This continuation project further explores the mechanisms of enamel development and mineralization in the human tooth. It is concerned with the ultrastructural localization and distribution of major enamel proteins, amelogenins and enamelins, in enamel organ cells and in extracellular matrix throughout human amelogenesis from early formation to maturation. These quantitative ultrastructural localization studies will be carried out using the high resolution protein-A gold immunocytochemical method and employing specific polyclonal and monoclonal antibodies against human amelogenin and against enamelin proteins which have recently been produced in our laboratory. These will reveal the mechanisms of synthesis secretion, degradation and resorption of each of these different enamel proteins and their structural relationship with the growing crystallite across the different stages of development. In addition, the primary structure of the major amelogenin and enamelin proteins will be further elucidated and their corresponding genes identified and localized using molecular biology techniques. A tooth enamel (ameloblast) cDNA library will be constructed using our human mRNA which we have now isolated and showed in Northern hybridization experiments to hybridize with a specifically synthesized amelogenin oligonucleotide and in cell-free translation to code for enamel proteins. The cDNA human amelogenin and enamelin clones will in turn be screened by our polyclonal and monoclonal antibodies as well as our specifically synthesized amelogenin oligonucleotide. Corresponding cDNA probes of amelogenin and enamelin will be isolated an sequenced to decipher the primary structures of amelogenin and enamelin. The corresponding cDNA probes will in turn be used t identify and localize the corresponding gene, as well as to prepare specific RNA probes for in situ hybridization studies to determine gene expression of these proteins throughout human amelogenesis. The final mineralization and maturation of surface mature enamel prior to, during, and after tooth eruption will be studied chemically, biochemically and immunologically by determining the changes in composition, concentration and distribution of both mineral and protein components using microsampling, microchemical and immunological methods developed and already used in our laboratory. Such information, together with our past studies, will further contribute to the detailed understanding of the basic mechanisms by which enamel develops and mineralizes in human teeth, and will provide an important baseline for studying normal and abnormal human amelogenesis.
