Culture of stromal cells derived from medullary cavity of human long bone in the presence of 1,25-dihydroxyvitamin D 3, recombinant human bone morphogenetic protein-2, or ipriflavone

Abstract

We previously showed that stromal cells derived from bone marrow specimens formed at the fracture site of human long bone differentiated during culture to polygonal cells and spindle cells, and polygonal cells, but not spindle cells, produced calcified matrix. To clarify the origin of polygonal and/or spindle cells, and factors necessary for differentiation of marrow stromal cells to osteogenic cells, we cultured stromal cells derived from the normal (unfractured) medullary cavity (SCN) as well as stromal cells from the medullary cavity distant from the fracture site (SCF). After 3 weeks of primary culture and 2 days of secondary culture, the cells were cultured in medium containing 1,25-dihydroxyvitamin D 3 (VD), recombinant human bone morphogenetic protein-2 (BMP), or ipriflavone (IF) for 3 weeks. For biochemical analysis, cells reaching confluence after 3 weeks of secondary culture were cultured with one of the factors for 3 days. Some of SCF cultured with VD or IF were transformed to polygonal cells, and showed high alkaline phosphatase (ALPase) activity and high osteocalcin and insoluble calcium production. Cloned polygonal cells from the SCF formed nodules and aggregates consisting of calcium. Other SCF cultured with VD or IF and SCF cultured with BMP were spindle shaped. Some spindle-shaped cells from SCF cultured with BMP or IF revealed high ALPase activity and high osteocalcin production, comparable with the spindle cells from the fracture site. However, spindle-shaped cells from SCF cultured with VD and other spindle-shaped cells from SCF cultured with BMP or IF showed low ALPase activity and low osteocalcin production. The results show that SCF probably contain at least three subpopulations: (a) cells that differentiate to polygonal cells by the influence of VD or IF; (b) cells that differentiate to the spindle cells by the influence of BMP or IF; and (c) cells that are not transformed by the influence of VD, BMP, or IF.