“体内生物反应器”构建组织工程化气管

Objective: In this study we propose a novel concept of "in-vivo bioreactor" and plan to test its effect on the reepithelialization process of tissue engineered trachea in a sheep model. Equipped with a perfusion system inside the scaffold, the "in-vivo bioreactor" connected to an extracorporeal pump system guarantees continuous medium flow inside the scaffold with the idea to maintain the survival of epithelial cells and to accelerate the revascularization process of the tissue engineered trachea after implantation. In the pilot exams we demonstrated the following advantages of this design: 1) It maintains the survival of epithelial cells seeded on the surface of the scaffold 2) It can be used to refresh seed cells 3) It serves as an efficient way for the delivery of growth factors.4) provide opportunity to establish the trachea regenerative model. Methods: We use acellular porcine tracheal matrix as degradable scaffold, pre-seed with skin keratinocytes and bone marrow stromal cells to reconstruct tissue engineered tracheal substitute to replace 6cm long trachea in ten sheep.The sheep will be divided into in-vivo bioreactor group and control group, 5 sheep in each group.In in-vivo bioreactor group, the porous catheters will be connected to two extracorporeal pumps to form an "in-vivo bioreactor" system which continuously administrates medium. In control group, the tissue engineered trachea will be implanted without any further support. Sheep will be monitored critically for respiratory symptoms and bronchoscopy exams will be performed every other week. All animals will be euthanized and the tracheal substitutes will be harvested after 3 months, assessments including scanning electronic microscopy, histological exams and immunochemistry staining to test the intactness of epithelial layer and the revascularization process. Expectation: The "in-vivo bioreactor" combines the in-vitro and in-vivo parts of tissue engineering research which have been traditionally separated. This novel design should be more practical in clinic since it can be applied immediately without an long perseeding in vitro reconstruction period. In our opinion the proposed scaffold design may improve revascularization and reepithelialization in tissue engineered tracheas.

临床上尚无气管替代品应用,组织工程化气管是研究热点，快速再血管化与再上皮化是急需解决的技术难题。我们提出"体内生物反应器"设计，通过可携带泵系统，在植入的组织工程化气管内部形成持续的培养液灌注，将传统组织工程研究中相互分离的体外种子细胞-支架材料复合物培养与体内组织工程替代品融合再生过程有机的结合在一起，体外预实验证明其具有维持种子细胞存活，实现种子细胞持续性接种，精确控制各类细胞因子浓度加速组织工程化器官替代品再血管化和再上皮化三大优点。本实验计划利用猪气管脱细胞基质作为可降解支架材料,自体表皮细胞和骨髓间充质干细胞为种子细胞,构建组织工程化气管修复10只羊6公分气管缺损，分为两组进一步在体内对比传统组织工程与 "体内生物反应器"方法。另外通过对"体内生物反应器"组灌注液中各类生长因子浓度在气管再生过程中的动态测定，建立气管再生数学模型，为下步干预和加速组织工程化气管再生提供理论依据。

在动物实验基础上，顺利完成亚洲首例组织工程化气管临床应用.组织工程化气管重建任然面临着缓慢的再血管化和替代品感染两个挑战。因此我们提出了“体内生物反应器”(in-vivo bioreactor)概念, 其基本设计原理是利用可携带式泵系统，在植入的组织工程替代品内部形成持续性营养液灌注来形成上皮器官再生。持续灌流液使种子细胞在再血管化前持续存活。与此同时，在灌流液中加入抗生素也能控制局部感染。.一个IIIA期肺鳞癌患者接受了5cm长的组织工程化气管替代手术，连接起了左基底段气管和隆突，得益于“体内生物反应器”的设计该患者避免了左肺切除术。持续的支架内林格氏液灌注持续了一个月，每两周进行一次原位周围全核细胞注射(TNCs)。.该患者最后康复。术后4个月定期的气管镜随访证实了完全的再血管化和再上皮化。废弃的灌流液显示了丰富的原位周围全核细胞（TNCs）分泌的再血管化生长因子。该患者随后接受了两个疗程化疗与30Gy的放疗，且未出现与组织工程气管替代品相关的并发症。.“体内生物反应器”设计“体内生物反应器”将患者本人当成是其组织工程化组织器官再生过程的生物反应器的同时将传统组织工程技术中绝对分离的体外细胞-支架材料培养和体内替代品整合再生过程有机的结合在一起。这一设计可以进行临床应用。我们也第一次证明了组织工程化气管替代品也可以耐受放化疗且适合用于肿瘤治疗。