MATRIX VESICLES AND CALCIFICATION

这项研究的长期目标是了解软骨内分泌是如何 钙化是骨骼发育和骨折的关键过程 治愈，工作。 由于孤立的基质囊泡（MV）启动 通过获得大量的Ca 2+和Pi来钙化，它们使 一个很好的模型，可以用来制造临床上有用的 用于刺激骨愈合的生物材料。 取得重大进展 在过去的赠款期间， 成矿我们已经确定并表征了Ca 2 + 在纯化MV磷脂酶A2方面取得了进展， 为克隆π转运体奠定了重要基础 我们有 揭示了MV脂质代谢和 表征MV的初始矿物相，并已完成 关于重组核心核设施的大量试点工作， 合成MV的形成。 基于这些发现，我们的主要目标 下一个资助期的任务是阐明这些脂质、蛋白质 和电解质相互作用以触发MV形成和矿化。 由于我们已经描述了Ca 2+转运蛋白、脂质和 核络合物的电解质成分，我们现在将 表征MV Pi离子转运体。 根据梅杰的发现， 在MV矿化过程中脂质成分的变化，我们现在将 描述了两种酶，似乎是负责这些 变化在过去的赠款期间，我们还获得了大量的 关于核络合物性质的光谱数据。 我们现在将使用这些数据来指导我们重建 合成复合物，以及使用分子推导其结构 仿真。 总的来说，这些信息将使我们能够 精确构建可植入的仿生材料， 骨愈合 我们的具体目标是：1）表征三个新的MV 蛋白质，似乎是关键的MV功能，2）进一步 阐明MV的形成，矿物质和代谢，和3）重建 并模拟MV的结构和功能。 具体来说，我们将 表征膜联蛋白，PS， Ca ~（2+）和Pi对MV形成的影响。 第一种矿物的性质 由MV形成的阶段和伴随的MV脂质组成的变化 将对矿化进行检查。 MV Na+依赖性Pi转运蛋白， 磷脂酶A2和磷脂碱交换酶将被 鉴定、克隆和测序，并表征其特性。 然后将合成MV核络合物，表征，并 通过计算机模拟建模。 最后，有了这些信息， 功能性MV将被重建并测试骨愈合情况 特性.

The long-range goal of this research is to understand how endochondral calcification, a process vital for skeletal development and fracture healing, works. Because isolated matrix vesicles (MV) initiate calcification by acquiring large amounts of Ca2+ and Pi, they make an excellent model on which to base the fabrication of clinically useful biomaterials for stimulating bone healing. Major progress was made during the past grant period in characterizing the mechanism of MV mineralization. We have identified and characterized the Ca2+ transporter, made progress in purifying the MV phospholipase A2 and have laid important groundwork in cloning the Pi transporter. We have uncovered important information on MV lipid metabolism and in characterizing the initial mineral phase of MV, and have done substantial pilot work on reconstitution of the nucleational core and formation of synthetic MV. Building on these findings, our major goals for the next grant period are to elucidate how these lipids, proteins and electrolytes interact to trigger MV formation and mineralization. Since we have already characterized the Ca2+ porter, lipid, and electrolyte components of the nucleational complex, we will now characterize the MV Pi ion porter. Based on the finding of major changes in lipid composition during MV mineralization, we will now characterize two enzymes that appear to be responsible for these changes. During this past grant period we have also acquired a large body of spectroscopic data on the nature of the nucleational complex. We will now use this data to guide us in our reconstitution of the synthetic complex, as well as to deduce its structure using molecular simulations. Collectively, this body of information will allow us to precisely construct an implantable biomimetic material for stimulating bone healing. Our specific aims are: 1) to characterize three new MV proteins that appear to be critical to MV function, 2) to further elucidate MV formation, minerals, and metabolism, and 3) to reconstitute and simulate MV structure and function. Specifically we will characterize critical interactions that occur between the annexins, PS, Ca2+ and Pi during MV formation. Also the nature of the first mineral phase formed by MV and changes in MV lipid composition that accompany mineralization will be examined. The MV Na+-dependent Pi-transporter, the phospholipase A2 and the phospholipid base-exchange enzyme will be identified, cloned and sequenced, and their properties characterized. The MV nucleational complex will then be synthesized, characterized, and modeled by computer simulation. Finally, with this information in hand, functional MV will be reconstituted and tested for bone-healing properties.