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Chondrocytes isolated from normal adult human articular cartilage were infected with a retroviral vector encoding a temperature-sensitive mutant of the simian virus 40 large tumor antigen and a linked geneticin (G418)-resistance marker. G418-resistant colonies were then isolated, ring-cloned, and expanded in serum-containing media. Several immortalized chondrocyte cell lines were established from the clones that survived, some of which have been maintained in continuous culture for over 2 years. Despite serial subcultures and maintenance as monolayers, these cells retain expression of markers specific for cells of the lineage, namely type II collagen and aggrecan, detected immunocytochemically. We also examined the phenotype of three of these immortalized cell lines (designated HAC [human articular chondrocyte]) using a pellet culture system, and in this report, we present evidence that a prototype of these lines (HAC-F cells) expresses markers normally associated with hypertrophic chondrocytes. When HAC-F cells were cultivated in centrifuge tubes, for periods of up to 63 days, at 39°C with mild and intermittent centrifugation they continued to express both lineage markers; total type II collagen/pellet remained stable, whereas there was a temporal decrease in cartilage-specific glycosaminoglycans content. In addition, in the presence of ascorbate but in the absence of a phosphate donor or inorganic phosphate supplement, the cells also begin to express a hypertrophic phenotype characterized by type X collagen synthesis and extensive mineralization of the extracellular matrix in late stage cultures. The mRNA encoding type X collagen was detected in the cell pellets by reverse transcriptase polymerase chain reaction as early as day 2, and anti-type X collagen immunoreactivity was subsequently localized in the matrix. The mineral was characterized by energy-dispersive X-ray microanalysis as containing calcium (Ca) and phosphorus (P) with a Ca:P peak height ratio close to that of mineralized bone tissue. The unexpected phenotype of this human chondrocyte cell line provides an interesting opportunity for studying chondrocyte maturation in vitro. (J Bone Miner Res 1998;13:432-442)

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Objective. To investigate the pattern of cytokine gene expression in human articular chondrocytes in culture in response to interleukin-lp (IL-lp). The effect of serum and variations in culture conditions was also studied.Methods. Messenger RNA was extracted from cells, reverse-transcribed to complementary DNA, and amplified by the polymerase chain reaction (PCR), using specific oligonucleotide primers. The PCR products were validated by restriction analysis with specific enzymes and by Southern blot analysis.Results. In cultured articular chondrocytes, ILlp, IL-la, granulocyte colony-stimulating factor (CSF), and granulocyte-macrophage CSF cytokine genes were expressed only after induction by IL-1p. However, IL-6, IL-8, and macrophage CSF genes were expressed constitutively. The expression of IL-lP was dose and time dependent.Conclusion. Using PCR, it was possible to demonstrate gene expression for several cytokines in human articular chondrocytes in culture. It was evident that some cytokine genes were expressed constitutively and some were inducible by IL-1p.The biological processes that contribute to joint destruction such as those which occur in rheumatoid arthritis (RA) and osteoarthritis involve interactions among many cytokines and growth factors. It is unlikely that any single cytokine is solely responsible for these events, since individual cytokines have overlapping activities and are produced together in response to particular stimuli. It is, however, important to know how individual cytokines contribute to each physiologic or pathologic process and how they interact with each other. Since many cytokines are involved in normal cellular processes, such as wound healing, bone turnover, proliferation, and differentiation, it may be that the imbalance in relative concentrations of cytokines produced locally precipitates the development of inflammation and joint destruction in pathologic conditions.Many cytokines have been studied in relation to their effect on and production by connective tissue cells. Of particular interest are interleukin-1 (IL-l), tumor necrosis factor a (TNFa), and interleukin-6 (IL-6), which are probably involved in destructive mechanisms in the joint (l), such as prostaglandin