Biomimetic Apoptotic Particles for Macrophage-driven Oral Bone Regeneration

用于巨噬细胞驱动的口腔骨再生的仿生凋亡颗粒

• 批准号: 10461732
• 负责人: Rahasudha Kannan
• 金额: $ 4.15万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2023-12-05
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10461732
• 关键词: Actins; Adult; Affect; Age; Aging; Apoptotic; Architecture; Behavior; Biochemical; Biocompatible Materials; Bioinformatics; Biological; Biomedical Engineering; Biomimetics; Biophysics; Bone Regeneration; Bone Transplantation; CCL2 gene; Cell Death; Cells; Coculture Techniques; Cues; Data; Dental; Dental Implants; Dentition; Eligibility Determination; Enzyme-Linked Immunosorbent Assay; Event; Gene Expression Profile; Gene Proteins; Genetic; Goals; Gold; Histology; Hydrogels; Immune system; Impaired wound healing; Impairment; Implant; In Vitro; Infection; Infiltration; Knockout Mice; Mediating; Mesenchymal; Microspheres; Morbidity - disease rate; Mus; Natural regeneration; Nature; Nuclear; Oral; Oral cavity; Osteoblasts; Osteogenesis; Outcome; Pain; Pathway interactions; Patients; Phagocytosis; Phenotype; Population; Process; Production; Proteins; Quality of life; Quantitative Reverse Transcriptase PCR; Role; Shapes; Signal Pathway; Signal Transduction; Site; System; Trauma; Treatment Cost; Work; Wound models; aging population; base; bone healing; bone quality; bone repair; clinical translation; clinically relevant; comorbidity; cost; design; experimental group; improved; insight; macrophage; mechanotransduction; microCT; mimicry; mouse model; novel; osteogenic; particle; permanent tooth; receptor; recruit; regenerative; regenerative therapy; repaired; response; restoration; single-cell RNA sequencing; stem; stem cells; success; tool; wound; wound healing

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.

抽象的 口腔中足够的骨质量、数量和伤口修复对于治疗资格也至关重要 种植牙的短期和长期成功。成功修复功能性牙列需要 了解内源性骨修复过程。经常被忽视，骨伤口的第一步 创伤后修复（例如拔牙）是细胞死亡和随后的凋亡细胞 (AC) 清除 （巨噬细胞胞吞作用）。由于胞吞作用，巨噬细胞分泌多种因子 促进再生，并迅速改变其行为以应对多种物理和生物因素 微环境线索。 CC 基序趋化因子配体 2 (CCL2) 由巨噬细胞分泌，介导 间充质干细胞/祖细胞（MSPC）募集到伤口部位。尽管之前的工作很大程度上 当前提案中的初步数据表明，重点关注驱动 CCL2 产生的生化信号 AC 吞噬的物理性质驱动细胞骨架事件和随后的机械传导 发信号。凋亡细胞的吞噬引起巨噬细胞形状、肌动蛋白组织和核的变化 可能会启动 CCL2 生产的架构。了解胞吞作用诱导的细胞内力和 由此产生的信号传导将为凋亡细胞模拟物（ACM）的设计提供信息，作为患者的再生疗法 其年龄或合并症损害了伤口愈合能力。该项目的目标是确定角色 胞吞作用诱导的巨噬细胞机械转导在骨修复中的作用并促进修复 使用 ACM 调节巨噬细胞行为，从而催化内源性骨伤口愈合反应。这 总体假设是巨噬细胞通过胞吞作用驱动促进成骨修复 生物物理信号传导，可以使用凋亡细胞模拟来重现以实现再生 优势。提出的两个目标是：1）将 CCL2 表达和巨噬细胞表型与 AC 连接起来 和 ACM 吞噬诱导的细胞和核骨架变化，以及 2) 优化和递送 ACM 促进巨噬细胞驱动的成骨并改善临床相关口腔骨伤口的骨修复 治愈模型。为了实现这些目标，首先需要建立允许巨噬细胞的体外共培养系统 将使用 AC 和 ACM 的吞没。这将是识别和验证基因和蛋白质的宝贵工具 与机械转导和成骨修复相关，这在两个实验中都发生了类似的改变 组。具有可调尺寸、刚度和可降解性的 ACM 将经过优化，以最大限度地提高 CCL2 分泌 它在 MSPC 招募中的作用。接下来我们将利用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 治疗将引起细胞骨架变化，从而导致 CCL2 生产并促进骨修复。该项目的成果将确定后研究中尚未充分探索的影响 胞吞作用巨噬细胞机械转导和修复活动，提供对骨的机制见解 再生，可用于为患者建立新的骨再生疗法。