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International Journal of Bioprinting OMT-loaded spinal cord scaffold

and shaken continuously for 3 h at room temperature. weight (Wt), degradation rate, and ratio to the original
They were shaken and rinsed with double-distilled water weight were determined.
for 3 h with water changes every hour. The above steps Degradation rate = (Wt − Wo)/Wo × 100%
were repeated once. The extracted spinal cord was placed
in sterile phosphate buffer saline (PBS, 0.01 mol/L, pH 7.2) 2.4. Cumulative release of OMT
and stored in a refrigerator at 4°C for later use. After setting The in vitro sustained release behavior of OMT was
the freeze-dryer program, the spinal cord decellularized determined by dialysis. The samples were placed in a
scaffold was freeze-dried for 24 h, after which it was stored dialysis bag (MWCO: 1000), then 50 mL PBS (pH 7.4)
for later use. solution was added and oscillated on a shaker set at 75 r/min
and a constant temperature of 37°C. At the set time node,
2.1.2. Spinal cord ECM hydrogel 2 mL of the released solution was removed and the same
The rat acellular spinal cord (ASC) scaffold was ground volume of fresh PBS was added. The concentration of OMT
and digested into a matrix solution using liquid nitrogen in the medium was determined by high-performance
and then mixed with polymer materials, such as water, liquid chromatography (HPLC).
gelatin, β-cyclodextrin, polyethylene glycol diacrylate, and
a photoinitiator. The mixed material was divided into two 2.5. Testing the mechanical properties of the
parts. One part was kept as the original polymer solution, scaffold
and the other was loaded with OMT to prepare a spinal Tensile and compression tests were carried out on each
cord ECM drug-loaded hybrid polymer solution. The group of scaffold samples at 37°C, and the strain rate
spinal cord ECM hydrogel was then irradiated with 300– was set at 2 mm/min. The same test was repeated three
410 nm ultraviolet light for 5–15 min. times.

2.1.3. Polycaprolactone microfiber 2.6. In vitro co-culture
The organic polymer PCL was dissolved in the organic 2.6.1. Seeding of neural stem cells
solvent trifluoroethanol to form a polymer solution. The The scaffolds were placed in 96-well plates, and the second-
polymer solution was transferred to an electrospinning generation neural stem cells (NSCs) were seeded with a
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machine to prepare PCL microfibers with the same pipette on the sterile scaffolds at a density of 1 × 10 cells/mL.
orientation. A control group was set.
The abovementioned spinal cord ECM hydrogels and 2.6.2. In vitro viability assay of NSCs
OMT-loaded supramolecular hydrogels were uniformly Cell viability was assessed using a calcein-AM/propidium
coated on PCL microfibers, which were then slowly curled iodide (PI) cell double-staining kit. NSCs (1 × 10 NSCs)
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lengthwise into a cylindrical-like structure (similar to the were resuspended in centrifuge tubes and seeded in
spinal cord morphology), about half the diameter of the 96-well plates. After 12 h, the medium was changed to
adult rat spinal cord and subjected to UV light irradiation. a proliferation medium. NSCs were seeded in 96-well
The 3D-printed spinal cord ECM hydrogel microfiber plates loaded with scaffold (control group) and scaffold
scaffolds loaded with OMT were obtained by crosslinking. + OMT (treated group). They were then double-stained
The prepared composite scaffolds were packaged, sterilized with calcein-AM and PI at 37°C for 15 min. Observation
with cobalt-60 gamma rays (3K GY), and stored at a low and photography were carried out with a laser confocal
temperature for future use (Figure 1). microscope.
2.2. Scanning electron microscopy 2.6.3. Immunofluorescence staining
A scanning electron microscope (SEM) (Zeiss, Germany) Cells were fixed with 4% paraformaldehyde fixative, rinsed
was used to observe the scaffold surface and cross-sectional three times with PBS at room temperature, and blocked with
morphology. The prestored composite scaffold was taken 10% goat serum blocking solution at room temperature for
out, and its surface was sprayed with gold, observed, and 1 h. Cells were incubated with primary antibodies (TUJ1,
photographed with a SEM at a voltage of 3 kV. MAP2, GFAP, dilution ratio: 1:200) overnight at 4°C
and then with the corresponding fluorescent secondary
2.3. Scaffold degradation test antibodies (dilution ratio: 1:800) at room temperature for
The degradation rate of the scaffolds was tested by weighing 1 h. The whole process was done in a dark room and away
them under dry conditions (Wo) and placing them in a from light. An appropriate amount of 4’,6-diamidino-2-
PBS solution at room temperature. The PBS solution was phenylindole (DAPI) dye solution was added for 30 min.
replaced every 3 days. The scaffolds were removed at 2, The cells were then observed and photographed under a
4, 6, and 8 weeks, frozen, and then freeze-dried. Their confocal microscope.

Volume 9 Issue 3 (2023) 107 https://doi.org/10.18063/ijb.692