3.4 Fabrication and physical properties of hybrid scaffold
3.4.1 The microstructure of PCL, PCL/SilMA, SilMA, SilMA/BMSCs
As the previous study, the crisscross structure of PCL scaffold forms the pore designs (Fig. 7a). It is widely known that inter-connected pore networks of a biological scaffold is a critical index affecting cell adhesion, proliferation and differentiation. SilMA hydrogel possessed highly 3D porous structure with moderate thickening of the hole wall (Fig. 7d, e). Compared with the surface of PCL (Fig. 7b), SilMA hydrogel is glossier (Fig. 7c). The surface of PCL coated with SilMA hydrogel clearly changed under the observation of SEM (Fig. 7f), the pores and gaps were filled with a thin layer of hydrogel, as well as the longitudinal section (Fig. 7g, h). After seeding cells to SilMA hydrogel, the surface was covered with underlying cells, which were mostly flat polygonal or long shuttle shapes (Fig. 7i, j). The cells were distributed in clusters and extended antennae to connect with each other.
3.4.2 Mechanical properties of hybrid scaffold
The morphology of each group was similar and there were no significantly differences in physical measurements between them. With the increase of stretched tension, the native trachea broke at the point of the cartilage rings, and remained in the original structural state. PCL and PCL/SilMA showed segmental changes with the samples were significantly elongated. During the stretched process, the tensile force reached the maximum value when the samples broke. The compression process could continue all the time, so in order to unify the standard, maximum load and elastic modulus were recorded at the 50% of the compression deformation. During three-point bending test, the data were recorded when the samples slided from the fulcrums on both sides. When PCL was coated with SilMA hydrogel, there were certain changes in structure, leading to better mechanical properties.
3.5 Trachealwindow-shape defect repair in vivo
3.5.1 Intraoperative anastomosis, postoperative examination and specimen acquisition
Last but not the least, this hybrid scaffold was applied to repair rabbit tracheal window-shape defects and seeded them with epithelia, BMSCs and KGN during the operation. As shown in Fig. 9a, the window-shape defect of 5 × 5 mm was established, with excision of two cartilage rings. Then, the hybrid scaffold seeding with epithelia was sutured the defect (Fig. 9b). After that, SilMA solution containing BMSCs and KGN was injected under the UV irradiation in the form of droplets to cover the outer surface of the patch (Fig. 9c).
Six rabbits survived well after the operation. There was slight sputum murmur in the first two days and disappeared from the fifth day. One rabbit died two weeks after operation, and a large amount of pleural effusion was detected through autopsy. Another died more than one month after operation and the other four all survived for two months, without sputum obstruction, stenosis, wheezing, cyanosis or infection. On the 30th and 60th day, X-ray examination demonstrated that there was no obvious stenosis or collapse (Fig. 9d, e). The airway, especially the point where the patch was sutured, was firstly examined via bronchoscopy, prior to acquisition of specimens. The picture of bronchoscopy on the 60th day (Fig. 9f) revealed that the transplantation site was covered with mucosa and maintained complete unobstruction of the lumen. It was consistent with the macroscopic appearance of the specimen, and there was no obvious abscess or necrosis at the patch site (Fig. 9g-i).
3.5.2 H&E staining analysis
H&E staining helped to recognize the transplantation site between the two segments of the cartilage ring of the graft, the morphology and structure of which were intact with few inflammatory cells infiltration. Residual SilMA hydrogel and undegraded PCL were also observed on the outside of the graft, while the continuous epithelial structures were detected from native area to patch site on the inside of the graft (Fig. 10a, c). What is more gratifying and unexpected is that a few vascular-like structures were observed near the repair site (Fig. 10b).
3.5.3 IHC and IF staining analysis
In order to further confirm that the tracheal transplantation site was covered by epithelial cells, IHC and IF staining were performed. IHC staining of CK-18 (Fig. 10d-f), which was specifi­cally expressed by epithelial cells, revealed that the continuous brown-col­ored antigen was expressed on the inner surface of the repair site, which was similar to normal trachea, indicating that the hybrid scaffold has good epithelization performance. Similarly, IF staining of CK-18(Fig. 10g-i), which was expressed by green fluorescence, confirmed the positive result again.