Inside-Out Tissue Engineering for Organ Fabrication

描述（由申请人提供）：器官衰竭的移植受到器官稀缺和需要终身免疫抑制的问题的限制。组织工程有望在人体外创造新的器官，但受到以下因素的阻碍：不能使组织构建物充分血管化，和2.）不能有效地将这些组织重新整合到体循环中。使用将细胞接种到可吸收基质上的经典组织工程方法已成功复制简单结构，但由于难以创建图案化血管网络，因此无法创建复杂的实质器官。为了解决这个问题，我们已经开发了一种新的方法，通过使用预先存在的微循环床作为组织工程的支架来设计器官水平复杂性的结构。由于这种方法以血管网络为基础开始，它以类似于胚胎发育或干细胞介导的组织再生的方式从“由内而外”构建自体组织。在我们之前的NIH资助期间，我们证明了在体外维持长时间（48- 72小时）的血管床，遗传修饰其生长环境，并有效地用祖细胞接种它们，创造功能性新器官单位用于重新植入的能力。基于这一成功，我们假设，更长时间的培养的微循环床将允许生成自体血管化的新器官，可以可靠地替代功能或生理作用的衰竭器官。在本提案中，具体目标1将定义离体3-14天的可靠存活力、生长和定向操作微循环床所需的灌注条件。在特定目标2中，将确定输注祖细胞以产生血管化新器官的最佳接种和分化条件。最后，具体目标3将研究重新植入的血管化新器官单位的耐久性和功能能力，以实现体内生理作用。我们相信这种“由内而外”的组织工程方法将有助于血管化的器官水平的结构，特别是解决关键问题，排除在其他组织工程范例的成功。 公共卫生相关性：组织工程有望创造新的器官，但进展受到组织结构血管化不良和这些组织无效重新整合到体循环中的限制。为了解决这个问题，我们已经开发了一种新的方法，通过使用预先存在的微循环床作为组织再生的自体支架来设计器官水平的构建体。我们的初步结果表明，可行的微循环床可以保持离体，与祖细胞接种，并转染，以产生治疗肽-所有关键的初始步骤产生血管化的器官水平的建设。

DESCRIPTION (provided by applicant): Transplantation for organ failure is limited by the problems of organ scarcity and the need for lifelong immunosuppression. Tissue engineering holds the promise to create new organs outside the human body but has been hindered by 1.) the inability to adequately vascularize tissue constructs and 2.) the inability to efficiently re-integrate these tissues into the systemic circulation. Classical approaches to tissue engineering using cells seeded onto resorbable matrices have had success in replicating simple structures but have been unable to create complex parenchymal organs because of the difficulty in creating patterned vascular networks. To solve this problem, we have developed a novel approach to engineer constructs of organ-level complexity by using pre-existing, explanted microcirculatory beds as the scaffold for tissue engineering. Since this approach starts with the vascular network as a foundation, it builds autologous tissue from the "inside-out," in a manner similar to embryonic development or stem cell mediated tissue regeneration. During our previous NIH funding period, we demonstrated an ability to sustain explanted vascular beds for extended periods ex vivo (48-72hrs), genetically modify their growth milieu, and efficiently seed them with progenitor cells creating functional neo-organ units for re-implantation. Based on this success, we postulate that more prolonged cultivation of explanted microcirculatory beds will permit generation of autologous vascularized neo-organs which can reliably substitute a functional or physiologic role for a failing organ. In this proposal, Specific Aim 1 will define the perfusion conditions necessary for reliable viability, growth, and directed manipulation of explanted microcirculatory beds for 3-14 days ex vivo. In Specific Aim 2, the optimal seeding and differentiation conditions of infused progenitor cells to generate vascularized neo-organs will be determined. Finally, Specific Aim 3 will investigate the durability and functional capacity of re-implanted vascularized neo-organ units to fulfill a physiologic role in vivo. We believe this "inside-out" approach to tissue engineering will facilitate the generation of vascularized organ-level constructs by specifically addressing the critical issues that preclude success in other tissue engineering paradigms. PUBLIC HEALTH RELEVANCE: Tissue engineering holds the promise to create new organs but progress has been limited by poor vascularization of tissue constructs and ineffective re-integration of these tissues into the systemic circulation. To address this problem, we have developed a novel approach to engineer organ-level constructs by using pre-existing, explanted microcirculatory beds as an autologous scaffold for tissue regeneration. Our initial results demonstrate that viable microcirculatory beds can be maintained ex vivo, seeded with progenitor cells, and transfected to produce therapeutic peptides - all critical initial steps toward generating vascularized organ- level constructs.