Light microscopic evaluation of the effects of resveratrol and/or nicotinamide on osteoblastic differentiation of MSCs in monolayer culture.

<p><i>a–d: Light microscopic demonstration of osteoid-tissue formation with von Kossa staining (A) or adipose-tissue formation with Oil Red O staining (B).</i> 21 days in monolayer culture. In cultures were stimulated with osteogenic induction medium (A,B: a) and with various concentrations of resveratrol (0.1 µM (A,B: b), 1 µM (A,B: c), 10 µM (A,B: d)calcium deposition was observed (A: a–d), but adipogenesis was negative (B: a–d). Light microscopy demonstrated that MSC cultures treated with osteogenic medium and with the sirtuin inhibitor nicotinamide (1 mM (A, B: e), 10 mM (A,B: f) and 100 mM (A,B: g)), did not differentiate to osteoblastic cells (A, e–g), but differentiated into adipocytes (B, e–g), exhibiting cytoplasmic lipid droplet accumulation in the presence of osteogenic induction medium. In another approach, MSCs were pre-treated with 1 µM resveratrol and then co-treated with various concentrations of nicotinamide (1 mM (A,B: h), 10 mM (A,B: i) and 100 mM (A,B: j)) in osteogenic medium. Pre-treatment of MSCs with 1 µM resveratrol and co-treatment with 1 and 10 mM nicotinamide inhibited adipogenic differentiation of MSCs (B: h–i), favoring osteoblastic differentiation (A: h–i). However, co-treatment with 100 mM nicotinamide resulted in adipogenesis (B: j), but not in osteogenesis (A: j). Magnification: ×200, <i>bar</i> 30 µm.