Prevention of Fracture Healing Failure in Alcohol Abusers

描述(由申请人提供)： 有长期酗酒史的骨折患者延迟愈合和不愈合的发生率较高。在骨折愈合的实验模型中，酒精抑制骨折部位的新骨形成，促进纤维化形成，未成熟软骨堆积，成骨细胞数量和功能减少，修复组织的硬度和强度降低。因此，酒精通过干扰直接(膜内)和间接(软骨内)新骨形成的细胞机制，促进骨折部位的瘢痕组织形成，而不是骨再生。在骨折后两周内，通过注射饮食抗氧化剂N-乙酰半胱氨酸(NAC)，通过皮下注射酒精，大鼠暴饮暴食，抑制新骨形成和导致骨折愈合缺陷，恢复到正常愈合。假设。我们推测，抗氧化剂N-乙酰半胱氨酸(NAC)的治疗将恢复骨折部位氧化应激和抗氧化反应的平衡，从而恢复正常的骨折愈合序列，包括初始炎症阶段、Wnt/β连环蛋白信号转导以及软骨内和膜内成骨，以防止慢性乙醇喂养大鼠骨折愈合失败。明确的目标。(1)测定“骨折”前后饮酒/禁酒和限制饮食摄入量对正常骨骼恢复的影响 未处理和NAC处理的大鼠的修复结果。(2)确定乙醇和减少饮食摄入是否会增加氧化应激，以及术后禁酒和/或NAC治疗是否会恢复正常骨折愈合过程中观察到的氧化-抗氧化剂平衡、炎症反应、Wnt/β连环蛋白信号、软骨内成骨和膜内成骨。研究设计。9个月龄的大鼠，相当于人类的平均年龄20岁，将每天喂食Lieber-DeCarli乙醇饮食，持续16周。在大鼠一侧股骨中段进行横形截骨术。股骨将通过内固定固定。这一模型将在给予酒精饮食和无酒精(对照)饮食的大鼠组中建立。在目标1中，将在术后16周通过机械测试来评估愈合的结果。此时老鼠的年龄将是17个月，相当于人类的大约60岁。我们将确定14天的NAC治疗是否可以防止乙醇引起的骨愈合抑制，以及是否仅仅通过手术后停止乙醇喂养并允许动物自由食用对照饮食也能达到这一结果。在目标2中，我们将获得初步数据来回答以下问题，这将为导致临床翻译的全面项目提供基础：(1)在与慢性酒精中毒相关的骨折愈合失败中，酒精诱导的氧化应激如何影响(A)对骨折愈合至关重要的炎性细胞及其细胞因子的募集？(2)在慢性酒精中毒时，NAC恢复Wnt/β连环蛋白信号和新骨形成的具体作用机制是什么？ 公共卫生相关性： 长期长期过度饮酒的人(“酗酒者”)会出现并发症，常常导致骨折愈合失败。实验研究表明，酒精在骨折愈合的早期阶段会导致细胞异常，从而导致这种失败。在拟议的项目中，我们将评估抗氧化剂N-乙酰半胱氨酸(NAC)在预防酒精滥用者骨折愈合失败方面的有效性。在最近的一项研究中发现 在大量的临床研究中，大量的临床研究证明，在大鼠股骨闭合性骨折中，酗酒后2周内给予NAC可以预防抑制新骨形成。在过去的20年里，NAC在临床上治疗各种疾病的有效性已经被证明。它耐受性好，有明确的作用机制，被公认为治疗半胱氨酸/谷胱甘肽缺乏症的安全解毒剂。

DESCRIPTION (provided by applicant): Fracture patients with a history of chronic alcohol abuse have a higher incidence of delayed unions and non-unions. In experimental models of fracture healing alcohol inhibits new bone formation in the fracture site and promotes fibrogenesis, accumulation of immature cartilage, decreased osteoblast number and function, and decreased stiffness and strength of the repair tissue. Thus, alcohol promotes scar tissue formation instead of bone regeneration in the fracture site by interfering with cellular mechanisms in both the direct ("intramembranous") and indirect (endochondral) pathways of new bone formation. Inhibition of new bone formation and the resulting deficiency in fracture healing in rats given ethanol by subcutaneous injection in a binge-drinking protocol was restored to normal healing by injection of the dietary antioxidant n-acetylcysteine (NAC) during two weeks after fracture. Hypothesis. We hypothesize that treatment with the antioxidant n-acetylcysteine (NAC) will restore the balance of oxidative stress and antioxidant response in the fracture site and thus restore the normal fracture healing sequence including the initial inflammatory phase, Wnt/beta catenin signaling, and endochondral and intramembranous ossification to prevent failure of fracture healing in chronically ethanol-fed rats. Specific Aims. (1) Determine the effects of alcohol consumption/ abstinence and restriction of diet intake before and after "fracture" on restoration of normal bone repair outcome in untreated and NAC-treated rats. (2) Determine whether oxidative stress is increased by both ethanol and reduced diet intake and whether post- surgery abstinence from ethanol and/or NAC treatment will restore the oxidant-antioxidant balance, inflammatory response, Wnt/beta catenin signaling, endochondral ossification, and intramembranous ossification observed in normal fracture healing. Research Design. Rats 9 months of age, which corresponds to average human age of 20 years, will be fed the Lieber-DeCarli ethanol diet daily for 16 weeks. Transverse osteotomy will be performed at middiaphysis of one femur of the rat. The femur will be immobilized by internal fixation. This model will be created in groups of rats that have been given the ethanol diet and ethanol-free (control) diet. In Aim 1, the outcome of healing will be evaluated at 16 weeks post surgery by mechanical testing. The age of the rat at this time will be 17 months, which corresponds approximately to human age of 60 years. We will determine if 14 days of NAC treatment prevents the ethanol-induced inhibition of bone healing and if this result is also achieved merely by stopping the ethanol feeding after surgery and allowing the animals to consume the control diet ad libitum. In Aim 2, we will obtain preliminary data to answer the following questions, which will provide the basis for a full-scale project leading to clinical translation: (1) In fracture healing failure associated with chronic alcoholism how does alcohol-induced oxidative stress affect (a) recruitment of inflammatory cells and their cytokine expressions that are critical for fracture healing? (b) Wnt/beta catenin signaling that mediates new bone formation? (2) What are the specific mechanisms of action of NAC that restore Wnt/beta catenin signaling and new bone formation in the context of chronic alcoholism. PUBLIC HEALTH RELEVANCE: Persons with a long history of chronic and excessive alcohol consumption ("alcohol abusers") experience complications often leading to failure of fracture healing. Experimental studies show that alcohol causes cellular abnormalities in the early stage of fracture healing leading to this failure. In the proposed project we will evaluate the efficacy of the antioxidant n-acetylcysteine (NAC) to prevent the failure of fracture healing in alcohol abusers. In a recent study it was found that inhibition of new bone formation in closed femoral fractures created in rats following binge alcohol exposure could be prevented by administration of NAC during 2 weeks after fracture.The efficacy of NAC for clinical treatment of a large variety of conditions has been documented in a large number of clinical studies during the past two decades. It is well tolerated, has well-defined mechanisms of action and is acknowledged as a safe antidote for cysteine/glutathione deficiency .