Influence of cartilage particle size and proteoglycan aggregation on immobilization of proteoglycans.

Mechanisms by which proteoglycan aggregates are retained within cartilage were studied using two approaches: mechanical fragmentation of cartilage and reassociation of proteoglycans within extracted cartilage. The extractability of proteoglycans from fresh bovine nasal cartilage with low ionic strength buffer was found to vary from 13 to 45% depending upon the degree of cartilage fragmentation. Forty per cent of those extracted from finely fragmented cartilage (5-20-mu diameter) were found to be in aggregates which contained "link" proteins. A method was developed to reintroduce proteoglycans, which had been extracted with guanidine hydrochloride, into cartilage from which they had been extracted. Stability of the newly formed associations was assessed by re-extraction with a guanidine hydrochloride gradient. Purified proteoglycans alone or reduced and alkylated proteoglycans formed associations which were disrupted with less than 2 M guanidine hydrochloride. Addition of "link" proteins resulted in associations which required 2-4 M guanidine hydrochloride to be re-extracted. Association of proteoglycans into Sepharose 4B particles gave extraction patterns similar to reassociation into extracted cartilage. These findings are consistent with the hypothesis that proteoglycans are immobilized within cartilage through the formation of aggregates and suggest that retention is dependent upon the integrity of the collagen mesh.