2.7.4. Mineralization assay (alizarin red S staining)

The modules were cultured in growth medium for 7 days, and then bone nodule formation was induced in osteogenic medium containing 0.1 mM dexamethasone, 10 mM β-glycerophosphate disodium salt solution, and 50 μg/mL l-ascorbic acid for 14 days. Matrix mineralization was analyzed by alizarin red S staining [39]. The modules were stained with 0.4% alizarin red S solution for 30 min and taken pictures. Then, the modules were extensively washed with PBS at room temperature, and the stain solution was destained in 10% cetylpyridinium chloride for 1 h and the washing solution was measured for absorbance at 562 nm for quantitation of mineralization status.