Page 117 - IJB-9-3

International Journal of Bioprinting OMT-loaded spinal cord scaffold

position on the animal operating table, and the limbs and on the animal operating table. Routine disinfection and
heads were fixed. A towel with routine disinfection (iodine) draping of towels were performed. After the onset of
was spread over the surgical site at the T10 level. The skin anesthesia, the skin of the rat chest was incised with a
along the posterior midline was cut, and the musculature scalpel, and the subcutaneous tissue was bluntly stripped
and soft tissues were bluntly dissected layer by layer to to expose the sternum and left rib. The pericardium was
expose the vertebral body. A laminectomy was performed opened. An indwelling needle with suitable size was used
at T8–T12 using a rongeur (corneoscleral forceps). This to pierce the left ventricle at the apex of the heart, and
was done under a microscope and under sterile conditions. the assistant helped to fix the needle to prevent it from
A cut was made 2 mm left of the spinal cord segment at the withdrawing. An incision of about 5 mm was made in the
level of T10 to form a 2.0-mm gap on the T10 spinal cord. right atrial appendage with ophthalmic scissors. First, a
A small suction device was used to aspirate the transected 250-mL syringe was used to extract physiological sodium
spinal cord tissue. The rat’s left hind limb twitched, the left chloride through the left ventricle. Perfusion was continued
foot was dorsiflexed, and the muscles were relaxed and until the fluid flowing from the right atrial appendage
weak, indicating that the modeling was successful. The became clear and transparent. At the same time, the rat’s
surgical port was flushed with 0.9% physiological sodium lung tissue and liver became pale and highly swollen. At
chloride. Following sufficient hemostasis after spinal cord this time, a 4% paraformaldehyde solution was used for
injury, the corresponding scaffolds of each group were continuous perfusion (whole-body muscle tremors were
immediately transplanted to the defect site. The wound was seen during the process, indicating the perfusion effect was
sutured layer by layer and sterilized again with iodophor. good). After the perfusion was complete, the spinal cord
Rats were resuscitated on an electric blanket and given a tissue was removed and placed in a 4% paraformaldehyde
dose of penicillin to prevent infection. solution. It was soaked in the test tube overnight. The next
day, it was removed for gradient dehydration treatment
2.8. Postoperative care and infection prevention with 15% and 30% sucrose solution or stored in a −80°C
After the surgery, the rats were marked and kept in separate freezer. Finally, the embedded tissue blocks were placed in
cages according to their group. Attention was given to the a cryostat for sectioning. The thickness of the slices was
general condition and activities of the rats after the surgery. 6–15 μm. The sections could be directly stained or stored
The rats were manually assisted daily with passive activities of in a −80°C freezer.
both lower limbs. This was to avoid joint stiffness and muscle
atrophy of the lower limbs so as not to affect the assessment 2.11. Hematoxylin–eosin (H&E) staining
of motor function. The rats’ bladders were manually Selected sections were rinsed three times with PBS for
massaged daily for 2 weeks to assist in urination and reduce 5 min each, hematoxylin staining solution for 5 min, tap
complications, such as urinary tract infection. Intramuscular water for 2 min, hydrochloric acid alcohol differentiation
injection of penicillin was given once a day for 1 week after solution for 3 s, tap water for 2 min, and ammonia water
surgery. Then, antibiotics or physiological sodium chloride for 3 s to blue. Sections were stained with eosin staining
solution were appropriately injected according to the rats’ solution for 2 min and rinsed with tap water for 2 min.
urine color, health status, and mental state. The litter was The sections were sequentially placed in 80% ethanol, 90%
cleaned up frequently to keep the cage dry. ethanol, and 100% ethanol twice for 1 min each time and
put in xylene two times for 5 min each time. The sections
2.9. Assessment of motor function were then placed in a fume hood to dry. After sealing with
Basso–Beattie–Bresnahan’s (BBB) score was used to neutral gum, they were observed under a microscope.
evaluate the recovery of hind limb motor function in rats
after SCI. Scores were performed weekly for 1–8 weeks 2.12. Nissl staining
after surgery. A score of 0 means complete loss of motor The washed sections were immersed in Nissl staining
function of the hind limbs, and a score of 21 means that solution in a staining box, which was placed in a 37°C
the motor function of the hind limbs was normal. The incubator, for 7 min. The sections were rinsed two times
behavioral assessment was independently completed by with PBS for 30 s each time, and placed in 95% ethanol
two experimenters who did not know the grouping. To for 30 s and in xylene solution two times for 5 min each
avoid the impact of bladder filling on exercise, the rats time. Then, the sections were placed in a fume hood to dry.
were massaged for urination before the measurement. After sealing with neutral gum, the sections were observed
under a microscope.
2.10. Heart perfusion, sampling, and sectioning
Eight weeks after the surgery, the experimental rats were 2.13. Immunofluorescence staining
anesthetized by 3% sodium pentobarbital (1 mL/kg) via The sections were selected and rinsed three times with PBS
intraperitoneal injection and fixed in the supine position for 5 min each time. The washed sections were placed in

Volume 9 Issue 3 (2023) 109 https://doi.org/10.18063/ijb.692