Mechanism of Nicotine Action in the Healing Skeleton

描述（由申请人提供）：在骨科文献中有充分的证据表明，吸烟对长骨骨折和脊柱融合手术后的愈合有负面影响。虽然这一临床问题的发病率和经济负担是显著的，但在阐明吸烟对骨骼愈合影响的潜在机制方面几乎没有取得进展。为了开始解决机制问题，我们提出测试一个新的假设，该假设预测尼古丁是香烟烟雾的关键成分，通过与间充质干细胞膜上表达的尼古丁乙酰胆碱受体（nACHR）相互作用影响愈合过程。更具体地说，我们假设nACHR的激活在干细胞中被招募参与再现软骨内成骨的愈合过程i)加速其通过软骨形成的进展ii)抑制或延迟其随后的进展到最终成熟。为了验证这一中心假设，我们计划解决3个具体目标。本实验将通过mRNA和蛋白分析以及第二信使信号分析来表征尼古丁和香烟烟雾提取物（CSE）对小鼠肢体芽间充质细胞nACHR表达和信号转导的影响。Specific Aim 2的实验将比较尼古丁和CSE对野生型和α 4 nACHR缺失小鼠MSCs体外软骨形成和软骨细胞成熟的影响。我们还将确定在这些细胞中看到的尼古丁引起的表型变化中特定信号事件的作用。最后，在具体目标3中，我们建议使用小鼠股骨骨折愈合的体内模型来比较尼古丁和香烟烟雾对软骨内骨形成的影响。暴露于尼古丁或香烟烟雾中的破碎野生型或α 4基因敲除小鼠将通过组织形态学、原位杂交、微ct和生物力学测试方法进行分析。完成这些目标将首次评估尼古丁作为香烟烟雾中对骨骼愈合产生负面影响的关键成分的关键作用。此外，拟议的实验旨在确定尼古丁在愈合过程中有害影响的潜在机制，这将为临床策略的发展铺平道路，减轻与吸烟相关的不良后果。

DESCRIPTION (provided by applicant): It is well documented in the orthopaedic literature that cigarette smoking has a negative impact on healing following long bone fracture and spinal fusion surgery. While the morbidity and financial burden of this clinical problem is significant, little progress has been made toward elucidating the underlying mechanisms that mediate the effects of smoking on skeletal healing. To begin addressing the question of mechanism, we propose to test a novel hypothesis which predicts that nicotine is a key component of cigarette smoke that affects the healing process by interacting with the nicotinic acetylcholine receptor (nACHR) expressed on mesenchymal stem cell membranes. More specifically, we hypothesize that activation of the nACHR in stem cells that are recruited to participate in healing processes that recapitulate endochondral ossification i) accelerates their progression through chondrogenesis and ii) inhibits or delays their subsequent progression to terminal maturation. To test this central hypothesis, we plan to address 3 Specific Aims. Experiments in Specific Aim 1 will characterize the impact of nicotine and cigarette smoke extract (CSE) on nACHR expression and signaling in mouse limb bud mesenchymal cells via mRNA and protein analysis and second messenger signaling assays. Experiments in Specific Aim 2 will compare the impact of nicotine and CSE on in vitro chondrogenesis and chondrocyte maturation in MSCs derived from wild type and alpha4 nACHR null mice. We will also determine the role of specific signaling events in the nicotine-evoked phynotypic changes seen in these cells. Lastly, in Specific Aim 3 we propose to compare the effects of nicotine and cigarette smoke on endochondral bone formation using the in vivo model of femur fracture healing in mice. Fractured wild type or alpha4 knockout mice exposed to nicotine or cigarette smoke will be analyzed via histomorphometric, in situ hybridization, microCT and biomechanical testing methods. Completion of these aims will for the first time evaluate the lynchpin role of nicotine as a key component in cigarette smoke that negatively influences skeletal healing. Furthermore, proposed experiments aimed at identifying the mechanism underlying the deleterious effects of nicotine during healing will pave the way toward the development of clinical strategies that alleviate the poor outcomes associated with smoking.