Application of Tubular Perfusion System (TPS) Generated Prevascularized Bone Tiss

描述（由申请人提供）：为了增加基于细胞的治疗策略的可行性，必须克服体外和体内营养转移限制。为了增强体外营养转运，我们实验室最近开发的一种新型生物反应器管状灌注系统（TPS）将在三维支架中动态培养人间充质干细胞（hMSCs）。该系统采用了一种优雅的设计来创造一个有效的细胞培养环境，而没有更复杂的灌注系统所具有的缺点。TPS设计由海藻酸珠包裹的hMSCs组成，海藻酸珠被紧密地包裹在管状生长室中。通过这个生长室灌注培养基可以增强营养物质的转移，同时使细胞暴露在剪切应力下。为了增强体内血管化，在植入前将在工程组织内模板化血管前网络。为了实现这一目标，TPS生物反应器将被优化以支持内皮细胞和hMSCs的共培养。为了研究这种增强体外营养转运和体内血管化的策略，我们建议首先研究TPS培养环境，特别是海藻酸盐珠的大小，珠的组成和介质灌注率，促进hMSC增殖和随后的成骨细胞分化。其次，我们建议研究内皮细胞共培养参数，特别是共培养比例，对血管前网络的发展以及hMSCs的增殖和分化的影响。第三，我们提出了一种合成聚合物套管系统，以支持体外培养组织的成功植入。这种策略允许功能性工程组织的体外培养，为组织的体内植入提供了一种优雅的方法，并促进了植入组织与宿主脉管系统的快速整合。这些研究的成功完成将证明这一基础技术在细胞为基础的设备中增强体外和体内营养转移的可行性。

DESCRIPTION (provided by applicant): In vitro and in vivo nutrient transfer limits must be overcome in order to increase the feasibility of cell based therapeutic strategies. To enhance in vitro nutrient transport, the tubular perfusion system (TPS), a novel bioreactor recently developed by our laboratory, will dynamically culture human mesenchymal stem cells (hMSCs) in three dimensional scaffolds. This system utilizes an elegant design to create an effective cell culture environment without the drawbacks often associated with more complicated perfusion systems. The TPS design consists of hMSCs encapsulated in alginate beads which are tightly packed in a tubular growth chamber. Perfusing media through this growth chamber enhances nutrient transfer while exposing the cells to shear stress. To enhance in vivo vascularization, a prevascular network will be templated within the engineered tissue prior to implantation. To accomplish this, the TPS bioreactor will be optimized to support a coculture of endothelial cells and hMSCs. To examine this strategy of enhanced in vitro nutrient transport and in vivo vascularization, we propose first to investigate the TPS culture environment, particularly alginate bead size, bead composition, and media perfusion rate, that promotes hMSC proliferation and subsequent osteoblastic differentiation. Second, we propose to investigate the impact of endothelial cell coculture parameters, specifically coculture ratio, on the development of a prevascular network as well as the proliferation and differentiation of hMSCs. Third, we propose to implement a synthetic polymer sleeve system to support successful implantation of the in vitro cultured tissue. This strategy allows for the in vitro culture of functional engineered tissue, provides an elegant method for the in vivo implantation of the tissue, and fosters rapid integration of the implanted tissue into the host vasculature. Successful completion of these studies will demonstrate the feasibility of this fundamental technology for enhanced in vitro and in vivo nutrient transfer within cell based devices. PUBLIC HEALTH RELEVANCE: Bone injuries resulting from trauma, tumor removal, or disease are often inadequately healed by the body's natural mechanisms. Current treatments for bone injuries have limited success. Regenerative medicine approaches often suggest successful in vitro culture of stem cells outside and rapid in vivo vascularization of the implanted tissue. To this end, we aim to develop strategies for the culture of mesenchymal stem cells, with a prevascularization network, using a novel bioreactor system.