In Vitro Cytotoxicity Evaluation of Sodium Hypochlorite, Chlorhexidine and EDTA

Objective: Canal disinfection is a crucial step in infection control, which is important for regenerative endodontic treatment. High antimicrobial effectiveness and low cytotoxicity are preferred traits of a proper canal irrigant used in the regenerative endodontic procedure, since it has less effect on the viability of dental stem cells such as DPSC and SCAP, which are beneficial to pulp regeneration. Sodium Hypochlorite (NaOCl), Chlorhexidine (CHX) and ethylenediaminetetraacetic acid (EDTA) are three common root canal irrigants. The objective of this study was to evaluate the cytotoxicity of NaOCl, CHX and EDTA on DPSC and SCAP, which are key stem cells in pulp regeneration.

Method: DPSC and SCAP were isolated and characterized respectively from mature and immature human third molars. The two stem cells passaged and expanded in vitro were treated with various concentrations of NaOCl (5.25% , 2.63%, 1.31% and 0.66%), CHX(2%, 1%, 0.5%, 0.25%) and EDTA(17%, 8.5%, 4.25%, 2.13%) for 30 minutes, 60 minutes and 120 minutes. After the treatment, the media containing irrigants was removed, the cytotoxic effect of NaOCl, CHX and EDTA was analyzed by CCK-8 assay. The statistical significance between the various groups was evaluated using the one-way analysis of variance (ANOVA).

Result: There was a significant (P<.05) decrease of cell viability in a dose-dependent manner, which indicates the cytotoxic effect of both NaOCl and EDTA when compared with the control group. Moreover, all the dilutions of NaOCl were significantly (P<.05) more cytotoxic than EDTA from the CCK-8 assays. The stem cells treated with all concentrations of CHX lacked any viable cells.

Conclusion: The study showed that irrigants alone affect the survivability of DPSC and SCAP in vitro and suggested that EDTA and low concentration of NaOCl might beneficial in regenerative treatment, while CHX was more cytotoxicity and might not be a good choice of irrigant used in this procedure.