Macrophages Modulate Osteoclast Activity through Plasmin Regulation

摘要 拔牙后适当的骨愈合对恢复功能至关重要。并发症会导致感染， 长期疼痛，患者的治疗选择较少。在愈合过程中，巨噬细胞被募集到该部位 炎症，在损伤后24小时达到峰值，并持续到第7天。此外，巨噬细胞 存在已显示支持骨形成和细胞吞噬作用（凋亡细胞的吞噬作用）， 与骨骼愈合有关。骨吸收是骨质疏松症早期阶段的另一个重要步骤， 伤口愈合用抗吸收剂治疗的患者有发生颌骨骨坏死（ONJ）的风险， 发展ONJ的可能性随着拔牙而显著增加。项目的初步数据 研究者显示纤溶酶原激活物抑制剂2（蛋白质：PAI 2，基因： 当巨噬细胞暴露于凋亡的骨细胞时，巨噬细胞中的SerpinB2），而当暴露于非凋亡的骨细胞时， 骨细胞PAI 2是丝氨酸蛋白酶抑制剂超家族的成员，抑制纤溶酶原激活剂，导致纤溶酶原激活剂的减少。 纤溶酶浓度。纤溶酶作为纤维蛋白溶解系统中的蛋白水解酶具有确定的作用。 纤溶酶还切割细胞外基质中的底物，并参与类骨质层的降解 覆盖骨表面，从而增加骨吸收的敏感性。这个项目的总体目标是 本研究旨在探讨纤溶系统与骨免疫学在口腔创伤愈合中的整合作用。是 假设吞噬巨噬细胞产生PAI 2，PAI 2抑制纤溶酶原激活 导致骨吸收减少和伤口愈合延迟。 提出了两个目的：1）确定巨噬细胞响应于 凋亡成骨细胞吞噬，和2）确定PAI 2缺乏对骨细胞活性的影响， 伤口愈合为了实现这些目标，首先，体外共培养系统，允许巨噬细胞 将使用凋亡骨髓细胞的吞噬。这将是一个宝贵的工具，以衡量增加 PAI 2基因和蛋白在受控环境中的变化。此外，我们将描述PAI 2是否是 细胞内的或分泌的。接下来，我们将使用PAI2来确定口腔窝骨愈合对PAI2的依赖性。 小鼠敲除模型。我们预期，由于纤溶酶的作用，破骨细胞的活性会受到影响。 引发骨吸收。因此，我们将重点评价这些样本的骨密度指标 活动了解骨微环境的恢复机制拓宽了我们的基本知识， 骨生物学和病理学，为未来的治疗干预提供早期靶点。建成后 本建议中描述的实验在确定PAI 2在骨生物学中的作用方面是新颖的， 内稳态，提供新的机制洞察骨微环境和信号通路。在 此外，这些研究将是建立科学经验的基础， 项目首席研究员的科学生涯。

Abstract Proper bone healing following tooth extraction is critical to restore function. Complications can lead to infection, prolonged pain, and fewer treatment options for patients. During healing, macrophages are recruited to the site of inflammation, which peaks 24 hours after injury and persists through day 7. Additionally, macrophage presence has been shown to support bone formation and efferocytosis (phagocytosis of apoptotic cells) is associated with bone healing. Osteoclastic resorption is another essential step in early stages of osseous wound healing. Patients treated with anti-resorptives are at risk for osteonecrosis of the jaw (ONJ), and the possibility of developing ONJ dramatically increases with tooth extraction. Preliminary data by the project investigator showed a significant up-regulation of plasminogen activator inhibitor 2 (protein: PAI2, gene: SerpinB2) in macrophages when they were exposed to apoptotic bone cells, and not when exposed to non- bone cells. PAI2, a member of the serpin superfamily, inhibits plasminogen activators resulting in reduced plasmin concentrations. Plasmin has an established role as a proteolytic enzyme in the fibrinolytic system. Plasmin also cleaves substrates in the extracellular matrix and is implicated in degradation of the osteoid layer covering the bone surface, thus increasing susceptibility to osteoclastic resorption. The overall goal of this project is to study the integration of the fibrinolytic system and osteoimmunology in oral wound healing. It is hypothesized that efferocytosing macrophages produce PAI2, which inhibits plasminogen activation resulting in decreased bone resorption and delayed wound healing. Two aims are proposed: 1) to identify the conditions of PAI2 production by macrophages in response to apoptotic osteoblast engulfment, and 2) to determine the effect of PAI2 deficiency on osteoclastic activity and wound healing. To accomplish these aims, first an in vitro co-culture system that allows for macrophage engulfment of apoptotic bone marrow cells will be used. This will be a valuable tool to measure increases in PAI2 gene and protein changes in a controlled environment. Furthermore, we will delineate whether PAI2 is intracellular or secreted. Next, we will determine the dependence of oral socket bone healing on PAI2 using a murine knock out model. We anticipate there to be an effect on osteoclast activity due to the role of plasmin in initiating bone resorption. Therefore, we will focus evaluation of these samples on measures of osteoclastic activity. Understanding bone microenvironmental mechanisms for recovery broadens our knowledge of basic bone biology and pathology, providing early targets for future therapeutic interventions. When completed, the experiments described in this proposal will be novel in establishing the role of PAI2 in bone biology and homeostasis, providing new mechanistic insights into the bone microenvironment and signaling pathways. In addition, these studies will be foundational in establishing scientific experiences that will launch a successful scientific career for the project principal investigator.