Interactive Cellular Modulation of Chondrogenic Differentiation in Vitro by Subpopulations of Chick Embryonic Calvarial Cells

Abstract The embryonic chick calvarium normally develops into an intramembranous bone without an intermediate cartilage stage, although cartilage-like calvarial cells have been observed in calcium-deficient chick embryos (Dev. Biol. 115, 215, 1986; Dev. Biol. 133, 221, 1989). To analyze the cellular basis of calvarial development, Incubation Day 14 embryonic chick calvarial cells were fractionated by Percoll gradient isopycnic centrifugation; after 12 days in monolayer culture, a subpopulation of cells (fraction F) was observed to differentiate into a rounded cellular morphology with refractile extracellular matrix. The cartilaginous nature of the extracellular matrix produced by fraction F cells was strongly suggested by the immunodetection of aggrecan and type II collagen, and Alcian blue staining. The other calvarial cell fractions (C, D, E) showed predominantly osteoblastic morphology, expressed alkaline phosphatase activity, and elaborated a collagen type I extracellular matrix. These findings suggest that a "chondrocyte-like" subpopulation of cells exist in the embryonic calvarium. To investigate how cellular interactions may influence the expression of osteogenic versus chondrogenic phenotypes by calvarial cells in vitro, the following cell type combinations were tested in high-density (20 × 106 cells/ml) micromass cultures: (1) total, unfractionated calvarial cells; (2) Percoll fractions (C to F) of calvarial cells; and (3) a highly chondrogenic cell type, Hamburger-Hamilton stage 23/24 chick limb bud mesenchymal cells. The micromass cocultures were set up either by mixing and plating two cell types to form the initial, single micromass or by plating the two cell types as separate, side-by-side cultures in the same culture well. The effects of interactions between cocultured calvarial and limb mesenchymal cells upon their respective diffrentiation fates were separately analyzed, on the basis of the number of Alcian blue-staining cartilage nodules in limb mesenchymal cells and [35S]-sulfate incorporation in both cell types. In cocultures with unfractionated or "osteoblast-like" fraction C and D calvarial cells, limb mesenchymal cells had decreased chondrogenesis. In separate cocultures with "chondrocyte-like" fraction F cells, limb mesenchymal cells exhibited enhanced chondrogenesis. Conditioned media from fraction C and D cells, and from fraction F cells, inhibited and enhanced limb mesenchymal cell chondrogenesis, respectively. Q35S]Sulfate incorporation was greater in (1) unfractionated and fractionated calvarial cells cocultured separately with limb mesenchymal cells, compared to calvarial cells cultured alone, and (2) fraction F cells, compared to other fractions or unfractionated calvarial cells. Interestingly, [35S]sulfate incorporation in fraction F cells was decreased when cocultured with fraction C, D, and E cells. Taken together, these findings show that (1) chondrogenesis in limb mesenchymal cells was inhibited by total, unfractionated and fraction C and D calvarial cells and enhanced by chondrocyte-like fraction F calvarial cells, and (2) the chondrocyte-like phenotype of the fraction F calvarial cells could be modulated by their interaction with chondrogenic (limb mesenchyme) or osteogenic (calvarial osteoblastic fractions) cells. It is proposed that in unfractionated calvarial cells in vitro, or in the calvaria developing in vivo, osteogenic differentiation predominates as a result of regulated inhibition of potential chondrogenic differentiation. As a corollary, aberrant chondrogenesis in the developing calvarium probably results from perturbation of such regulatory mechanisms.