The goal of this project is to advance our understanding of enamel formation by overcoming an apparent impasse in the field. Although the need for multi-protein studies is clearly recognized, in vitro studies have heavily focused on amelogenin as the key enamel matrix protein. However, the two other matrix proteins ameloblastin and enamelin occur in much lower abundance in the matrix and have extensive posttranslational modifications. Their low abundance precludes purification from animal teeth and attempts to express these proteins in recombinant form have failed because they are not properly post-translationally modified. This has resulted in a limited view of enamel formation because in vitro studies have difficulty addressing the role ameloblastin and enamelin play in enamel formation. Mouse models have shown that both ameloblastin and enamelin are critical for enamel formation and that no enamel forms if either protein is absent. Therefore, a critical need exists to better understand ameloblastin and enamelin function in enamel development. We address this problem by proposing three specific aims and working hypotheses. 1) Enamelin and/or ameloblastin facilitate the conversion of amorphous calcium phosphate (ACP) to hydroxyapatite (HAP) and their cleavage products become part of a support structure for growing crystallites. 2) In the absence of amelogenin, the enamelin and/or ameloblastin proteins still regulate the mineral phase. 3) The inability to remove matrix proteins from Klk4 knockout enamel prevents proper ion movement that then compromises enamel maturation. We will test these hypotheses by characterizing the matrix architecture in situ relative to mineral phase at the mineralization front in wild type and amelogenin ablated mice. Likewise, we will analyze pH, expression levels of ion exchangers and their protein levels, and compare the enamel matrix composition between Klk4 KO mouse versus wild type using mass spectrometry LC-MS/MS. Taking advantage of recent advances in microscopy techniques, we can now perform in situ fluorescence microscopy of specifically labeled enamel matrix proteins correlated with scanning electron microscopy in undermineralized samples. This allows visualization of both protein and crystallites and to extend the scale of analytical resolution from micrometers to nanometers for the study of protein and mineral phase. Importantly, we will be able to address questions of protein distribution around crystallites within prisms and identify mineral phase by further extending the analytic resolution to an atomic scale through in situ atom probe analyses. To examine and visualize the three dimensional organization of matrix and developing crystallites in situ with unprecedented depth of field, we will perform high resolution helium ion microscopy. The realization of this project will substantially advance the field of enamel research by integrating the distribution of all three structural matrix proteins relative to forming mineral into a new concept of enamel formation. This information provides the basis for understand how hierarchically organized composite materials form, which is necessary knowledge for tissue engineering efforts focused on regenerating functional tooth tissues.
Tooth enamel forms in a matrix consisting of three proteins, amelogenin, enamelin, and ameloblastin but the latter two are not well studied since their phosphate and sugar groups prevent them to be purified or produced in bacteria in sufficient quantities. This project takes advantage of recent technological advances in high- resolution imaging and atom probe elemental analysis to test specific hypotheses on the matrix architecture and the role of matrix removal and pH fluctuations during the late stages of mineralization in the mouse model. The realization of this project will provide much needed new insights on the role of enamel proteins to form healthy dental enamel and, to understand the mechanisms for forming hierarchically organized composite materials using biomimetic tissue engineering approaches.
|Bidlack, Felicitas B; Xia, Yan; Pugach, Megan K (2017) Dose-Dependent Rescue of KO Amelogenin Enamel by Transgenes in Vivo. Front Physiol 8:932|