Diabetes, Macrophages and Bone Regeneration

项目总结/摘要 软组织和硬组织伤口愈合在2型糖尿病（T2 DM）中均受损。糖尿病的负面影响 骨内种植体的骨折愈合、骨再生和骨整合。复杂的生理 与糖尿病相关的变化通常表现为对创伤和修复的免疫反应， 巨噬细胞在决定结果方面起着重要作用。最近的发现表明， 免疫系统与骨生理紧密相连，免疫调节是影响骨修复的关键 间充质干细胞（MSC）的相互作用。然而，一个基本的知识差距 在T2 DM存在的情况下，存在关于控制这种相互作用的性质和机制。 我最近发表的研究表明，糖尿病小鼠巨噬细胞的条件培养基损害 MSC的成骨分化。我的研究还表明巨噬细胞分泌表型依赖的 细胞外囊泡（EV）影响骨修复水平。我假设糖尿病巨噬细胞 EV介导骨生成的特异性旁分泌控制。为了验证这一假设，我提出了两个独立的，但 主题相关的目标。在目标1中，我将描述和比较野生型小鼠巨噬细胞EV 在结构和功能水平上对糖尿病小鼠巨噬细胞EV（wtEV）和糖尿病小鼠巨噬细胞EV（dbEV）进行了比较，并定义了 定量分析这些EV对MSC成骨分化的影响。我将展示巨噬细胞的作用 通过干扰Argonaute 2中的miRNA功能（参与RISC复合体），miRNA货物对骨诱导的影响 形成和miRNA功能）敲减MSC。此外，我还将验证dbEV中是否含有miRNAs， 通过从DICER产生EV（成熟miRNA所需的）对骨生成过程产生负面影响 形成）敲除巨噬细胞。在目标2中，我将在dbEV中鉴定出显著不同的miRNA，它们的靶点是 通过生物信息学的方法研究骨发生过程中的基因和信号通路。我将评估 通过利用增加或消除骨诱导的mimics/miRNAs， 识别miRNAs的作用。我将在miRNA相互作用的水平上研究选定的靶基因，以证实 直接影响基因调控和这些关键miRNAs对骨诱导途径的下游作用。 在翻译方面，我将通过工程改造候选miRNA（启动子）来产生功能工程EV（FEE）。 骨生成），其将dbEV效应拯救到MSC EV中。FEE将在结构上具有特点， 在体外功能上。进一步利用颅骨骨缺损模型评价所选miRNAs的功能 FEE对糖尿病小鼠骨愈合的影响。总的来说，这些机制研究将探讨的意义， 巨噬细胞EV介导的免疫调节发生在巨噬细胞和MSC之间， 在T2 DM的情况下骨愈合。这些研究将完善糖尿病病理学知识， 治疗干预的潜在目标。

Project Summary/Abstract Both soft and hard tissue wound healing are impaired in type 2 diabetes (T2DM). Diabetes negatively impacts fracture healing, bone regeneration and osseointegration of endosseous implants. The complex physiological changes associated with diabetes are often manifest in immunological responses to wounding and repair where macrophages play a prominent role in determining outcomes. Recent discoveries have demonstrated that the immune system is tightly linked to bone physiology and immunomodulation of bone repair is affected by key interaction involving macrophages and mesenchymal stem cells (MSCs). Yet, a fundamental knowledge gap exists with respect to the nature of and the mechanisms that govern this interaction in the presence of T2DM. My recent published study has revealed that the conditioned medium from diabetic mouse macrophages impairs osteogenic differentiation of MSCs. My studies also show that macrophages secrete phenotype-dependent extracellular vesicles (EVs) that affect the level of bone repair. Here, I hypothesize that diabetic macrophage EVs mediate specific paracrine control of osteogenesis. To test this hypothesis, I propose two independent but thematically related aims. In Aim 1, I will characterize and compare wild type mouse macrophage EVs (wtEVs) and diabetic mouse macrophage EVs (dbEVs) at the structural and functional level and define the effects of these EVs on MSC osteogenic differentiation quantitatively. I will demonstrate the role of macrophage miRNAs cargo on osteoinduction by interfering miRNA function in Argonaute 2 (involved in RISC complex formation and miRNA function) knockdown MSCs. In addition, I will validate that dbEVs contain miRNAs that negatively influence the process of osteogenesis by generating EVs from DICER (required for mature miRNA formation) knockout macrophages. In Aim 2, I will identify significantly distinct miRNAs in dbEVs, their target genes and signaling pathways involved in osteogenesis by bioinformatic approach. I will evaluate the functionality of these miRNAs on osteoinduction by utilizing mimics/antagomirs that increase or eliminate the effects of identified miRNAs. I will study selected target genes at the level of miRNA interaction to affirm the direct effects on gene regulation and downstream effects of these key miRNAs on osteoinductive pathways. Translationally, I will generate functionally engineered EVs (FEEs) by engineer the candidate miRNAs (promote osteogenesis) that rescue dbEV effects into MSC EVs. The FEEs will be characterized structurally and functionally in vitro. Further I will utilize calvarial bone defect model to evaluate the function of selected miRNAs within FEEs on bone healing in diabetic mice. Overall, these mechanistic studies will explore the significance of the macrophage EV-mediated immunomodulation that occurs between macrophages and MSCs in the context of bone healing in the presence of T2DM. These studies will refine knowledge of diabetic pathology and provide potential targets for therapeutic intervention.