3D Acellular Vascular Beds: Characterization and Re-endothelialization

描述(申请人提供)：拟议研究的目标是从天然组织开发一种三维(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，或可用作体内坏死组织的独立移植物。为了创建血管结构，PI之前开发的脱细胞程序将被用于从高度血管化的肺组织中去除免疫原性细胞成分，同时保留微血管细胞外基质。PI之前已经开发了一种脱细胞程序，以准确地保留周围神经的复杂微结构。根据Co-I之前的工作，这个无细胞的血管床随后将被人间充质干细胞(HMSCs)重新内皮化，hMSCs已被反式分化为内皮细胞表型。脱细胞组织提供了极好的临床机会；事实上，目前许多再生疗法的例子都是利用天然的无细胞组织(例如，Cook Biotech的SIS产品、LifeCell的Allderm和AxoGen的Avance；注：Avance神经移植物基于PI对神经的脱细胞过程)。此外，骨髓间充质干细胞具有很大的临床转化潜力，因为它们具有免疫特权，或者可以从患者体内分离并进行体外扩增。在具体目标1中，高度血管化的肺组织将使用PI先前的去细胞方案进行去细胞处理，然后用于基质保存、细胞去除和体内免疫反应。特别关注的将是保护由大血管和毛细血管组成的3D血管互联网络。脱细胞方法能有效地维持神经中直径为5-10微米的基底膜，支持这种方法可以维持由相同基质蛋白组成的毛细血管网络的假说。在特定目标2中，通过将转分化的人骨髓间充质干细胞注射到组织的血管轴中，脱细胞的血管床将重新内皮化。正如之前由Co-I所做的那样，人MSCs将在聚乙二醇交联纤维蛋白基质(聚乙二醇化纤维蛋白)中向内皮细胞系进行转分化。这些细胞在3D无细胞血管结构内扩张和形成管腔的能力将被评估。一旦接种，这些转分化的hMSCs将受到脉动流的影响，以使细胞适应在天然血管系统中发现的生理压力。该方案中开发的血管构造物可用于促进多种组织类型中临界大小(&gt；100微米)组织的血管形成和组织再生，并可用作研究转分化hMSCs特性的模型系统。 与公共卫生相关：随着器官等待名单继续超过每年捐赠者的数量，替代移植疗法的必要性变得越来越重要。然而，研究人员已经证明，为了维持厚度超过100-200微米的组织的生存能力，血管连接是必要的。到目前为止，还没有一种有效的方法来重建这种血管连接。我们提出的研究目标是从天然组织中开发三维血管床，最终可以用于需要立即血管化的组织工程应用的支架，或者可以用作体内坏死组织的独立移植物。为了创建血管结构，我们将使用我们之前开发的去细胞程序，以准确地保留周围神经复杂的微结构，从高度血管化的肺组织中去除免疫原性细胞成分，同时保留微血管细胞外基质。随后，血管床将用已转分化为内皮细胞表型的人间充质干细胞(MSCs)重新内皮化。MSCs提供了巨大的临床转化潜力，因为它们是免疫特权的，或者可以从患者体内分离出来。此外，脱细胞组织提供了临床机会；事实上，目前大多数基于支架的再生疗法都使用天然的无细胞组织(例如，Cook Biotech的SIS产品、LifeCell的Allderm和AxoGen的Avance；注：Avance神经移植物基于PI对神经组织的脱细胞过程)。

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).