Cells were stained using an Alizarin red staining kit according to the manufacturer’s instructions (Solarib, China). In brief, the cells were fixed with 4% paraformaldehyde for 30 min and were rinsed with PBS. Then, the cells were stained with 0.1% (w/v) Alizarin red for 15 min. The matrix mineralization deposition appeared red, and the stained cells were photographed after washing with deionized water for 15 min. 10% ice acetic acid desorbed alizarin red for 15 min. The samples in each treatment group were added with 100 µL of ammonia and reacted at room temperature for 5 min. OD values were then determined at 405 nm.
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