ANALYSIS OF TYPE-I, TYPE-II, TYPE-III, TYPE-IX AND TYPE-XI COLLAGENS SYNTHESIZED BY FETAL BOVINE CHONDROCYTES IN HIGH-DENSITY CULTURE

Objective: This study was undertaken in order to examine phenotypic mo dulation of the chondrocytes more closely in high-density culture cond itions and to clarify the role of ascorbate. Levels of five collagen t ypes were analyzed qualitatively and quantitatively, and their distrib ution was observed in the cell layer and the culture medium. Design: T ypes I, II, III, IX and XI collagens, synthesized by fetal bovine chon drocytes in high-density culture, were analyzed qualitatively and quan titatively by direct measurement of radiolabeled collagens separated b y sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by specific radioimmunoassays. Results: Under the experimental co nditions used in this study (0.6x10(6) cells/cm(2)), chondrocytes did not proliferate in the absence of ascorbate, whereas a twofold increas e in cell number was observed in the presence of ascorbate at day 14. Cartilage-specific collagens (types II, IX and XI) were synthesized th roughout the culture period (up to 47 days), as was type III collagen, which appeared as early as day 1 and was essentially present in the c ulture medium. Partial dedifferentiation of chondrocytes was demonstra ted by the synthesis of type I collagen, which was detected by day 2 i n culture medium containing ascorbate, and by day 6 without ascorbate. After 33 days of culture, a threefold increase in type I collagen syn thesis was observed in culture medium with ascorbate, reaching 66% of the type II collagen content of the cell layer. One month of culture m arked the onset of a progressive decrease in the synthesis of all coll agen types. Conclusions: Under these high-density culture conditions, fetal bovine chondrocytes undergo a time and ascorbate-dependent progr am of partial dedifferentiation. This system provides a simple model f or studying the initial mechanisms of chondrocyte dedifferentiation.