准静态平面均匀流场下组织工程皮肤毛细血管网形成及功能研究

Vascularization of engineered constructs is one of the biggest challenges for the development of tissue engineering. The traditional method is to transplant the constructs on the animal for the vascularization. Recently, a new microfliudic technology is emerging and shows the capillary-like network in the tissue engineered constructs can be perfusable in the perfusional flow especially under the normal physiologic range. We have established a novel perfusion method called the quasi-static flat homogeneous flow field that is consistent with the perfusion condition of the normal physiologic range. So we pose a new view that the quasi-static flat homogeneous flow field is suited to induce the capillary-like network to finish the process of mature and perfusable. So in this project, on the base of the composite chitosan tissue engineered skin established before, endothelial cells will be inoculated into the dermal layer of the composite chitosan tissue engineered skin and perfused under the quasi-static flat homogeneous flow field to observe the perfusable and mature of the capillary-like network including the total length, total number and average length of vessel network, and average number of branches per vessel network on the cross section, the mature markers on immunohistochemical staining, the anastomosed and interconnected network with the perfusion of red blood cells, resin cast and fluorescent materials. Then the different perfusion velocity will be studied and the prevascularization of needle-based subtractive molding will be added in this method. Finally, transplantational test will be done on the immunodeficiency animal mice to observe the time and number of anastomosed vessel networks. This project will be provided the evidence that our bioreactor as a important platform to improve and develop the capillary-like network into mature and perfusable and become the functional vessel.

组织工程面临的最大挑战之一是工程化组织的血管化问题，传统方法是采用动物移植来解决，近年来微流控技术研究表明在灌注条件下能够使毛细血管网实现灌通，尤其是符合生理范围的参数更好，前期我们建立的准静态平面均匀流场符合其灌注的生理参数，因此我们提出“准静态平面均匀流场体外能够实现毛细血管网的灌通和成熟”的新观点。本研究拟在复方壳多糖组织工程皮肤的基础上，真皮层中再加入内皮细胞，置于准静态平面均匀流场中行动态培养，再联合应用微针预包埋技术，探讨适宜的灌注速度，观察真皮层中毛细血管网的可灌流功能情况，包括组织切片计数毛细血管网的数量、长度、分枝数，免疫组化分析毛细血管网的成熟，环氧树脂灌注塑形、鼠红细胞和荧光物质分别灌注了解毛细血管网吻合和互通情况，进而移植到免疫缺陷鼠观察与机体血管的吻合时间和吻合数量，为证明生物反应器能够使毛细血管网样结构进一步发育成熟成具有灌流作用的功能性血管提供重要实验证据。

组织工程面临的最大挑战之一是工程化组织的血管化问题，缺乏有效的血管网，移植后不易从机体获得营养，导致组织不能存活。本研究以血管生成和细胞共培养技术为基础，在复方壳多糖真皮基质凝胶中预混成纤维细胞和血管内皮细胞，表面再接种角质形成细胞，形成双层组织工程皮肤，在准静态平面均匀流场中进行动态培养，观察真皮层中毛细血管网的可灌流功能。首先需要摸索了一种适合三种种子细胞共培养的培养基，我们对现有的一些培养基进行改良筛选，选出了一种相对好的共培养基；其次，对复方壳多糖真皮基质凝胶配方进行改良，结果表明在真皮层中能够形成较多血管管腔，HE和免疫荧光表明内皮细胞在真皮层能够实现血管化，说明三种细胞的共培养环境得到了一定改善；再次我们以复方壳多糖组织工程真皮为研究对象，对三个灌注速度即50、100、200ml/min进行了对比研究，从细胞形态、葡萄糖乳酸代谢、羟脯氨酸含量、最大抗张强度及真皮内细胞数量等几个指标来分析，表明100 ml/min的灌注流速更适合培养复方壳多糖组织工程真皮。