The in vitro osteogenetic characteristics of primary osteoblastic cells from a rabbit calvarium

Previously, we showed that recombinant human bone morphogenetic protein-2 (rhBMP-2) increased bone augmentation beyond the skeletal envelope within a titanium cap in a rabbit calvarium; many cuboidal osteoblastic cells were observed histologically. These results suggested that the new osteoblastic cells might have differentiated and matured via stimulation by rhBMP-2. To date, however, no studies have reported the characteristics of osteoblastic cells derived from adult rabbit calvarium, after addition of rhBMP-2. To determine the effects of rhBMP-2 on osteoblastic cells, we observed morphological characteristics and alkaline phosphatase activity of osteoblastic cells from an adult rabbit calvarium. The expression of proteins in the BMP signaling pathway and extracellular matrix were analyzed, and mineralized nodule formation was assessed. The alkaline phosphatase activity increased significantly after rhBMP-2 stimulation. The protein levels of phosphorylated-Smad1, Runx2, osteocalcin, osteopontin, and type I collagen were augmented by rhBMP-2 stimulation using Western blotting or ELISA; rhBMP-2 also stimulated mineralized nodule formation with alizarin red staining. The results suggest that primary osteoblastic cells derived from a rabbit calvarium have osteogenetic characteristics in vitro, underscoring the potential use of these cells as a model for studying bone formation. These cells may play an important role in in vivo bone augmentation in a rabbit experimental model.