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Effect of bioactive endodontic cements on stem cells from human exfoliated deciduos teeth (shed)

Grant number: 19/10516-0
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): February 01, 2020
Effective date (End): January 31, 2021
Field of knowledge:Health Sciences - Dentistry
Principal Investigator:Luciana Lourenço Ribeiro Vitor
Grantee:Laura Giraldi Ferrari
Home Institution: CENTRO UNIVERSITARIO SAGRADO CORACAO/IASCJ

Abstract

This study will aim to evaluate the effect of bioactive endodontic cements on viability, migration and phenotypic expression of stem cell from human exfoliated deciduous teeth (SHED). SHED will be obtained from a primary culture, from caries-free deciduous teeth indicated for extraction due to orthodontic reasons, of children aged from 5 to 7 years, good general health. SHED will be characterized through flow cytometry by positive (CD105 PerCP-Cy5.5, CD73 APC, CD90 FITC) and negative expression (CD45, CD34, CD11b, CD19, HLA-DR PR) of cell surface markers. The materials will be divided into the following groups to analyze the biological properties of the materials: Group 1 - new MTA endodontic cement (MTA HP Repair); Group 2 - bioceramic cement (Bio-C Sealer); Control Group - conventional MTA. The culture medium will be conditioned with the cements at a final concentration of 1 mg/mL MEMa supplemented with 10% fetal bovine serum (FBS), aliquoted and frozen. To analyze the cell viability a density of 1 x104 SHED will be plated, let to adhere, submitted to cell cycle synchronization, and exposed to the conditioned media with the bioactive endodontic cements according to each experimental group. After 24, 48, and 72 h, cell viability will be evaluated by MTT assay. To evaluate the effect of the different bioactive endodontic cements on SHED migration, after plating and cell confluency, a "wound" will be created with the aid of 200-ul tip. Next, SHED will be incubated inside the conditioned media with the bioactive cement for 48 hours and evaluated at 0, 24h, and 48h through a microscope. The analysis of the phenotypic expression will be executed concomitantly by the total protein production and the alkaline phosphatase activity, after 24 h of incubation with the conditioned media and 7 days with osteogenic medium, by spectrophotometry at 655 nm and 590 nm, respectively. Means and standard deviation will be tested regarding data normality. If the data have normal distribution, intragroup and intergroup comparisons will be performed by two-way ANOVA, followed by Tukey test. If the data have a normal distribution, the intragroup comparison will be performed by the Friedman test and the intergroup comparisons by the Kruskal-Wallis test. The level of significance of 5% will be adopted to detect statistical differences.