Modular Assembly Approach to Engineer Prevascularized Large 3D Tissue Constructs

描述(由申请人提供)： 组织工程学在创造功能组织方面有着巨大的希望，这些组织可以取代人类患病或丢失的组织。近年来，三维(3D)组织培养在再现体内细胞微环境和组织结构方面优于传统的二维(2D)细胞培养已达成共识。人们已经发现，许多工程化组织只有在3D系统中开发时才具有功能。尽管三维组织工程的重要性已经得到认可，并且已经做出了巨大的努力，但开发具有临床相关尺寸的大型和可存活的三维组织的进展有限。制造这种组织产品的一个主要挑战是在大的和血管结构的内部区域没有足够的传质。虽然由预制的多孔支架制备的大型支架在体外可以通过灌流培养增强传质，但这些支架植入体内后，传质不足的问题仍然存在。另一方面，一些原位形成的水凝胶允许被包裹的内皮细胞在植入后形成与宿主血管吻合的毛细血管网络，但没有大孔的水凝胶结构不能进行灌流培养，因此其大小受到限制。缺乏方法来创造大的可灌流的水凝胶结构，支持血管形成前的体外内皮细胞的形态形成，限制了我们解决3D组织工程中质量传递不足的问题的能力。这项R21应用的目的是使用模块化组装方法来开发包含内皮毛细血管网络的大而多孔的水凝胶构建体，并检测这些构建体中细胞的移植后存活。这项工作的中心假设是，具有良好控制的形态和负载内皮细胞和其他感兴趣细胞的纤维蛋白微凝胶可以通过在生理允许的条件下发生的明智的选择的化学反应，在原位模块化地组装成大的、多孔的结构，并且这种组装的结构可以在体外灌流培养，并发展成支持高种植后细胞存活的预血管的、多孔的结构。本项目的具体目标是：(1)设计、制造和鉴定负载人脐静脉内皮细胞(HUVECs)和人骨髓间充质干细胞(HMSCs)的模块化组装的大孔纤维蛋白水凝胶；(2)体外灌流培养大孔载细胞结构，并对毛细血管的形态发生和细胞活力进行表征；(3)移植前多孔结构的植入，并对体内毛细血管网络的功能和移植细胞的存活进行表征。本申请中提出的方法将提供一个平台，以创建厘米大小的包含毛细血管网络的多孔结构，该网络在植入后与宿主血管系统进行吻合，并允许体液的间质流动。该项目的成功完成将解决质量传递不足的问题，这一问题阻碍了临床相关尺寸的功能性3D组织产品的创造。(摘要结束)

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)