Dexamethasone Stimulates Osteogenesis in Chick Periosteumin Vitro

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Abstract
Folded periosteal explants derived from 16-day old chick embryo calvariae differentiate and form bone when cultured for 6 days in chemically defined, hormone-supplemented medium or on plasma clots. We studied the effect of dexamethasone on generation of cells with osteoblastic phenotype in such cultures. Bone cell phenotype was evaluated by determination of alkaline phosphatase (AP) activity. Cellular proliferation was assessed by measuring ornithine decarboxylase (ODC) activity [3H]thymidine uptake, and radioautography of cultures that had incorporated [3H]thymidine. Cultures were exposed to various medium concentrations of dexamethasone (10-6 M, 10-7 M, 10-8 M) from the outset or from the second or fourth day of culture onwards. In cultures continuously exposed to dexamethasone and maintained in chemically defined media or on plasma clots, dexamethasone increased AP activity measured at day 6. This effect was maximal at 10-7 M dexamethasone. Cultures exposed to dexamethasone after day 2 in culture also showed increased AP activity, but only in the cultures maintained on plasma clots. There was no stimulation of AP activity when dexamethasone was added at day 4 of culture with either medium, thus suggesting that the effect of glucocorticoids depends on the stage of differentiation of the cultures. In addition of AP stimulation, dexamethasone also stimulated ODC activity. Since ODC activity has been associated with mesenchymal cell proliferation, this suggested that dexamethasone stimulated the proliferation of similar cells in the cultured periostea. Measuring [3H]thymidine uptake in and performing autoradiography of control cultures and cultures treated with dexamethasone confirmed that stimulation of proliferation did occur and located this proliferation within the cell layer adjacent to the bone surface. These results demonstrate that dexamethasone stimulates in vitro osteogenesis, and that this effect appears to be mediated through stimulation of progenitor cell proliferation. In addition, our data indicate that factors in the clot medium modulate the responsiveness of the precursor cell population.