Ultrastructural and Protein Aspects of Apatite Formation in Vertebrate Hard Tissues

Abstract

The central dark line (CDL) which is one of the 100 planes of hydroxyapatite and represents the site of initiation of crystal growth, was observed not only in the crystallites of enamel but in dentin, bone and baleen as well. The area of CDL probably contains high concentration of carbonate ions. It was demonstrated in all the hard tissues tested that the earliest appearance of mineral structure is composed of a ribbon- or disc-shaped precursor mineral plate intimately surrounded by an organic envelope. The CDL appears at the center of the mineral portion as the first step in crystal growth. Carbonic anhydrase activity was observed in the area where crystal nucleation occurs in developing enamel, dentin, cartilage and bone. The enzyme probably contributes the carbonate ion to the crystal nucleation site where the carbonate-rich CDL is being formed. By means of immunoblotting method it has been shown that carbonic anhydrase is one of the major protein constituents of first formed enamel. The similarities in the mineralization process among the apatite forming tissues suggest that the matrices of these hard tissues contain similar groups of proteins, probably derived from basic cytoplasmic proteins. Some of these proteins such as troponin T and I, actin and myosin, were in fact identified in developing enamel matrix by means of the immunoblotting technique. The presence of troponins and actin were also demonstrated in calcifying dentin. Calmodulin was shown to be present only in the later (maturing) stage of enamel. These cytoplasmic proteins contained in the calcifying matrix are in a somewhat altered condition compared with their original form in the cytoplasm. The alteration is probably due to the results of the complicated process of degradation and recombination that occurs in these proteins.