Cells isolated from fetal rat calvaria by isopycnic separation express different collagen phenotypes

Calvaria from 20-21 day old fetuses were obtained under sterile conditions and the endo- and exoperiosteum stripped off. Cells were dispersed by sequential collagenase-DNase treatment and suspended in 0.5% low Tm agarose in the presence of DMEM supplement with 10% FCS. After 4-5 days of incubation some 30% of these cells showed active synthesis of metachromatic extracellular matrix. Cells from skin, muscle and periosteum failed to show metachromatic matrix positive colonies to a comparable extent. The phenotypic expression of these cells was determined by analysis of collagen types. Eleven day old cultures were incubated in the presence of [3H]-proline plus beta-aminopropionitrile and ascorbic acid and the collagen extracted analyzed by polyacrylamide electrophoresis of their intact chains or CNBr-derived peptides. The results show that anchorage independence is a requirement for calvaria cells to express type II collagen. Type I collagen was preferentially expressed in monolayer culture or when pre-attached to a substrate before being cultured in agarose. Type II collagen was the predominant collagen when cells were cultured in agarose. Further characterization of cell populations was achieved by isopycnic centrifugation in a percoll gradient. Cell fractions were tested for their collagen phenotype when cultured in agarose. Cells recovered from densities 1.04 g/ml or higher synthesized type II collagen, while cells with densities lower than 1.04 g/ml synthesized mainly type I collagen. Isopycnic centrifugation appears to be a novel method for separation of phenotypically different cells from a heterogeneous population in fetal calvaria. The high density cell fractions may represent a mixture of pre-chondrocytes as well as pluripotential cells.