Integrative Tissue Engineering in the Spinal Cord

描述(由申请人提供)：一种综合的、组织工程学方法治疗脊髓损伤将在大鼠横断模型中进行评估。以聚(乳酸-乙醇酸)(PLGA)为原料，研制出一种具有可控微结构的可生物降解植入物。将通过扫描电子显微镜、共聚焦显微镜和X射线微计算机断层扫描对植入物的结构进行评估和表征。软骨素酶ABC(C-ABC)是一种分解创伤后富含蛋白多糖的瘢痕的酶，将被包裹在PLGA微球中，并将在体外对其生物活性和释放动力学进行评价。将研究将C-ABC结合到聚合物植入物中的技术，并将描述C-ABC从植入物中的分布和释放。雪旺细胞将从新生大鼠的坐骨神经中提取，在细胞培养中扩增，并悬浮在Matrigel中，以整合到聚合物植入物中。成年SD大鼠在接受胸椎8~9节段椎板切除和脊髓横断手术前麻醉。各种植入物将被插入椎管，硬脑膜将闭合在椎管上。脊髓将在多个时间点收获，纵向分割，并进行组织学、免疫组织化学和扫描电子显微镜(SEM)的固定。组织修复的形态评估将在组织切片上进行，而功能分析将用于神经恢复的评估。将对试验组和对照组进行评估，以确定每个植入组件及其不同组合对轴突再生和功能恢复的影响。

DESCRIPTION (provided by applicant): An integrative, tissue-engineering approach to treat spinal cord injury will be evaluated in a rat transection model. A biodegradable implant with controlled microarchitecture will be developed and fabricated from poly(lactic-co-glycolic acid) (PLGA). Implant architecture will be evaluated and characterized with scanning electron microscopy, confocal microscopy, and X-ray micro-computed tomography. Chondroitinase ABC (C-ABC), an enzyme shown to break down the proteoglycan-rich scar that results from injury, will be encapsulated in PLGA microspheres, and its bioactivity and release kinetics will be evaluated in vitro. Techniques for incorporating C-ABC into the polymer implant will be investigated, and the distribution and release of C-ABC from the implant will be described. Schwann cells will be harvested from neonatal rat pup sciatic nerves, expanded in cell culture, and suspended in Matrigel for incorporation into the polymer implant. Adult Sprague-Dawley rats will be anesthetized before receiving thoracic 8-9 laminectomies and spinal cord transections. Various implants will be inserted into the spinal canals and the dura closed over them. Spinal cords will be harvested at multiple time points, split longitudinally, and fixed for histology, immunohistochemistry, and scanning electron microscopy (SEM). Morphologic evaluation of tissue repair will be performed on tissue sections, while functional assays will be employed for assessment of neurological recovery. Experimental groups and controls will be evaluated for determining the effects of each implant component, as well as their various combinations, on axon regeneration and functional recovery.