Treatment of proteoglycan aggregates with physeal enzymes reduces their ability to inhibit hydroxyapatite proliferation in a gelatin gel

Abstract

In vitro, cartilage proteoglycans (PGs) are effective inhibitors of hydroxyapatite formation and growth. Their inhibitory ability decreases with decreasing PG size and charge density. It has been suggested that the enzyme-mediated alteration in the size and conformation of PGs in the growth plate may similarly facilitate the calcification process. In this study, a gelatin gel system was used to monitor hydroxyapatite formation and growth in the presence of proteoglycan aggregates, before and after enzyme treatment. To reproduce the physeal degradation cascade, an enzyme preparation was used that contained all of the growth plate enzymes. At a concentration of 500 micrograms/ml, the untreated proteoglycan aggregates reduced the amount of mineral formed by 30%. When the aggregates were treated with the heat-inactivated enzyme, the same extent of inhibition was found. In contrast, treating the aggregates with the crude growth plate enzyme preparation removed all the inhibitory ability, such that 500 micrograms/ml of proteoglycan preparation yielded 10% more mineral than the controls. Treatment of the aggregates with chondroitinase ABC and trypsin, similarly removed all the inhibitory ability. These data, suggest that enzymatic degradation of proteoglycans may contribute to the regulation of growth plate calcification.