Cytotoxicity and ROS production in cells by root canal sealers

Objectives: Endodontic sealers release chemicals that may interact with connective apical tissues. Here, we hypothesized that sealers might cause cytotoxicity in transformed human pulp cells (tHPC) via the generation of reactive oxygen species (ROS).

Methods: Specimens of Acroseal (Septodont, France), Epiphany (Pentron Clinical, USA), AH Plus (Dentsply Maillefer, Brazil) and Castor oil bean sealer (COB) (Poliquil, Brazil) were set for 6h in 100% humidity at 37°C, and then extracted in culture medium (MEMalpha with 10% FBS, 91.6 mm2 sample surface/ml) for 24h at 37°C. Exposure of tHPCs to dilutions of the extracts was stopped after 24h, and cell survival was determined photometrically after staining the cells with crystal violet. Concentrations leading to 50% cell survival (EC50) were calculated from dose-response curves. The generation of ROS was determined after a 1h exposure of the cells to extracts. ROS was detected by staining the cells with 2',7'-dichlorofluorescin diacetate, and fluorescence was measured by flow cytometry (FACS). Differences between cell survival rates and fluorescence levels (n=4) were statistically analyzed (Mann-Whitney-U test, p<0.05). Results: Extracts of the root canal sealers decrease cell survival in a dose-related manner, except for COB. The cytotoxic potencies of the materials varied at least 15-fold, and the ranking of the cytotoxic effects based on EC50 values was as follows: Acroseal > Epiphany and AH Plus >> and COB. Extracts of the sealers produced cellular ROS levels in tHPC in a dose-related manner. The amounts of ROS were increased about 7-fold by undiluted extracts of Epiphany, and the ranking of the potencies to induce ROS production was as follows: Epiphany > AHPlus > Acroseal. ROS production was not detected with COB.

Conclusions: Acroseal, Epiphany and AHPlus presented different cytoxicity levels, and COB sealer did not have negatively influence in cell viability and ROS production.