Abstract
Introduction: This study focused on the optimization of sodium hypochlorite-EDTA irrigation in terms of the viability and morphology of dental pulp stem cells (DPSCs) and the effects of an optimized EDTA protocol alone or prepared with nanobubble (NB) water on cell behavior. Methods: In the first part, human dentin discs were conditioned with the following protocols: (1) Sodium hypochlorite followed by phosphate-buffered saline (PBS), (2) Irrigation protocol from group 1 followed by EDTA, (3) Irrigation protocol from group 2 followed by PBS, (4) Sodium hypochlorite followed by EDTA, (5) Irrigation protocol from group 4 followed by PBS. DPSC viability and morphology were determined. In the second part, dentin discs were conditioned with the (1) optimized protocol in the first part, (2) EDTA prepared using NB water, (3) ultrasonic-activated EDTA, or (4) ultrasonicactivated EDTA prepared using NB water. Transforming growth factor beta release and DPSC viability, morphology, and migration were determined using the enzyme-linked immunosorbent assay, the water-soluble tetrazolium salt-1 cell viability assay and live-dead assay, and the transwell migration assay, respectively. Data were analyzed using Kruskal-Wallis or one-way analysis of variance andpost hoc tests. Results: The highest cell viabilitywas observed in group 3 followed by group 5 (P<.05) in which PBS was used as a final rinse. Irrigation protocol from group 3 was used for the subsequent experiments. Ultrasonic-activated EDTA improved transforming growth factor beta release, viability, andmigration of the cells compared with EDTA (P<.05). The preparation of EDTA with NBs did not change the biological properties of the EDTA-conditioned dentin (P>.05). Conclusions: Removing the residual EDTA using PBS improved the cell viability on the dentin surface. Ultrasonic activation enhanced the growth factor release and biological properties, whereas the preparation of EDTA with NBs showed a similar effect to regular EDTA without compromising the cellular effect.