3D Acellular Vascular Beds: Characterization and Re-endothelialization

描述（由申请人提供）：拟议研究的目标是从天然组织中开发三维（3D）血管床，最终可用于需要立即血管化的组织工程应用的支架，或可作为体内坏死组织的独立移植物。为了创建血管结构，PI先前开发的一种脱细胞程序将用于从高度血管化的肺组织中去除免疫原性细胞成分，同时保存微血管细胞外基质，以准确地保存周围神经的复杂微结构。根据Co-I先前的工作，这种脱细胞血管床随后将用人间充质干细胞（hMSCs）重新内皮化，这些干细胞已被转分化为内皮细胞表型。脱细胞组织提供了良好的临床机会；事实上，目前许多再生疗法都是利用天然的脱细胞组织（例如，Cook Biotech的SIS产品、LifeCell的AlloDerm产品和AxoGen的Avance产品；注意：Avance神经移植是基于PI的神经脱细胞过程）。此外，间充质干细胞为临床转化提供了巨大的潜力，因为它们具有免疫特权，或者可以从患者身上分离出来并在体外扩增。在Specific Aim 1中，高度血管化的肺组织将使用PI先前的脱细胞方案进行脱细胞，随后进行基质保存，细胞去除和体内免疫反应的表征。特别的重点将是保存大血管和毛细血管的三维血管互联网络。脱细胞方法在维持5-10微米直径的神经基层上是有效的，这支持了该方法可以维持由相同基质蛋白组成的毛细血管网络的假设。在Specific Aim 2中，通过将转分化的人间充质干细胞注射到组织的血管轴中，将脱细胞血管床重新内皮化。人类间充质干细胞将在聚乙二醇（PEG）交联纤维蛋白基质（聚乙二醇化纤维蛋白）中向内皮细胞谱系转分化，如Co-I先前所做的那样。这些细胞在三维无细胞血管结构内扩张和形成管腔的能力将被评估。一旦播种，这些转分化的hMSCs将受到脉动流的影响，为细胞在天然血管系统中发现的生理应激做好准备。本研究中构建的血管结构可用于促进多种组织类型中临界尺寸缺陷（bbb100微米）组织的血管化和再生，也可作为研究转分化hMSCs特性的模型系统。

DESCRIPTION (provided by applicant): The goal of the proposed research is to develop a three-dimensional (3D) vascular bed from natural tissues, which can ultimately be used in scaffolds for tissue engineering applications requiring immediate vascularization or which can be used as stand-alone grafts for necrotic tissues in the body. To create the vascular construct, a decellularization procedure, which the PI has previously developed to accurately preserve the intricate micro-architecture of peripheral nerve, will be used to remove the immunogenic cellular components from highly vascularized lung tissue and to simultaneously preserve the microvessel extracellular matrix. This acellular vascular bed will be subsequently re-endothelialized with human mesenchymal stem cells (hMSCs) that have been trans differentiated into an endothelial cell phenotype, based on previous work of the Co-I. Decellularized tissues offer excellent clinical opportunities; in fact, many examples of current regenerative therapies utilize natural, acellular tissues (e.g., SIS products from Cook Biotech, AlloDerm from LifeCell, and Avance from AxoGen; note: the Avance nerve graft is based on the PI's decellularization processes for nerve). In addition, MSCs offer great potential for clinical translational because they are immune-privileged or they can be isolated from the patient and expanded ex vivo. In Specific Aim 1, highly vascularized lung tissue will be decellularized using the PI's previous decellularization protocol and subsequently characterized for matrix preservation, cellular removal, and in vivo immune response. Particular focus will be on preserving the 3D vascular interconnected network of large vessels and capillaries. The decellularization method has been effective in maintaining basal laminae of 5-10 microns diameter in nerve, supporting the hypothesis that this method can maintain capillary networks composed of the same matrix proteins. In Specific Aim 2, the decellularized vascular bed will be re-endothelialized by injecting transdifferentiated human MSCs into the vascular axis of the tissue. Human MSCs will be transdifferentiated in a poly(ethylene glycol) (PEG) crosslinked fibrin matrix (PEGylated fibrin) towards endothelial lineages, as performed previously by the Co-I. The ability of these cells to expand and form lumen inside the 3D acellular vascular construct will be evaluated. Once seeded, these transdifferentiated hMSCs will be subjected to pulsatile flow, to precondition the cells for physiological stresses that are found in the native vascular system. The vascular constructs developed in this proposal could be used to promote vascularization and regeneration of tissues in critically-sized defects (>100 microns) in a multitude of tissue types, as well as be used as a model system to investigate the properties of transdifferentiated hMSCs. PUBLIC HEALTH RELEVANCE: As the organ wait list continues to exceed the number of donors each year, the need for alternative therapies to transplantation is becoming increasingly important. Yet, researchers have demonstrated that a vascular connection is necessary in order to maintain viability in tissues beyond 100-200 microns thick. To date, there is no effective manner in which to recreate this vascular connection. The goal of our proposed research is to develop three-dimensional vascular beds from natural tissues, which can ultimately be used in scaffolds for tissue engineering applications requiring immediate vascularization or which can be used as stand-alone grafts for necrotic tissues in the body. To create the vascular construct we will use a decellularization procedure, which we have previously developed to accurately preserve the intricate micro-architecture of peripheral nerve, to remove the immunogenic cellular components from highly vascularized lung tissue and to simultaneously preserve the microvessel extracellular matrix. The vascular bed will be subsequently re- endothelialized with human mesenchymal stem cells (MSCs) that have been transdifferentiated into an endothelial cell phenotype. MSCs offer great potential for clinical translational because they are immune- privileged or they can be isolated from the patient. In addition, decellularized tissues offer clinical opportunities; in fact, most examples of current scaffold-based regenerative therapies utilize natural, acellular tissues (e.g., SIS products from Cook Biotech, AlloDerm from LifeCell, and Avance from AxoGen; note: the Avance nerve graft is based on the PI's decellularization processes for nerve tissue).