3D Acellular Vascular Beds: Characterization and Re-endothelialization

3D 无细胞血管床：表征和再内皮化

• 批准号: 8096092
• 负责人: CHRISTINE E SCHMIDT
• 金额: $ 18.05万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8096092
• 关键词: Affect; Alternative Therapies; Anastomosis - action; Angiogenic Factor; Architecture; Basal lamina; Biological Models; Biological Preservation; Blood Vessels; Blood capillaries; Caliber; Cell Culture Techniques; Cell Survival; Cells; Cellularity; Clinical; Complex; Defect; Dimensions; Endothelial Cells; Engineering; Ethylene Glycols; Excision; Extracellular Matrix; Fibrin; Fractals; Future; Gene Expression; Goals; Histocompatibility Testing; Histology; Human; Immune; Immune response; Immunohistochemistry; Implant; Liquid substance; Mesenchymal Stem Cells; Methods; Microfilaments; Modeling; Morphology; Necrosis; Nerve; Nerve Tissue; Nutrient; Organ; Patients; Penetration; Perfusion; Peripheral Nerves; Phenotype; Physiological; Procedures; Process; Proliferating; Property; Prostaglandins I; Proteins; Protocols documentation; Pulsatile Flow; Rattus; Research; Research Personnel; Shapes; Source; Stress; Structure of parenchyma of lung; Surgical Anastomosis; System; Techniques; Technology; Thick; Tissue Engineering; Tissues; Translating; Transplantation; Vascular System; Vascularization; Waiting Lists; Western Blotting; Work; base; capillary; capillary bed; cooking; crosslink; ethylene glycol; experience; fluid flow; immunogenic; immunogenicity; implantation; in vivo; preconditioning; regenerative; regenerative therapy; scaffold; shear stress; tissue regeneration; two-dimensional; vascular bed

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).

描述（由申请人提供）：拟议研究的目标是利用天然组织开发三维（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，或可用作体内坏死组织的独立移植物。为了创建血管结构，PI先前开发了一种脱细胞程序，用于准确保存周围神经的复杂微结构，该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，并同时保留微血管细胞外基质。根据 Co-I 之前的工作，该无细胞血管床随后将用已转分化为内皮细胞表型的人间充质干细胞 (hMSC) 重新内皮化。脱细胞组织提供了极好的临床机会；事实上，当前再生疗法的许多例子都利用天然的脱细胞组织（例如，Cook Biotech 的 SIS 产品、LifeCell 的 AlloDerm 和 AxoGen 的 Avance；注：Avance 神经移植物基于 PI 的神经脱细胞化过程）。此外，间充质干细胞具有巨大的临床转化潜力，因为它们具有免疫特权，或者可以从患者体内分离出来并进行离体扩增。在具体目标 1 中，高度血管化的肺组织将使用 PI 之前的去细胞化方案进行去细胞化，并随后进行基质保存、细胞去除和体内免疫反应的表征。特别关注的是保存大血管和毛细血管的 3D 血管互连网络。脱细胞方法可有效维持神经直径5-10微米的基底层，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。在具体目标 2 中，通过将转分化的人类 MSC 注射到组织的血管轴中，脱细胞血管床将重新内皮化。人类 MSC 将在聚乙二醇 (PEG) 交联纤维蛋白基质（聚乙二醇化纤维蛋白）中向内皮谱系转分化，如 Co-I 之前进行的那样。将评估这些细胞在 3D 无细胞血管结构内扩张和形成管腔的能力。一旦接种，这些转分化的 hMSC 将受到脉动流的作用，以预处理细胞以应对天然血管系统中发现的生理应激。该提案中开发的血管构建体可用于促进多种组织类型中临界尺寸缺陷（> 100微米）的血管化和组织再生，并可用作研究转分化hMSC特性的模型系统。 公共卫生相关性：随着器官等待名单每年继续超过捐赠者的数量，对移植替代疗法的需求变得越来越重要。然而，研究人员已经证明，为了维持厚度超过 100-200 微米的组织的活力，血管连接是必要的。迄今为止，还没有有效的方法来重建这种血管连接。我们提出的研究的目标是利用天然组织开发三维血管床，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，我们将使用我们之前开发的脱细胞程序，以准确保留周围神经的复杂微结构，从高度血管化的肺组织中去除免疫原性细胞成分，并同时保留微血管细胞外基质。随后，血管床将被已转分化为内皮细胞表型的人间充质干细胞（MSC）重新内皮化。间充质干细胞具有巨大的临床转化潜力，因为它们具有免疫特权或者可以从患者体内分离出来。此外，脱细胞组织提供了临床机会；事实上，当前基于支架的再生疗法的大多数例子都利用天然的脱细胞组织（例如，Cook Biotech 的 SIS 产品、LifeCell 的 AlloDerm 和 AxoGen 的 Avance；注：Avance 神经移植物基于 PI 的神经组织脱细胞化过程）。