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To induce mineralization, the cells were cultured using complete DMEM containing osteogenic factors: 10 nM dexamethasone, 10 mM β-glycerophosphate, and 50 μg/mL ascorbic acid. Calcium precipitation was detected on day 20 of culture using Alizarin Red S staining (A5533-25G, Sigma-Aldrich, Beijing, China) and quantified as previously described [17]. The experiments were repeated at least three times.
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