MATRIX PROTEIN CONTROL OF CRYSTAL NUCLEATION AND GROWTH

本项目的目的是确定釉质基质蛋白作为釉质矿物质形成的潜在调节剂的结构-功能关系。我们的工作假设是，可溶性亲水性釉质蛋白（如釉蛋白）与不溶性基质成分（如釉原蛋白聚集体）提供了适当的表面特性，以诱导磷酸钙的成核。在适当浓度的矿物质离子存在下，这导致形成非常薄的釉质矿物质带。这些釉质带在厚度和宽度上的生长立即被疏水蛋白质（例如溶液中的釉原蛋白）抑制，所述疏水蛋白质吸附在形成晶体的特定表面上。进一步假设，在组织成熟过程中，这些矿物质条带的生长开始受到釉质蛋白酶对这些吸收的蛋白抑制剂的降解的控制。为了更好地了解基质蛋白如何控制釉质等组织中的矿化，我们提出使用重组釉原蛋白和釉蛋白来确定主要釉质基质蛋白在羟基磷灰石（HA）和磷酸八钙（OCP）晶体上的吸附等温线。本研究选择HA和OCP作为釉质矿形成的原型，进一步研究了重组釉原蛋白和釉蛋白对HA晶种生长的抑制作用，并将这些结果与蛋白质吸附的程度联系起来。我们将进一步评估重组釉质蛋白酶（例如enameylsin和EMPS 1）降解吸收到HA和OCP表面上的釉质蛋白的能力，因为这可能是釉质蛋白酶可以控制矿化的重要机制。我们进一步建议评估重组釉原蛋白和釉蛋白的能力，以促进磷酸钙成核在体外，使用琼脂糖凝胶和蛋白质共价连接到琼脂糖凝胶珠，并影响晶体形态。从长远来看，这些研究可能对开发可用于治疗受损或患病牙齿的新仿生方法很重要。

The purpose of this project is to determine the structure-function relationship of enamel matrix proteins as potential regulators of enamel mineral formation. Our working hypothesis is that soluble hydrophilic enamel proteins (e.g. enamelin) in association with insoluble matrix components (e.g. amelogenin aggregates) provide the appropriate surface characteristics to induce the nucleation of calcium phosphates. In the presence of appropriate concentrations of mineral ions, this results in the formation of very thin ribbons of enamel mineral. The growth of these enamel ribbons in thickness and width is immediately inhibited by hydrophobic proteins (e.g. amelogenins in solution) which absorb onto specific faces of the forming crystals. It is further hypothesized that the onset of growth of these mineral ribbons during tissue maturation is controlled by the degradation of these absorbed protein inhibitors by enamel proteinases. To gain a better understanding how matrix proteins can control mineralization in a tissue like enamel, we gain a better understanding how matrix proteins can control mineralization in a tissue like enamel, we proposed to determine the adsorption isotherms for the major enamel matrix proteins onto hydroxyapatite (HA) and octacalcium phosphate (OCP) crystals, using recombinant amelogenin and enamelin. HA and OCP were chosen for study as prototypes for forming enamel mineral. We further propose to determine the inhibitory effect of adsorbed recombinant amelogenin and enamelin on HA seeded crystal growth and relate these findings to the extent of protein adsorption. We will further assess the ability of recombinant enamel proteinases (e.g. enameylsin and EMPSl) to degrade enamel proteins which are absorbed onto HA and OCP surfaces, since this may be an important mechanism by which enamel proteinases can control mineralization. We further propose to assess the ability of recombinant amelogenin and enamelin to promote calcium phosphate nucleation in vitro, using agarose gels and proteins covalently linked to sepharose beads, and to influence crystal morphology. Long term, these studies may be important to the development of new biomimetic approaches which could be used to treat damaged or diseased teeth.