Modular Assembly Approach to Engineer Prevascularized Large 3D Tissue Constructs

描述（由申请人提供）： 组织工程在创造功能性组织方面具有巨大的前景，这些功能性组织可以替代人类患病或丢失的组织。近年来，三维组织培养在再现体内细胞微环境和组织结构方面上级传统的二维细胞培养，这一点已成为共识。已经发现，许多工程组织只有在3D系统中开发时才有功能。尽管认识到3D组织工程的重要性和已经做出的巨大努力，但开发具有临床相关尺寸的大且可行的3D组织的进展受到限制。产生这种组织产品的一个主要挑战是在大血管构造的内部区域中的质量传递不足。尽管在体外通过灌注培养可以增强由预成型多孔支架制备的大结构中的传质，但是在这些结构植入体内后，传质不足仍然是一个问题。另一方面，一些原位形成的水凝胶允许包封的内皮细胞形成毛细血管网络，所述毛细血管网络在植入后经历与宿主脉管系统的吻合，但是没有大孔的水凝胶构建体不能进行灌注培养，使得它们的尺寸受到限制。缺乏方法来创建大的可灌注的水凝胶结构，支持在体外内皮毛细血管形态发生血管化前限制了我们的能力，以解决问题的质量转移不足的三维组织工程。该R21申请的目的是使用模块化组装方法来开发含有内皮毛细血管网络的大型多孔水凝胶结构，并检查这些结构中细胞的植入后存活。这项工作的中心假设是，具有良好控制的形态并负载有内皮细胞和其他感兴趣的细胞的纤维蛋白微凝胶可以通过在生理允许条件下发生的明智选择的化学反应原位模块化地组装成大的多孔结构，并且这种组装的结构可以在体外灌注培养并发育成预血管化的，支持高植入后细胞存活的多孔结构。本课题的具体目标是：（1）设计、制备和表征负载人脐静脉内皮细胞（HUVECs）和hMSCs的模块化组装大孔纤维蛋白水凝胶;（2）在灌注下体外培养负载大孔细胞的构建体并表征毛细血管形态发生和细胞活力;（3）植入预血管化的多孔结构和表征毛细血管网络的体内功能和移植细胞的植入后存活。本申请中提出的方法将提供一个平台，以创建包含毛细血管网络的厘米级多孔结构，所述毛细血管网络在植入后与宿主脉管系统进行吻合，并允许体液的间质流动。该项目的成功完成将解决质量转移不足的问题，这阻碍了临床相关尺寸的功能性3D组织产品的创建。(End摘要）

DESCRIPTION (provided by applicant): Tissue engineering holds great promise in creating functional tissues that can replace diseased or lost tissues of human beings. Recently, consensus has been reached that three-dimensional (3D) tissue culture is superior to traditional two-dimensional (2D) cell culture in recapitulating the in vivo cell microenvironments and tissue structures. It has been found that many engineered tissues are functional only when they are developed in 3D systems. Despite the recognized importance of 3D tissue engineering and the tremendous efforts that have been made, the progress of developing large and viable 3D tissues of clinically relevant sizes has been limited. One major challenge in creating such tissue products is insufficient mass transfer in the interior region of large and a vascular constructs. Although mass transfer in large constructs prepared from preformed porous scaffolds can be enhanced in vitro through perfusion culture, insufficient mass transfer remains a problem after these constructs are implanted in vivo. On the other hand, some in situ forming hydrogels allow encapsulated endothelial cells to form capillary networks that undergo an anastomosis with the host vasculature after implantation, but hydrogel constructs without large pores cannot be perfusion-cultured so that their size is limited. Lack of methods to create large perfusable hydrogel constructs supporting in vitro endothelial capillary morphogenesis for prevascularization limits our ability to address the problem of insufficient mass transfer in 3D tissue engineering. The objective of this R21 application is to use a modular assembly approach to develop large, porous hydrogel constructs containing endothelial capillary networks and to examine postimplantation survival of the cells in these constructs. The central hypothesis of this work is that fibrin microgels having well-controlled morphology and laden with endothelial cells and other cells of interest can be modularly assembled into large, porous constructs in situ through a judiciously selected chemical reaction occurring under physiologically permissive conditions and such assembled constructs can be perfusion- cultured in vitro and develop into prevascularized, porous constructs that support high postimplantation cell survival. The Specific Aims of this project are: (1) design, fabrication, and characterization of modularly assembled large porous fibrin hydrogels laden with human umbilical vein endothelial cells (HUVECs) and hMSCs; (2) in vitro culture of large porous cell-laden constructs under perfusion and characterization of capillary morphogenesis and cell viability; (3) implantation of prevascularized porous constructs and characterization of in vivo function of the capillary networks and postimplantation survival of transplanted cells. The method proposed in this application will provide a platform to create centimeter-sized porous constructs containing capillary networks that undergo anastomosis with the host vasculature after implantation and allow interstitial flow of body fluids. Successful accomplishment of this project will address the problem of insufficient mass transfer that hampers creation of functional 3D tissue products of clinically relevant sizes. (End of Abstract)