Exosomes in Alveolor Bone Remodeling and Root Resorption

描述(申请人提供)：破骨细胞(吸收骨和牙齿的细胞)和成骨细胞(形成骨的细胞)之间的信号调节正常和病理性的骨和牙根吸收。Exosome是一种小的(直径30-120 nm)细胞外小泡，参与细胞间的信号传递，被认为是潜在的疾病标志物。它们从细胞中释放出来，在细胞表面或内化后与靶细胞相互作用，影响靶细胞的活动。通过对与牙根吸收相关的牙周沟液(GCF)中蛋白质含量变化的蛋白质组学分析，发现了许多在外体中发现的蛋白质。破骨细胞是直接导致牙根吸收的细胞，在体外培养时可以释放外切体。当破骨细胞积极吸收骨时，破骨细胞的外切体的组成发生变化，当加入骨化三醇刺激的小鼠骨髓培养物时，破骨细胞来源的外切体刺激破骨细胞的形成，包括破骨细胞和成骨细胞。这些数据启发了这一提议的中心假设：破骨细胞释放的外体参与骨重建的调节，并作为正常和病理性骨和牙齿吸收的标志物。这一低风险/高回报方案的目标是通过开发技术来分离破骨细胞释放的外切体的亚群，研究分离的外切体的组成，并测试它们的生物学活性，从而在细胞培养中检验这一假说。这有助于建立一个框架，研究外切体在调节骨重建中的作用，并寻找方法将外切体用作牙根和骨吸收的生物标志物。本项目的第一个目标是在体外分离和鉴定破骨细胞释放的外切体。使用抗体(以前用于外切体分离)和凝集素(新型)的亲和层析，以及标准的外切体分离技术，将分离具有不同组成的破骨细胞来源的外切体的亚群。分离的外切体将用电子显微镜、一维和二维质谱进行表征。初步数据表明，吸收破骨细胞的外切体可能具有独特的组成和活性。第二个目的是测试破骨细胞来源的外切体对原代颅骨成骨细胞的调节活性。来自破骨细胞的外体将被添加到分离培养的和小鼠颅骨成骨细胞中.外切体对成骨细胞的影响将在细胞分化、存活、激活和活性的特征分析中得到检验。研究团队在所有建议的方法方面都有丰富的经验。该项目的成功完成将为外周小体在调节骨重建中的作用提供新的研究基础。由于在这方面没有文献报道，我们提出的工作将提供新的基础数据，将导致1)创新的治疗方式治疗牙科疾病和2)未来的翻译工作，使用GCF中的Exosome作为生物标志物，早期检测牙根吸收和病理性牙槽骨吸收。

DESCRIPTION (provided by applicant): Signaling between osteoclasts (the cells that resorb bone and teeth) and osteoblasts (the cells that form bone) regulates normal and pathological bone and root resorption. Exosomes are small (30-120 nm in diameter) extracellular vesicles involved in cell-cell signaling that are being explored as potential disease markers. They are released from cells and interact with target cells, either at the cell surface or after being internalized, to affect the target cell's activity. Proteomic analysis of changes in the protein content of gingival crevicular fluid (GCF) associated with root resorption in humans by mass spectroscopy identified many proteins that have been found in exosomes. Osteoclasts, the cells directly responsible for root resorption, were then shown to release exosomes when cultured in vitro. The composition of the exosomes from osteoclasts changed when osteoclasts were actively resorbing bone, and osteoclast-derived exosomes stimulated osteoclast formation when added to calcitriol-stimulated mouse marrow cultures, which contain both osteoclasts and osteoblasts. These data inspired the central hypothesis of this proposal: Exosomes released by osteoclasts are involved in the regulation of bone remodeling and serve as markers for normal and pathological bone and tooth resorption. The goals of this low risk/high reward proposal are to test this hypothesis in cell culture by developing techniques to isolate subsets of exosomes released by osteoclasts, to study the composition of the isolated exosomes, and to test their biological activities. This can help build a framework for studying exosomes' role in regulating bone remodeling and for finding ways to use exosomes as biological markers for root and bone resorption. The first aim of this project is to isolate and characterize exosomes released from osteoclasts in vitro. Affinity chromatography using antibodies (previously used in exosome isolation) and lectins (novel), together with standard exosome isolation techniques will isolate subsets of osteoclast-derived exosomes with different compositions. The isolated exosomes will be characterized by electron microscopy and one and two- dimensional mass spectroscopy. Preliminary data suggest that exosomes from resorbing osteoclasts may have unique composition and activities. The second aim is to test the regulatory activity of the osteoclast- derived exosomes on primary calvarial osteoblasts. Exosomes from osteoclasts will be added to separate cultures of and mouse calvarial osteoblasts. The effects of exosomes on osteoblasts will be examined in well- characterized assays of cell differentiation, survival, activation, and activity. The team of researchers has extensive experience in all of the proposed methods. Successful completion of this project will provide a basis for novel studies of the role of exosomes in regulating bone remodeling. Because no reports in literature have studied exosomes in this context, our proposed work should provide new fundamental data that will lead to 1) innovative therapeutic modalities to treat dental diseases and 2) future translational effort using exosomes in GCF as biomarkers for the early detection of root resorption and pathological alveolar bone resorption.