Dose-dependent effect of the ascorbic acid and FBS supplementation during osteogenic differentiation of bMSC. (A) Culture of bMSC under high concentrations of ascorbic acid (0.1 mM) in combination with 10% or 5% FBS resulted in higher (P < 0.05) OC mRNA and lower NANOG mRNA expression compared to other treatments. (B) ALPL activity decreased (P < 0.05) when acid ascorbic was eliminated or reduced to 0.001 mM. (C) von Kossa (VK) staining evidenced higher matrix mineralization in bMSC cultures supplemented with 0.1 mM ascorbic acid in combination with 10 and 5% FBS. Data are shown as mean ± standard errors. Superscripts (a,b,*) represent significant (P < 0.05) differences between treatment and sampling days. Bar scale: 500 μm.