Enhanced endothelialization for tissue engineering

增强组织工程内皮化

• 批准号: 7011892
• 负责人: Kytai Truong Nguyen
• 金额: $ 21.13万
• 依托单位: UNIVERSITY OF TEXAS ARLINGTON
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7011892
• 关键词: bioassay; biodegradable product; bioengineering /biomedical engineering; biomaterial development /preparation; biotechnology; cardiovascular prosthesis; cell adhesion; cell line; cell proliferation; enzyme linked immunosorbent assay; fibronectins; growth factor receptors; immunocytochemistry; lactates; low density lipoprotein; medical implant science; nitric oxide synthase; polymers; surface coating; tissue engineering; transfection; vascular endothelial growth factors; vascular endothelium; von Willebrand factor; western blottings

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.

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，例如血管移植物和支架。但是，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的内皮化来减少这些并发症。我们的假设是，通过将假体材料与内皮细胞（EC）粘附（纤连蛋白）（纤连蛋白）和生长因子（血管内皮生长因子，VEGF）合并，可以通过遗传因子（VEGF受体受体，KDR/FLK-1）融合生长因子（血管内皮生长因子，VEGF），可以通过内皮细胞（EC）粘附（纤连蛋白）和生长因子（血管内皮生长因子，VEGF）纳入假体材料（EC）粘附（纤连蛋白）和生长因子（血管内皮生长因子，VEGF），这是可能的。具体而言，目标＃1是使用不同的固定技术将纤连蛋白和VEGF掺入PLLA表面上：涂料，交联和静电自组装（ESA）。将使用PICOGREEN DNA分析研究这些表面上的细胞粘附和增殖，而在生理流动条件下将使用体外平行流动系统研究细胞在生理流动条件下的保留。为了评估种子细胞是否维持EC的功能特征，我们将确定：（1）乙酰化LDL（AC-LDL）的细胞摄取，（2）组胺介导的Von Willerbrand因子（VWF）（VWF）的释放，以及（3）使用VWF（内皮标记）和内皮层（内皮标记）表达，使用Noosch（Eciccate）eCnos（Ecicc）（Eciccate）。 AIM＃2是使用Lipofectamine（TM）转染试剂开发具有VEGF受体（KDR/FLK-1质粒）的转基因EC。转导基因的表达将使用蛋白质印迹证实。目标＃3是在AIM＃1中开发的最佳表面上进行转基因EC并评估其性能。 EC的粘附，保留，增殖和功能将如AIM＃1所述进行评估。提出的可生物降解的PLLA材料将具有多种好处，因为这些材料将通过创建容器壁保护新内皮细胞来刺激血管的愈合，因此将来可以用作修饰血管组织工程表面的一种方式。我们认为，在我们策略中使用的粘附和生长因子的结合以及生长因子受体的基因转移的结合将改善内皮化，从而减少人体对假体的不良反应。

项目成果

Enhanced endothelialization on surface modified poly(L-lactic acid) substrates.

• DOI: 10.1089/ten.tea.2010.0129
• 发表时间: 2010-07
• 期刊: Tissue engineering. Part A
• 作者: Hao Xu;R. Deshmukh;R. Timmons;K. Nguyen