The effects of insulin-like growth factor-I (IGF-I) and insulin on bone matrix synthesis and bone cell replication were studied in cultured 21-day-old fetal rat calvariae. Histomorphometry techniques were developed to measure the incorporation of [2,3-3H]proline and [methyl-3H]thymidine into bone matrix and bone cell nuclei, respectively, using autoradiographs of sagittal sections of calvariae cultured with IGF-I, insulin, or vehicle for up to 96 h. To confirm an effect on bone formation, IGF-I was also studied for its effects on [3H]proline incorporation into collagenase-digestible protein (CDP) and noncollagen protein and on [3H]thymidine incorporation into acid-precipitable material (DNA). IGF-I at 10(-9)-10(-7) M significantly increased the rate of bone matrix apposition and CDP after 24 h by 45-50% and increased cell labeling by 8-fold in the osteoprogenitor cell zone, by 4-fold in the osteoblast cell zone, and by 2-fold in the periosteal fibroblast zone. Insulin at 10(-9)-10(-6) M also increased matrix apposition rate and CDP by 40-50%, but increased cell labeling by 2-fold only at a concentration of 10(-7) M or higher and then only in the osteoprogenitor cell zone. When hydroxyurea was added to IGF-I-treated bones, the effects of IGF-I on DNA synthesis were abolished, but the increase in bone matrix apposition induced by IGF-I was only partly diminished. In conclusion, IGF-I stimulates matrix synthesis in calvariae, an effect that is partly, although not completely, dependent on its stimulatory effect on DNA synthesis.

PTH was studied for its effects on bone formation in cultured rat calvariae. 0.01-10 nM PTH stimulated [Hjthymidine incorporation into DNA by up to 4.8-fold. Although continuous treatment with PTH for 24-72 h inhibited [3Hlproline incorporation into collagen, transient (24 h) treatment enhanced 1H1-proline incorporation into collagen 24-48 h after the hormone was removed. The collagen stimulated by PTH was type I and the effect was observed in the periosteum-free bone and was not blocked by hydroxyurea. Furthermore, treatment with 1-100 nM PTH for 24 h increased insulin-like growth factor (IGF) I concentrations by two to fourfold, and an IGF I antibody prevented the PTH stimulation of collagen synthesis, but not its mitogenic effect. In conclusion, continuous treatment with PTH inhibits calvarial collagen, whereas transient treatment stimulates collagen synthesis, and the stimulatory effect is mediated by local production of IGF I.

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