Transmission electron microscopic (TEM) studies of the effects of resveratrol on osteoblastic differentiation of MSCs in high-density culture.

Abstract

<p><i>A: a–d: 14 days in high-density culture.</i> Cultures were stimulated with osteogenic induction medium (a) and with various concentrations of resveratrol (0.1 µM (b), 1 µM (c), 10 µM (d)). TEM revealed details of ultrastructural changes that the MSCs underwent while differentiating into osteoblasts (Os). Cells contained high levels of nuclear euchromatin and a large number of sub-cellular organelles (mitochondria, rough ER, Golgi apparatus). Large quantities of thick extracellular matrix fibrils (arrows) were observed in the extracellular space (arrows). However, no significant differences in osteogenesis were observed at the ultrastructural level between resveratrol-treated and control MSC cultures. Magnification: 5000×, bar = 1 µm. <i>A: e–g: 14 days in high-density culture.</i> MSC cultures were treated with osteogenic medium and with the sirtuin inhibitor nicotinamide (1 mM (e), 10 mM (f) and 100 mM (g)). TEM clearly demonstrated that MSCs differentiated into adipocytes (F), exhibiting cytoplasmic lipid droplet accumulation (*) in the presence of osteogenic induction medium. The adipocytes produced high quantities of ECM (arrows) and were embedded in this well organized matrix. Magnification: 5000×, bar = 1 µm. <i>A: h–j: 14 days high-density culture.</i> MSCs were pre-treated with 1 µM resveratrol for 4 h and then co-treated with various concentrations of nicotinamide (1 mM (h), 10 mM (i) and 100 mM (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, favoring osteoblastic differentiation. However, co-treatment with 100 mM nicotinamide resulted in adipogenesis. Magnification: 5000×, bar = 1 µm. B: Adipocyte differentiation in the cultures was estimated by counting 100 cells from 20 different microscopic fields. The number of adipocytes was highest in cultures stimulated with 100 mM nicotinamide alone. However, cells pre-treated with resveratrol and co-treated with nicotinamide at 1 or 10 mM but not at 100 mM nicotinamide significantly decreased the number of adipocytes compared to the chemical by itself (*).</p

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