EFFECTS OF PROTEOGLYCAN MODIFICATION ON MINERAL FORMATION IN A DIFFERENTIATING CHICK LIMB-BUD MESENCHYMAL CELL-CULTURE SYSTEM

In the presence of 4 mM inorganic phosphate, differentiating chick lim b-bud mesenchymal cells plated in micromass cultures form a mineralize d matrix resembling that of chick calcified cartilage. To test the hyp othesis that cartilage proteoglycans are inhibitors of cell mediated m ineralization, the synthesis, content, and turnover of proteoglycans w ere altered in this system, and the extent of mineralization and prope rties of the mineral crystals examined. In all cases where the proteog lycan synthesis or proteoglycans present were modified to provide fewe r or smaller molecules, mineralization was enhanced. Specifically, whe n proteoglycan synthesis was blocked by treatment with 10(-10) M retin oic acid, extensive mineral deposition occurred on a matrix devoid of both proteoglycans and cartilage nodules. The crystals, which formed r apidly, were relatively large in size based on analysis by X-ray diffr action or FT-IR microspectroscopy, and were more abundant than in cont rols. When 2.5 or 5 mM xylosides were used to cause the synthesis of s maller proteoglycans, the extent of mineral accretion was also increas ed relative to controls; however, the matrix was less affected, and th e extent of mineral deposition and the size of the crystals were not a s markedly altered as in the case of retinoic acid. Modification of ex isting proteoglycans by either chondroinase ABC or hyaluronidase treat ment similarly resulted in increased mineral accretion (based on Ca-45 uptake or total Ca uptake) relative to cultures in which the proteogl ycan content was not manipulated. Crystals were more abundant and larg er than in control mineralizing cultures. In contrast, when proteoglyc an degradation by metalloproteases was inhibited by metal chelation wi th o-phenanthroline, the Ca accretion at early time points was increas ed, but as mineralization progressed, Ca accumulation decreased. These data provide evidence that in this culture system, proteoglycans are inhibitors of mineralization. (C) 1997 Wiley-Liss, Inc.