| Popis: |
Matrix vesicles (MVs) are involved in the initial deposition of hydroxyapatite (HAp) during bone mineralization, but their mechanism of action is not yet fully understood.In vitrostudies propose two pathways by which MVs can trigger HAp precipitation: the first is mediated by their enhanced phosphatase activity, and the second suggested to depend on structural components present in MVs to mediate nucleation directly from soluble ions without the requirement of phosphatase activity. However, the relevance of these two pathways for bone mineralization and the relationship between MVs and forming mineral in suchin vitroexperiments remains unclear. Here, we used near-native cryoTEM nanoscale imaging in combination with bulk characterizations to disentangle the content and action of MVs duringin vitromineralization. We show that MVs isolation by conventional ultracentrifugation results in heterogeneous dispersions containing non-vesicular particles, including collagens and proteoglycans, in addition to bilayered vesicles. The separation of phosphatase-enriched MVs from non-vesicular particles and comparative mineralization experiments demonstrated that the ability of MVs to induce fast mineralization, independently of phosphatase activity, depends on the presence of non-vesicular particles. Therefore, we conclude that the primary pathway by which MVs trigger mineralization is through the action of their resident phosphatase enzymes, with the direct mineral nucleation to be a secondary event consequential of their membrane components. Lastly, we observed mineral formation restricted to the extravesicular space or in close proximity to the membrane interface, suggesting that the relationship between MVs and forming mineral is more intricate than previously understood. |
