Evaluation of Calcium Release from Pulp-capping Material Using International Organization for Standardization Guidelines

Aims and Objectives: Cytotoxicity assays are one of the methods used to assess the biocompatibility of dental materials. The objective of this study was to determine if surface area/volume ratios designated by the International Organization for Standardization (ISO) for biological evaluation of medical devices affects cytotoxicity and calcium release in an endodontic pulp-capping materials. Material and Methods: Human periodontal ligament fibroblasts (PDLFb) were treated with extractables derived from the calcium silicate pulp-capping material TheraCal LC at pellet surface area/culture medium volume ratios of 0.2, 0.5, and 1.0 cm 2 /mL. A colorimetric cytotoxicity assay (MTT) and phase-contrast microscopy was used to assess biocompatibility against the fibroblasts. The concentration of calcium leached into culture medium from TheraCal LC at pellet surface area/culture medium volume ratios of 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, and 6.0 cm 2 /mL after 24 hours was determined by colorimetric assay. Results: Both MTT assays and microscopy demonstrate that at a pellet surface area/culture medium volume ratio of 1.0 cm 2 /mL TheraCal LC extractable challenge to fibroblasts results in a significant decrease in cell viability ( P < 0.001) compared to control. TheraCal LC calcium release into culture medium varied with the surface area/volume ratio, with concentrations ranging from 2 to 8 times the calcium concentration in culture medium. Conclusion: Pellet surface area/culture medium volume ratios impacted parameters of TheraCal LC used in in vitro studies to determine biocompatibility with oral tissues. The ISO guidelines should be followed for all in vitro cytotoxicity studies of dental materials.