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

描述（由申请人提供）： 为了提高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。为了提高体外营养转运能力，本实验室新近开发的新型生物反应器--管状灌注系统（tubular perfusion system，TPS）将在三维支架中动态培养人骨髓间充质干细胞（human mesenchymal stem cells，hMSCs）。该系统采用优雅的设计来创建有效的细胞培养环境，而没有通常与更复杂的灌注系统相关的缺点。TPS设计由包封在藻酸盐珠中的hMSC组成，藻酸盐珠紧密包装在管状生长室中。通过该生长室灌注培养基增强了营养转移，同时使细胞暴露于剪切应力。为了增强体内血管化，在植入之前将血管前网络模板化在工程化组织内。为了实现这一点，将优化TPS生物反应器以支持内皮细胞和hMSC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，特别是藻酸盐珠大小，珠组成和培养基灌注率，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的影响，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。第三，我们建议实施合成聚合物套管系统，以支持体外培养组织的成功植入。该策略允许功能性工程化组织的体外培养，提供了用于组织的体内植入的优雅方法，并且促进植入的组织快速整合到宿主脉管系统中。这些研究的成功完成将证明这种基本技术在基于细胞的装置内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.