Chronic exposure to lipopolysaccharides as an in vitro model to simulate the impaired odontogenic potential of dental pulp cells under pulpitis conditions

Author(s)

Soares, Igor Paulino Mendes ; Anselmi, Caroline ; Pires, Maria Luiza Barucci Araujo ; Ribeiro, Rafael Antonio de Oliveira ; Leite, Maria Luisa ; Soares, Diana Gabriela ; Costa, Carlos Alberto De Souza ; Hebling, Josimeri

• Document type: Journal article
• Source: Journal of Applied Oral Science; v. 31, p. 12-pg., 2023-01-01.

Abstract

Simulating a bacterial-induced pulpitis environment in vitro may contribute to exploring mechanisms and bioactive molecules to counteract these adverse effects. Objective: To investigate the chronic exposure of human dental pulp cells (HDPCs) to lipopolysaccharides (LPS) aiming to establish a cell culture protocol to simulate the impaired odontogenic potential under pulpitis conditions. Methodology: HDPCs were isolated from four healthy molars of different donors and seeded in culture plates in a growth medium. After 24 h, the medium was changed to an odontogenic differentiation medium (DM) supplemented or not with E. coli LPS (0 - control, 0.1, 1, or 10 & mu;g/ mL) (n=8). The medium was renewed every two days for up to seven days, then replaced with LPS-free DM for up to 21 days. The activation of NF-KB and F-actin expression were assessed (immunofluorescence) after one and seven days. On day 7, cells were evaluated for both the gene expression (RT-qPCR) of odontogenic markers (COL1A1, ALPL, DSPP, and DMP1) and cytokines (TNF, IL1B, IL8, and IL6) and the production of reactive nitrogen (Griess) and oxygen species (Carboxy-H2DCFDA). Cell viability (alamarBlue) was evaluated weekly, and mineralization was assessed (Alizarin Red) at 14 and 21 days. Data were analyzed with ANOVA and post-hoc tests (& alpha;=5%). Results: After one and seven days of exposure to LPS, NF-KB was activated in a dose-dependent fashion. LPS at 1 and 10 & mu;g/mL concentrations downregulated the gene expression of odontogenic markers and up-regulated cytokines. LPS at 10 & mu;g/mL increased both the production of reactive nitrogen and oxygen species. LPS decreased cell viability seven days after the end of exposure. LPS at 1 and 10 & mu;g/mL decreased hDPCs mineralization in a dose-dependent fashion. Conclusion: The exposure to 10 & mu;g/mL LPS for seven days creates an inflammatory environment that is able to impair by more than half the odontogenic potential of HDPCs in vitro, simulating a pulpitis-like condition. (AU)

• FAPESP's process: 21/13096-2 - Bioactivity of flavonoids on LPS-challenged human pulp cells and association with fibrous scaffolds for modulating dentinogenesis in a degenerative inflammatory environment
• Grantee: Igor Paulino Mendes Soares
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 19/16473-1 - Polycaprolactone-nanohydroxyapatite nanofiber scaffolds for regeneration of the pulp-dentin complex: synthesis, characterization and biological behavior in human teeth culture
• Grantee: Josimeri Hebling Costa
• Support Opportunities: Regular Research Grants