Micro-Thickness Collagen Membranes in Tissue Engineering

描述（由申请人提供）：动脉硬化冠状动脉搭桥治疗的一个主要问题是许多患者缺乏合适的自体替代血管。该提案探索了新技术，以创建组织工程血管（TEBV），可以作为冠状动脉的现成替代品。动脉壁（中膜）的大部分厚度由排列在同心层中的血管平滑肌细胞（VSMC）组成，这些细胞提供机械强度和收缩性。在目标1中，我们将构建TEBV介质，其中包含我们最近开发的新型生物相容性胶原膜（CM）支架。CM的高内在强度和体外预对准VSMC的能力（通过微槽）可以将TEBV制造时间大大缩短到几天，而不是其他方法所需的几周或几个月。填充有汇合、对齐的人冠状动脉SMC（HCASMC）的CM将围绕芯轴缠绕以形成多层管。在移除心轴之后，将用人冠状动脉内皮细胞接种TEBV管腔，并且TEBV随后在营养液浴中生长，其中营养液也通过TEBV管腔循环。在特定的培养间隔后，将评估TEBV的：（i）VSMC的细胞外基质（ECM）沉积，（ii）CM支架的结构变化，（iii）VSMC的存活、定向和分化状态，（iv）管腔的功能性内皮化，和（v）机械（爆裂）强度。弹性蛋白质弹性蛋白在正常动脉壁中是丰富的，并且其在大多数TEBV中的缺乏导致弹性的缺乏和VSMC的过度增殖，这使TEBV管腔变窄。最终，希望使用患者自身的VSMC来限制移植物排斥。不幸的是，成人VSMC合成很少或没有弹性蛋白。为了解决这个问题，目的2评估ECM沉积，细胞存活和取向，和机械性能（爆裂强度，应力/应变响应）的TEBV，纳入成年大鼠主动脉SMC产生的弹性蛋白水平升高的结果表达（通过病毒转导）的蛋白聚糖多功能蛋白聚糖3。该提案旨在开发新的方法和技术，并将其应用于人类健康的一个重要问题（冠状动脉疾病）。此外，这项工作的数据将作为进一步开发TEBV的基础。因此，本申请非常适合R21机制的目的。

DESCRIPTION (provided by applicant): A major problem in coronary bypass treatment for arteriosclerosis is the lack of suitable autogenous replacement vessels in many patients. This proposal explores new technologies to create tissue-engineered blood vessels (TEBVs) that can serve as readily available replacements for coronary arteries. The bulk of the thickness of an artery wall (the media) is comprised of vascular smooth muscle cells (VSMCs) arranged in concentric layers that provide mechanical strength and contractility. In Aim 1, we will construct TEBV media that incorporate a novel, biocompatible, collagen membrane (CM) scaffold that we developed recently. The CMs' high intrinsic strength and capacity to prealign VSMCs in vitro (via microgrooving) could dramatically shorten TEBV fabrication times to a matter of days, rather than the weeks or months required by other methods. CMs populated with confluent, aligned human coronary artery SMCs (HCASMCs) will be wrapped around a mandrel to form a multilamellar tube. Following removal of the mandrel, the TEBV lumen will be seeded with human coronary artery endothelial cells and the TEBV subsequently grown in a bath of nutrient fluid with nutrient fluid also circulated through the TEBV lumen. After specific intervals of culture, the TEBVs will be evaluated for: (i) deposition of extracellular matrix (ECM) by the VSMCs, (ii) structural changes in the CM scaffolds, (iii) survival, orientation and state of differentiation of the VSMCs, (iv) functional endothelialization of the lumen, and (v) mechanical (burst) strength. The resilient protein elastin is abundant in normal artery walls and its absence in most TEBVs results in a lack of elasticity and excessive proliferation of VSMCs which narrows the TEBV lumen. Ultimately, it would be desirable to use the patient's own VSMCs to limit graft rejection. Unfortunately, adult VSMCs synthesize little or no elastin. To address this problem, Aim 2 evaluates ECM deposition, cell survival and orientation, and mechanical properties (burst-strength, stress/strain response) of TEBVs that incorporate adult rat aortic SMCs that produce elevated levels of elastin as a consequence of expression (via viral transduction) of the proteoglycan versican 3. This proposal seeks to develop new approaches and technologies and apply them to an important problem in human health (coronary artery disease). Moreover, data from this work will serve as a foundation for further development of TEBVs. Accordingly, this application is well suited to the purpose of the R21 mechanism.