VASCULAR TISSUE ENGINEERING: RATIONAL DESIGN USING MODELING

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 组织工程学为心血管疾病的治疗带来了希望。对于组织工程化的心血管结构，人们普遍认为无血栓形成的内皮细胞(EC)表面将是有利的。因此，许多研究试图描述EC与血液的反应性，即EC的“血栓性”。不幸的是，大多数EC的体外研究都采用了抗凝血液、静态或非生理性血流条件，以及相对较短的血液暴露时间。因此，这些结果与体内应用的相关性仍然不确定。同样，在组织工程构建的发展中，关于EC在体内的反应性的研究很少，也没有报道将可在体外测量的ECs的特性与体内反应相关联的系统研究。尽管如此，组织工程界(以及医疗器械和制药行业)现在已经认识到，进一步发展的一个关键障碍是缺乏能够合理设计结构的预测性动物模型，即能够基于体外关键细胞活动的识别和选择性操作来优化体内构建性能的临床前模型。因此，我们的目标是，对于与血液接触的内皮细胞表面，建立可在体外预适应的内皮细胞的促止血和抗止血特性之间的关系，以及体内血栓形成和血管愈合的生理反应。为了实现这一目标，体外可变预处理的构建物的止血性能将与体内结果相关，从而证明合理设计的实用性。建议的研究将使用在体外从全血中容易分离的内皮祖细胞(EPC)，以及相关的在体狒狒血栓形成和血管移植物内膜增生模型。为了特别增强或抑制调节凝血、血小板和其他止血功能的EPC特性，EPC将生长在涂有不同基质蛋白(胶原或弹性蛋白)的常规ePTFE上，并接受不同的血流动力学预处理(静态和正常剪切)。由内皮祖细胞协调的重要止血、炎症和促有丝分裂活动将使用功能、生化、分子和组织学分析进行评估。然后，我们将评估EPC的特性如何影响：1)生理流动条件下天然非抗凝血液中的血栓形成(房室分流模型)，以及2)手术放置后EPC构建物的愈合(主动脉-髂骨移植模型)。尽管结果可能建议新的治疗策略，但我们的主要目标并不是开发改进的血管移植或证明使用内皮祖细胞是合理的。相反，通过证明体外细胞预适应可以预测地改善体内的结果-这一原理可以扩展到其他细胞类型和动物模型-这些结果将验证总体上的合理设计，并鼓励使用其他结构和试验床的研究。最终，这些技术将使开发用于人类的生物结构成为可能。公共卫生相关声明(由申请者提供)：正在开发用于治疗心血管疾病的组织工程替代品。现在必须实现的一个关键进展是在临床前动物模型中建立结构的重要属性--可以在体外识别、测量和优化的属性--与体内包括血栓形成和血管愈合在内的生理反应之间存在预测关系。目前的研究将使用临床相关的灵长类动物模型进行，将建立与血液接触的血管管道的这种相关性，从而验证这些设备的合理设计，并鼓励其他预测性试验台的发展。这些技术对于安全有效的生物假体的开发和临床应用至关重要。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Tissue engineering holds promise for the treatment of cardiovascular disease. For tissue-engineered cardiovascular constructs it is widely believed that a nonthrombogenic endothelial cell (EC) surface will be advantageous. Many studies have therefore attempted to characterize the reactivity of EC with blood, i.e., EC "thrombogenicity". Unfortunately, most in vitro studies with EC have employed anticoagulated blood, static or nonphysiologic blood flow conditions, and relatively short blood exposure times. Consequently, the relevance of these results for in vivo applications remains uncertain. Similarly, in the development of tissue-engineered constructs there have been few studies of EC reactivity in vivo, and no systematic studies have been reported that correlate the properties of ECs that are measurable in vitro with in vivo responses. Nonetheless, it is now recognized within the tissue engineering community (and medical device and drug industries in general) that a key impediment to further progress is the lack of predictive animal models that will enable the rational design of constructs, i.e., preclinical models that will enable optimization of construct performance in vivo based on the identification and selective manipulation of key cellular activities in vitro. Our goal is therefore to establish, for blood-contacting EC surfaces, relationships between pro- and anti- hemostatic properties of ECs that can be preconditioned in vitro, and physiologic responses of thrombosis and vascular healing in vivo. To achieve this goal, the hemostatic properties of constructs variably preconditioned in vitro will be correlated with in vivo outcomes, thereby documenting the utility of rational design. The proposed studies will employ baboon endothelial progenitor cells (EPCs) that are readily isolated from whole blood in vitro, and relevant in vivo baboon models of thrombosis and vascular graft intimal hyperplasia. To specifically enhance or inhibit EPC properties that regulate coagulation, platelets, and other hemostatic functions, EPCs will be grown on conventional ePTFE coated with different matrix proteins (collagen or elastin) and subjected to variable hemodynamic preconditioning (static vs. normal shear). Important hemostatic, inflammatory, and mitogenic activities that are orchestrated by EPCs will be assessed using functional, biochemical, molecular, and histologic assays. We will then assess how EPC properties affect: 1) thrombus formation in native, non-anticoagulated blood under physiologic flow conditions (AV shunt model), and 2) healing of EPC-constructs following surgical placement (aorto-iliac graft model). Although the results may suggest new therapeutic strategies, it is not our primary goal to develop an improved vascular graft or to justify the use of EPCs. Rather, by documenting that cellular preconditioning in vitro can predictably improve outcomes in vivo - a principle that can be extended to other cell types and animal models - these results will validate rational design in general, and encourage studies with other constructs and test beds. Ultimately, these technologies will enable the development of biological constructs for use in man. Public Health Relevance Statement (provided by applicant): Tissue-engineered substitutes are being developed for the treatment of cardiovascular disease. A key advancement that must now be realized is to establish in preclinical animal models that predictive relationships exist between important properties of constructs - properties that can be identified, measured, and optimized in vitro - and in vivo physiological responses including thrombosis and vascular healing. The present studies, to be performed using clinically relevant primate models, will establish such correlations for blood-contacting vascular conduits, thereby validating rational design for these devices and encouraging the development of other predictive test beds. These technologies are critical for the development and clinical application of safe and effective biological prostheses.