Synthesis of Cartilage Matrix by Mammalian Chondrocytes 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 105 cells/cm 2) 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. Hyaline cartilage is a specialized connective tissue whose major function depends on the state of hydration and the structural arrangement of a vast extracellular matrix. As a tissue, cartilage is characterized by a rather homogeneous cell population, which produces structural macromolecules that are the biochemical expressions of this cell's phenotype. The chondrocyte establishes a specialized microenvironment, the territorial matrix , and, in contrast to the majority of cells found in other tissues, exists without direct cell-cell contact. Each cell can be thought of as a functional unit of cartilage and, as such, is ultimately responsible for the turnover of the extracellular matrix of the entire tissue. For these reasons, cartilage represents an attractive and suitable tissue in which development and differentiation can be studied on the biochemical level (27). Such studies have focused largely on avian cartilages, especially for chondrocyte culture in vitro (4, 8-10, 12, 14, 16, 23). Embryonic chick chondrocytes and mesenchyme have been used extensively in tissue culture experiments to study the process of chondrocytic differentiation (4, 14, 23, 27) and the synthesis of specific matrix components, especially collagen types and proteoglycans (8-10, 12, 26, 27). These culture systems have provided many insights into the mechanisms involved in chondrogenesis and have supplied a framework for the understanding of biosynthetic processes of matrix-specific macromolecules and phenotypic stability (1, 2, 5, 26). Mammalian chondrocytes have been isolated for culture from different cartilages including the Swarm rat chondrosar-This system has provided an opportunity to gain insight into the biosynthesis and assembly of proteoglycan aggregates by …