Chondrocytes isolated from bovine articular cartilage were plated at high density and grown in the presence or absence of ascorbate. Collagen and proteoglycans, the major matrix macromolecules synthesized by these cells, were isolated at times during the course of the culture period and characterized. In both control and ascorbate-treated cultures, type II collagen and cartilage proteoglycans accumulated in the cell-associated matrix. Control cells secreted proteoglycans and type II collagen into the medium, whereas with time in culture, ascorbate-treated cells secreted an increasing proportion of types I and III collagens into the medium. The ascorbate-treated cells did not incorporate type I collagen into the cell-associated matrix, but continued to accumulate type II collagen in this compartment. Upon removal of ascorbate, the cells ceased to synthesize type I collagen. Morphological examination of ascorbate-treated and control chondrocyte culture revealed that both collagen and proteoglycans were deposited into the extracellular matrix. The ascorbate-treated cells accumulated a more extensive matrix that was rich in collagen fibrils and ruthenium red-positive proteoglycans. This study demonstrated that although ascorbate facilitates the formation of an extracellular matrix in chondrocyte cultures, it can also cause a reversible alteration in the phenotypic expression of those cells in vitro.

We describe the isolation and the ultrastructural characteristics of adult bovine articular chondrocytes in vitro. Slices of bovine articular cartilage undergo sequential digestions with pronase and collagenase in order to release cells. Chondrocytes are plated at high density (1 x 10(5) cells/cm2) in culture dishes or roller bottles with Ham's F-12 medium, supplemented with 10% fetal bovine serum. Before culture, chondrocytes are freed of surrounding territorial matrix. Within the first few days of culture they re-establish a territorial matrix. As time progresses, chondrocytes synthesize both territorial and extraterritorial matrices. The matrices are rich in collagen fibrils and ruthenium red-positive proteoglycans. These features are most apparent in mass roller cultures in which aggregates of cells and matrix appear as long streaks and nodules. This morphology reveals an organization of chondrocytes and their matrices that is similar to that of the parent articular cartilage in vivo.

The in vitro phenotype of bovine articular chondrocytes is described. Chondrocytes plated at high density in roller-bottle and dish cultures were maintained in vitro. The major matrix macromolecules, collagen and proteoglycan, synthesized by these cells were characterized during the course of the culture period. The chondrocytes synthesized mainly Type II collagen, which was found predominantly in the cell-associated matrix. The media contained a mixture of Type II and Type III collagens. Type I collagen was detectable in neither the medium nor the cell-associated matrix. The proteoglycan monomers found in media and cell-associated matrix had the same hydrodynamic sizes as monomers synthesized by cartilage slices or those extracted from adult articular cartilage. The majority of proteoglycans synthesized by the cells were found in high molecular weight aggregates which were readily recovered from the media and were extractable from cell-associated matrix with low ionic strength buffers. The results demonstrate the long-term in vitro phenotypic stability of the bovine articular chondrocytes. The advantages of the in vitro system as a model for studying the effects of external agents, such as drugs and vitamins, are discussed.

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.

The localization of chick embryonic lysozyme was determined by two techniques: by studying the rate of release from the tissue during sequential enzymatic digestion and by immunocytochemistry. Both techniques indicate that, in this tissue, lysozyme is primarily extra-cellular. Cartilage lysozyme was isolated and partially characterized and found to be identical with egg white lysozyme in its immunologic and enzymatic behavior. In addition, a method for the isolation of large numbers of viable chondrocytes is described.

Some characteristic enzymatic activities were determined in chick embryonic cartilage and compared with the analogous activities in bone and liver. Chondrocytes were isolated, broken by sonication, and subjected to subcellular fractionation to yield a nuclear pellet, the mitochondrial, lysosomal, and microsomal fractions, and the high speed supernatant solution. It was established that these fractions are characterized by enzymatic activities usually associated with similar fractions in other tissues, but with some quantitative differences. Lysozyme, a particulate-associated enzyme in other tissues, was not detected in any subcellular fraction even by the sensitive technique of microzone electrophoresis and is therefore considered to be primarily extracellular in cartilage.

Embryonic mouse femoral cartilage, like the epiphyseal cartilage of the calf scapula, contains large amounts of lysozyme. The addition of egg white lysozyme to organ cultures of embryonic mouse femurs induces unique alterations in the gross and microscopic morphology of the femurs. The sites of these alterations are precisely related to the natural distribution of lysozyme in calf scapula. If the exogenous lysozyme is withdrawn from the culture, the morphological changes disappear, accompanied by a resumption or derepression of growth. The effect on growth is evident only in 17-day embryos. These observations support the idea that lysozyme has a physiological role in cartilage, perhaps related to a regulatory mechanism in bone formation.