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

摘要/项目摘要 骨或牙本质吸收失调与许多口腔疾病有关，包括牙周炎和 外部牙根吸收，仅举几例。目前，这些疾病的标准诊断依赖于 放射学所见或计算机断层扫描。然而，这些方法通常会检测到 在严重的组织损伤发生的晚期出现的问题，通常会导致牙齿脱落。 此外，成像技术可能不能表明“碎裂”细胞的活动是持续的还是历史的。外切体是 多囊泡体融合后由不同细胞类型释放的内分泌性小泡 与质膜结合。由于它们的蛋白质、脂肪和核酸含量密切反映了 对于亲本细胞的性质和状态，外切体被认为是重要的信息来源。在这里我们 目前的初步数据表明：1)外切体的组成与 吸收骨的碎裂细胞(破骨细胞)与吸收牙本质的碎裂细胞(破牙细胞)，以及2)外体 参与碎裂细胞活动的蛋白质可以在从以下来源收集的牙周沟液中鉴定出来 重新吸收牙齿。因此，我们假设外切体的生化组成存在差异。 在骨和牙本质吸收过程中释放，允许区分 破牙和破骨功能。我们将通过追求以下两个具体目标来检验这一假设： 1)体外分离和鉴定破骨细胞和破牙细胞来源的外体；2) 建立以外切体为基础的检测口腔疾病的临床可行性 骨或牙本质吸收增加。总而言之，这个项目的总体目标是优化 在同时测试口服液来源的外切体的能力的同时进行外切体分析的程序 区分体内破骨细胞活性部位和破牙细胞活性部位。这项研究将为 开发新的、更安全和更便宜的测试来诊断和监测牙周的进展 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.