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被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、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来减少这些并发症。我们的假设是，通过结合内皮细胞（EC）粘附（纤维连接蛋白）和生长因子（血管内皮生长因子，VEGF）的假体材料，以及在假体表面植入带有生长因子受体（VEGF受体，KDR/flk-1）的转基因EC，可以实现假体表面的快速内皮化。具体来说，目标1是使用不同的固定技术将纤维连接蛋白和VEGF结合到PLLA表面：涂层，交联和静电自组装（ESA）。细胞在这些表面的粘附和增殖将使用PicoGreen DNA测定法进行研究，而细胞在生理流动条件下的保留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，我们将确定：(1)细胞对乙酰化LDL （ac-LDL）的摄取，(2)组胺介导的von Willerbrand因子（vWF）的释放，以及(3)vWF（内皮标志物）和内皮构成型NO合成酶（ecNOS）的表达。目的2是使用Lipofectamine（tm）转染试剂开发带有VEGF受体（KDR/flk-1质粒）的转基因内皮细胞。转导基因的表达将用Western blotting进行确认。目标#3是在目标#1中开发的最佳表面上播种转基因ec，并评估其性能。EC的粘附、保留、增殖和功能将按照目标1的描述进行评估。提出的可生物降解pla材料将有几个好处，因为这些材料将通过创建血管壁保护的新内皮细胞来刺激血管愈合，因此可以在未来用作血管组织工程修饰表面的一种方法。我们相信，结合粘附因子和生长因子受体的表面修饰以及我们策略中使用的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。

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