细胞膜片技术促进3D打印组织工程气管再上皮化并行犬原位移植的实验研究

Tissue-engineering tracheal graft is a viable solution to overcoming the shortage of graft material for tracheal transplantation. In previous project, we have developed a composite structural (PLLACL copolymer rigid layer and cell-supporting soft layer) tissue-engineered trachea via 3D bioprinting. We then successfully performed orthotopic transplantation in animal experiment and subsequent ex vivo validation of its physical and biological characteristics. However, epithelial cells were found easily damaged during 3D bioprinting. Misalignment of printed seed cells resulted in a lack of intercellular connection. To overcome these problems in the present proposed study, we introduce Cell Sheet technique, attempting to prevent epithelial cells loss from digestion or bioprinting procedures, thus maintaining extracellular matrix integrity and a robust intercellular connection for epithelial layer of tissue-engineered trachea. Subsequently, we transfer the epithelial layer to the interior of 3D bioprinted tracheal scaffold, to forge a triple-layered tissue-engineered trachea (PLLACL rigid layer/hydrogel-chondrocyte layer/Cell Sheet epithelial layer). We assess the physical and biological characteristics of bioprinted trachea segment, then study its re-epithelialization and revascularization potential through animal orthotopic transplantation. The proposed study serves as theoretical prototype and laboratorial evidence to future clinical applications.

目前气管移植缺乏成熟的替代物，组织工程气管是最有前景的解决方案。在前期国自然基金资助下，我们使用3D生物打印技术成功构建出一种复合结构的组织工程气管(PLLACL硬材料层和细胞软材料层)，并在动物体内进行原位移植，随后通过体内外实验证实其良好的理化及生物学性能。然而，我们发现上皮细胞在制备过程中易受损耗，打印后排列不均一且缺乏胞间连接，值得进一步探索和优化。为此，本课题引入细胞膜片(Cell Sheet)技术，使上皮细胞免于酶消化及打印损伤，保留完整的细胞外基质及胞间连接，构建组织工程气管的上皮层。随后将其迁移贴附于3D生物打印气管支架内表面，成功构建出三层结构组织工程气管(PLLACL硬材料层/水凝胶软骨细胞层/Cell Sheet上皮细胞层)，并对其理化和生物学性能进行评估。最后在动物体内行原位移植，并动态观察其再上皮化及再血管化情况，为将来进一步的临床应用提供理论基础和实验依据。

目前气管移植缺乏成熟的替代物，亟待开发具备功能性气道上皮层的组织工程气管。为此，本课题完成下述工作：① 引入细胞膜片(Cell Sheet)技术，利用聚N-异丙基丙烯酰胺涂层的温敏培养皿进行气道上皮细胞膜片制备，使上皮细胞免于酶消化及打印损伤，并保留完整的细胞外基质及胞间连接；② 采用甲基丙烯酰胺明胶（Gelatin methacryloyl, GelMA）作为软骨细胞生物打印墨水，筛选最优打印参数，构建活性气管软骨层；③ 利用聚己内酯（PCL）材料，以3D plotting 技术完成补片硬材料骨架层的构建，并结合活性气管软骨层与上皮细胞膜片，成功构建出三层结构组织工程气管(PCL硬材料层/水凝胶软骨层/Cell Sheet上皮细胞层)，并通过原位犬原位移植验证其生物安全性与临床价值。本课题为构建具备天然功能的组织工程气管提供了丰富的理论基础与技术支持，有助于气管替代物产品的进一步开发与临床转化。