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

项目概要/摘要 颌骨骨坏死（ONJ）是一种潜在的严重，衰弱的条件，影响口的病人 患有癌症或骨质疏松症的患者服用抗吸收药物，如唑来膦酸（ZOL）或狄诺塞单抗， 并同时具有口腔风险因素，例如拔牙、牙周炎或根尖周感染。临床 ONJ（1-3期）定义为患者颌骨中存在暴露的死骨超过8周 没有放射治疗史也没有转移到颌骨的癌症相反，早期ONJ（0期） 无骨外露，表现为间歇性疼痛和非特异性影像学表现。是否 坏死的颌骨及其死亡的骨细胞存在于早期阶段，而口腔粘膜仍然覆盖着颌骨。 是否发生在潜在组织中，或仅在暴露后发生，仍有待确定。此外， ONJ中影响骨细胞的细胞死亡尚未完全阐明。了解骨细胞死亡是否是 阶段0中已经存在的特征可能代表了寻找ONJ新疗法的关键的第一步。的确， 早期药物干预，避免骨细胞死亡，同时口腔风险因素被删除， 预防ONJ。知识上的差距限制了在ONJ的早期阶段识别ONJ并进行干预以阻止其发展的能力。 进展骨细胞凋亡在各种骨骼疾病的病理生理学中的关键作用， 得到证实。然而，很少有人注意到坏死性凋亡，一种特殊的“调节”形式的细胞， 由炎症引发的死亡，例如与牙周炎和根尖周感染相关的死亡。不像 细胞凋亡、坏死性凋亡增强免疫应答和炎症。值得注意的是，ZOL治疗的大鼠 ONJ显示骨细胞坏死性凋亡增加，但不凋亡。药理学抑制剂靶向 已经开发了坏死性凋亡的特定调节成分。其中一些正在进行临床试验， 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）骨细胞死亡的发生与特定的放射学、细胞学和 分子特征;和3）坏死性凋亡是ONJ中涉及的细胞死亡的主要类型。我们的方法 是使用患有局部牙周炎的老鼠，通过口腔检查可以很容易地监测到， 与ZOL一起确定： 目标1： 骨细胞死亡的时间和类型影响骨细胞的早期阶段， ONJ和骨细胞死亡与放射学、细胞和分子学结果的时间关系; 目标2： 应用细胞凋亡和坏死性凋亡在大鼠ONJ发病中的作用 药理学抑制剂。研究结果将在时间上和机制上定义ONJ的前驱症状， 在人类中可能是0期，支持靶向治疗的发展，以阻止骨细胞死亡， 为坏死性凋亡对ONJ的作用建立直接证据，并为治疗ONJ建立体内概念验证。 抑制其信号传导途径组分阻止ONJ进展的潜力。

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.