Cytotoxicity and anti-inflammatory effects of zinc ions and eugenol during setting of ZOE in immortalized human oral keratinocytes grown as three-dimensional spheroids

Abstract

ObjectivesThe objective of this study is to assess the cytotoxic and anti-inflammatory effects of ZOE cement during setting in two-dimensional (2D) or three-dimensional (3D) cultures of immortalized human oral keratinocytes (IHOKs) with determining the extract components responsible for these effects. MethodsExtracts of mixed ZOE at different stages of setting were analyzed by a digital pH meter, ICP-MS, and GC–MS. Serial concentrations of extract and their mixture of ZnCl2, ZnSO4·H2O, and eugenol liquid were added to the 2D and 3D IHOK cultures to determine the half maximal effective concentration in investigating the cause of cytotoxicity by means of WST assay and to investigate mRNA expression levels of inflammatory cytokines by RT-PCR. ResultsZn2+ and eugenol (4–19ppm) were detected in the extracts. In the early setting stage, significant cytotoxicity was observed in the 2D and 3D IHOK cultures (P<0.05). The EC50 of Zn2+ from ZnCl2 was 5–44ppm in both cultures, whereas the EC50 of eugenol was not detectable under 100ppm. Along with the lower levels of inflammatory cytokine gene expressions in the extract, treatment of the 2D IHOKs with Zn2+ alone and treatment of the 3D IHOKs with Zn2+ plus eugenol resulted in significantly lower expression levels of IL-1β, IL-6, and IL-8 (P<0.05). SignificanceThe cytotoxic effect of ZOE on IHOKs was greater during the setting stage owing to the presence of Zn2+. The anti-inflammatory response to ZOE was induced by a combination of Zn2+ and eugenol. Cytotoxic and anti-inflammatory effects differed between the 2D and 3D IHOK cultures.