Objective: Bone marrow stromal cells (BMSC) represent an interesting target for novel strategies in the gene and cell therapy of skeletal pathologies, involving BMSC in vitro expansion/transfection and reinfusion.

Materials and Methods: Stromal cells were obtained from healthy donors. For the first 2 weeks, culture medium was supplemented only with human recombinant fibroblast growth factor 2 (FGF-2) to promote cell proliferation and maintain cells in a more immature state. Confluent cultures were detached with trypsin-EDTA. Cells were replated for the in vitro differentiation experiments and for determination of BMSC growth kinetics. Cultures were stimulated with appropriate inductive media and the chondro-/osteo-/adipo-diferentiations were tested by staining with alizarin red, alcian blue, Sudan black and by immunostaining for osteocalcina or collagen II.

Results: After the first passage, BMSC had a markedly diminish proliferation rate and gradually lost their multiple differentiation potential. Their bone-forming efficiency in vivo was reduced by about 36 times at first confluence as compared to fresh bone marrow.

Conclusion: Culture expansion causes BMSC gradually to lose their early progenitor properties. Both the duration and the conditions of culture could be crucial to successful clinical use of these cells and must be considered when designing novel therapeutic strategies involving stromal mesenchymal progenitor manipulation and reinfusion. There are numerous potential applications of this novel strategy, for example: reconstruction of extensive long-bone defects, osteochondral defect repair, treatment of bone cyst, bioactivable scaffolds, etc.