MATRIX BASED MINERAL ENAMEL-BIOMIMETICS

PROJECT SUMMARY/ABSTRACT The goal of this proposal is to provide insight into the structure, dynamic interactions and multifunctionality of the enamel protein ameloblastin (Ambn), and hence to contribute to the fundamental knowledge as relevant to enamel biomineralization. Ambn is multifunctional with critical roles in the regulation of mineral formation, cell differentiation, cell polarization, and cell–matrix adhesion. We recently identified a novel, highly-conserved amphipathic helix (AH) cell binding domain, located within the sequence encoded by exon 5, and adjacent to the C-terminus of the well-established Ambn self-assembly/co-assembly domain. Our preliminary data using two novel CRISPR-Cas9 gene editing mouse models in which different parts of the AH domain are deleted, show that the AH plays essential roles in amelogenesis. Analysis of enamel from homozygous strains with different deletion mutations on AH domain revealed distinct enamel phenotypes that exhibited hypomineralization with different severity. These observations along with recent published reports from our laboratory have led us to hypothesize that Ambn is a matricellular protein, and its complex role in amelogenesis relies on a multitargeting property centered in the region of the sequence encoded by exon 5. Ambn interacts with ameloblast cells via the AH domain and functions to anchor the mineralizing extracellular matrix to enamel-forming cells. To test our hypothesis, we seek to implement a mechanistic approach to interrogate the structure and function of the multitargeting motif in Ambn, including the AH domain. This will be achieved by 1) conducting a detailed investigation of enamel formation in two novel Ambn AH mutant mouse models in vivo (Aim I), 2) examining Ambn-cell interactions in 3-D cell culture (Aim II), and 3) completing an in vitro investigation of interacting domains in the Ambn sequence involved in its self-assembly, amelogenin (Amel), other potential targets, and cell membrane interactions (Aim III). Overall, our studies will advance understanding of the molecular function of Ambn in enamel biomineralization and of inherited enamel disease, elucidate genetic factors leading to caries susceptibility, and contribute to our efforts to regenerate dental hard tissues.