Selective emergence of differentiated chondrocytes during serum-free culture of cells derived from fetal rat calvaria.

Abstract

Cells dispersed from the chondrocranial portions of fetal rat calvaria proliferated and performed specialized functions during primary culture in a chemically defined medium. Mature cultures were typified by multilayered clusters of redifferentiating cartilage cells. Flattened cells that lacked distinguishing features occupied areas between the clusters. Alkaline phosphate-enriched, ultrastructurally typical chondrocytes within the clusters were encased in a dense extracellular matrix that stained prominently for chondroitin sulfate proteoglycans. This matrix contained fibrils measuring 19 nm in diameter, which were associated with proteoglycan granules that preferentially bound ruthenium red. A progressive increase in the number of cells indicated the proliferation of certain elements in the primary culture. The cells in primary culture were biochemically as well as morphologically heterogeneous since they were found to synthesize type I and type II collagens. Homogeneous populations of redifferentiated chondrocytes were recovered as floating cells and were shown to express the chondrocyte phenotype in secondary culture. Subcultured cells synthesized type II collagen and its precursors almost exclusively and incorporated 35SO4 into proteoglycan monomer and aggregates to a greater degree than the cells in primary culture. The pattern of proteoglycan monomer and aggregate labeling resembled that of intact cartilage segments and bovine articular chondrocytes. Skin fibroblasts harvested from the same rat fetuses failed to proliferate when maintained under identical conditions. Hence, exogenous hormones, growth factors, and protein are not required for chondrocyte growth and maturation.

Chondrogenic potential of skeletal cell populations: Selective growth of chondrocytes and their morphogenesis and development in vitro

“…For example, promotion of chondrogenesis by an absence of serum in growth medium is consistent with studies showing that either embryonic calvarial cell cultures or undifferentiated limb bud cells in serum-free conditions become chondrogenic rather than osteogenic (Cole et al, 1992;Rifas et al, 1982;Toma et al, 1997;Wong and Tuan, 1995). Unlike many other cell types, chondrocytes appear to be specifically adapted to grow in serum-free conditions in vitro (Ballock and Reddi, 1994;Bruckner et al, 1989;Quarto et al, 1992).…”

“…In addition, specific experimental manipulation in vivo such as that causing calcium deficiency by growing chick embryos in a shell-less environment has been shown to give rise to cartilage formation within the calvaria (Jacenko and Tuan, 1986). Such changes to either cartilage or bone during vertebrate development would not be unanticipated considering that cell populations derived from embryonic calvaria from several species exhibit the potential to differentiate into one of several multiple cell lineages including those of osteoblasts (Aronow et al, 1990;Gerstenfeld et al, 1987), adipocytes (Bellows et al, 1994;Beresford et al, 1992;Kodama et al, 1982;Yamaguchi and Kahn, 1991), myoblasts (Grigoriadis et al, 1988;Yamaguchi and Kahn, 1991), and chondrocytes (Asahina et al, 1993;Rifas et al, 1982;Toma et al, 1997). These results indicate that, although not normally observed, there is a subset of cells within the cell populations of the cranial bones that retains chondrogenic potential and can express a cartilage phenotype in vitro.…”

“…These results indicate that, although not normally observed, there is a subset of cells within the cell populations of the cranial bones that retains chondrogenic potential and can express a cartilage phenotype in vitro. The selection of cells having a cartilage phenotype in cultured calvarial cell populations has been shown to occur as a result of a variety of experimental conditions including treatment of the cultures with osteogenic protein (OP-1) (Asahina et al, 1993) or corticosteroids (dexamethasone) (Grigoriadis et al, 1988); growth of the cells on gelatin bone matrix (Terashima and Urist, 1975); or growth of the cells in the absence of serum (Cole et al, 1992;Rifas et al, 1982;Toma et al, 1997). These observations suggest that the epigenetic factors that regulate normal lineage progression of skeletal cells and presumably direct the changes so noted might be both identified and studied in vitro.…”

Pleiotropic effects of the steroid hormone 1,25-dihydroxyvitamin D3 on the recruitment of mesenchymal lineage progenitors in fetal rat calvaria cell populations

“…However, RC cell populations in culture are a heterogeneous mixture of cell types including different differentiation stages of osteoblasts, chondrocytes and adipocytes (Rifas et al 1982, Aubin 1998, Bellows & Heersche 2001. The fact that the clonally-derived RC cell line RCJ 3·1 is capable of differentiating into cartilage, fat, muscle and bone indicates that mesenchymal stem or multipotential progenitor cells are also present in RC cell populations (Grigoriadis et al 1988(Grigoriadis et al , 1990).…”

Bone morphogenetic protein-2 does not alter the differentiation pathway of committed progenitors of osteoblasts and chondroblasts

“…Since calvariae have been thought to be formed through the process of intramembranous ossification, cells isolated from the calvariae are used for studying the regulatory mechanism of osteoblast differentiation. However, the chondroblasts also appear in cultures of calvarial cells (Rifas et al 1982, Bellows et al 1989), although it is not known whether osteoblast progenitor cells can differentiate into chondroblast lineage cells.…”