Extracellular Matrix Vesicles and Intercellular Communication of Osteoblast-lineage Cells

项目总结 自体骨移植是目前治疗严重骨缺损和慢性骨质疏松症的金标准 骨不连的治疗效果有限，需要辅助性治疗来促进整体的骨愈合。 胞外囊泡的种类很多，是一种很有前途的无细胞、膜结合的新型囊泡。 再生医学领域的治疗。专门针对骨再生和修复领域的是基质 小泡：由矿化能力强的细胞释放的小的细胞外小泡，锚定在 细胞外基质，是通过软骨内成骨形成矿化骨的必要成分。 它们通过生长板中的小而非编码的microRNA参与细胞信号传递，这表明它们 可能在骨骼中扮演类似的角色。先前的研究表明，软骨细胞衍生的基质囊泡 富含microRNA和其他有助于其影响增殖能力的细胞信号分子 以及靶细胞的分化，然而成骨细胞衍生基质的作用和作用机制 囊泡，尤其是骨骼中的囊泡，而不是生长板，仍有待阐明。有了这些信息， 基质囊泡提供的骨发育和再生的生物中介可能转化为新的 骨科病理学的治疗潜力，包括临界大小的骨缺损和骨折不愈合。 因此，我们假设由成骨细胞系细胞产生的基质小泡，作为成骨细胞系细胞的一个特定子集 细胞外小泡，利用其携带的microRNA促进成骨分化和增殖，驱动 成骨，并有助于骨缺损的愈合。首先，我们的目标是确定成骨细胞- 衍生的基质小泡和另一类细胞外小泡--外小体。接下来，我们的目标是确定 以基质泡microRNA货物为靶点的通路。最后，我们的目标是确定 成骨细胞来源的基质囊泡调节成骨和骨缺损的愈合。为了检验这些目标，我们将 使用成骨细胞系细胞来获得基质小泡，并表征蛋白质的表达、大小和形态。 我们还将使用体外实验模型来评估通路参与和评估共培养反应。 有成骨细胞样细胞。为了测试治疗潜力，我们将使用一种可翻译的体内小鼠模型 长骨缺损处和双正交可注射水凝胶以输送基质囊泡。我们希望能找到 成骨细胞来源的基质小泡是一类特殊的细胞外小泡，含有microRNA小体， 靶向规范的Wnt途径激活细胞信号并导致成骨细胞分化增加 在体外共培养时，靶细胞的数量。我们的体内模型有望显示出改善的愈合 使用水凝胶输送的基质囊泡进行治疗。所有这些共同证明了角色基质囊泡 发挥协调的努力，形成新的骨及其生存能力，作为一种治疗选择，以改善骨骼愈合。

PROJECT SUMMARY Autologous bone grafting, the current gold standard for treating critical sized bone defects and chronic nonunions, has limited success, leaving a need for adjunct treatments to contribute to overall bone healing. Extracellular vesicles, of which there are many types, are a promising new cell-free, membrane-bound therapeutic in the field of regenerative medicine. Specific to the field of bone regeneration and repair are matrix vesicles; small extracellular vesicles released by mineralization-competent cells that become anchored in the extracellular matrix and are a necessary component of mineralized bone formation via endochondral ossification. They are involved in cellular signaling via small, non-coding microRNA in the growth plate, suggesting that they may play a similar role in bone. Previous research indicates that chondrocyte-derived matrix vesicles are enriched with microRNA and other cell signaling molecules that contribute to their ability to influence proliferation and differentiation of target cells, however the role and mechanism of action of osteoblast-derived matrix vesicles, particularly in bone as opposed to the growth plate, remains to be elucidated. With this information, the biological mediation of bone development and regeneration that matrix vesicles provide may translate into novel therapeutic potential for orthopedic pathologies, including critical size bone defects and fracture nonunions. Therefore, we hypothesize that matrix vesicles produced by osteoblast-lineage cells, as a specific subset of extracellular vesicle, use their microRNA cargo to enhance osteogenic differentiation and proliferation, drive osteogenesis, and aid in bone defect healing. First, we aim to determine the relationship between osteoblast- derived matrix vesicles and another class of extracellular vesicle, the exosome. Next, we aim to determine the pathways targeted by matrix vesicle microRNA cargo. Finally, we aim to determine the extent to which osteoblast-derived matrix vesicles modulate osteogenesis and bone defect healing. To test these aims, we will use osteoblast-lineage cells to derive matrix vesicles and characterize protein expression, size, and morphology. We will also use in vitro experimental models to assess pathway involvement and evaluate co-culture response with osteoblast-like cells. To test the therapeutic potential, we will use a translatable in vivo model of a mouse long bone defect and a biorthogonal injectable hydrogel to deliver matrix vesicles. We expect to find that osteoblast-derived matrix vesicles are a specialized subclass of extracellular vesicle with microRNA cargo that targets the canonical Wnt pathway to activate cellular signaling and leads to increased osteoblastic differentiation of target cells when co-cultured in vitro. Our in vivo model is expected to demonstrate improved healing upon treatment with hydrogel-delivered matrix vesicles. All of which together demonstrates the role matrix vesicles play in the coordinated effort to form new bone and their viability as a therapeutic option to improve bone healing.