2.6 In vivo experiment
2.6.1 Establishment of a rabbit model for tracheal partial window-shape defect
Adult male New Zealand white rabbits (n=6) were selected as recipients. Intravenous inhalation combined anesthesia was performed during the operation with isoflurane inhalation by mask, follow by ethyl carbamate (Aladdin, Shanghai, China) injection (20%, 2.5 mL/kg) through the ear vein. Keep continuous inhalation anesthesia until the operation was done. Surgical procedures refer to previous study,[20] a median vertical neck skin incision was made to dissociate the fascia and neck muscles, and the irrelevant tissues around the trachea were dissected layer by layer carefully, in order to prevent bleeding and retain as much fascia as pos­sible. After exposing the cervical trachea completely, the window-shape defect with a size of 5 × 5 mm was established at the position of 2 cm away from the cricoid cartilage. The tracheal patch, which was hybridized by PCL and SilMA hydrogel loaded with autologous tracheal epithelia, was used to repair the defect and was sutured intermittently with 4-0 absorbable surgical sutures. After suturing, the hydrogel carrier was injected in the form of droplets with ingredients of BMSCs (5×104/100 μl) and Kartogenin (10 mM, MCE, New Jersey, USA) on the outer surface of the patch. Next, the tissues, including fascia and muscles, were sutured tightly. In the end, the skin sutures were operated via the continuous suturing method. In the first 2 days postoperatively, the recipients were treated with daily intramuscular injection of penicillin (5×104 U/kg, Mingyue, Suzhou, China).
2.6.2 Bronchoscopy, H&E, IHC and IF staining
After surgery, the experimental rabbits were examined daily for 60 days or until death. On the 30th day, X-ray was performed to observe whether there is obvious airway stenosis. After 60 days, the rabbits were anesthetized with inhalation anesthesia as described above, then the airway patency and repair patch could be observed and photographed under bronchoscopy (Jiechong International Trade Co., Ltd, Shanghai, China). Afterwards, they were euthanized by aeroembolism through ear veins and the patch specimen could be obtained. Specimens were washed three times with PBS gently and embedded in O.C.T. compound, and then sectioned successively at 6 mm thickness using freezing microtome. H&E staining was performed to observe the tissue structures of the patch. To confirm the sub-regional structural morphology, especially the epithelial cells’ creeping condition and the differentiation effects of BMSCs on the patch, IHC and IF staining of cytokeratin-18 (CK-18) (Absin, Shanghai, China) and type-II colla­gen antibodies (Huabio, Woburn, USA) (dilution ratio was 1:200) were performed.