Figure legends
Figure 1: Schematic illustration of the designs of the hybrid scaffold combining 3D printed polycaprolatone coated with Silk Fibroin Methacryloyl, epithelium, BMSCs and Kartogenin for reconstruction of rabbit tracheal window-shape defect.
Figure 2: The macroscopic appearance of SilMA in different status (A: curing ring; B: pure 10% SilMA; C, D: 15%, 20% SilMA after culturing in DMEM-F12 medium; E: freeze-dried status of Fig. D).
Figure 3: The swelling ratio and remaining mass of SilMA in different concentration and different status (A, D: The swelling ratio and remaining mass of pure SilMA; C: The degradation process of pure SilMA; B, E: The swelling ratio and remaining mass of freeze-dried SilMA). Data represent mean ± SD; **P < 0.01.
Figure 4: A1: At day 4, almost no RBCs left and BMSCs grew well and fast; A2: The 2nd passage BMSCs; A3: Flow cytometry analysis revealed BMSCs matched the characteristics of MSCs that CD29, CD44, and CD90 were highly expressed and few cells were positive in CD34. The epithelial cells were cultured by the tissue extracted from autologous trachea mucosa. B1, B2: At day 5, the cells gathered into clusters; B3, B4: Cells reached 90% confluence at day 13. (Magnification: A1, A2: ×40; B1, B3: ×100; B2, B4: ×200).
Figure 5: A: CCK-8 test of SilMA in different concentration. B represented the H&E staining of 15% SilMA and C represented 20%, red arrows indicate the alive cells (Magnification: B, C: ×100; b, c: ×200). D represented the quantified column chart. Data represent mean ± SD; *P < 0.05, **P < 0.01.
Figure 6: The appearance and Giemsa staining of BMSCs around the scaffold of each group after inoculation for 48 h. In vitro cytotoxicity test shows 20% SilMA has good cytocompatibility (Magnification: a-l: ×40).
Figure 7: Scanning electron microscopy (SEM) observation of PCL, 20% SilMA, PCL+20% SilMA and SilMA+BMSCs (Magnification: a: ×50, d and g: ×500, b, c, e, f, h and i: ×1000, j: ×3000).
Figure 8: Mechanical test shows hybrid scaffold has favorable biomechanical properties. In the upper left corner of the figure, from left to right are native trachea, PCL scaffold and hybrid scaffold (PCL+20% SilMA).
Figure 9: a-c: Intraoperative images of tracheal partial window-shape defect and repair. Black arrow indicates the SilMA hydrogel droplet containing BMSCs and KGN; d, e: X-ray images of 1, 2 months after operation; f: Bronchoscopic images of the luminal area of the graft at 2 months, white arrow indicates the transplantation site; g-i: Gross view of native trachea and the patch after tracheal reconstruction for 2 months. Red arrow and white dotted boxes indicate the transplantation patch.
Figure 10: a-c: H&E staining (S: SilMA hydrogel; P: PCL; C: cartilage rings); d-f: Immunohistochemistry staining of CK-18; g-i: Immunofluorescence staining of CK-18. Red arrow indicates the vascular-like structures, white dotted lines mark the anastomosis, green arrows indicate the native tracheal epithelium and yellow arrows show the nascent epithelial cell layer on the inner surface of the repair site (Magnification: a, d: ×40, e: ×100, b, c, f: ×200, g, h, i: ×200).