Safranin o staining

All samples were harvested, fixed in 10% formalin, and decalcified in 10% EDTA. The tissues were then sliced into 5-μm sections and stained with hematoxylin and eosin (H&E). The newly formed cartilage and cartilaginous matrix were examined using commercial kits for Alcian Blue staining (Cat No. G2541, Solarbio), Safranin-O staining (Cat No. G1470, Solarbio), and Toluidine Blue staining (Cat No. G3661, Solarbio). The expression of COL-2 in the tissue sections was determined using immunohistochemical staining assays by COL-2 antibody (Cat No. bs-10589R, Bioss, Beijing, China). The degree of tissue regeneration was graded in a blinded manner by 5 graders according to the International Cartilage Repair Society (ICRS) scoring system as described previously [37 (link), 38 (link)].

Tracheae were decellularized with seven cycles of a detergent–enzymatic treatment protocol as previously described13 (link); namely, the tracheae were incubated in sterile filtered water for 48 h at 4 °C in 4% sodium deoxycholate (Sigma) diluted in distilled water and rotated continuously for 4 h at 37 °C, and in DNase-I (2 kU mL⁻¹; Sigma) in 1 mol/L NaCl (Sigma) with continuous rotation for 3 h at 37 °C. After the washing steps, the trachea samples were stored in PBS containing 1% antibiotic and antimycotic solution at 4 °C overnight; the following day, the next cycle was initiated. To determine the decellularization efficacy, a Movat pentachrome stain kit (Leagene, Beijing, China) was employed to evaluate the connective tissues, including the cartilage, elastic fibres, collagen, reticular fibres, and muscle. Sections were also stained with 4′-6-diamidino-2-phenylindole (DAPI; KeyGEN, Nanjing, China) to detect nuclear material. Safranin O staining (Solarbio, Beijing, China) was performed to evaluate the glycosaminoglycan expression. An immunohistochemical analysis of type II collagen (antibodies all from ABM, Carlsbad, USA) was performed to evaluate the type II collagen expression.