Diabetes, Macrophages and Bone Regeneration

项目摘要/摘要 2型糖尿病(T2 DM)患者软硬组织创面愈合均受损。糖尿病带来的负面影响 骨内种植体的骨折愈合、骨再生和骨整合。复杂的生理学 与糖尿病相关的变化通常表现在对创伤和修复的免疫反应中， 巨噬细胞在决定预后方面发挥着重要作用。最近的发现表明， 免疫系统与骨生理密切相关，骨修复的免疫调节受关键 巨噬细胞和间充质干细胞(MSCs)的相互作用。然而，一个根本性的知识差距 存在关于T2 DM存在时的这种相互作用的性质和管理机制。 我最近发表的研究表明，糖尿病小鼠巨噬细胞的条件培养液会损害 骨髓间充质干细胞的成骨分化。我的研究还表明，巨噬细胞分泌表型依赖的 影响骨修复水平的细胞外小泡(EV)。在这里，我假设糖尿病巨噬细胞 EVS介导了成骨的特异性旁分泌控制。为了检验这一假设，我提出了两个独立但 主题相关的目标。在目标1中，我将对野生型小鼠巨噬细胞EV进行表征和比较 (WtEVS)和糖尿病小鼠巨噬细胞EVS(DBEVS)的结构和功能水平，并定义 这些EV对MSC成骨分化的影响是定量的。我将演示巨噬细胞的作用 MiRNAs在干扰ArgAerte2中miRNA功能诱导成骨方面的作用(参与RISC复合体 形成和miRNA功能)击倒间充质干细胞。此外，我将验证DBEV是否包含符合以下条件的miRNA 通过从DICER产生EV(成熟的miRNA所必需的)对成骨过程产生负面影响 形成)敲除巨噬细胞。在目标2中，我将在它们的目标DBEV中识别显著不同的miRNAs 用生物信息学方法研究参与成骨的基因和信号通路。我会评估一下 这些miRNAs通过利用增加或消除骨诱导的模拟/反配子的功能 已识别的miRNAs的影响。我将在miRNA相互作用的水平上研究选定的靶基因，以确认 这些关键miRNAs对基因调控的直接影响和对骨诱导通路的下游影响。 在翻译上，我将通过设计候选miRNAs(推广)来生成功能工程的电动汽车(费用 成骨)，将DBEV效应恢复到MSC EV中。这些费用将在结构上和 在体外的功能上。此外，我将利用颅骨骨缺损模型来评估选定的miRNAs的功能 在糖尿病小鼠的骨愈合费用范围内。总体而言，这些机械论研究将探索 巨噬细胞EV介导的巨噬细胞与间充质干细胞之间的免疫调节 在存在T2 DM的情况下的骨愈合。这些研究将提炼对糖尿病病理的知识，并提供 治疗干预的潜在靶点。

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.