MATRIX PROTEIN CONTROL OF CRYSTAL NUCLEATION AND GROWTH

本项目的目的是确定牙釉质基质蛋白作为牙釉质矿物形成的潜在调节因子的结构-功能关系。我们的工作假设是，可溶性亲水性珐琅质蛋白（如珐琅质）与不溶性基质成分（如淀粉原蛋白聚集体）相结合，提供了适当的表面特征，以诱导磷酸钙成核。在适当浓度的矿物离子存在下，这导致形成非常薄的珐琅矿物带。这些珐琅带在厚度和宽度上的生长立即被疏水蛋白（如溶液中的淀粉原蛋白）所抑制，这些疏水蛋白会吸收到形成晶体的特定表面上。进一步的假设是，在组织成熟过程中，这些矿物带的生长是由釉质蛋白酶对这些吸收的蛋白质抑制剂的降解控制的。为了更好地理解基质蛋白如何控制牙釉质等组织中的矿化，我们提出利用重组淀粉原蛋白和搪瓷素确定主要的牙釉质基质蛋白在羟基磷灰石（HA）和八磷酸钙（OCP）晶体上的吸附等温线。选择HA和OCP作为形成牙釉质矿物的原型进行研究。我们进一步提出确定吸附重组淀粉原蛋白和搪瓷素对HA种子晶体生长的抑制作用，并将这些发现与蛋白质吸附的程度联系起来。我们将进一步评估重组牙釉质蛋白酶（如enameylsin和EMPSl）降解被吸收到HA和OCP表面的牙釉质蛋白的能力，因为这可能是牙釉质蛋白酶控制矿化的重要机制。我们进一步建议评估重组淀粉原蛋白和漆包素在体外促进磷酸钙成核的能力，使用琼脂糖凝胶和与磷酸钙珠共价连接的蛋白质，并影响晶体形态。从长远来看，这些研究可能对新的仿生方法的发展很重要，这些方法可以用于治疗受损或患病的牙齿。

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.