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

项目摘要 高能创伤常与周围神经损伤有关。这些多发伤的病例数 [4]而重建一条纯感觉神经1-2厘米的缝隙可能是 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 嫁接。几乎没有可牺牲的感觉神经可供使用。它们往往数量不足，而且 收获会导致永久性供体部位严重的感觉丧失。一个现成的替代方案，具有类似或 需要更好的性能。目前，最有希望的替代方案是加工后的同种异体神经移植。 为再生轴突提供了理想的支架，修复短感觉神经缺损效果良好。 然而，在较长的运动神经缺陷中，功能结果令人失望，并进一步改善了 同种异体移植是必要的。在这项研究中，我们的目标是通过提供更多的 未分化或“雪旺细胞样”脂肪来源间充质干细胞的生物支持 (AMSCs)和/或由包绕筋膜瓣提供的外科血管生成。我们将关联泛函 通过观察基因表达、干细胞存活的变化来评估肌肉大小、重量和力量 以及移行、神经电生理学、组织形态计量学和免疫反应的程度。 我们之前已经描述并验证了准确量化大鼠破伤力和 兔，并显示了其相对于坐骨神经功能指数(SFI)的优越性。 同种异体神经移植修复1 cm大鼠坐骨神经缺损处的运动功能优于中空神经导管。 并发现用糖胺糖苷基质填充胶原神经导向器几乎没有额外的价值。 直接注射血管内皮细胞生长因子(VEGFR)在改善运动功能方面无效。 [11]我们还在大鼠和兔体内分离了脂肪来源的间充质干细胞(AMSCs)，并 分化它们以产生“雪旺细胞样”的基因表达模式。我们已经开发出一种 优化处理的同种异体神经移植(OPA)，使用弹性酶最大限度地减少细胞碎片，并找到冷藏 避免冻结造成的不利结构变化。我们还用荧光素酶标记了AMSCs，使用 由此产生的生物发光在体内对它们进行成像。这些初步研究为以下工作提供了必要的背景 拟议中的调查。 我们的目标有四个：1)研究移植到去细胞同种异体神经上的AMSCs的分化是否不同 关于移植物的存活、迁移、“雪旺细胞样”基因表达和免疫反应，以及 以确定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 血管生成；3)测试AMSCs与外科血管生成是否在所有这些方面具有协同作用 措施。4)最后，我们将询问AMSC和外科血管生成相对于自体移植物的黄金表现如何 “标准”。

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’.