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Acquired pellicle engineering: in vitro evaluation of the protective potential of a recombinant hybrid protein against initial erosion

Grant number: 23/11617-0
Support Opportunities:Scholarships in Brazil - Scientific Initiation
Effective date (Start): December 01, 2023
Effective date (End): November 30, 2024
Field of knowledge:Health Sciences - Dentistry
Principal Investigator:Marília Afonso Rabelo Buzalaf
Grantee:Anna Clara de Mello Zonta
Host 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

A new recombinant hybrid protein deserves to be studied, due to its practicality to be produced when it occurs with the detailed production of CaneCPI-5 and the statherin derived peptide. Thus, producing a protein in a functional way, with the peptide derived from statherin in fusion with cystatin, enables its production in bacteria, which is cheaper and more viable. It should be mentioned that the peptide and CaneCPI-5 bind to different sites on the hydroxyapatite, which may guarantee greater protection against the initial deposit. Thus, the aim of the present study will be to analyze the protective effect and define concentrations of the different recombinant hybrid protein constructs by modifying the acquired pellicle for protection against the initial erosion of enamel in vitro. 119 samples of enamel (4 X 4 mm) will be made. Subsequently, the samples will be randomized and divided into 7 groups, according to the treatments (n/group=17): 1) Deionized water (negative control); 2) CaneCPI-5 0.1 mg/mL (positive control); 3) Commercial solution with SnCl2/NaF/AmF (800 ppm Sn+ 500 ppm F-, pH 4.5, Erosion Protection® - GABA, (positive control); 4) Hybrid protein CaneCPI-5 + statherin derived peptide at the N- and C- terminus 0.05mg/ml; 5) Hybrid protein CaneCPI-5 + statherin derived peptide at the N- and C- terminus 0.1 mg/mL; 6) Hybrid protein CaneCPI-5 + statherin derived peptide at the N- and C- terminus 0.5 mg/mL; 7) Hybrid protein CaneCPI-5 + statherin derived peptide at the N- and C - terminus 1.0 mg/mL. Initially, the samples will be treated individually (250 µL) with the respective solutions for 3 min, 37 ºC, under constant pressure. Then, the acquired pellicle will be prepared individually (250 µL) for 2 h, 37 ºC, under constant pressure (stimulated saliva will be collected from 9 volunteers). Subsequently, the erosive challenge will be performed individually (1 mL) with 0.65 % citric acid (pH = 2.5) for 1 min at 37 °C under constant inspiration. These procedures will be performed once a day, for 3 consecutive days. Surface microhardness (SHM) analyzes will be performed and changes to the SMH (SHM baseline - post-erosion SMH) will be included. Data will be analyzed using GraphPad Prism software. First, the data will be checked for normality (Kolmogorov-Smirnov test) and homogeneity (Bartlett test), to select the appropriate statistical test. The significance level will be 5%.

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