Human three-dimensional dental pulp organoid model for toxicity screening of dental materials on dental pulp cells and tissue.

Abstract

To establish a 3D model for screening the biocompatibility of dental materials/drugs on dental pulp cells and tissue. Human dental pulp cells (hDPC) and endothelial cells (EC) were mixed with or without human dental pulp derived extracellular matrix (hDP-ECM) according to several protocols and cultured in 3D plates to fabricate 3D organoids. Cell viability and proliferation in organoids were evaluated using Live/Dead cell viability assay and ATPase assay. Organoids were fixed, cut and stained with a H&E staining kit. The expressions of DSPP, DMP-1, CD31, vWF and COL1A in 3D organoids were evaluated using immunofluorescence. To assess the feasibility of 3D organoids on drug/material toxicity screening, the organoids were treated with lipopolysaccharides (LPS) or iRoot BP. Then, cell viability and apoptosis were assessed. The expressions of IL-6, TNF-α, and IL-1β were compared in LPS-treated and non-treated organoids. Alizarin Red S staining was used to evaluate calcium deposit formation in organoids. Data were analysed using one-way anova followed by Tukey's post hoc comparison. The 3D spheres/organoids were formed at day 1 or day 2. Cells in 3D organoids maintained a high viability rate and low proliferation activity. The level of CD31 increased significantly (p<.05) when EC were added to coculture with hDPC. The expressions of odontogenesis-associated proteins (DSPP, COL1A) upregulated (p<.05) with the addition of hDP-ECM. Level of IL-6 expression and rates of dead and apoptotic cells in 3D organoids were increased significantly (p<.05) in response to LPS. Calcium deposit formation was observed in iRoot BP-treated organoids. Coculture of hDPC and EC in the presence of hDP-ECM formed functional dental pulp organoids. The experimental model provides an alternative tool for toxicity screening of dental pulp capping agents and dental pulp regeneration research.