Effect of resveratrol or/and nicotinamide on extracellular matrix, Runx2 and PPAR-γ expression during osteogenesis of MSC and pre-osteoblastic cells in high-density cultures.

Whole cell lysates (500 ng of protein per lane) were probed with antibodies for collagen type I (a), for the osteogenic specific transcription factor Runx2 (b) and for the adipogenic specific transcription factor PPAR-γ (c) in MSC (A) and in pre-osteoblastic cells in high-density culture (B). Cultures were treated with 0.1, 1 and 10 µM resveratrol alone, or with 1, 10 and 100 mM nicotinamide alone or pre-treated with 1 µM resveratrol for 4 hours and then co-treated with 1, 10, 100 mM nicotinamide or left untreated for 2 weeks with osteogenic induction medium in high-density cultures. Untreated cultures (without resveratrol or nicotinamide) produced collagen type I (a, A–B) and Runx2 (b, A–B) in both cultures. Incubation with nicotinamide reduced collagen type I and Runx2 production and increased the expression of PPAR-γ in a concentration dependent manner in MSC-cultures (c, A) and decreased the expression of PPAR-γ in a concentration dependent manner in pre-osteoblastic cultures (III, B). However, pre-treatment with resveratrol inhibited the adverse effects of nicotinamide and the osteoblasts produced large amounts of collagen type I and Runx2. Synthesis of the housekeeping protein β-actin was unaffected (d, A–B).