Cytotoxicity and ROS production by pulp capping materials in cells

Objectives: Pulp capping materials could release toxic components that may interact with pulp tissue. Castor oil bean cement (COB) is a new material that has been used as endodontic sealer, and it is a candidate material for direct pulp capping. We hypothesized that these materials might promote cytotoxicity in pulp cells (tHPCs) via the generation of reactive oxygen species (ROS).

Methods: Specimens of calcium hydroxide (HYDRO C, Dentsply-Brazil), MTA gray (Angelus-Brazil), MTA white (Angelus-Brazil), and COB (Ricinus communis-Poliquil-Brazil) were mixed and subsequently extracted in culture medium (MEMalpha, 10% FBS, 91.6 mm2 sample surface/ml) for 24h at 37°C. Vitrebond (3M ESPE) served as a positive control. 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 were 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: The ranking of the cytotoxic effects based on EC50 values was as follows: Vitrebond > HYDRO C > MTA gray > MTA white >> COB. Extract of COB even induced cell proliferation, but HYDRO C decrease cell survival in a dose-related manner. ROS production was not detected with any pulp-capping material. Vitrebond increased ROS production about sevenfold.

Conclusions: Our results suggest that the cytotoxic action of some pulp capping materials could have clinical relevance. Especially COB, and MTA (gray and white) did not negatively influence cell viability and ROS production. Therefore, COB has a good potential for being used in deep cavities or directly in contact with pulp tissue.