Novel Enamel Proteins for Biomineralization Applications

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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. Unlike enamel, the mineralization of bone and dentin occurs in a collagen-based matrix, but the mineral formed is very similar to that of enamel. The localization of AMTN coincides perfectly with the formation of the dense enamel surface layer. Our hypothesis is therefore that AMTN, particularly in its phosphorylated form, is able to promote HA mineralization in enamel, but also in dentin and bone. To test this hypothesis, the current proposal has three main objectives as follows:***1. Determine the role of phosphoserine in AMTN-mediated mineralization***In the first part of this objective we will use a transgenic mouse model we have developed and published to determine whether native AMTN is modified by serine phosphorylation . In the second part we will develop protocols for the recombinant production of AMTN that is phosphorylated at evolutionary conserved 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. Evaluate the mineral-inducing capacity of AMTN and p-AMTN ex vivo***AMTN and (p)AMTN-containing chitosan and collagen matrices will be applied to demineralized enamel and dentin slices prepared from extracted human teeth, respectively, and the degree of remineralization and the nature of the mineral formed will be determined.***3. Evaluate the mineral-inducing capacity of AMTN and p-AMTN in vivo***AMTN and (p)AMTN-containing chitosan and collagen matrices will be applied to demineralized rat molar enamel and calvarial bones. As above, the degree of remineralization and and the nature of the mineral formed will be determined.***Significance:***If successful, the defined mineral-inducing properties of AMTN and p-AMTN will be widely applicable to achieve remineralization and integration of restorative and implant materials into mineralized tissues**