Abstract
Structural and chemical changes in the sulfated proteoglycans synthesized by "aging" cultures of dispersed chondrocytes were compared to those synthesized by freshly excised, intact cartilage explants in organ culture from chickens of various ages. In vitro, chondrocytes isolated from embryonic chick vertebral cartilages synthesized a proteoglycan monomer characteristic of cartilage (type IV). Monomers synthesized between 3 days and 6 weeks in culture: (i) showed a decrease in average molecular size, which could be correlated, in part, with a decrease in the average size of the chondroitin sulfate chains; (ii) were able to interact with hyaluronic acid to form proteoglycan aggregates regardless of size; (iii) maintained a relatively constant 6S/4S disaccharide ratio of 2.2 for chondroitin sulfate; and (iv) maintained a constant keratan sulfate composition of 5 to 10%. Sulfated proteoglycans synthesized by freshly excised embryonic tibia cartilaginous head and by tibial articular cartilages of 1- and 6-year-old chickens were compared. With age: (i) the type IV monomer size decreased; (ii) the chondroitin sulfate chain size decreased; (iii) the type IV monomers retained their ability to interact with hyaluronic acid; (iv) the 6S/4S disaccharide ratio decreased from 1.7 to 0.6; (v) the keratan sulfate composition increased from 7 to 40%. Although monomer sizes decrease in both the culture and explant systems, in other respects the differences are considerable. These findings suggest that the changes in sulfated proteoglycans observed in subcultured monolayers of chondrocytes must be interpreted with caution when attempting to relate them to the changes that occur in aging in vivo chondrocytes.
Highlights
Structural and chemical changes in thesulfated pro- 2 for reviews)
Monomers This report focuses on the question of whether the strucsynthesized between 3 days and 6 weeks in culture: (i) tural andchemical changes in the typIeV-sulfated proteoglyshowed a decrease in average molecular size, which couldbe correlated, in part, with a decrease in the average size of the chondroitin sulfate chains; (ii) were able to interact with hyaluronic acid to form proteoglycan aggregates regardless of size; (iii) maintained a relatively constant 6S/4S disaccharide ratio of 2.2 for can characteristicof cartilage [10,11,12,13,14] that occurs in vitro are qualitatively equivalent to thoksenown to occur in vivo
Synthesized by both the chondrocyte cultures weresignificantly retarded (Kav= 0.29). These results indicate that the organ-cultured vertebral cartilage type IV monomers have a larger average molecular size than the bovine nasal cartilage proteoglycans,whereas the type IV monomers synthesized by 10-day-oldsecondary passage or 6th-passage chondrocytes in vitro are significantly smaller
Summary
From the Laboratoryof Biochemistry, National Instituteof Dental Research, National Instituteosf Health, Bethesda, Maryland 20205. The precipitated material was collected by centrifugation (10,000rpm X 15 min), resuspended in 4 ml of 4 M guanidine HC1/50 mM sodium acetate, pH 5.8,containing protease inhibitors, 0.1 M 6-aminohexanoic acid, 0.005 M benzamidine, and 0.05 M EDTA, and a few hours later, the material was precipitated again with ethanol/potassium acetate overnight at 4°C This precipitation was repeated a 3rd time. Organ-cultured, intact embryonic, and adult chicken cartilages were labeled for 6 h with ["Slsulfate, washed five times with cold Eagle's minimal essential medium, and extracted in 10 volumes of Gdn-HC1solution by shaking overnight at 4'C. Teoglycans from both the dispersed chondrocyte cultures and from the organ-cultured embryo vertebral and adult cartilages were isolated as follows.After labeling with [35]sulfate,medium and cells were separated.
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