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问题和终身免疫抑制的需要的限制。组织工程有希望在人体外创造新的器官，但受到了1。不能使组织结构充分血管化和2.)无法有效地将这些组织重新整合到体循环中。使用种植在可吸收基质上的细胞进行组织工程的经典方法在复制简单结构方面取得了成功，但由于难以创建图案化的血管网络，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来设计器官水平的复杂性结构，使用预先存在的、可移植的微循环床作为组织工程的支架。由于这种方法以血管网络为基础，它从“内向外”构建自体组织，其方式类似于胚胎发育或干细胞介导的组织再生。在我们之前的NIH资助期，我们展示了一种能力，可以在体外较长时间(48-72小时)维持移植的血管床，从基因上改变它们的生长环境，并有效地将祖细胞接种到它们身上，创建功能良好的新器官单位用于重新植入。基于这一成功，我们推测，对移植的微循环床进行更长时间的培养将允许产生能够可靠地替代衰竭器官的功能或生理作用的自体血管新生器官。在这项提案中，具体目标1将确定体外3-14天内移植微循环床可靠存活、生长和定向操作所需的灌流条件。在具体目标2中，将确定输注的祖细胞产生血管新生器官的最佳种植和分化条件。最后，特殊目标3将调查重新植入的血管新生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具体解决阻碍其他组织工程范例成功的关键问题，促进血管器官水平结构的产生。

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.