Characterization of a programmed necrosis process in 3-dimensional cultures of dental pulp fibroblasts

Le Clerc J, Perard M, Pellen-Mussi P, Novella A, Tricot-Doleux S, Jeanne S, Perez F. Characterization of a programmed necrosis process in 3-dimensional cultures of dental pulp fibroblasts. International Endodontic Journal, 46, 308-316, 2013. Aim To analyse and compare the expression of nemosis markers in human lung and dental pulp fibroblasts and to determine whether this process differs by the type of mesenchymal cell. Methods Human dental pulp fibroblasts were obtained from unerupted third molars. Sound lung and pulpal fibroblasts were cultured in vitro as spheroids to determine the expression of the nemosis hallmark cyclooxygenase-2 (COX-2) mRNA using RT-PCR and the concentrations of vascular endothelial growth factor (VEGF) and hepatocyte growth factor/scatter factor (HGF/SF) proteins using an ELISA test. Cell viability within spheroids was also compared with spheroid diameters over time. Results Increased expression of COX-2 and VEGF was found in all spheroids compared with corresponding monolayers. Although HGF/SF was highly expressed in MRC5 cells, dental pulp fibroblasts aggregates maintained only a basal level compared with monolayer cultures. Further, the observed progressive loss of viable cells explained the decreased diameters of spheroids over time. The results demonstrate that nemosis occurs in sound lung and pulpal fibroblasts. This cell death also displays differences between these two different cell types, as they do not produce the same growth factors quantity release. Conclusions The nemosis process occurred in human dental pulp fibroblasts and is different between the two cell types studied. This in vitro experimental nemosis model could become an interesting inflammatory tool. More investigations are needed to compare nemosis process in dental pulp fibroblast and inflammation during pulpitis.