Osteocyte Death in Osteonecrosis of the Jaw in Rice Rats: Role of Necroptosis and Temporal Relationship with Radiographic, Molecular and Histopathologic Findings

项目摘要/摘要 颌骨坏死(ONJ)是一种影响患者口腔的潜在严重、衰弱的疾病。 癌症或骨质疏松症患者服用了抗吸收药物，如唑来膦酸(ZOL)或地诺舒单抗， 并同时有口腔危险因素，如拔牙、牙周炎或根尖周感染。临床 ONJ(1-3期)的定义是患者颌骨外露死骨超过8周。 没有放射治疗或颌骨转移癌的病史。相比之下，早期ONJ(阶段0) 缺乏裸露的骨骼，表现为间歇性疼痛和非特异性的放射学表现。无论是 坏死性颌骨及其死亡的骨细胞存在于早期阶段，而口腔粘膜仍覆盖着 下面的组织，或只有在暴露后才发生，仍有待确定。进一步，类型(S) 细胞死亡对ONJ中骨细胞的影响尚未完全阐明。了解骨细胞死亡是否 阶段0中已经存在的一个特征可能代表着为ONJ寻找新疗法的关键的第一步。的确， 在去除口腔危险因素的同时避免骨细胞死亡的早期药物干预可能 预防ONJ。知识上的差距限制了在ONJ的最早阶段识别ONJ并进行干预以阻止其 进步。骨细胞凋亡在各种骨骼疾病的病理生理学中的关键作用 已经被证实了。然而，鲜有人注意到坏死性下垂，这是一种特殊的“调节”细胞形式。 由炎症引发的死亡，如与牙周炎和根尖周炎有关的炎症。不像 细胞凋亡、坏死性下垂可增强免疫反应和炎症反应。值得注意的是，ZOL治疗了发育不良的大鼠 ONJ显示骨细胞坏死性下垂增加，但未见细胞凋亡。药物靶向抑制物 坏死性上睑下垂的特定调节成分已经开发出来。其中一些目前正在进行临床试验，以治疗 炎症性疾病。因此，我们假设：1)骨细胞死亡发生在骨暴露之前 ONJ；2)骨细胞死亡与特定的放射学、细胞学和 分子特征；3)坏死性下垂是ONJ的主要细胞死亡类型。我们的方法 是使用患有局限性牙周炎的大米鼠，这种牙周炎很容易通过口腔检查进行监测，并开始治疗 与ZOL一起确定： 目标1： 骨质疏松症早期影响骨细胞的细胞死亡时间和类型(S) ONJ和骨细胞死亡与放射学、细胞和分子表现的时间关系；以及 AIM2： 坏死性下垂和细胞凋亡在大米大鼠临床ONJ发生发展中的作用 药理上的抑制剂。研究结果将在时间和机械上定义ONJ的前驱症状， 在人类中可能处于0期，支持开发靶向治疗以阻止骨细胞死亡，以及 为坏死性下垂对ONJ的作用建立直接证据，并在体内验证治疗的概念 抑制其信号通路成分阻止ONJ进展的可能性。

PROJECT SUMMARY/ABSTRACT Osteonecrosis of the jaw (ONJ) is a potentially severe, debilitating condition affecting the mouth of patients with cancer or osteoporosis who have taken antiresorptive drugs, like zoledronic acid (ZOL) or denosumab, and concurrently have an oral risk factor such as tooth extraction, periodontitis, or periapical infection. Clinical ONJ (stages 1-3) is defined by the presence of exposed dead bone in the jaw for longer than 8wks in patients with no history of radiation therapy or metastatic cancer to the jaws. In contrast, early-stage ONJ (stage 0) lacks exposed bone, demonstrating intermittent pain and nonspecific radiographic findings. Whether the necrotic jaw bone with its dead osteocytes is present in the early stages, while the oral mucosa still covers the underlying tissues, or occurs only after it becomes exposed, remains to be determined. Further, the type(s) of cell death affecting osteocytes in ONJ is not completely elucidated. Understanding whether osteocyte death is a feature already present in stage 0 may represent a key first step to finding new therapies for ONJ. Indeed, early pharmacologic interventions that avert osteocyte death while oral risk factors are being removed could prevent ONJ. Gaps in knowledge limit the ability to identify ONJ in its earliest stages and intervene to stop its progression. The critical role of osteocyte apoptosis in the pathophysiology of various skeletal conditions has been substantiated. However, little attention has been paid to necroptosis, a specific “regulated” form of cell death triggered by inflammation, such as that associated with periodontitis and periapical infection. Unlike apoptosis, necroptosis enhances immune responses and inflammation. Notably, ZOL treated rats developing ONJ showed increased necroptosis, but not apoptosis, in osteocytes. Pharmacologic inhibitors targeting specific regulatory components of necroptosis have been developed. Some are now in clinical trials to treat inflammatory diseases. Thus, we hypothesize that: 1) osteocyte death occurs before bone exposure in ONJ; 2) osteocyte death occurs in temporal association with specific radiologic, cellular, and molecular features; and 3) necroptosis is the dominant type of cell death involved in ONJ. Our approach is to use rice rats with localized periodontitis that can be easily monitored by oral exams and start treating them with ZOL to determine: Aim 1: the timing and type(s) of cell death affecting osteocytes in the early stages of ONJ and the temporal relationship of osteocyte death to radiographic, cellular, and molecular findings; and Aim2: the contribution of necroptosis and apoptosis in the development of clinical ONJ in rice rats using pharmacologic inhibitors. The study outcomes will define temporally and mechanistically the prodrome of ONJ, potentially stage 0 in humans, supporting the development of targeted therapies to halt osteocyte death, and establish direct evidence for the role of necroptosis to ONJ and in vivo proof of concept for the therapeutic potential of inhibiting components of its signaling pathway from halting ONJ progression.