MATRIX VESICLE MEDIATED MATRIX CALCIFICATION

描述（由申请人提供）：自从40多年前在钙化软骨基质中首次观察到这些神秘的颗粒以来，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研究中，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最初形成的地方。然而，当这些组织矿化时，发现绝大多数矿物质与胶原原纤维有关，胶原原纤维完全渗透并包裹在矿物质中。在所提出的MV引发基质矿化机制中，一个关键问题是解释MV内形成的矿物晶体如何引发胶原钙化。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，每个模型都为这个问题提供了可能的答案。第一个模型侧重于MV酶在改变无机磷酸盐（Pi）和无机焦磷酸盐（PPi）的相对浓度方面的作用，使细胞外环境允许矿物直接在基质分子上成核。第二个模型预测MV在内部使矿物成核，释放出小晶体，这些晶体为基质的矿化埋下种子。第三种模型说明MV在特定的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加以区分。第一个实验旨在确定MV是否必须接近胶原原纤维才能正确地矿化它们。如果需要接近性，那么第一个模型不太可能是正确的。接下来的实验将检验MV结合对于胶原矿化是必要的这一命题。这些实验将使我们能够区分第二种和第三种模型。另一组实验将检验MV相关酶重塑胶原原纤维以促进晶体进入的可能性。这些数据将支持模型2或模型3，这取决于重塑酶是MV相关的还是在基质中是自由的。最后，我们将构建MV的合成模型，并分别测试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增强。

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.