Teeth are tissue composites in which each tissue produces a unique matrix whose protein component(s) direct biomineralization. The outer covering of teeth is a highly ordered, acellular bioceramic called enamel. Enamel is built upon dentin, similar but distinctive from bone. We predict that hierarchical gene expression for dental proteins results in a structural hierarchy. In this application we will acquire information about enamel structural hierarchy needed to produce an enamel biomimetic. The protein template directing the inorganic phase will be analyzed in vitro using recombinant produced an enamel proteins. The unique interface between enamel and dentin, the dentin enamel junction (DEJ), plays a critical role in the biomechanical function of the tooth. Presently there is a paucity of information about DEJ development, the proteins contributing to formation and the organization of these proteins yielding an interface between materials of such dissimilar biomechanical properties. To produce an enamel biomimetic requires the ability to recapitulate the DEJ. We will test the hypothesis that DEJ formation is dependent upon the expression of specific genes encoding structural proteins that undergo admixture at the interface and this mixture at the interface and this mixture is essential to bonding enamel to dentin. We will modulate the DEJ interface and the bulk enamel in transgenic animal using tissue specific promoters to express selected protein at the DEJ and within enamel. The complementary approach of targeted gene deletion will be used to ablate the contribution of selected structural proteins. Genetically stable lines of mice will be created whose teeth will reflect novel morphologic and structural features. A first generation enamel tissue replacement, complete with DEJ, will be produced using immortal ameloblast-like cells organized into a tissue-like three dimensional scaffold yielding an enamel biomimetic complete wit DEJ. Collaborative research with Dr. Baer and Dr. Sarikaya will identify biomechanical properties of teeth from control mice as well as from ~gain or loss of function~ mice. Four specific aims are proposed: 1) Determine the effect(s) of selected tooth specific proteins on mineral deposition and composition in vitro; 2) Using transgenic animals as part of a gain of function test, determine the effects of excessive amounts of selected tooth specific proteins on tooth biomineralization in vivo; 3) Alter the dentin enamel junction and/or bulk enamel using homologous recombination to reduce or eliminate a tooth specific protein in vivo as part of a loss of function test; 4) Produce artificial enamel by providing a three-dimensional framework for enamel biomineralization under the control of reconstituted enamel organ epithelia. These experiments, coupled to collaborative interactions with material scientist at University of Washington and Case Western Reserve University will enable a rational design for a human enamel biomimetic device.