Re-expression of differentiated proteoglycan phenotype by dedifferentiated human chondrocytes during culture in alginate beads.

The proteoglycans (PGs) synthesised by normal human articular chondrocytes and a chondrocyte cell line cultured in monolayer and alginate beads were compared. Chondrocytes became dedifferentiated after serial subcultures in monolayer, exhibited a fibroblastic morphology and synthesised a large proportion of lower molecular weight, dermatan sulphate containing PGs. When transferred into alginate beads, the cells quickly regained their spherical shape and actively incorporated [3H]thymidine and [35S]sulphate during 70 days of culture. This resulted in a continuous increase in their DNA content and a rapid deposition of PGs for the first 25 days of culture, which then remained stable. Immediately after dedifferentiated chondrocytes were encapsulated into alginate beads, they began to synthesise a population of PGs with normal monomer size and an increased ability to form aggregates. The monomer size of newly synthesised PGs remained unchanged during extended periods of culture, but their ability to form aggregates and the ratios of chondroitin-6-sulphate to chondroitin-4-sulphate in their glycosaminoglycan chains gradually increased for the first 25 days before reaching normal values. Parallel experiments with HCS-2/8 cells, derived from a human chondrosarcoma, showed that they followed a similar pattern of development in alginate culture. The ability of their newly synthesised PGs to form aggregates increased with time and their sulphation pattern also gradually became normal. These results showed that culture in alginate promoted redifferentiation of dedifferentiated articular chondrocytes and assisted differentiation of HCS-2/8 chondrocytes. However, complete redifferentiation took a period of several weeks, after which synthesis of normal aggregating PGs was maintained.