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

Bone marrow stromal cells (BMSCs) for osteoblast differentiation studies ca n be obtained by gradient isolation techniques or by directly plating a fil tered cell suspension. We compared these two procedures to evaluate whether this step is critical in order to obtain a high number of differentiated c olonies. Isolated primary rat BMSCs were cultured in vitro with or without insulin-like growth factor II (IGFII), basic fibroblast growth factor (b-FG F), epidermal growth factor (EGF) or transforming growth factor beta1 (TGF beta1), and histochemically and biochemically analysed at different time po ints. The gradient procedure produced a significantly higher number of colo nies capable of osteoblastic differentiation. The growth factors had differ ent effects. In particular, b-FGF and EGF significantly increased the numbe r of Alizarin red S positive colonies, while IGFII and TGF beta1 exerted in hibitory effects. Nodules obtained on day 21 showed some alkaline phosphata se positive cells and were Von Kossa-positive. These data demonstrate that more differentiated colonies are obtainable from BMSCs isolated by the grad ient procedure.