Regulation of DNA Synthesis in Chick Calvaria Cells by Factors from Bone Organ Culture

Embryonic chick bones growing in organ culture release a substance into the culture medium which stimulates bone formation in previously untreated bones. This 'conditioned' medium also enhances proliferation of monolayer cultures of chick calvaria cells in serum-free medium. The active principle is nondialyzable, indicating a molecular weight greater than 12,000 daltons. Dialysis also separates the mitogenic activity from a low-molecuar-weight inhibitor. The amount of the mitogen found in conditioned medium increases as the rate of the bone resorption increases in response to treatment with parathyroid hormone or 1,25-dihydroxyvitamin D₃. Maximal stimulation of DNA synthesis in calvaria cells is evident with conditioned medium obtained 3 to 5 days after treatment of bone cultures with parathyroid hormone. The cells must be treated with the conditioned medium continuously for 20 hr in order to obtain peak enhancement of DNA synthesis; there is no detectable effect in the first 8 hr. In contrast, the inhibitor acts within 4 hr. The data suggest that the stimulatory factor acts to increase cell proliferation by promoting entry of cells into the S phase of mitosis. We conclude that this stimulator is a locally produced regulator of bone formation, probably acting via an increased proliferation in osteoblast precursor cells.