Bridging the gap: Angiogenesis and stem cell seeding of processed nerve allograft

弥合差距：加工神经同种异体移植物的血管生成和干细胞播种

• 批准号: 10381652
• 负责人: Alexander Y. Shin
• 金额: $ 34.78万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10381652
• 关键词: 3-Dimensional; Adipose tissue; Affect; Allografting; Autologous; Autologous Transplantation; Axon; Biological; Bioluminescence; Blood Vessels; Blood capillaries; Brachial plexus structure; Caliber; Cells; Clinical; Collagen; Combined Modality Therapy; Cryopreservation; Defect; Development; Distal; Elastases; Electrophysiology (science); Engineering; Epigastric; Evaluation; Excision; Freezing; Gene Expression; Gene Expression Profile; Goals; Gold; Growth Factor; Harvest; Human; Image; Immune response; Implant; In Vitro; Inferior; Injury; Investigation; Label; Length; Longevity; Luciferases; Measures; Mesenchymal Stem Cells; Modeling; Morbidity - disease rate; Motor; Multiple Trauma; Muscle; Natural regeneration; Nerve; Nerve Regeneration; Nerve Transfer; Operative Surgical Procedures; Oryctolagus cuniculus; Outcome; Outcome Measure; Patients; Performance; Peripheral Nerves; Peripheral nerve injury; Process; Production; Rattus; Recovery of Function; Schwann Cells; Sensory; Site; Source; Supporting Cell; Surgical Flaps; Techniques; Testing; Time; Tissues; Trauma; Undifferentiated; Up-Regulation; Vascular Endothelial Growth Factors; Vascular blood supply; Weight; afferent nerve; angiogenesis; cell motility; density; functional outcomes; improved; in vivo; indexing; migration; motor function improvement; motor recovery; nerve autograft; nerve gap; peripheral nerve repair; process optimization; reconstruction; scaffold; sciatic nerve; stem cell differentiation; stem cell survival; stem cells

Project Summary High energy trauma is commonly associated with peripheral nerve injuries. These poly-trauma cases number in the tens of thousands annually.[4] While reconstruction of a 1-2 cm gap in a purely sensory nerve may be bridged with engineered nerve conduits, longer and larger diameter mixed nerves require autologous nerve grafts. Few expendable sensory nerves are available to use. They are often insufficient in quantity and harvest results in significant permanent donor site sensory loss. A readily available alternative with similar or better performance is needed. Currently, the most promising alternative is the processed nerve allograft. Providing an ideal scaffold for regenerating axons, it yields good outcome in short sensory nerve defects. However, in longer motor nerve defects functional outcomes are disappointing and further improvement of the allograft is necessary. In this study, we aim to improve results of such allografts by providing additional biological support in the form of undifferentiated or “Schwan cell-like” adipose-derived mesenchymal stem cells (AMSCs) and/or surgical angiogenesis provided by an enveloping fascial flap. We will correlate functional evaluation of muscle size, weight and strength with observed changes in gene expression, stem cell survival and migration, nerve electrophysiology, histomorphometry, and extent of immunologic response. We have previously described and validated the means to accurately quantify tetanic force in the rat and rabbit, and have shown its superiority to the sciatic function index (SFI).[5-7] We have demonstrated processed nerve allograft to provide better motor recovery than hollow nerve conduits in 1 cm rat sciatic nerve defects, and found filling of collagen nerve guides with a glycosaminoglycoside matrix to have little additional value.[8-10] Direct delivery of vascular endothelial growth factor (VEGF) was ineffective in improving motor recovery in this model.[11] We have also isolated adipose-derived mesenchymal stem cell (AMSCs) in rats and rabbits, and differentiated them to produce “Schwann cell-like” patterns of gene expression. We have developed an optimized processed nerve allograft (OPA) using elastase to minimize cellular debris and found cold storage to avoid adverse structural changes created by freezing. We have also labeled AMSCs with luciferase, using the resulting bioluminescence to image them in vivo. These preliminary studies provide the needed background for the proposed investigation. Our goals are four-fold: 1) to ask if differentiation of AMSCs seeded onto a decellularized nerve allograft differ with respect to survival, migration, “Schwann cell-like” gene expression and immune response to the graft, and to determine if any observed differences effect motor recovery; 2) to similarly evaluate the effect of surgical angiogenesis; 3) to test whether AMSCs combined with surgical angiogenesis are synergistic in all of these measures. 4) Finally, we will ask how AMSC and surgical angiogenesis perform relative to the autograft ‘gold standard’.

项目摘要 高能量创伤通常与周围神经损伤有关。这些多发性创伤病例 数以万计。[4]虽然在纯粹的感觉神经中重建1- 2cm的间隙可能是可行的， 采用工程化神经导管桥接，更长、更大直径的混合神经需要自体神经 移植物可供使用的消耗性感觉神经很少。它们往往数量不足， 收获导致显著的永久性供体部位感觉丧失。一种现成的替代品，具有类似或 需要更好的性能。目前，最有前途的替代方法是处理过的神经同种异体移植物。 它为轴突再生提供了理想的支架，在短感觉神经缺损中产生了良好的结果。 然而，在较长的运动神经缺损中，功能结果是令人失望的， 需要同种异体移植。在这项研究中，我们的目标是通过提供额外的， 未分化或“许旺细胞样”脂肪来源的间充质干细胞形式的生物支持物 （AMSC）和/或由包膜筋膜瓣提供的外科血管生成。我们将函数 通过观察基因表达、干细胞存活的变化评估肌肉大小、重量和力量 和迁移、神经电生理学、组织形态计量学和免疫应答程度。 我们之前已经描述并验证了准确定量大鼠强直力的方法， 家兔，并已显示出其优越性坐骨神经功能指数（SFI）。[5-7]我们已经证明了 在1cm大鼠坐骨神经缺损中，同种异体神经移植物比中空神经导管提供更好的运动恢复， 并发现用糖胺糖苷基质填充胶原蛋白神经导管几乎没有附加价值。[第8-10页] 直接输送血管内皮生长因子（VEGF）在改善运动恢复方面是无效的。 模型[11]我们还分离了大鼠和兔的脂肪间充质干细胞（AMSC）， 分化它们以产生“雪旺细胞样”的基因表达模式。我们已经开发了一个 使用弹性蛋白酶优化处理过的神经同种异体移植物（OPA），以最大限度地减少细胞碎片，并发现冷藏， 避免因冻结而产生的不利结构变化。我们还用荧光素酶标记了AMSC， 从而产生生物发光以在体内对其成像。这些初步研究提供了必要的背景， 拟议的调查。 我们的目标有四个方面：1）询问接种在脱细胞神经同种异体移植物上的AMSC的分化是否与 关于存活、迁移、“雪旺细胞样”基因表达和对移植物的免疫应答，以及 以确定任何观察到的差异是否影响运动恢复; 2）类似地评估手术的效果， 3）测试AMSC与手术血管生成的组合是否在所有这些血管生成中是协同的; 措施4)最后，我们将询问AMSC和手术血管生成相对于自体移植物的金是如何表现的 标准。