Osteoblast Differentiation and Mineralization Assays

Primary calvarial osteoblasts were seeded at a density of 5×10⁴ cells per well in a 24-well plate. For the drug treatment, cells were pre-treated with 10 μM Dex for 24 h, then treated with 10⁻⁶ M VK2 without or with 5 mM 3-MA for 48 h as indicated. After treatment, cells were cultured under osteogenic conditions (complete DMEM containing 20 μM ascorbic acid and 10 mM β-glycerophosphate) with osteogenic media replaced every other day. Total cellular proteins were extracted on the 7^th day of osteogenesis for western blot analysis of proteins involved in osteoblast differentiation. Alkaline Phosphatase (ALP) activity was measured after 7 days of differentiation using the ALP Staining Kit (Beyotime Institute of Biotechnology; Jiangsu, China). For mineralization and bone nodule formation, cells were differentiated under osteogenic conditions for 21 days, fixed and then stained with Alizarin Red S (ARS) solution (Solarbio Science & Technology). The absorbance at 520 nm for ALP and at 570 nm for ARS staining was detected using a microplate reader.