Study of the effect of cellular activation by pathogen or Danger Associated Molecular Patterns on apical papilla cells viability and differentiation in vitro

• Grant number: 15/12218-6
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: November 01, 2015
• End date: October 31, 2016
• Field of knowledge: Health Sciences - Dentistry - Endodontics
• Principal Investigator: Carla Renata Sipert
• Grantee: Pamela Rocha Lopes de Almeida
• Host Institution: Faculdade de Odontologia (FO). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

The tooth development is usually impaired by inflammatory processes resulting from dental trauma or root canal infection. The role of apical papilla in root development has been demonstrated by the literature; however, the mechanisms involved in the modulation of this tissue by bacterial byproducts or necrotic cells resulting from caries or trauma are still unknown. Therefore, the aim of this project is to investigate the cytotoxicity and odontogenic differentiation of cultured human apical papilla cells under challenge with bacterial byproducts such as lipopolysaccharide (LPS) or/and lipoteichoic acid (LTA) or with necrotic cells supernatant (SN) isolated or combined. Primary cultures of apical papilla cells (n = 3) will be established based on an explant technique. Cells will be plated for evaluation of the cytotoxicity of increasing concentrations of LPS, LTA, SN, LPS + LTA, LPS + LTA + SN for 1, 3 and 5 days. Medium alone will be used as control. The constitutive and the modulation of PAMPs and DAMPs under this context will be assessed by means of RT-qPCR. The higher concentrations unable to compromise cell viability will be used to activate the cells prior to differentiation assay. Cells will be kept in contact with stimulators or medium only for 5 days and then subjected to odontogenic differentiation for 14 and 28 days. As negative control, naïve cells will be kept with conventional proliferation medium. The cellular differentiation will be assessed by calcium deposition by using alizarin red S staining. Data will be analized by using one-way analysis of variance setting p < 0.05.