Cetylpyridinium chromide

To evaluate the calcified nodules formed from human dental stem cells, the ARS staining assay was performed on Days 7 and 14. The hDPSCs were inoculated at a density of 0.5 × 10⁴ cells/well. Over 14 days, hDPSCs were incubated using all experimental disk eluates. The hDPSCs cultured in OM without experimental eluates were used as the control group. The cells were fixed with 4% paraformaldehyde solution for 20 min and stained with 2% ARS solution (ScienCell, Carlsbad, CA, USA) on Days 7 and 14. To stain, 10% cetylpyridinium chromide (Sigma-Aldrich) was applied for 15 min. Each group was measured in six independent experiments.

The ARS staining assay was used to evaluate the formation of calcium nodules in hBMSCs. Each experimental disk was incubated in osteogenic medium and kept in an incubator at 37 °C and 100% humidity for 7 days, producing a concentration of 5 mg/mL experimental eluate for each tested material. The superficial fluid was purified by 0.20-μm filters (Minisart; Sartorius Stadium Biotech, Göttingen, Germany). hBMSCs were sprayed at a density of 2.0 × 10⁴ cells/well in a 24-well cell culture plate, then incubated for 14 days in the experimental material eluates. Emdogain at an initial concentration of 30 mg/mL was added to groups 2, 4, 6, and 8, and diluted in osteogenic medium to a final concentration of 100 μg/mL. The cells were fixed with a 4% paraformaldehyde and a 2% ARS solution (ScienCell, Carlsbad, CA, USA) for 20 min. The staining was performed for 15 min with 10% cetylpyridinium chromide (Sigma-Aldrich). Finally, an absorbance microplate reader was used to measure the optical density at 560 nm. Each group was measured in sextuplicate.

Calcified nodule formation ability of hBMSC was evaluated after 7 and 14 days of incubation using Alizarin Red-S (ARS) staining assay. After production of all disks, we measured the weight of each disk, adjusted the osteogenic medium volume according to the weight, and produced eluate stock as a 50 mg/mL concentration. In order to release disk components into the osteogenic medium, all disks with media were stored in an incubator at 100% humidity and 37 °C for 7 days. Supernatant of each eluate was filtered using a 0.2 μm membrane filter, diluted 1/10, and prepared to a final concentration of 5 mg/mL.
The hBMSCs were sprayed at a density of 2.0 × 10⁴ cells/well and incubated for 14 days using each retrograde filling cement eluate. On days 7 and 14, the cells were treated with 2% ARS solution (ScienCell, Carlsbad, CA, USA) for 20 min and stained with 10% cetylpyridinium chromide (Sigma-Aldrich, St. Louis, MO, USA) for 15 min. Each group was measured in sextuplicate.