An elevated number of differentiated osteoblast colonies can be obtained from rat bone marrow stromal cells using a...

Bone marrow stromal cells (BMSCs) for osteoblast differentiation studies can be obtained by gradient isolation techniques or by directly plating a filtered cell suspension. We compared these two procedures to evaluate whether this step is critical in order to obtain a high number of differentiated colonies. Isolated primary rat BMSCs were cultured in vitro with or without insulin-like growth factor II (IGFII), basic fibroblast growth factor (b-FGF), epidermal growth factor (EGF) or transforming growth factor beta 1 (TGF beta 1), and histochemically and biochemically analysed at different time points. The gradient procedure produced a significantly higher number of colonies capable of osteoblastic differentiation. The growth factors had different effects. In particular, b-FGF and EGF significantly increased the number of Alizarin red S positive colonics, while IGFII and TGF beta I exerted inhibitory effects. Nodules obtained on day 21 showed some alkaline phosphatase positive cells and were Von Kossa-positive. These data demonstrate that more differentiated colonies are obtainable from BMSCs isolated by the gradient procedure.