Biomimetic Apoptotic Particles for Macrophage-driven Oral Bone Regeneration

摘要 口腔中足够的骨骼质量、数量和伤口修复也是治疗合格的关键。 作为牙种植体的短期和长期成功。成功修复有功能的牙列需要 了解内源性骨修复过程。经常被忽视的是骨伤的第一步 创伤后的修复，如拔牙，是指细胞死亡和随后的凋亡细胞(AC)清除 巨噬细胞的吞噬作用。吞噬作用的结果是，巨噬细胞分泌多种因子， 促进再生，并迅速改变他们的行为，以应对大量的物理和生物 微环境线索。巨噬细胞分泌的CC基序趋化因子配体2(CCL2)介导 间充质干细胞/祖细胞(MSPC)向创面募集。尽管以前的工作基本上是 目前提案中的初步数据表明，关注于驱动CCL2生产的生化信号 AC吞噬的物理性质驱动细胞骨架事件和随后的机械转导 发信号。吞噬凋亡细胞导致巨噬细胞形态、肌动蛋白组织和核的改变 可能启动CCL2生产的体系结构。了解泡泡细胞吞噬诱导的细胞内力和 由此产生的信号将为设计凋亡细胞模拟物(ACM)作为患者的再生疗法提供信息 其年龄或合并症损害伤口愈合能力。这个项目的目标是确定角色 泡腾诱导的巨噬细胞机械转导在骨修复中的作用及促进修复 巨噬细胞使用ACM的行为，从而催化内源性骨伤愈合反应。这个 总体假设是巨噬细胞通过胞吐作用促进成骨修复 生物物理信号，可以使用凋亡细胞拟态来重现再生 优势。提出的两个目标是：1)将CCL2表达和巨噬细胞表型与AC联系起来 和ACM吞噬引起的细胞和核骨架的变化，以及2)优化和传递ACM到 促进巨噬细胞驱动的成骨和改善临床相关口腔骨创伤的骨修复 治愈模式。为了实现这些目标，首先是一个允许巨噬细胞的体外共培养系统 将使用AC和ACM吞没。这将是鉴定和验证基因和蛋白质的有价值的工具。 与机械转导和成骨修复相关，在两个实验中都有类似的变化 组。具有可调大小、硬度和可降解性的ACM将被优化，以最大限度地释放CCL2。 它在MSPC招聘中的作用。接下来，我们将使用CCL/R2基因敲除小鼠模型来证实其作用 CCL2在骨再生中的作用我们预计ACM治疗将导致细胞骨架改变，从而导致CCL2 生产和促进骨修复。该项目的成果将确定未被充分开发的后 吞噬巨噬细胞的机械转导和修复活动，提供对骨骼的机械洞察 再生，可用于为患者建立新的骨再生疗法。

ABSTRACT Adequate bone quality, quantity, and wound repair in the oral cavity are crucial for treatment eligibility, as well as short and long-term success of dental implants. Successful restoration of a functional dentition requires an understanding of the endogenous bone repair process. Often overlooked, one of the first steps in osseous wound repair after trauma, such as in a dental extraction, is cell death and subsequent apoptotic cell (AC) clearance (efferocytosis) by macrophages. As a result of efferocytosis, macrophages secrete a variety of factors that facilitate regeneration, and swiftly alter their behavior in response to a multitude of physical and biological microenvironmental cues. CC-motif chemokine ligand 2 (CCL2), which is secreted by macrophages, mediates mesenchymal stem/progenitor cell (MSPC) recruitment to the wound site. Although previous work has largely focused on biochemical signals that drive CCL2 production, preliminary data in the current proposal suggests that the physical nature of AC engulfment drives cytoskeletal events and subsequent mechanotransductive signaling. Engulfment of apoptotic cells induces changes in macrophage shape, actin organization, and nuclear architecture that likely initiates CCL2 production. Understanding efferocytosis-induced intracellular forces and resulting signaling will inform the design of apoptotic cell mimics (ACM) as a regenerative therapy for patients whose age or co-morbidities impair wound healing capacity. The goals of this project are to determine the role of efferocytosis-induced macrophage mechanotransduction in bone repair and to promote reparative macrophage behavior using ACM, hence catalyzing the endogenous osseous wound healing response. The overall hypothesis is that macrophages promote osteogenic repair through efferocytosis-driven biophysical signaling, which can be recapitulated using apoptotic cell mimicry for a regenerative advantage. The two aims proposed are: 1) to connect CCL2 expression and macrophage phenotype with AC and ACM engulfment-induced changes to the cyto- and nucleoskeleton, and 2) to optimize and deliver ACM to promote macrophage-driven osteogenesis and improve bone repair in a clinically relevant oral osseous wound healing model. To accomplish these aims, first an in vitro co-culture system that allows for macrophage engulfment of AC and ACM will be used. This will be a valuable tool to identify and validate genes and proteins associated with mechanotransduction and osteogenic-repair that are similarly altered in both experimental groups. ACM with tunable size, stiffness, and degradability will be optimized to maximize CCL2 secretion given its role in MSPC recruitment. Next, we will use CCL/R2 genetic knockout mouse models to confirm the role of CCL2 in bone regeneration. We anticipate ACM treatment will induce cytoskeletal changes that result in CCL2 production and promote bone repair. The outcomes of this project will identify underexplored effects of post- efferocytosis macrophage mechanotransduction and reparative activity, providing mechanistic insights into bone regeneration, which can be used to establish new bone regeneration therapies for patients.