冷凍保存骨髄細胞を用いた培養骨移植による骨形成能の評価

Abstract

To investigate the osteogenic capacity of cryopreserved marrow cells, rat bone marrow cells obtained from femora were culturedin a standard medium for ten days. The cultured cells were released by trypsin treatment and stored at -196-C (liquid nitrogen) in a preservative culture medium containing lO％ dimethylsulfoxide. After 3 months, the cryopreserved cells were thawed and subcultured in porous hydroxyapatite (HA; Interpore500). The medium consisted of Eagle-MEM containing 15％ fetal bovine serum, antibiotics, ascorbic acid, b-glycerophosphate and dexamethasone. After 2 weeks of the subculture, the composites of the cells and porous HA were subcutaneously implanted into syngeneic rats. These implants were harveSted 2 and 4 weeks postimplantation and prepared for histological, biochemical and gene expression analyses. These implants showed bone formation together with active osteoblasts in many pore regions. Northern hybridization showed osteoblast marker of alkaline phosphatase (ALP) and osteocalcin in cryopreserved marrow cells. The course of ALP activities and amounts of osteocalcin derived ftom cryopreserved marrow cells were comparable to those derived from fresh marrow cells. These results indicate the osteogenic capacity of cryopreserved marrow cells in porous HA.