CHARACTERIZATION OF HUMAN OSTEOBLASTIC CELLS - INFLUENCE OF THE CULTURE CONDITIONS

Human osteoblastic cells were isolated enzymatically from adult human spongy bone and grown in MEM-Ham F12 1:1 medium supplemented with 2% U ltroser (USM). They were subcultured and examined for osteoblast featu res by morphological, histological, and biochemical approaches. The ce lls had a characteristic polyhedral morphology and produced a high lev el of alkaline phosphatase (ALKP). Confluent cultures were uniformly s tained for ALKP and flow cytometry analysis with fluorescein diphospha te gave a single peak signal, reflecting a highly positive population, distinct from cultures of fibroblasts, The ALKP activity was stimulat ed by 1,25 (OH)(2) vitamin D-3. CD 44 was strongly expressed in these cultures, although osteoblasts are negative in vitro and osteocytes ar e positive. The main collagen synthesized was type I collagen and oste ocalcin was produced after stimulation by vitamin D-3. 10 mM beta GP i nduced mineralization and microprobe analysis of the crystals showed a composition close to hydroxyapatite. Changing the culture conditions to MEM-10% calf serum acted on cell behavior: it reduced the productio n of these biochemical markers of osteoblasts and the morphology becam e fibroblastlike with more rapid cell multiplication. The parameter mo st affected by the change in culture medium was ALKP, which was select ed as the determinant criterion for defining an osteoblast culture. AL KP activity was then used to characterize a culture of cells seeded in a collagen gel.