Osteogenesis with cryopreserved marrow mesenchymal cells.

ABSTRACT

Rat marrow cells were collected from the femurs of 7-week-old male rats (Fischer 344), cultured in 75-cm2 flasks for 10 days, released with trypsin, and then frozen and stored at -196 degrees C in liquid nitrogen. Three months later, the cryopreserved marrow cells were rapidly thawed and cultured in porous hydroxyapatite (HA) blocks in osteogenic medium containing 10 mM sodium beta-glycerophosphate, vitamin C phosphate (82 microg/mL), and 10 nM dexamethasone. After 2 weeks of subculture, cultured cells-HA constructs were subcutaneously implanted into syngeneic rats. The constructs were harvested 2 and 4 weeks postimplantation and examined by histological, biochemical, and genetic analyses. Histological examination showed extensive bone formation in the HA pores. High alkaline phosphatase (ALP) activity and high osteocalcin content were detected in the constructs. Expression of ALP and osteocalcin mRNA was observed at both 2 and 4 weeks. These results indicate that artificial bone prepared with cryopreserved cells had a marked osteogenic capacity.