Characterization of osteoblasts derived from bone marrow stromal cells in a modified cell culture system.

Bone marrow is a complex tissue composed of hematopoietic and stromal stem cells with the potential to differentiate into adipogenic, fibroblastic, reticular, osteogenic and chondrogenic lineages. Identification of differentiation markers during transformation of stromal cells into osteoblasts in a time-dependent manner may be informative for cell-based tissue engineering. Therefore, we investigated the effects of osteogenic medium (OM) on the proliferation and differentiation of rat bone marrow stromal cells (BMSCs). BMSCs from adult male rat tibia and femur were collected and cultured in alpha-MEM medium with 10% fetal bovine serum, penicillin, streptomycin and gentamycin. After three days of culture, the medium covering the adherent cells in culture was changed to OM containing dexamethasone, Na-beta-glycerophosphate and ascorbic acid. As a control, cell culture was also continued in the original medium for the same time period. Differentiated osteoblast cells were collected after 7, 10, 14, 21 and 30 days of culture, fixed with 4% paraformaldehyde and their immunolabelling for osteoblast markers osteonectin (ON) and osteocalcin (OC) was assessed using an indirect immunoperoxidase technique. Immunolabelling of ON and OC was detectable from day 10 of culture, began to increase on day 14, and increased steadily through to day 21. Labelling was highest on day 30 and was more intense in cells cultured with OM compared to the culture without OM. The control cells cultured in the absence of OM produced negligible levels of both markers. In conclusion, our culture system facilitated differentiation of BMSCs into osteoblasts featuring osteoblast markers, and these cells may be useful in autologous bone implant for the treatment of bone wound healing.