Enhanced endothelialization for tissue engineering

描述（由申请人提供）：包括聚（l-乳酸）（PLLA）在内的可生物降解聚合物已用于开发心血管假体，如血管移植物和支架。然而，与植入物相关的血栓形成、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，假体表面上的快速内皮化是可能的，通过将假体材料与内皮细胞（EC）粘附（纤连蛋白）和生长因子（血管内皮生长因子，VEGF）结合，并通过在假体表面上接种具有生长因子受体（VEGF受体，KDR/flk-1）的遗传修饰的EC。具体而言，目标#1是使用不同的固定化技术将纤连蛋白和VEGF结合到PLLA表面上：涂覆、交联和静电自组装（ESA）。这些表面上的细胞粘附和增殖将使用PicoGreen DNA测定法进行研究，而生理流动条件下的细胞保留将使用体外平行流动系统进行研究。为了评估接种的细胞是否保持EC的功能特征，我们将使用免疫细胞化学确定：（1）乙酰化LDL（ac-LDL）的细胞摄取，（2）组胺介导的von Willerbrand因子（vWF）的释放，以及（3）vWF（内皮标志物）和内皮组成型NO合酶（ecNOS）的表达。目的#2是使用Lipofectamine（tm）转染试剂开发具有VEGF受体（KDR/flk-1质粒）的遗传修饰的EC。将使用蛋白质印迹法确认转导基因的表达。目标#3是在目标#1中开发的最佳表面上接种遗传修饰的EC并评估其性能。EC粘附、保留、增殖和功能性将按照目标#1中所述进行评估。所提出的可生物降解的PLLA材料将具有几个益处，因为这些材料将通过产生血管壁保护性新内皮来刺激血管愈合，因此可以在未来用作血管组织工程的表面改性的方式。我们相信，结合粘附和生长因子以及我们策略中使用的生长因子受体的基因转移的表面改性将改善内皮化，从而减少身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.