OSTEOGENESIS IN CULTURES OF LIMB MESENCHYMAL CELLS

In previous studies osteoblasts developed in cultures derived from phenotypically unexpressive stage 24 chick limb mesenchymal cells. From this study initial cell plating densities may provide environmental conditions deterministic to a particular limb phenotype. Quantitative microscopic studies, histochemical localization of calcium phosphate and EM indicate that osteoblasts develop in cultures derived from stage 24 limb mesenchymal cells. Of the cells from stage 24 limbs, 1-3% are associated with mineral deposits when plated at initial high densities (5 .times. 106 cells per 35 mm culture dish), while more than 50% of the cells are associated with cartilage by day 9. Cultures plated at intermediate seeding densities (between 2.0 and 2.5 .times. 106 cells per 35 mm culture dish) have minimal cartilage development, and approximately 20% of the cells are associated with mineral by day 9. Cultures prepared from stage 31 limb mesenchymal cells form well-developed bone nodules with both osteoblasts and osteocytes present, but not cartilage. It is clear from these observations and from a consideration of the initiation of osteogenesis in vivo that the initiation of bone development in the limb is not associated with cartilage development. Based on these studies and observations on the effect of nutrient factors on phenotypic expression in culture, an hypothesis is presented relating differential vascularization and nutrient flow to the determination of limb phenotypes in vivo.