MINERAL MATRIX RELATIONS IN CALCIFYING TISSUES

本申请的长期目标是获得 脊椎动物骨骼组织的组织和化学：骨， 软骨和肌腱。 正常人和老年人钙化的研究 异常样品将检查矿物晶体之间的相互作用， 细胞外基质和胶原蛋白，囊泡， 某些磷酸化蛋白质在矿物成核，生长和 发展 矿化作用将首先在正常雏鸡中进行研究， 大鼠、小鼠和火鸡作为脊椎动物模型系统， 包括佝偻病、骨质疏松症、钙化症、 钙过敏症和骨硬化症。 超微结构，生化， 免疫细胞化学和生物物理方法将被应用于确定 矿物和有机基质存在、性质、位置和关系 各组织中的成分。 具体来说，假设 测试是：某些磷蛋白位于细胞外有机 矿物质的沉积需要脊椎动物组织的基质 钙化。 这些磷蛋白与胶原蛋白有关 和/或细胞外囊泡以提供关键成核位点， 矿物形成 矿物的生长和发育 成核受到立体化学和物理化学的严格控制， 约束 检验这一假设的确切目的是：1） 在钙化体系中表征大分子立体化学 胶原纤维及其空间和结构关系， 磷蛋白或细胞外囊泡使用常规和高 电压电子显微镜，三维计算机图像，计算机图像 重建、显微断层扫描和模拟视频技术; 2） 用电子显微镜识别矿物沉积部位 衍射-暗场成像，高分辨电子探针 显微分析和地形成像结合常规和 无水样品制备和3）确定空间和时间 矿物质和磷蛋白、胶原蛋白和其他蛋白之间相互作用 有机基质成分通过上述成像的相关性 方法. 这些数据将有助于了解正常结构， 组织和细胞外的生理功能 矿化组织基质与脊椎动物的基本事件 钙化

The long term objective of this application is to obtain new information of the organization and chemistry of vertebrate skeletal tissues: bone, cartilage, and tendon. Investigation of calcification in normal and abnormal samples will examine interaction between mineral crystals and extracellular matrix and the precise role of collagen, vesicles, and certain phosphorylated proteins in mineral nucleation, growth and development. Mineralization will be studied initially in normal chicks, rats, mice, and turkeys as vertebrate model systems and later in examples of abnormal calcification including rickets, osteoporosis, calcergy, calciphylaxis, and osteopetrosis. Ultrastructural, biochemical, immunocytochemical, and biophysical methods will be applied to determine the presence, nature, location, and relation of mineral and organic matrix components in the respective tissues. Specifically, the hypothesis to be tested is: Certain phosphoproteins located in extracellular organic matrices of vertebrate tissues are required for the deposition of mineral in calcification. These phosphoproteins are associated with collagen and/or extracellular vesicles to provide critical nucleation sites for mineral formation. Growth and development of mineral subsequent to nucleation is under strict control of stereochemical and physicochemical constraints. The precise aims for examining this hypothesis are to 1) characterize in the calcifying systems the macromolecular stereochemistry of collagen fibrils and their spatial and structural relationship with phosphoproteins or extracellular vesicles using conventional and high voltage electron microscopy, three-dimensional computer image reconstruction, microscopic tomography, and simulated video techniques; 2) identify microscopic sites of mineral deposition through electron diffraction-dark field imaging, high resolution electron probe microanalysis, and topographic imaging in conjunction with conventional and anhydrous sample preparation and 3) determine the spatial and temporal interaction between mineral and phosphoproteins, collagen, and other organic matrix constituents through correlation of the above imaging methods. The data will contribute to knowledge of normal structure, organization, and physiological functions of cells and extracellular matrices of mineralizing tissues and fundamental events of vertebrate calcification.