目的 利用猪耳廓软骨细胞制备软骨细胞膜片,与3D打印聚酰胺带孔管状支架结合,构建管状软骨组织,用于气管软骨的缺损重建。方法 用酶消化法分离获得原代猪耳廓软骨细胞,以5×105cells/m L的密度将第2代软骨细胞接种于6 cm细胞培养皿上,在软骨细胞膜片培养环境中培养约14 d,获得完整软骨细胞膜片;4层软骨细胞膜片依次卷叠于3D打印的聚酰胺(PA12)带孔管状内支架上,以构建管状软骨组织复合物;体外静态培养12周或裸鼠皮下培养8周,对形成的管状软骨组织行大体观察及石蜡切片组织学染色鉴定。结果 体外静态培养12周后,半透明管状软骨组织形成,组织学染色可见少量分泌的软骨细胞外基质,质软,弹性差;体内培养8周后,白色管状软骨组织形成,其组织学染色可见大量软骨细胞外基质分泌,质较硬,有一定弹性。结论 利用软骨细胞膜片复合3D打印聚酰胺支架,可构建组织工程管状软骨,有望将其用于气管软骨缺损的修复。

Objective To construct tubulous cartilage by porcine auricular cartilage-derived chondrocyte-sheets combing with 3D printed supporter for repairing tracheal cartilage defects. Methods Primary chondrocytes were isolated from porcine auricular cartilage with methods of trypsin enzyme digestion and collagenase enzyme digestion. After proliferation in vitro, the chondrocytes of passage 2 were seeded on culture dish with a high density of 5 ×105 cells/mL, and intact chondrocyte-sheets were detached after cultured in a specific cell sheet culture environment for 14 days in vitro. Four detached chondrocyte-sheets were rolled onto a 3D printed supporter of PA material one by one to construct a tubulous cartilage-like complex. Twelve weeks’ static culture in vitro as well as 8 weeks' subcutaneous culture in nude mouse were performed. Gross inspection and paraffin section staining were applied for evaluation. Results Both in-vitro and in-vivo culture contributed to formation of tubulous cartilage with specific mechanical property. The cartilage extracellular matrix secretion and mechanical property of tissue engineered tubulous cartilage tissue cultured in vivo were better than that cultured in vitro. Conclusion It is feasible to construct tissue engineered tubulous cartilage tissue by chondrocyte-sheets and 3D printed PA supporter for tracheal cartilage repair.