MINERALIZATION STUDIES RELATED TO ORAL BIOLOGY

拟议的研究将集中在基质囊泡（MV），发挥作用， 在牙齿和骨骼的矿化中起起始作用。 mv被 亚显微镜下，细胞外，膜投资颗粒，作为 牙本质钙化的起始部位，生长软骨和 发育中的骨骼 我们的实验室参与了第一次鉴定， MV的分离和表征。 我们和其他人提供了 有证据表明，MV磷酸酶，包括碱性磷酸酶（ALP）， 参与MV矿化。 此外，这些磷酸酶是 整合到MV膜中，在MV膜下矿物质首先出现 提示ALP和MV的其他成分的关键作用 膜启动钙化。 我们的具体目标是 为了增加对分子和结构的理解 组织MV膜和SAP，重点是识别和 定位主要组成蛋白，检测MV的功能 磷酸酶在孤立MV钙化机制中的作用， 培养骨髓细胞对MV生物合成调控 骨祖细胞 建议的新研究包括：1）进一步分析的组成， 分离的MV以完成主要蛋白质的鉴定; 2） 主要MV成分分配到膜与囊泡 SAP使用高分辨率EM免疫定位结合生化 通过表面活性剂释放膜附着蛋白质， 洗涤剂的总释放; 3）进一步表征 MV磷酸酶在整个软骨MV矿化中的作用与 在骨细胞或骨髓骨祖细胞的培养物中产生MV。 已知可促进MV的药物，如1，250 H2 D3、地塞米松或氟化物 将检测ALP活性对MV钙化的积极影响; 4)进一步的研究将描述MV生物发生的机制， 培养的大鼠软骨细胞、成骨细胞和骨祖细胞 骨髓基质被已知的促进骨形成的试剂修饰 包括糖皮质激素、氟化物和维甲酸 酸 这是一项关于牙齿和骨骼 开始形成矿化。 矩阵的新知识 囊泡钙化可以应用于广泛的主题，包括 发生异常钙化的特定疾病。

The proposed study will focus on matrix vesicles (MVs) which play an initiating role in the mineralization of teeth and bones. MVs are submicroscopic, extracellular, membrane-invested particles that serve as the initial site of calcification in dentin, growth cartilage and developing bone. Our lab was involved in the first identification, isolation and characterization of MVs. We and others have provided evidence that MV phosphatases, including alkaline phosphatase (ALP), are involved in MV mineralization. Furthermore, these phosphatases are integrated into the MV membrane under which mineral first appears suggesting a critical role for ALP and other components of the MV membrane in initiating calcification. Our specific aims are directed toward an increased understanding of the molecular and structural organization of the MV membrane and sap with emphasis on identifying and localizing major constitutive proteins, testing the function of MV phosphatases in the calcification mechanism of isolated MVs, and studying the regulation of MV biogenesis by cultured bone cells and marrow osteoprogenitor cells. Proposed new studies include: 1) further analysis of the composition of isolated MVs to complete the identification of major proteins; 2) assignment of major MV constituents to the membrane versus the vesicle sap using high resolution EM immunolocalization coupled with biochemical release of membrane-attached proteins by surface active agents versus total release by detergents; 3) further characterization of the role of MV phosphatases in the mineralization of MVs from whole cartilage versus MVs generated in cultures of bone cells or marrow osteoprogenitor cells. Agents such as 1,250H2D3, dexamethasone, or fluoride, known to boost MV ALP activity, will be tested for a positive effect on MV calcification; 4) further studies will characterize the mechanism of MV biogenesis by cultured rat chondrocytes, osteoblasts and osteoprogenitor cells of the marrow stroma as modified by agents known to promote osseous differentiation in vitro including glucocorticoids, fluoride and retinoic acid. This is a fundamental study of the mechanism by which dental and skeletal forms of mineralization are initiated. New knowledge of the matrix vesicle calcification can be applied to a broad range of topics including specific diseases in which abnormal calcification occurs.