Enamel-derived modulators of biomineralization

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。我们早些时候已经确定釉质蛋白(amelotin，AMTN)是一种釉质特异性蛋白质，并在之前的拨款期限内证明，AMTN促进骨骼和牙齿的矿物质羟基磷灰石(HA)的形成。这项建议的主要目标是优化和利用AMTN的矿化诱导特性，用于牙釉质和其他矿化组织的再矿化。背景：大部分牙釉质形成于一种无细胞有机基质中，主要由磷酸化蛋白质釉原蛋白(AMEL)组成(90%)。这种基质引导形成高度有序的HA晶棒交织网络，使牙釉质具有显著的机械性能。然而，牙釉质的表面在结构上是独特的，充分矿化和致密，类似于一层矿物“清漆”，而不是矿化的网状结构。AMTN的定位与釉质表面层的形成完全一致。我们在最近广泛的工作中证明了AMTN促进牙釉质中的HA，但在其他矿化组织中也是如此。我们还表明，AMTN衍生的多肽的磷酸化可以增强它们的矿物质促进特性。基于这项工作，目前的建议有三个主要目标如下：*1.评估AMTN丝氨酸磷酸化在促进矿化中的作用*我们将优化在进化保守的丝氨酸残基(PAMTN)处磷酸化的AMTN的重组生产方案。PAMTN的矿物诱导性能将在体外进行测定，并与我们已经常规生产的未经修饰的AMTN进行比较。*2.生产含有AMTN蛋白质或多肽的HA纳米颗粒*我们将生产作为AMTN及其衍生多肽的载体的小HA纳米颗粒，包括未经修饰和磷酸化的版本，并对其进行详细的表征。我们期望这些纳米颗粒能够增强AMTN和多肽的矿物质促进特性，更容易地整合到原有的矿物中，延长蛋白质和多肽的保质期。3.在体外和体内测试AMTN-HA纳米颗粒的矿化诱导性能*HA-AMTN纳米颗粒将应用于牙釉质损伤和脱矿牙本质的体外实验。同样，脱矿的大鼠牙釉质和头盖骨将被HA-AMTN纳米颗粒处理，以评估其在体内的效果。再矿化的程度以及形成的矿物的程度和性质将被确定。*意义：如果成功，HA-AMTN纳米颗粒确定的矿物诱导特性将广泛适用于实现再矿化，并将修复和植入材料整合到矿化组织中。**

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.**