Ultrastructural distribution of acidic glycosaminoglycans associated with matrix vesicle-mediated calcification in mouse progenitor predentine.

The ultrastructural localization of acidic glycosaminoglycans, presumed to be proteoglycans, was examined during initial matrix vesicle-mediated calcification in dentine, by using ruthenium red (RR) staining, high iron diamine thiocarbohydrazide silver proteinate (HID-TCH-SP) staining, and an enzymatic digestion method. Progenitor predentine 2-10 micron width of developing mouse molar tooth germs was used throughout the present study. The outer surface membrane of the intact matrix vesicles had a strong affinity for RR. The RR positive materials appeared beaded and extended perpendicularly from the vesicle membrane. They tended to disappear with the disruption of the vesicular membrane, which resulted from overextension due to needle-like, crystal-like structures. The HID-TCH-SP stain deposits, approximately 10 nm in diameter, were densely distributed around the intact matrix vesicles, though few were found inside them. Some matrix vesicles that were presumably disrupted, however, contained smaller stain deposits. On the outer surface membrane of the disrupted vesicles, HID-TCH-SP stain deposits were fewer in number. The results obtained from enzymatic degradation studies showed that the anionic materials on the outer surface membrane of the matrix vesicles were represented by chondroitin-4-sulfate and/or chondroitin-6-sulfate. We suggest that chondroitin sulfates attached to the outer leaflet of the vesicular membrane play an important role during the incipient stage of the matrix vesicle-mediated calcification process.