In vitro Study on the Osteogenetic Capacity of Expanded Human Marrow Mesenchymal Cells - For Development of Advanced Bio-Artificial Bone

摘要

Marrow mesenchymal cells contain stem cells and can differentiate into various types of cells with the capacity to regenerate tissue. Cell proliferation can be substantially enhanced by subculture. However, repeated subculture is known to decrease the differentiation potential as proliferation occurs. We examined human marrow mesenchymal cells to determine the number of passages through which these cells maintained their potential to differentiate into osteogenic cells that could be expected to have an efficient intensifying effect on bone regeneration, ha a 64-year-old man with lumbar spondylosis, 3 ml of marrow fluid was collected from the ilium at surgery. Primary culture was done in T75 flasks and standard medium (MEM containing 15% bovine fetal serum). , Two weeks later, the cultures were trypsinized to prepare a cell suspension. Then 10% of the cell suspension was seeded into a T75 flask and subculture was done in the standard medium. The remaining 90% of the cell suspension was seeded into 6-wellplates. Incubation was carried out in an osteogenic medium that was prepared by adding 10 nM dexamethasone, ascorbic acid and beta-glycerophosphate to the standard medium [Dex (+) cultures]. Two weeks later, the calcium level and ALP activity were measured. A similar procedure was repeated up to the fifth subculture and the subcultured marrow mesenchymal cells were examined for their capacity for differentiation into osteogenic cells. There was significant ALP activity in the first to fifth Dex (+) subcultures, cells up to the second subculture were shown to have the potential to differentiate into osteogenic cells. Calcium was detected in the first and second subcultures. These data indicate that a small volume of marrow fluid could be used for extended osteogenetic therapy and that a large amount of artificial bone with stable osteogenetic capacity could be produced.