ALP staining and quantification

MC3T3-E1 subclone 14 was inoculated in a 12-well plate. After the cells adhered, they were replaced with osteogenic induction medium (HUXMA-90021; Cyagen, USA) and cultured for 2 weeks. The medium was changed once every 3 days. The ALP color-development kit (C3206; Beyotime, China) was used according to the provided directions to assess ALP staining. Pre-stained cells were washed three times with PBS (10010023; Thermo Fisher Scientific, USA) to remove any residual culture medium; fixed with 95% alcohol at room temperature for 15 min, and washed with PBS 3 times to remove excess alcohol. 5-Bromo-4-Chloro-3-Indolyl Phosphate/ Nitroblue tetrazolium chloride (BCIP/NBT) staining working solution was added, ensuring that the sample was fully covered. The cells were incubated at room temperature in the dark for 12 h. The BCIP/NBT dyeing working solution was removed by washing with distilled water 1–2 times to halt the color reaction. After drying, the images were captured under a microscope (Olympus, BX53; Melville, NY, USA). To quantify the ALP staining, we dissolved stained cells in 10% (w/v) cetylpyridinium chloride (500 uL/wells, 6-cell plate) for 10 min, and the absorbance of the extracted solution (200 μL/cell, 96-cell plate) was measured at 562 nm (n = 3). The staining experiment was repeated three times. Data are presented as mean ± SD (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; # no significance).