REEXPRESSION OF CARTILAGE-SPECIFIC GENES BY DEDIFFERENTIATED HUMAN ARTICULAR CHONDROCYTES CULTURED IN ALGINATE BEADS

We have used the three-dimensional culture system in alginate beads to redifferentiate human articular chondrocytes which were first expande d on a plastic support. After 15 days in alginate beads, electron micr oscopy showed that cells had synthesized an extracellular matrix conta ining collagen fibrils. Electrophoretic analysis of proline-labeled ce lls demonstrated that redifferentiated chondrocytes synthesized mainly type II collagen and its precursors (pro alpha(1)II, p(c) alpha(1)II, and p(n) alpha(1)II). After pepsin digestion a small amount of collag en type XI was also detected. These results were confirmed by Northern blot analysis of total RNAs. Hybridization with collagen cDNA probes coding for the alpha(1),(II) and alpha 1(I) chains of collagen types I I and I showed that chondrocytes cultured in alginate expressed mainly alpha(1)(II) mRNA, whereas alpha(1)(I) mRNA transcripts were almost u ndetectable. Such a result was observed even after several passages on plastic flasks, suggesting that dedifferentiated cells were able to r evert to a chondrocytic phenotype in this three-dimensional system. Ho wever, SV 40-transformed chondrocytes were not able to redifferentiate in alginate as no alpha(1)(II) mRNAs were detected. Total RNA was con verted into cDNA by reverse transcription and amplified by polymerase chain reaction. This technique was employed to amplify mRNAs specific for collagen type II and type X and the large aggregating proteoglycan aggrecan. Two transcripts resulting from an alternative splicing of t he complement regulatory protein (CRP)-like, domain of aggrecan were o riginally identified in chondrocytes in monolayers. Like intact cartil age, chondrocytes in alginate expressed only the larger transcript wit h the CRP domain, whereas the two transcripts were equally expressed i n SV40-transformed chondrocytes. Thus, the alginate system appears to represent a relevant model for the redifferentiation of human chondroc ytes, especially when only a small cartilage biopsy is available, and could prove useful for pulse-chase studies of patients with skeletal c hondrodysplasias. However it was unable to restore the chondrocytic ph enotype in virally transformed cells. (C) 1994 Academic Press, Inc.