MATRIX VESICLE MEDIATED MATRIX CALCIFICATION

描述（由申请人提供）：自40多年前在钙化软骨基质中首次观察到这些神秘颗粒以来，基质囊泡（MV）在硬组织矿化中的作用一直存在争议。在这些研究和许多最近的调查中，已经表明MV存在于许多矿化组织的基质中，包括软骨，骨和牙本质，并且它们通常被视为矿物晶体首先形成的部位。当这些组织矿化，然而，绝大多数的矿物质被发现与胶原纤维，这是完全渗透和包裹在mineral.A提出的机制MV启动基质矿化的关键问题是要解释如何在MV启动胶原钙化形成的矿物晶体。这就是这项建议所针对的问题。为了回答这个问题，我们提出了三个假设模型，每个模型都提供了一个可能的答案。第一个模型集中在MV酶在改变无机磷酸盐（Pi）和无机焦磷酸盐（PPi）的相对浓度以使细胞外环境允许矿物质直接在基质分子上成核中的作用。第二个模型预测MV在内部使矿物成核，释放小晶体，并且这些晶体为基质的矿化提供种子。第三种模型特异性地表明MV在特定位点与胶原原纤维结合，并且当结合时，它们促进晶体进入胶原原纤维。提出了三个具体目标来测试这些模型并区分它们。第一个实验旨在确定MV是否必须接近胶原纤维以正确矿化它们。如果需要接近，则第一模型不太可能是正确的。接下来的实验将检验MV结合是胶原矿化所必需的这一命题。这些实验将使我们能够区分第二和第三模型。另一组实验将检查MV相关酶重塑胶原纤维以促进晶体进入的可能性。根据重塑酶是MV相关的还是游离在基质中，这些数据将支持模型2或模型3。最后，我们将构建MV的合成模型，并测试MV蛋白和脂质的功能，以更好地区分所提出的假设。当这些实验完成后，我们对MV如何引发基质钙化的认识将大大提高。

DESCRIPTION (provided by applicant): The role of matrix vesicles (MV) in hard tissue mineralization has been debated since these enigmatic particles were first observed more than 40 years ago in the matrix of calcifying cartilage. In those studies, and many more recent investigations, it has been shown that MV are present in the matrix of many mineralizing tissues, including cartilage, bone and dentin, and that they are often seen as the site where mineral crystals first form. When these tissues mineralize, however, the vast majority of the mineral is found associated with collagen fibrils, which are completely permeated and encased in mineral. A key question in the proposed mechanism of MV initiated matrix mineralization is to explain how mineral crystals formed within MV initiate collagen calcification. This is the problem at which this proposal is aimed. To answer this question, we propose three hypothetical models which each provide a possible answer to the question. The first model focuses on the role of MV enzymes in altering the relative concentration of inorganic phosphate (Pi) and inorganic pyrophosphate (PPi) to make the extracellular milieu permissive for mineral to nucleate on matriix molecules directly. The second model predicts that MV nucleate mineral internally, release small crystals and that these crystals seed the mineralization of the matrix. The third model specificies that MV bind to collagen fibrils at specific sites, and that while bound, they facilitate crystal entry into the colagen fibrils. Three Specific Aims are proposed to test these models and distinguish amongst them. The first experiments are designed to decide whether MV must come into proximity with collagen fibrils to correctly mineralize them. If proximity is required, then the first model in unlikely to be correct. The next experiments will examine the proposition that MV binding is necessary for collagen mineralization. These experiment will allow us to distinguish between the 2nd and 3rd models. Another set of experiments will examine the possibility that MV associated enzymes remodel the collagen fibrils to facilitate crystal entry. Depending on whether the remodeling enzymes are MV associated or are free in the matrix, these data will support either model 2 or model 3. Finally, we will construct synthetic models of MV, and test the function of MV proteins and lipids in isolation and together to better distinguish among the proposed hypotheses. When these experiments are completed, our knowledge of how MV initiate matrix calcification will be greatly enhanced.