An ultrastructural study of osteogenesis in chick periosteum in vitro

We described a chick periosteal osteogenesis model at the light microscopic level and biochemically. To confirm that morphologically identifiable bone is being formed, we performed an ultrastructural study of this model. Samples of freshly dissected periostea, as well as whole calvariae, were fixed in glutaraldehyde. Cultured tissues were subsequently harvested and similarly fixed at 2, 4, and 6 days of culture. Examination of freshly dissected periostea showed that these tissues contained many fibroblast-like cells within a loosely arranged connective tissue. There was no evidence of osteoid or mineral in this tissue. Whole calvarial explants contained well-mineralized bone surrounded by an osteoid seam. At 2 days in culture there was an increase in cellular organization as well as extracellular matrix production. No mineral was detected. By 4 days a non-mineralized osteoid matrix was present, which consisted of densely packed, highly oriented, crossbanded collagen fibrils. Early spherical mineral deposits were seen. The osteoid was surrounded by a multilayer of osteoblast-like cells. The 6-day cultures all contained well-mineralized matrix. The mineral was identified by electron diffraction to be apatite. The data suggest that this in vitro model accurately recapitulates the highly ordered and controlled sequence of events leading to de novo bone formation.