ADULT STEM CELL SUPPRESSION DUE TO ALCOHOL INDUCED MICROENVIRONMENT ALTERATIONS

描述(申请人提供)：酗酒和酗酒影响至少1400万美国人。长期饮酒与骨质疏松症和腰椎骨折发生率增加有关。后外侧脊柱融合术是酒精所致骨质疏松中常见的腰椎压缩骨折的标准治疗方法。长期接触酒精会抑制骨折愈合，并导致脊柱融合失败的发生率显著增加。2003年，与翻修脊柱融合手术相关的费用高达每例72,000美元。再生医学是一种利用干细胞促进组织愈合的新型治疗方法。脂肪组织来源的基质细胞(ASCs)在体内和体外都能促进成骨。干细胞和微环境之间的复杂相互作用对于促进成骨是必要的，而长期酒精暴露引起的两者中任何一种的变化都可能限制它们的联合疗效。基于这一认识，我们假设：(A)慢性酒精摄入降低了ASCs的数量、扩张率和胸膜潜在能力；(B)受者长期酒精暴露抑制或阻止了正常ASCs在合适的生物材料载体中应用，从而抑制或阻止了脊柱融合的加速；以及(C)从慢性酒精暴露受试者身上获取的ASCs，并应用于合适的生物材料载体，不能加速正常受者的脊柱融合。这些假说将在以下特定目标下进行检验：目的1.确定慢性酒精摄入对大鼠ASCs的影响。研究将对正常大鼠和暴露于已建立的慢性酒精摄入模型的大鼠的细胞进行研究。在体外扩增和传代的干细胞的数量和行为将用标准程序进行量化。目的2.研究正常ASCs对慢性酒精暴露和非慢性酒精暴露大鼠脊柱融合的影响。研究将用从没有酒精暴露的受试者中获得的ASCs植入有慢性酒精暴露的受试者和没有长期酒精暴露的受试者。放射照片、显微CT、RT-PCR、成分分析、组织学和免疫组织化学的结果测量将提供有关正常干细胞在正常微环境中诱导成骨的能力的信息，而不是慢性酒精暴露改变的微环境。目的3.研究慢性酒精暴露大鼠分离的ASCs对慢性酒精暴露大鼠和非慢性酒精暴露大鼠脊柱融合的影响。研究将用从慢性酒精暴露的受试者身上获取的ASCs植入有慢性酒精暴露的受试者和没有长期酒精暴露的受试者。与AIM 2相同的结果衡量标准将提供有关长期酒精暴露改变的干细胞在正常与长期酒精暴露改变的微环境中诱导成骨能力的信息。这项研究的结果将极大地促进人们对酒精诱导的干细胞和抑制骨形成的基质改变的认识。这项研究的结果将大大有助于目前关于酒精诱导的干细胞和基质改变的知识，这些改变抑制了骨形成。公共卫生相关性：阐明在面对慢性酒精滥用时导致脊柱融合失败的病理生理学将提供解决这一重要医学问题的机制。此外，应用ASC是一种有前景的治疗方法，可以加速与酒精暴露无关的天然成骨细胞前体数量减少的患者的骨折愈合。

DESCRIPTION (provided by applicant): Alcoholism and alcohol abuse affect at least 14 million Americans. Chronic alcohol consumption is associated with osteoporosis and an increased rate of lumbar spinal fractures. Posterolateral spinal fusion is the standard treatment for lumbar compression fractures common in ethanol-induced osteoporosis. Chronic alcohol exposure inhibits fracture healing and results in a significantly higher incidence of spinal fusion failures. Costs associated with revision spinal fusion surgery were as high as $72,000 each in 2003. Regenerative medicine is a novel therapeutic modality that employs stem cells to promote tissue healing. Adipose tissue-derived stromal cells (ASCs) promote osteogenesis both in vivo and in vitro. Complex interactions between stem cells and the microenvironment are necessary to enhance osteogenesis, and alterations in either from chronic alcohol exposure can limit their combined efficacy. Based on this knowledge we postulate: (A) That chronic alcohol ingestion reduces the number, rate of expansion, and pleuripotential capacity of ASCs; (B) That chronic alcohol exposure in the recipient inhibits or prevents the acceleration of spinal fusion by application of normal ASCs in a suitable biomaterial carrier; and (C) That ASCs harvested from subjects with chronic alcohol exposure and applied in a suitable biomaterial carrier do not accelerate spinal fusion in normal recipients. These hypotheses will be tested with the following Specific Aims: Aim 1. To determine the effect of chronic alcohol ingestion on ASCs in a rat model. Studies will be performed on cells harvested from normal rats and rats exposed to an established model of chronic alcohol ingestion. The number and behavior of stem cells expanded and passaged in vitro will be quantified with standard procedures. Aim 2. To determine the effect of normal ASCs on spinal fusion in rats with and without chronic alcohol exposure. Studies will be performed with ASCs harvested from subjects with no alcohol exposure implanted into subjects with and without chronic alcohol exposure. Outcome measures of radiographs, micro-CT, RT-PCR, compositional analysis, histology, and immunohistochemistry will provide information surrounding the ability of normal stem cells to induce osteogenesis in normal microenvironments versus microenvironments altered by chronic alcohol exposure. Aim 3. To determine the effect of ASCs harvested from rats with chronic alcohol exposure on spinal fusion in rats with and without chronic alcohol exposure. Studies will be performed with ASCs harvested from subjects with chronic alcohol exposure implanted into subjects with and without chronic alcohol exposure. Outcome measures identical to those of Aim 2 will provide information surrounding the ability of stem cells altered by chronic alcohol exposure to induce osteogenesis in normal versus microenvironments altered by chronic alcohol exposure. Results from this investigation will substantially advance knowledge surrounding alcohol-induced stem cell and matrix alterations that inhibit bone formation. Results from this investigation will contribute substantially to current knowledge surrounding alcohol-induced stem cell and matrix alterations that inhibit bone formation. PUBLIC HEALTH RELEVANCE: Elucidation of the pathophysiology contributing to failure of spinal fusion in the face of chronic alcohol abuse will provide mechanisms to address this important medical problem. Additionally, ASC application is a promising treatment that may accelerate fracture healing in patients with reduced numbers of native osteoblast precursors unrelated to alcohol exposure.