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终身免疫抑制的限制。组织工程有希望在人体外创造新的器官，但一直受到以下方面的阻碍：1)无法充分地使组织结构血管化；2)无法有效地将这些组织重新整合到体循环中。组织工程的经典方法是将细胞植入可吸收基质，在复制简单结构方面取得了成功，但由于难以创建有图案的血管网络，因此无法创建复杂的实质器官。为了解决这个问题，我们开发了一种新的方法，通过使用预先存在的、外植的微循环床作为组织工程的支架，来工程构建器官水平的复杂性。由于这种方法以血管网络为基础，它以类似于胚胎发育或干细胞介导的组织再生的方式，从“内向外”构建自体组织。在我们之前的NIH资助期间，我们展示了在体外长时间维持外植血管床的能力（48-72小时），对其生长环境进行遗传修饰，并有效地将祖细胞植入其中，产生用于再植入的功能新器官单位。基于这一成功，我们假设外植微循环床的长期培养将允许产生自体血管化的新器官，可以可靠地替代功能或生理功能衰竭的器官。在本提案中，Specific Aim 1将定义3-14天体外移植微循环床可靠生存、生长和定向操作所需的灌注条件。在特异性目标2中，将确定输注祖细胞产生血管化新器官的最佳播种和分化条件。最后，特异性目标3将研究再植入血管化新器官单位的耐久性和功能能力，以实现体内的生理作用。我们相信这种“由内而外”的组织工程方法将通过专门解决阻碍其他组织工程范式成功的关键问题，促进血管化器官水平构建的产生。

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.