Osteogenic Differentiation of Human Mesenchymal Stem Cells in Vitro

INTRODUCTION: Some patients require bone grafts prior to application of endosseous implants and for certain surgical procedures. Collection of bone grafts has typically been invasive and painful, sometimes requiring general anesthesia and inpatient care. A promising alternative is to aspirate patient's bone marrow, expand adult mesenchymal stem cells and differentiate them into osteoblasts that can be used as the autologous graft.

OBJECTIVE: This is a pilot study to show that human mesenchymal stem cells (hMSC's) obtained from volunteers of different age can be expanded and differentiated in vitro into osteoblasts using a commercial growth medium supplemented with autologous serum.

METHODS: Our study involved three human subjects with insignificant medical histories, a 19-year-old female and two males 28 and 66 years old. About 15-20 ml of bone marrow aspirate was collected from the iliac crest and about 60 ml of serum was prepared from venous blood. The cells were cultivated in plastic flasks or Petri dishes in the MSC Basal Medium (Lonza, Inc.) supplemented with L-glutamine (2 mM), Penicillin (10 units/mL), Streptomycin (10 microg/mL) and 10 % subject's non-inactivated serum. Lonza's osteogenic medium contained dexamethasone, ascorbic acid and betaglycerophosphate. Cell counts were used for measurement of growth. Osteoblasts were characterized by immunofluoresecent staining of surface markers, by alkaline phosphatase stain (intracellular enzyme activity) and by Von Kossa stain showing extracellular mineral deposits.

RESULTS: We found practically the same growth and differentiation characteristics of hMSC's obtained from all three subjects. Practically all cells became positive for osteogenic markers.

CONCLUSIONS: It has been shown that hMSC's aspirated from the iliac crest of three volunteers could be successfully isolated, expanded, and differentiated into osteoblasts regardless of age and using commercial medium supplemented with subject's serum.