Ars staining

Cells were seeded at 1.5 × 10⁴ cells/well in 48-well plates and cultured in a complete medium for 24 h. Then, the old medium was replaced with the osteogenic/odontogenic medium with or without CBD (0, 0.1, 0.5, and 2.5 µM) and TNF-α (50 ng/mL), respectively. The medium was refreshed every 3 days. On day 14, after removing the old medium and washing it twice with PBS, the cells were fixed with 4% paraformaldehyde for 30 min. The cells were washed twice with PBS and stained by ARS staining (Solarbio, Beijing, China) for 5 min at room temperature. The microscope was used for observation and photography. For semiquantitative analysis, OD values of the solution at 562 nm wavelength were recorded after the mineralized nodules were completely eluted by 10% cetylpyridinium chloride (CPC) solution (Sigma-Aldrich, St Louis, MO, USA) for 30 min.

MC3T3-E1 cells were cultured in 24-well plates for a 21-day period in osteogenic medium [29 (link)]. After rinsing twice with PBS on day 21, the cells were subjected to 15 min fixation using 4% PFA (Solarbio, Beijing, China). The fixed cells were rinsed three times with deionized water, followed by 30 min of ARS staining (Solarbio, Beijing, China). The stained mineralized nodules were monitored using an optical microscope (Olympus Optical Co. Ltd.).