Oral Mucosa in Bisphosphonate-associated Osteonecrosis of the Jaw

描述（由申请人提供）：双膦酸盐相关性颌骨骨坏死（ONJ）对患者的生活质量产生不利影响，并产生显著的发病率。它的特点是发现暴露的牙槽骨在口腔。接受高剂量静脉注射双膦酸盐（帕米膦酸盐和唑仑膦酸）治疗的癌症患者发生ONJ的风险高于接受低剂量口服双膦酸盐治疗的骨质疏松症患者（0.00001 - 0.0001），发生率为1% - 10%。人们一直认为ONJ的原发病变位于骨，与骨转换的过度抑制有关。然而，目前尚不清楚为什么这些病变以下颌骨或上颌骨的口腔粘膜覆盖丧失为主要临床特征。对这种症状的一种可能解释是，双膦酸盐在骨骼中积聚的浓度足以对口腔黏膜产生直接毒性，导致软组织病变愈合失败，从而导致ONJ的发病机制。此外，拔牙是ONJ发生的主要原因，尽管其他原因如牙周病、牙种植手术、外生牙和假牙不合适也是ONJ发生的主要因素。此外，最近有报道称双膦酸盐具有抗血管生成作用。由于血管的充分重建是伤口愈合的前提，抑制血管生成可能导致软组织病变愈合失败，从而导致ONJ的发病机制。尽管这些研究结果表明，双膦酸盐对口腔黏膜的影响参与了ONJ的发生和/或进展，但静脉注射双膦酸盐对口腔黏膜的影响尚未在体内研究。我们的总体假设是静脉注射双膦酸盐可能抑制口腔黏膜的愈合，并有助于ONJ的发生和/或进展。我们计划通过测试以下目标来阐明双膦酸盐与口腔黏膜之间的关系。在Aim 1中，我们将通过基础组织学和活/死细胞活力测定，确定静脉注射双膦酸盐唑来膦酸对大鼠拔牙后伤口愈合过程中口腔黏膜的细胞毒性作用。目的是证明唑来膦酸在拔牙后口腔黏膜愈合过程中的细胞毒作用。在Aim 2.1中，我们将使用免疫荧光法确定唑来膦酸在拔牙后愈合过程中是否会改变口腔黏膜血管的重建。在Aim 2.2中，我们将进一步研究血管重建的改变是否由于血管生成和缺氧相关基因表达的抑制。目的是在体内验证唑来膦酸在拔牙后口腔黏膜愈合中的抗血管生成作用。如果成功，该实验将推进研究，填补目前静脉注射双膦酸盐对口腔软组织作用的分子机制表征方面的知识空白，从而可能随后导致对双膦酸盐相关ONJ发生和/或进展的病理生理学的理解。

DESCRIPTION (provided by applicant): Bisphosphonate-associated osteonecrosis of the jaw (ONJ) adversely affects the quality of life and produces significant morbidity in afflicted patients. It is characterized by the finding of exposed alveolar bone in the oral cavity. The risk of ONJ in patients with cancer treated with high doses of intravenous bisphosphonates (Pamidronate and Zolendronic acid) is higher, ranging from 1 to 10 %, compared to patients with osteoporosis treated with low dose, oral bisphosphonates (0.00001 to 0.0001 %). It has been assumed that the primary lesion for ONJ lies in bone and is related to over-suppression of bone turnover. However, it is unclear why these lesions present with loss of the oral mucosal covering of the mandible or maxilla as the primary clinical feature. A possible explanation of this symptom is that bisphosphonates are accumulated in bone in concentrations sufficient to be directly toxic to the oral mucosa and result in the failure of healing of soft tissue lesions, leading to the pathogenesis of ONJ. In addition, tooth extraction is the dominating event preceding ONJ, although other causes such as periodontal disease, dental implant procedures, exostoses, and ill-fitting dentures are also preceding factors for ONJ. Moreover, an anti-angiogenic effect of bisphosphonates has been reported recently. Since sufficient reconstitution of vasculature is prerequisite for wound healing, suppression of angiogenesis may result in the failure of healing of soft tissue lesions leading to the pathogenesis of ONJ. Although, these findings suggest involvement of bisphosphonate on oral mucosa in the development and/or progression ONJ, the effect of intravenous bisphosphonates on oral mucosa has not been studied in vivo. Our overall hypothesis is that intravenous bisphosphonates may inhibit healing of oral mucosa and contribute to the development and/or progression ONJ. We plan to elucidate the relationship between bisphosponates and the oral mucosa by testing the following Aims. In Aim 1, we will determine the cytotoxic effect of intravenous bisphosphonate, zoledronic acid, on oral mucosa, in vivo, during the wound healing process after tooth extraction in a rat model, by utilizing basic histology and the live/dead cell vitality assay. The aim will demonstrate the cytotoxic effect of zoledronic acid during healing of oral mucosa after tooth extraction. In Aim 2.1, we will determine if reconstitution of the vasculature in the oral mucosa is altered by zoledronic acid during the healing process after a tooth extraction, using immunofluorescence method. In Aim 2.2 we will further investigate whether the alternation of vasculature reconstitution is due to suppression of angiogenic and hypoxia related gene expression. The aim will demonstrate the anti-angiogenic effect of zoledronic acid during healing of oral mucosa after tooth extraction, in vivo. If successful, the proposed experiments will advance research that will fill a current knowledge gap in characterization of molecular mechanisms of action of intravenous bisphosphonates on oral soft tissues that may subsequently lead to the understanding of pathophysiology of development and/or progression of bisphosphonate-associated ONJ. PUBLIC HEALTH RELEVANCE: Bisphosphonate-associated osteonecrosis of the jaw (ONJ) is characterized by the finding of exposed alveolar bone in the oral cavity and adversely affects the quality of life and produces significant morbidity in afflicted patients. The underlying pathogenesis of ONJ is yet to be clearly elucidated. The proposed experiments will advance research that will fill a current knowledge gap in characterization of molecular mechanisms of action of intravenous bisphosphonates on oral soft tissues that subsequently lead to the understanding of pathophysiology of development and/or progression of bisphosphonate-associated ONJ.