EXPRESSION OF TYPE X COLLAGEN AND MATRIX CALCIFICATION IN 3-DIMENSIONAL CULTURES OF IMMORTALIZED TEMPERATURE-SENSITIVE CHONDROCYTES DERIVEDFROM ADULT HUMAN ARTICULAR-CARTILAGE

Chondrocytes isolated from normal adult human articular cartilage were infected with a retroviral vector encoding a temperature-sensitive mu tant 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 immortal ized chondrocyte cell lines were established from the clones that surv ived, some of which have been maintained in continuous culture for ove r 2 years. Despite serial subcultures and maintenance as monolayers, t hese cells retain expression of markers specific for cells of the line age, namely type II collagen and aggrecan, detected immunocytochemical ly. We also examined the phenotype of three of these immortalized cell lines (designated HAC [human articular chondrocyte]) using a pellet c ulture system, and in this report, we present evidence that a prototyp e of these lines (HAC-F cells) expresses markers normally associated w ith hypertrophic chondrocytes, When HAC-F cells were cultivated in cen trifuge tubes, for periods of up to 63 days, at 39 degrees C with mild and intermittent centrifugation they continued to express both lineag e markers; total type II collagen/pellet remained stable, whereas ther e was a temporal decrease in cartilage-specific glycosaminoglycans con tent. In addition, in the presence of ascorbate but in the absence of a phosphate donor or inorganic phosphate supplement, the cells also be gin to express a hypertrophic phenotype characterized by type X collag en synthesis and extensive mineralization of the extracellular matric in late stage cultures, The mRNA encoding type X collagen was detected in the cell pellets by reverse transcriptase polymerase chain reactio n as early as day 2, and anti-type X collagen immunoreactivity mas sub sequently localized in the matrix. The mineral was characterized by en ergy-dispersive X-ray microanalysis as containing calcium (Ca) and pho sphorus (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 matu ration in vitro.