciences - Dentistry - Social and Preventive Dentistry
Principal researcher:Marília Afonso Rabelo Buzalaf
Grantee:Talita Mendes Oliveira Ventura
Home Institution: Faculdade de Odontologia de Bauru (FOB). Universidade de São Paulo (USP). Bauru , SP, Brazil
Associated research grant:19/26070-1 - Modulation of acquired enamel pellicle to control dental mineral loss: unravelling mechanisms to make therapies possible, AP.TEM

Abstract

The Acquired Enamel Pellicle (AEP) is one of the protective factors against Erosive Tooth Wear (ETW) and dental caries. Among the proteins of the acquired pellicle with the potential to bind to the enamel and acid-resistant properties, we have recently identified cystatin, hemoglobin (Hb) and statherin. The modification of the AEP protein profile by incorporating proteins can interfere with the initial bacterial colonization of the biofilm, changing its structure. Therefore, this modification is promising not only for the control of ETW, but also for the control of dental caries. In this sense, the present research project, inserted in the "acquired pellicle Engineering", intends to evaluate the effect of enriching saliva with CaneCPI-5, peptide derived from statherin (Stn15pSpS) or hemoglobin (Hb), isolated or combined, associated or not to the epigallo catechin gallate (EGCG), in thickness, viscoelastic properties, interfacial load and AEP morphology. The real-time analysis of the interaction between the studied proteins, associated or not with EGCG, and hydroxyapatite, will be done using a quartz crystal scale with dissipation monitoring (QCM-D). For this, a Q-Sense E4 system will be used, with hydroxyapatite sensors (QXS327). The isolated or combined proteins, in concentrations determined in previous experiments, associated or not with EGCG (1.25 mM), will be included in a pool of human saliva, which will be added to the hydroxyapatite sensor cell, in a flow of 200 µL/min, for 5 min. Then, the flow will be reduced to 10 µL/min and the adsorption will be monitored for 55 min. In order to remove saliva, water will be added to the cell, at a flow of 200 µL/min for 5 min, after which the flow will be reduced to 10 µL/min for 25 min. The elution of the adsorbed pellicle will be done by the addition of 0.5% sodium dodecyl sulfate, at a flow of 200 µL/min for 5 min. The Q-Tools software will be used to obtain the thickness, modulus of elasticity and viscosity of the AEP. The analysis of the AEP interfacial load will be done during a BEPE internship at Saarland University, using an electrokinetic device developed in-house. The AEP will be formed for 90 min from human saliva plus the proteins of interest, isolated or combined, associated or not with EGCG, along a transmission channel formed by the surfaces of SiO2 substrates. The magnitude and signal of the measured transmission current will be determined by the spatial distribution of charges and hydrodynamic penetration in the AEP layer. Finally, the morphology and thickness of the AEP formed in situ for 30 min after treatment of the bovine enamel with the isolated or combined proteins, associated or not with the EGCG (mouthwash for 1 min), submitted to the erosive challenge with 1% citric acid pH 2,5 for 10 seconds or HCl 0.01 M pH 2.0 for 10 seconds, will be evaluated by Transmission Electron Microscopy (TEM). The data will be checked for normality and homogeneity, using the appropriate statistical test (parametric or not, p <0.05). These steps are crucial for a product to be developed, based on one of these proteins or a combination of them, associated or not with EGCG, that will be used on a large scale as a new approach for the control of ETW and dental caries, based on "acquired pellicle Engineering" procedures. (AU)

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