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.