2.9. Alizarin Red S Staining

BMSCs groups consisting of 2 × 10⁴ cells were sowed on 24-well plates and grown with an osteogenic medium containing 2 mg/mL of glycerophosphate disodium salt hydrate, 38 μg/mL of dexamethasone, 10 mM of ascorbic acid 2-phosphate (Sigma-Aldrich Co.), 200 mM of L-glutamine (Sigma-Aldrich Co.), and 15% fetal bovine serum (Gibco). On Days 8 and 16, the cells were rinsed twice with PBS (Welgene), followed by fixation with 4% paraformaldehyde, then cleansed twice with deionized water. The sample was stained using alizarin red S (Sigma-Aldrich Co.) at room temperature for 30 min.

To eliminate the staining of non-specific binding, the cells were rinsed three times with deionized water. The solubilization of bound dye was performed using 10 mM of sodium phosphate comprising 10% cetylpyridinium chloride, then quantitated at 562 nm by a spectrophotometer. The inverted microscope was utilized for morphological evaluation (CKX41SF, Olympus Corporation, Tokyo, Japan). A quantitative analysis of alizarin red S was accomplished using image analysis and processing software (ImageJ, National Institutes of Health, Bethesda, MD, USA).