Novel Enamel Proteins for Biomineralization Applications

生物矿化是骨骼和牙齿等重要器官形成和维持的基础，但这一过程的详细机制尚不清楚。我们之前已经确定AMTN （AMTN）是一种珐琅质特异性蛋白，并在之前的授权任期内证明AMTN促进羟基磷灰石（HA）的形成，羟基磷灰石是骨骼和牙齿的矿物质。***本提案的总体目标是优化和利用***AMTN的矿物诱导特性，用于牙釉质和其他矿化组织的再矿化。***背景：大部分牙釉质是在非细胞有机基质中形成的，主要由（90%）磷酸化的淀粉原蛋白（AMEL）组成。这种基质引导形成高度组织的HA晶棒交织网络，从而赋予搪瓷卓越的机械性能。然而，牙釉质的表面结构独特，完全矿化且致密，类似于矿物“清漆”层，而不是矿化的网状物。与牙釉质不同，骨和牙本质的矿化发生在胶原基质中，但形成的矿物质与牙釉质非常相似。AMTN的定位与致密牙釉质表层的形成完全吻合。因此，我们的假设是，AMTN，特别是其磷酸化形式，能够促进牙本质和骨骼中的HA矿化。为了验证这一假设，目前的提案有三个主要目标：***1。确定丝氨酸在AMTN介导的矿化中的作用***在本目标的第一部分，我们将使用我们开发和发表的转基因小鼠模型来确定天然AMTN是否被丝氨酸磷酸化修饰。在第二部分中，我们将开发重组生产进化保守残基（pAMTN）磷酸化的AMTN的方案。将在体外测定pAMTN的矿物诱导性能，并与我们已经常规生产的未经改性的AMTN进行比较。***2。评价AMTN和p-AMTN体外诱导矿物质的能力***将AMTN和含(p)AMTN的壳聚糖和胶原基质分别应用于拔牙制备的脱矿牙釉质和牙本质切片，并测定再矿化程度和形成的矿物质性质。评价AMTN和p-AMTN在体内的诱导矿化能力***AMTN和(p)AMTN含壳聚糖和胶原基质将应用于脱矿大鼠磨牙釉质和颅骨。如上所述，再矿化的程度和形成的矿物的性质将被确定。***意义：***如果成功，AMTN和p-AMTN所定义的矿物诱导特性将广泛应用于实现修复体和种植体材料在矿化组织中的再矿化和整合**

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**