Abstract: 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.

Key words: Cell sheet technology, Chondrocyte sheets, 3D printed supporter, Tubulous cartilage tissue