Enhanced endothelialization for tissue engineering

描述(申请人提供)：包括聚L-乳酸(PLLA)在内的可生物降解聚合物已被用于开发心血管假体，如血管移植物和支架。然而，植入物相关的血栓、炎症和再狭窄仍然是这些假体通畅的主要障碍。我们的长期目标是通过快速内皮化减少这些并发症。我们的假设是，通过将具有内皮细胞(EC)黏附的假体材料(纤维连接蛋白)和生长因子(血管内皮生长因子，VEGF)结合在假体表面，以及在假体表面种植带有生长因子受体(VEGF受体，KDR/Flk-1)的转基因ECs，可以在假体表面快速内皮化。具体地说，第一个目标是使用不同的固定技术将纤维连接蛋白和血管内皮生长因子结合到PLLA表面：涂层、交联和静电自组装(ESA)。细胞在这些表面上的黏附和增殖将使用PicoGreen DNA分析进行研究，而细胞在生理流动条件下的保留率将使用体外平行流动系统进行研究。为了评估种子细胞是否保持内皮细胞的功能特征，我们将检测：(1)细胞对乙酰化低密度脂蛋白(Ac-LDL)的摄取；(2)组胺介导的von Willerbrand因子(VWF)的释放；(3)用免疫细胞化学方法检测血管内皮细胞标志物(VWF)和内皮组成型一氧化氮合酶(EcNOS)的表达。目的#2利用脂质体转染剂构建血管内皮生长因子受体基因修饰的内皮细胞(KDR/Flk-1)。转导基因的表达将通过Western blotting得到确认。目标3是在目标1中开发的最佳表面上种植转基因内皮细胞，并评估它们的性能。EC的粘附性、保持性、增殖性和功能性将按照目标1中的描述进行评估。建议的可生物降解的PLLA材料将具有几个好处，因为这些材料将通过创建保护血管壁的新内皮来促进血管愈合，从而可以在未来作为一种修改血管组织工程表面的方法使用。我们相信，在我们的策略中使用的结合黏附和生长因子的表面修饰和生长因子受体的基因转移将改善内皮化，从而减少人体对假体的不良反应。

DESCRIPTION (provided by applicant): Biodegradable polymers including poly(l-lactic acid) (PLLA) have been used to develop cardiovascular prostheses such as vascular grafts and stents. However, implant-associated thrombosis, inflammation, and restenosis are still a major obstacle for the patency of these prostheses. Our long-term goal is to reduce these complications by rapid endothelialization. Our hypothesis is that rapid endothelialization on the prosthetic surfaces is possible by incorporation of the prosthetic materials with endothelial cell (EC) adhesion (fibronectin) and growth factors (vascular endothelial growth factor, VEGF) and by seeding of genetically modified ECs with growth factor receptors (VEGF receptors, KDR/flk-1) on the prosthetic surfaces. Specifically, aim #1 is to incorporate fibronectin and VEGF onto PLLA surfaces using different immobilized techniques: coating, cross-linking, and electrostatic self-assembly (ESA). Cell adhesion and proliferation on these surfaces will be investigated using PicoGreen DNA assays, whereas cell retention under physiological flow conditions will be studied using an in vitro parallel flow system. To assess whether seeded cells maintain functional characteristics of ECs, we will determine: (1) the cellular uptake of acetylated LDL (ac-LDL), (2) histamine-mediated release of von Willerbrand factor (vWF), and (3) the expression of vWF (endothelial marker) and endothelium constitutive NO synthase (ecNOS) using immunocytochemistry. Aim #2 is to develop genetically modified ECs with VEGF receptors (KDR/flk-1 plasmids) using Lipofectamine(tm) transfection reagent. Expression of transduced genes will be confirmed using Western blotting. Aim #3 is to seed genetically modified ECs on the optimal surface developed in Aim #1 and evaluate their performance. EC adhesion, retention, proliferation, and functionality will be assessed as described in Aim #1. The proposed biodegradable PLLA materials will have several benefits as these materials will stimulate the vessel healing by creating a vessel wall-protecting neoendothelium and thus can be used in the future as a way to modify surfaces for vascular tissue engineering. We believe that surface modification with the combination of adhesion and growth factors and gene transfer of growth factor receptors used in our strategies will improve endothelialization, and thereby reduce the body's adverse reactions toward the prostheses.