Evidence for Changes in Protein Polysaccharide Associated with the Onset of Calcification in Cartilage

Past work has suggested that protein polysaccharide may play a role in the calcification of cartilage. Recent electron microscopic studies on noncalcified cartilage have indicated that protein polysaccharide in cartilage matrix is represented by granules associated with collagen fibers. The present work has been designed for comparison of the matrix of noncalcified cartilage to that of calcified cartilage, with particular reference to these granules. Small blocks of tibia from 16-day embryos were fixed in cacodylate-buffered glutaraldehyde and postfixed in either phosphate- or Veronal-buffered osmium tetroxide. Special care was taken to maintain the pH above 7.0 at all times. For electron microscopy the tissues were dehydrated, embedded in Epon 812, sectioned, and stained with uranyl acetate or lead citrate. A marked decrease in the size of granules in the matrix of calcified cartilage compared to noncalcified cartilage was noted. Associated with the decrease in the size of granules was a condensation of matrix components and the presence of an amorphous electron-opaque material that was not seen in noncalcified areas. These results are interpreted to represent either a drop in concentration or a change in state of protein polysaccharide with the onset of calcification in cartilage.