Further biochemical and molecular characterization of primary rat parietal bone cell cultures

Primary bone cell cultures are used widely to examine the regulation of bone metabolism by growth factors and hormones. Characterization of this model system is now being conducted at the molecular level to define modulation of gene expression. Cells were obtained from rat parietal bone by sequential collagenase digestions. Cell populations were evaluated for bone-related products, including collagen isoform expression and mRNA levels, alkaline phosphatase activity, and osteocalcin production. Serum-deprived, confluent cultures of the first and second collagenase-released populations produced a lower percentage of total protein as collagen than the third, fourth, and fifth populations, while co-culturing the third through fifth populations resulted in the highest level. Collagen typing on SDS-polyacrylamide gels revealed an abundance of mature type I collagen in all cell populations; type III collagen synthesis was undetectable by this method. This is in contrast to the presence of cytoplasmic mRNA for both type I and type III collagen in all cell populations, suggesting post-transcriptional modulation of type III collagen synthesis. The expression of alkaline phosphatase and osteocalcin was highest in cultures of later released cells, indicating that these cell populations display phenotypic characteristics associated with cells of the osteoblast lineage.