Cytotoxicity of a Calcium Hydroxide Formulation on Cultured Human Cells

Objectives: The objective of this study was to determine if cells stressed by known cytotoxic or inflammatory agents were more susceptible to the deleterious effects of a calcium hydroxide formulation used in pulp capping and as a liner. Methods: Dycal (Dentsply) was mixed at a 1:1 base to catalyst ratio and samples were placed into plastic wells with a diameter of 5 mm and a thickness of 6 mm. Five samples were placed in 50 ml of culture medium for 48 hours to allow soluble materials to leach into the medium. Adult human dermal fibroblasts (Zen Bio, Research Triangle Park, NC) were treated for 24 hours with 0.001% chlorhexidine, 0.2% ethanol, 5 μg/ml E. coli LPS, or 0.05 mM nicotine. Cells were subsequently treated with the calcium hydroxide solution for an additional 24 hours. Cytoxicity was measured using MTT, WST, and total and secreted LDH colorimetric assays (Sigma, St. Louis, MO and Biovision, Mountain View, CA). In addition, mitotic activity was evaluated using a colorimetric histone H3 phosphorylation assay (Active Motif, Carlsbad, CA). Data were analyzed using ANOVA with Tukey’s multiple comparison post-test and significance at p < 0.05. Results: For all assays, measured values for cells treated with a known cellular stress agent plus calcium hydroxide solution were significantly lower compared to control (p < 0.05). However between treatments, there were no significant differences observed for any assay. Conclusions: Calcium hydroxide in a formulation used in dental clinical procedures is highly cytotoxic to cultured cells, as evidenced by several cellular assays. However, cells that are stressed by other known toxic agents, including chlorhexidine, ethanol, bacterial LPS, and nictotine do not appear to exacerbate the deleterious cellular effects of the calcium hydroxide.