Development of an exosome-based diagnostic platform for periodontal disease and root resorption

摘要/项目总结 骨或牙本质吸收失调与许多口腔疾病有关，包括牙周炎和 外牙根吸收等等目前，这些疾病的标准诊断依赖于 放射学发现或计算机断层扫描（CT）扫描。然而，这些方法通常检测出 在晚期出现严重的组织损伤问题，通常导致牙齿脱落。 此外，成像技术可能无法显示“碎屑”细胞活动是正在进行的还是历史性的。外来体是 核内体起源的小泡，在多泡体融合后由不同类型的细胞释放 与细胞膜结合。由于它们的蛋白质、脂质和核酸含量密切反映了 外泌体与它们的亲本细胞的性质和状态有关，外泌体被认为是重要的信息来源。这里我们 目前的初步数据表明，1）从一个人脱落的外泌体的组成存在差异， 吸收骨的碎屑细胞（破骨细胞）与吸收牙本质的碎屑细胞（破牙本质细胞），以及2）外泌体 参与碎屑细胞活性的蛋白质可以在从牙龈组织收集的龈沟液（GCF）中鉴定， 正在吸收的牙齿因此，我们假设外泌体的生化组成存在差异， 在骨和牙本质吸收过程中释放，这将允许区分 破牙和骨质增生功能。我们将通过以下两个具体目标来检验这一假设： 1)体外分离和表征来自破骨细胞和破牙本质细胞的外泌体，以及2） 建立基于外泌体的测定在检测口腔状况中的临床可行性， 存在上调的骨或牙本质再吸收。总之，本项目的总体目标是优化 外泌体分析的程序，同时测试口腔流体来源的外泌体的能力， 在体内区分破骨细胞活性位点和破牙细胞活性位点。该研究将为 开发新的，更安全，更便宜的测试来诊断和监测牙周疾病的进展 疾病和牙根吸收。

ABSTRACT / PROJECT SUMMARY Dysregulated bone or dentin resorption is associated with a host of oral diseases including periodontitis and external root resorption, to name a few. Currently, standard diagnosis of these conditions relies on radiographic findings or computed tomography (CT) scans. Nevertheless, these methods usually detect the problem at an advanced stage where significant tissue damage had occurred, oftentimes leading to tooth loss. Moreover, imaging techniques may not indicate if `clastic' cell activity is ongoing or historical. Exosomes are small vesicles of endosomal origin that are released by different cell types after fusion of multi-vesicular bodies with the plasma membrane. Due to their protein, lipid and nucleic acids contents, which closely reflect the nature and state of their parental cells, exosomes are considered an important source of information. Here we present preliminary data demonstrating that 1) there is a difference in composition of exosomes shed from clastic cells resorbing bone (osteoclasts) vs. clastic cells resorbing dentin (odontoclasts), and 2) exosomal proteins involved in clastic cell activity can be identified in gingival crevicular fluid (GCF) collected from resorbing teeth. Thus, we hypothesize that there is a difference in the biochemical composition of exosomes released during the processes of bone and dentin resorption that would allow for distinction between odontoclastic and osteoclastic function. We will test this hypothesis by pursuing the following two specific aims: 1) To Isolate and characterize exosomes derived from osteoclasts and odontoclasts in vitro, and 2) To establish the clinical feasibility of an exosome-based assay in the detection of oral conditions where upregulated bone or dentin resorption is present. In summary, the overall goal of this project is to optimize procedures for exosome analysis while at the same time testing the capability of oral-fluid derived exosomes to distinguish osteoclast-active sites from odontoclast-active sites in vivo. The study will provide a basis for the development of new, safer and less expensive tests to diagnose and monitor the progression of periodontal disease and dental root resorption.