This project further explores the mechanisms of enamel development and mineralization in human deciduous teeth. It is concerned with the composition and the detailed physical, chemical and biochemical changes taking place on a molecular level in each of the two main enamel components - protein and mineral - across the different stages of enamel development from early formation during fetal life to complete maturation during postnatal life. The distribution and changes in the concentration of three important trace elements, fluoride, magnesium and carbonate, affecting enamel composition, stability and well being will also be determined across the different stages (forming, maturing and mature) and throughout the entire period of development and mineralization in each of the different human deciduous teeth (maxillary and mandibular incisors, canines and molars). Such information will contribute to our detailed understanding of the basic mechanism by which enamel develops and mineralizes in human deciduous teeth and will provide an important baseline for studying normal and abnormal human amelogenesis. The first part of the project will be concerned with the elucidation of the changes in nature, structure, and chemical composition of the mineral component as well as the changes in F, Mg, and CO3 content across the different stages of development employing microdissecting methods, X-ray diffraction, infra-red spectroscopy and micro-analytical methods, some of which have recently been developed and published by the principal investigator to study enamel mineralization in great detail both in the longitudinal dimension (cervical margin - incisal tip), and width dimension (enamel surface - DEJ). The second part of this project will be concerned with the isolation, purification, identification and characterization of human amelogenins and enamelins, (the two different classes of enamel matrix proteins associated with enamel development and mineralization) and with their distribution within and across the different stages (both in a longitudinal dimension and across the width of enamel) which will be determined quantitatively using micro-dissection methods, sequential extraction methods followed by further purification with PAGE SDS and urea electrophoresis, isoelectric focusing, aminoacid analysis, HPLC, ion exchange and molecular exclusion columns and with the use of specific polyclonal and monoclonal antibodies. One important aspect of this work will be to raise specific antibodies against human enamelins and amelogenins. Finally we intend to carry out sequencing of purified human enamelin (66K) and amelogenin (26K) proteins believed to be involved in the nucleation and control of crystal growth.
