dipocytes, accumulating cytoplasmic lipid droplets and exhibiting welldeveloped rough endoplasmic reticulum and mitochondria. Pre-treatment of MSCs with resveratrol and co-treatment with nicotinamide promoted osteogenic differentiation. However, the inhibition of adipogenesis by resveratrol was concentration dependent. Pre-treatment of MSCs with 1 mM resveratrol and co-treatment with 100 mM nicotinamide resulted in adipogenesis. Incubation of pre-osteoblastic MC3T3-E1 cells with the osteogenic induction medium or/and resveratrol resulted in osteogenesis. However, in contrast to MSCs, treatment of preosteoblastic MC3T3-E1 cells with nicotinamide, led to apoptosis instead of to formation of adipocytes. Pre-treatment of preosteoblastic MC3T3-E1 cells with resveratrol and co-treatment with nicotinamide promoted osteogenic differentiation. Statistical evaluation of the data clearly highlighted changes in the number of cells with fat vacuole accumulation before and after nicotinamide-treatment in MSC-osteogenesis high-density cultures. Co-treatment with resveratrol decreased the number of adipocytes with accumulated fat vacuoles. Effect of resveratrol or/and nicotinamide on extracellular matrix, Runx2 and PPAR-c expression during MSCosteogenesis and in pre-osteoblastic cell-osteogenesis To TAK 438 free base web confirm the morphological results described above and to 
demonstrate more precisely the identity of the osteogenesis or adipogenesis by MSCs or pre-osteoblastic cell cultures, whole cell extracts were probed for collagen type I, Runx2 and PPAR-c. High collagen type I content was detected by immunoblotting in the osteogenic-induced control cultures. Treatment of MSCs with osteogenic induction medium and 0.1, 1 and 10 mM resveratrol in high-density cultures resulted in a stimulation of collagen type I production and expression of Runx2. MSC cultures treated with 9 Resveratrol Promotes Osteogenesis of MSCs nicotinamide alone at various concentrations showed a significant downregulation of synthesis of collagen type I and Runx2, but upregulation of PPAR-c and this was in a concentration-dependent manner. In contrast to this, pretreatment of MSCs with resveratrol followed by stimulation with the sirtuin inhibitor, nicotinamide resulted in an inhibition of nicotinamide-induced effects on collagen type I production and Runx2 during MSCosteogenesis and downregulated PPAR-c in high-density cultures. However, 1 mM resveratrol could not completely inhibit the blocking effect of 100 mM nicotinamide on the synthesis of collagen type I and Runx2 during osteogenesis and downregulated PPAR-c in high-density culture. Synthesis of the house-keeping 10460232” protein b-actin remained unaffected. To see that the nicotinamide-induced inhibition of Runx2 and stimulation of PPAR-c and adipogenesis during MSC-osteogenesis occurs also transiently during osteogenesis with pre-osteoblastic cells, we compared the effects of resveratrol or/and nicotinamide on protein expression profiles of MSC and pre-osteoblastic MC3T3-E1 cells during the osteogenesis in high-density culture to further confirm their differentiation capacities. Pre-osteoblastic MC3T3-E1 cells produced large quantities of collagen type I in presence of 0.1, 1 and 10 mM resveratrol and Runx2 expression 25909282” was also stimulated. High collagen type I content was also detected in the osteogenic-induced control cultures. Pre-osteoblastic cells treated with nicotinamide alone at various concentrations showed a significant downregulation of