In Vitro Mineralization Staining Assay

In vitro mineralization was performed as previously described [35]. For the in vitro Alizarin red-stain (ARS; Sigma Aldrich) assay, BMMSCs were seeded at a concentration of 5 × 10⁴ in 12-well culture plates under osteogenic induction medium (α-MEM+15% FBS supplemented with 2 mM β-glycerophosphate, 100 nM dexamethasone, and 50 μg/mL ascorbic acid) with or without NaHS for 21 days. At the end of the experiment, cells were washed with PBS, fixed with 70% ethanol for 35 minutes and were stained with 2% ARS, pH 4.2, for 20 minutes at room temperature. Briefly, absorbance was read at 510 nm calorimetrically for quantification of mineralized nodules. For the Von Kossa measurement, the BMMSCs were washed with PBS and fixed with 70% ethanol. The cells were then rinsed with distilled water and thereafter incubated under UV light in the presence of a 5% silver nitrate solution. After 20 minutes, the cells were washed with distilled water and incubated with a 5% sodium thiosulfate solution. All images were taken on a phase contrast microscope.