VESICLES AND ENDOCHONDRAL BONE FORMATION

本研究的总体目标是探讨 软骨内骨钙化。 具体来说，我们将测试假设 关于基质囊泡在软骨矿化中的功能， 利用一种新的技术，通过融合这些囊泡， 合成磷脂脂质体。 这些杂交或“重组”囊泡 是研究基质囊泡功能的有用试剂，因为它们可以 构建成含有确定的囊内介质和增强的 膜内容物的选定蛋白质，包括碱性磷酸酶， 长期以来被认为在矿物质代谢中起重要作用的酶。 该提议的一个目的是构建杂交囊泡，其在以下方面不同： 膜的酸性和中性磷脂含量，并评估 这些杂化膜吸收Ca 2+，PO 4和矿化的能力， 体外 此外，我们将构建重组囊泡， 囊泡内pH或离子含量，我们将确定这些是否 施加的离子和pH梯度可以促进矿物的引发， 由囊泡形成。 最后，我们将构建杂交囊泡 富含碱性磷酸酶、蛋白脂质和 钙-磷脂-磷酸盐复合物，所有这些都已被证明， 促进体外矿化。 这些研究的结果将允许 批判性评价长期持有的理论，基质囊泡行动，并 可能会导致对这些神秘的角色的新理解， 颗粒在硬组织的形成。

The overall goal of this research is to investigate basic mechanisms of calcification in endochondral bone. Specifically, we will test hypothesis concerning the function of matrix vesicles in cartilage mineralization, utilizing a new technology for modifying these vesicles byfusing them with synthetic phospholipid liposomes. These hybrid or "recombinant" vesicles are useful reagents for the study of matrix vesicle function, as they can be constructed to contain a defined intravesicular medium and an enhanced membrane content of selected proteins including alkaline phosphatase, an enzyme long considered to play an important role in mineral metabolism. One aim of this proposal is to construct hybrid vesicles which differ in membrane content of acidic and neutral phospholipids, and to assess the ability of these hybrid membranes to take up Ca2+, PO4 and to mineralize in vitro. Further, we will construct recombinant vesicles which differ in intravesicular pH or ion content, and we will ascertain whether these imposed ion and pH gradients can facilitate the initiation of mineral formation by the vesicles. Finally, we will construct hybrid vesicles enriched in alkaline phosphatase, proteolipid and calcium-phospholipid-phosphate complex, all of which have been shown to enhance mineralization in vitro. The results of these studies will permit critical evaluation of long held theories of matrix vesicle action, and are likely to lead to a new understanding of the role of these enigmatic particles in the formation of hard tissues.