Bone cell culture in a three-dimensional polymer bead stabilizes the differentiated phenotype and provides evidence that osteoblastic cells synthesize type III collagen and fibronectin.

We report a novel method to culture chick embryo osteoblasts in vitro. Primary cells were grown from explants of calvaria and then cultured within alginate polymer beads. Enriched cultures of primary osteoblasts were obtained because these cells grow readily within alginate beads but other cell types present in the initial outgrowth from calvarial fragments, such as fibroblasts, do not. A reproducible bone cell phenotype was observed in calvarial cells cultured in the alginate polymer for as long as 8 months. Alginate is a uronic acid monomer that reversibly polymerizes based on the presence or absence of divalent cations. Osteoblasts derived from the alginate beads elaborated and mineralized an extracellular matrix in vitro that contained fibronectin, type III collagen, and type I collagen. The synthesis and deposition of these matrix molecules was also demonstrated in the chick embryo calvaria in vivo. Together, these in vitro and in vivo observations provide the first evidence that type III collagen and fibronectin colocalize with type I collagen during the development of avian membranous bone. They also indicate that the phenotype of chick embryo osteoblasts can be expanded to include the synthesis of fibronectin and type III collagen.