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骨折延迟愈合和骨不连的发生率较高。在骨折愈合的实验模型中，酒精会抑制骨折部位的新骨形成，促进纤维形成、未成熟软骨的积累、成骨细胞数量和功能减少以及修复组织的刚度和强度降低。因此，酒精通过干扰新骨形成的直接（“膜内”）和间接（软骨内）途径中的细胞机制，促进骨折部位的瘢痕组织形成，而不是骨再生。抑制新骨形成和由此产生的缺陷，在骨折愈合的大鼠给予乙醇皮下注射在暴饮暴食协议恢复正常愈合的饮食抗氧化剂N-乙酰半胱氨酸（NAC）注射骨折后两周内。假说.我们假设，抗氧化剂N-乙酰半胱氨酸（NAC）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顺序，包括初始炎症阶段，Wnt/β连环蛋白信号传导，软骨内和膜内骨化，以防止长期乙醇喂养大鼠骨折愈合失败。具体目标。(1)确定“骨折”前后饮酒/戒酒和限制饮食摄入对恢复正常骨骼的影响 未处理和NAC处理大鼠的修复结果。 (2)确定氧化应激是否因乙醇和减少饮食摄入而增加，以及术后戒酒和/或NAC治疗是否会恢复正常骨折愈合中观察到的氧化剂-抗氧化剂平衡、炎症反应、Wnt/β连环蛋白信号传导、软骨内骨化和膜内骨化。研究设计。9月龄的大鼠（对应于20岁的平均人类年龄）将每天喂食Lieber-DeCarli乙醇饮食，持续16周。将在大鼠一侧股骨的中段进行横向截骨术。股骨将通过内固定术固定。将在给予乙醇饮食和无乙醇（对照）饮食的大鼠组中创建该模型。在目标1中，将在术后16周通过力学测试评价愈合结局。此时大鼠的年龄为17个月，约相当于人类的60岁。我们将确定14天的NAC治疗是否阻止了乙醇诱导的骨愈合抑制，以及是否仅通过在手术后停止乙醇喂养并允许动物随意食用对照饮食也可以实现该结果。在目标2中，我们将获得初步数据来回答以下问题，这将为导致临床转化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）炎症细胞的募集及其细胞因子表达，这对骨折愈合至关重要？(b)介导新骨形成的Wnt/β连环蛋白信号？(2)在慢性酒精中毒的情况下，NAC恢复Wnt/β连环蛋白信号传导和新骨形成的具体作用机制是什么？

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.