Mechanical Conditioning of Tissue Engineered Blood Vessels for Atherosclerosis

组织工程血管的机械调节治疗动脉粥样硬化

• 批准号: 8398690
• 负责人: Elaine L Lee
• 金额: $ 5.16万
• 依托单位: BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-17 至 2016-01-16
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8398690
• 关键词: Actins; Alcian Blue; American Heart Association; Architecture; Arteries; Atherosclerosis; Biochemical; Biological Assay; Blood Vessels; Carbohydrates; Cardiac; Cell Culture Techniques; Cells; Chronic; Collagen; Collagen Fiber; Complex; Custom; Devices; Dimensions; Elasticity; Elastin; Electrophoresis; Engineering; Excision; Exhibits; Extracellular Matrix; Extracellular Matrix Proteins; Fellowship; Fiber; Genetic Transcription; Glycosaminoglycans; Growth; Hydroxyproline; Immunohistochemistry; Implant; Individual; Inflammatory Response; Laboratories; Lead; Measures; Mechanics; Membrane; Morphology; Myosin Heavy Chains; Periodic acid Schiff stain method; Physiological; Property; Proteins; Recovery; Research; Reverse Transcriptase Polymerase Chain Reaction; Scanning Electron Microscopy; Schiff' s reagent; Smooth Muscle; Smooth Muscle Actin Staining Method; Smooth Muscle Myocytes; Smooth Muscle Myosins; Staining method; Stains; Stenosis; Stretching; Structure; Surface; Technology; Tensile Strength; Testing; Tissue Engineering; Tissues; Transmission Electron Microscopy; Trichrome stain; Western Blotting; artery stenosis; cell injury; cellular imaging; conditioning; falls; fastins; fluorophore; implantation; monolayer; nutrition; pre-doctoral; protein expression; scaffold

DESCRIPTION (provided by applicant): Atherosclerosis is the chronic inflammatory response that leads to stenosed blood vessels. Highly organized and multilaminate in structure, native blood vessels have complex 3D spatial organization with anisotropic contractile properties. Although cell sheet engineering technologies have been devised to allow recovery of aligned cells without damage, native mechanical properties using mechanical conditioning may also be replicated. A device was created using an elastic membrane that can mechanically condition cells for cellular alignment and secretion of proteins and extracellular matrix (ECM), and then allow for a thermoresponsive spontaneous, nondamaging detachment of those cells and ECM fully intact for a scaffold-free cellular implant. We aim to engineer functional tissue engineered blood vessels by creating cell sheet monolayers with controlled ECM growth, which are subsequently stacked and rolled to create a durable blood vessel for implantation. The central hypothesis of this proposal is that layered sheets of pre-conditioned vascular smooth muscle cells results in mechanical architecture and function that more closely mimics native blood vessel mechanics than layered cell sheets without prior conditioning. Aim 1: Nonconditioned or anisotropically conditioned vascular smooth muscle cell sheets will cause cellular alignment comparable to current cell sheet engineering technologies. Cells will be mechanically conditioned at 10% cyclic elongation strain. Immunostaining will be used to identify actin fibers for cellular orientation analysis. We can also measure cell sheet shrinkage after removal from our thermoresponsive surfaces that allow nondamaging cell detachment. Aim 2: Cellular sheets of vascular smooth muscle cells secrete higher protein and extracellular matrix content as a result of mechanical conditioning. Conditioned and nonconditioned cells will be evaluated qualitatively by immunohistochemistry and quantitatively by RT-PCR and Western analysis, to determine RNA and protein expression levels of vascular proteins. Biochemical assays will also be performed to obtain a quantitative ECM profile. Aim 3: Conditioned cell sheets have architecture and mechanical properties more similar to native blood vessels than nonconditioned cell sheets. We will examine individual layers and layered constructs using transmission electron microscopy (TEM) to examine architecture. Mechanical testing using an Instron machine can measure for elasticity and tensile strength. PUBLIC HEALTH RELEVANCE: Atherosclerosis is the chronic inflammatory response that leads to plaque formation in blood vessels. Our research aims to engineer functional tissue engineered blood vessels that have the mechanical properties of the native blood vessel to replace stenosed arteries. We aim to do this by stretching vascular smooth muscle cells to influence secretion of vascular proteins.

描述（由申请人提供）：动脉粥样硬化是导致血管狭窄的慢性炎症反应。天然血管具有高度组织化和多层结构，具有复杂的3D空间组织，具有各向异性的收缩特性。虽然已经设计了细胞片工程技术以允许在没有损伤的情况下恢复对齐的细胞，但是也可以复制使用机械调节的天然机械性质。使用弹性膜创建了一种装置，该弹性膜可以机械调节细胞的细胞排列和蛋白质和细胞外基质（ECM）的分泌，然后允许这些细胞和ECM的热响应性自发、非损伤性脱离，完全完整地用于无支架细胞植入物。我们的目标是通过创建具有受控ECM生长的细胞片单层来工程化功能性组织工程血管，随后将其堆叠并卷起以创建用于植入的耐用血管。该提议的中心假设是，预处理的血管平滑肌细胞的分层片导致比没有预先处理的分层细胞片更接近地模仿天然血管力学的机械结构和功能。目标1：非条件化或各向异性条件化的血管平滑肌细胞片将导致与当前细胞片工程技术相当的细胞排列。细胞将在10%循环伸长应变下进行机械调节。将使用免疫染色来鉴定肌动蛋白纤维以进行细胞取向分析。我们还可以测量从我们的温敏表面去除后的细胞片收缩，从而允许无损伤的细胞分离。目的2：血管平滑肌细胞的细胞片层分泌更高的蛋白质和细胞外基质含量，作为机械调节的结果。 将通过免疫组织化学定性评价条件化和非条件化细胞，并通过RT-PCR和Western分析定量评价条件化和非条件化细胞，以确定血管蛋白的RNA和蛋白质表达水平。还将进行生化测定以获得定量ECM谱。目的3：条件细胞片具有比非条件细胞片更类似于天然血管的结构和机械性质。我们将使用透射电子显微镜（TEM）检查单个层和分层结构来检查架构。使用Instron机器的机械测试可以测量弹性和拉伸强度。 公共卫生相关性：动脉粥样硬化是导致血管中斑块形成的慢性炎症反应。我们的研究旨在设计具有天然血管机械性能的功能性组织工程血管，以取代狭窄的动脉。我们的目标是通过拉伸血管平滑肌细胞来影响血管蛋白的分泌。