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.