Enamel-derived modulators of biomineralization

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Biomineralization is the basis for formation and maintenance of such important organs as bones and teeth, but the detailed mechanisms of this process are poorly understood. We have earlier identified amelotin (AMTN) as an enamel-specific protein and have demonstrated during the previous grant tenure that AMTN promotes the formation of hydroxyapatite (HA), the mineral of bones and teeth. The overarching goal of this proposal is to optimize and harness the mineral-inducing properties of AMTN for remineralization of enamel and other mineralized tissues. Background: The bulk of dental enamel is formed in an acellular organic matrix consisting predominantly (90%) of the phosphorylated protein amelogenin (AMEL). This matrix guides the formation of a highly organized interwoven network of HA crystal rods, which give enamel its remarkable mechanical properties. The surface of enamel, however, is structurally distinct, fully mineralized and compact, and resembles a mineral "varnish" layer rather than a mineralized mesh work. The localization of AMTN coincides perfectly with the formation of this enamel surface layer. We have demonstrated in extensive recent work that AMTN promotes HA in enamel, but also in other mineralized tissue. We have also shown that phosphorylation of AMTN-derived peptides can enhance their mineral-promoting properties. Based on this work, the current proposal has three main objectives as follows: 1. Evaluate the role of AMTN serine phosphorylation in promoting mineralization We will optimize protocols for the recombinant production of AMTN that is phosphorylated at evolutionary conserved serine residues (pAMTN). The mineral inducing properties of pAMTN will be determined in vitro and compared with those of unmodified AMTN, which we already produce routinely. 2. Produce HA nanoparticles that include AMTN protein or peptides We will produce small HA nanoparticles as carriers for AMTN and derived peptides, both in unmodified and phosphorylated versions, and characterize them in detail. We expect these nanoparticles to enhance the mineral-promoting properties of AMTN and peptides, integrate more readily into pre-existing mineral and prolong the shelf-life of proteins and peptides. 3. Test the mineral-inducing properties of AMTN-HA nanoparticles in vitro and in vivo HA-AMTN nanoparticles will be applied to enamel lesions and demineralized dentin obtained from extracted teeth in vitro. Similarly, demineralized rat molar enamel and calvarial bones will be treated with HA-AMTN nanoparticles to evaluate their effect in vivo. The degree of remineralization and the extent and nature of the mineral formed will be determined. Significance: If successful, the defined mineral-inducing properties of HA-AMTN nanoparticles will be widely applicable to achieve remineralization and integration of restorative and implant materials into mineralized tissues.