Blood Outgrowth Endothelial Cell Seeding of Heart Valve Leaflets

描述(申请人提供)：这项拟议的研究将研究一种治疗心脏瓣膜疾病的新方法，方法是使用三叶聚氨酯假体替换心脏瓣膜，这些假体是使用血液生长内皮细胞(BOEC)内皮种植的。我们将研究由Abied(马萨诸塞州丹弗斯)制造的ABioCor聚亚安酯阀门(ABV)。ABV已经在7000多名人类受试者中使用。BOEC种植的ABV被认为是有效的，可以抵抗导致聚氨酯心脏瓣膜衰竭的主要原因，包括叶表面的血栓形成、钙化和氧化降解。目的1.合成和表征一种新型的双组分光活化复合体，用于胆固醇修饰的聚氨基甲酸乙酯心脏瓣膜，在高流量和高剪切条件下假想增加BOEC的粘附性。我们的合成方法将使用两步法：1)可光激活的聚烯丙基胺-二苯甲酮化合物，称为PBPC，激活聚氨酯心脏瓣膜小叶的表面，随后连接到光连接的PBPC上；2)胆固醇衍生的聚烯丙胺，用硫醇反应基团和离子基团进行生物化学修饰--该络合物的第二种成分称为CPB。这些研究的目标将是创建一套候选的PBPC-CPB配方，用于AIMS 2和3的研究。目标2.体外研究用PBPC-CPB改性的聚氨酯表面以促进BOEC粘附性。这些研究将评估不同候选体外循环的黏附机制、BOEC表型稳定性、内皮功能和激活。然后，我们将研究用铅CPB化合物修饰的ABV和以二尖瓣血流模拟为种子的BOEC。综上所述，这些研究将用于了解黏附机制，并指导候选PBPC-CPB的配方变化，从而在下文描述的目标3中实现最佳的体内细胞黏附。目的3.BOEC种子的体内研究(使用绵羊)，将探索PBPC-CPB的最佳配方。两个体内模型系统将被研究：1)一个主动脉纽扣模型植入物，将在2周的研究中评估BOEC的粘附性、表型稳定性和血栓抗性。还将研究外植体的细胞覆盖范围、内皮表型稳定性、非内皮细胞类型的存在、内皮激活以及血小板和纤维蛋白血栓沉积。2)对ABV表面经PBPC-CPB、BOEC接种和未修饰对照的绵羊进行为期150天的二尖瓣置换术，以评估BOEC的粘附性和表型稳定性，以及预防血栓、钙化和氧化降解的治疗效果。公共卫生相关性：项目叙述心脏瓣膜疾病影响数百万人，只能通过心脏手术治疗。此外，目前的人工心脏瓣膜经常会因血栓形成和原发材料失效而失效。这项提议将通过研究种植有自体血液生长的内皮细胞的三叶型聚氨酯瓣膜来研究心脏瓣膜置换的新方法。

DESCRIPTION (provided by applicant): The proposed research will study a novel approach for treating heart valve disease via the use of heart valve replacements with trileaflet polyurethane prostheses that are endothelial seeded using blood outgrowth endothelial cells (BOEC). We will investigate the AbioCor polyurethane valve (ABV), manufactured by Abiomed (Danvers, MA). The ABV has been used in more than 7,000 human subjects. BOEC seeded ABV are hypothesized to be efficacious for resisting the principal causes of polyurethane heart valve failure, which include thrombosis, calcification and oxidative degradation of leaflet surfaces. Aim 1.Synthesize and characterize a novel two component photoactivated complex for surface modifying polyurethane heart valve leaflets with cholesterol to hypothetically increase BOEC adhesion under high flow and high shear. Our synthetic methods will use a two step approach using: 1) A photo- activatable polyallylamine-benzophone compound, referred to as PBPC, to activate the surface of polyurethane heart valve leaflets for the subsequent attachment to the photo-linked PBPC of, 2) a cholesterol-derivatized polyallylamine, biochemically modified with thiol-reactive groups and ionogenic groups--this 2nd component of the complex is referred to as CPB. The goal of these studies will be to create a candidate set of PBPC-CPB formulations for investigation in Aims 2&3. Aim 2. In vitro investigations of polyurethane surfaces modified with PBPC-CPB to promote BOEC adhesion. These studies will assess adhesion mechanisms, BOEC phenotype stability, endothelial function and activation with various candidate CPB. We will then study ABV modified with lead CPB compounds and BOEC seeded with mitral valve flow simulations. Taken together, these studies will be used to both understand adhesion mechanisms and guide formulation changes in candidate PBPC-CPBs that will result in optimal in vivo cell adhesion in Aim 3 described below. Aim 3. In vivo studies (using sheep) of BOEC seeding that will investigate optimal PBPC-CPB formulations. Two in vivo model systems will be studied: 1) An aortic button model implant that will assess BOEC adhesion, phenotype stability and thrombo-resistance in 2 week studies. Explants will also be studied for extent of cell coverage, endothelial phenotype stability, the presence of nonendothelial cell types, endothelial activation, and platelet and fibrin thrombus depositon. 2) Mitral valve replacements in sheep with ABV surface modified with PBPC-CPB and BOEC seeded and nonmodified controls will be carried out for 150 days with the goal of assessing both BOEC adhesion and phenotype stability, and therapeutic efficacy for preventing thrombus, calcification and oxidative degradation. PUBLIC HEALTH RELEVANCE: Project Narrative Heart valve disease affects millions and is only treatable by cardiac surgery. In addition current artificial heart valves fail frequently due to thrombosis and primary material failure. This proposal will investigate a novel approach for a heart valve replacement by studying a trileaflet polyurethane valve that is seeded with autologous blood outgrowth endothelial cells.