Characterization of osteoblast-like cells from normal adult rat femoral trabecular bone

Abstract

Osteoblast-like cell cultures have been established from the trabecular surfaces of normal adult rat femoral trabecular bone. The cultured cells responded to stimulation by parathyroid hormone (rPTH), with a rise in intracellular cAMP in excess of 25-fold while failing to respond to incubation with sCT. Furthermore, the osteoblast-like cells exhibited a high level of alkaline phosphatase expression, both histochemically and biochemically. Incubations with 1,25(OH)2 vitamin D3 increased the alkaline phosphatase activity by 50% and stimulated bone Gla-protein (BGP) synthesis. When the cell layers were supplemented with both 50 micrograms/ml ascorbic acid and 10 mM beta-glycerophosphate and allowed to grow past confluency for 3 weeks, they formed calcified ridges and multilayered nodules. Confirmation of the mineralization of an extracellular matrix was made by von Kossa staining. This simple isolation technique now facilitates the availability of normal adult rat osteoblastic cells for investigation of bone and mineral metabolism.