4.5. Osteogenic Differentiation Assays

The ability of culture-expanded MSCs to differentiate into the osteogenic lineage was validated according to earlier described methods.^2,67 In brief, cells were plated in 12-well plates at 50000 cells/cm² and cultured in DMEM for 24 h. Culture medium was then aspirated and replaced with osteogenic differentiation medium consisting of DMEM supplemented with 10% FBS, 1%AA, 0.25 mM ascorbic acid (#A4544 Sigma-Aldrich), 0.1 μM dexamethasone (#D4902 Sigma-Aldrich), and 10 mM β-glycerophosphate (#G9891 Sigma-Aldrich). Treatment-containing medium was replaced two times per week. The early osteogenic differentiation marker, alkaline phosphatase, was assessed in cell culture medium after 7 days using an Alkaline Phosphatase Assay Kit (#ab83369 Abcam). After 3 weeks, osteogenic differentiation was assessed by staining with Alizarin Red staining solution (#TMS-008 C Millipore Sigma). The cells were fixed with 10% phosphate-buffered formalin for 30 min at room temperature (RT) and stained with Alizarin Red staining solution for 45 min at RT. Stained monolayers were visualized by phase-contrast microscopy using an inverted microscope (Nikon, Melville, NY). Differentiation was quantified as previously described.⁶⁸ In brief, cells were destained using 10% cetylpyridinium chloride (#855561 Sigma-Aldrich), and collected samples were analyzed using a microplate reader at 570 nm.