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International Journal of Bioprinting 3D printed topographically fabricated micron track peripheral nerve conduit

2.4. Cytocompatibility assay and the wound was closed, and the rat was allowed to
To assess cytocompatibility, we cultured RSC96 cells recover.
(ATCC, CRL-2764) in a CO incubator and then
2
inoculated RSC96 onto sterilized MTC. After 1, 3, and 7 2.9. Immunofluorescence
days of culture, RSC96 were stained using the LIVE/DEAD The stained sections were used to identify and quantify
activity/cytotoxicity kit. Then, the viability and growth of the presence and distribution of regenerated axons in the
RSC96 were analyzed by fluorescence images captured by peripheral nerve sections. The intensity and distribution
inverted fluorescence microscopy (ZEISS), where live and of the NF200 staining indicated the degree of axonal
dead cells were stained green and red, respectively. regeneration, and the length and density of the stained
axons provided information on the extent of nerve repair.
2.5. Schwann cell’s directed migration and
proliferation 2.10. Toluidine blue stain
To evaluate the effect of MTC on the proliferation of To assess the quality and quantity of regenerated myelin,
primary Schwann cells (harvested from rat), we inoculated we performed toluidine blue staining of tissue sections that
the isolated primary cells on sterilized MTC and placed were fixed and dehydrated. The sections were then stained
them in a complete medium for culture. We stained with a toluidine blue solution that binds to the acidic
with the Red Cell Chromatin Kit from Bestbio Biologics components of the tissue, including the myelin sheath. The
(product number: BB-441256). Nine photographs were stained sections were then dehydrated and mounted for
randomly selected in n > 3 culture dishes to count the microscopic analysis.
orientation angle to determine the Schwann cell’s directed 2.11. Neurophysiological test
migration. A neurophysiological test (NT) is a test that measures the
speed of electrical signals traveling through the nerves.
2.6. Hemolysis test Electrodes were placed on the skin over the nerve being
To test the hemocompatibility of MTC, 50, 100, and tested, and a small electric shock was applied. The speed
200 µg of MTC samples were immersed in 1 ml of PBS, and strength of the nerve’s response to the shock were
and rabbit red blood cells were added and incubated at measured to determine if there was any nerve damage or
37°C to observe the extent of hemolysis. If the substance dysfunction.
caused complete hemolysis, the tube would appear clear. If
it caused partial hemolysis, the tube would appear cloudy. 2.12. Gastrocnemius characterization
To test the maintenance of the target muscle in each group,
2.7. Antibacterial experiment we used MTC, CC, MTC@NT3, and autologous nerve
To evaluate the antimicrobial properties of MTC, we graft (Autologous group) for peripheral nerve repair,
prepared a bacterial culture. The bacteria were cultured respectively. After 8 and 12 weeks, the gastrocnemius
on an agar plate until fully grown (Escherichia coli: ATCC muscle was harvested to measure the mean and cross-
25922, Staphylococcus aureus, ATCC 6538). The MTC was sectional areas, and Masson’s staining was performed to
cut into 5-mm diameter discs and placed on the surface obtain and analyze the data.
of the agar plates, ensuring they were in contact with the
bacterial cultures. The plates were incubated at 37°C for 1 2.13. Sciatic function index
and 3 days. The diameter of the clear area around the plate The Sciatic function index (SFI) is used to assess the degree
(i.e., the zone of inhibition) was measured using a ruler or of impairment of the sciatic nerve, the largest nerve in
caliper. The size of the inhibition zone is proportional to the body. The CatWalk instrument was used to test the
the antibacterial activity of the MTC. A larger inhibition calculated SFI (SFI = 109.5(ETS–NTS)/NTS–38.3(EPL–
zone indicates stronger antimicrobial activity, while a NPL)/NPL+13.3(EIT–NIT)/NIT–8.8) (N, normal foot; E,
smaller inhibition zone or no zone of inhibition indicates injured lateral foot; PL, footprint length; TS, toe width; IT,
weaker or no antimicrobial activity. middle toe width).

2.8. In vivo experiment 2.14. Statistical analysis
The rat sciatic nerve injury model is commonly used The images obtained in this experiment were analyzed by
for studying nerve regeneration and repair. Rats were ImageJ software. All numerical data were analyzed using
anesthetized, and a small incision was made in the skin Graph Prism Program, Version 7.0 (GraphPad Software,
of the lateral thigh. The sciatic nerve was then exposed, Inc., La Jolla, CA, USA) with mean ± SEM (Standard
and the nerve was transected to create a 15-mm defect. Error of Mean). Differences among multiple groups were
After the injury, the wound was repaired with CC, MTC, analyzed by one-way analysis of variance (ANOVA).
MTC@NT3, or autologous nerve graft, respectively, When p > 0.05, Tukey’s post hoc test was applied in the

Volume 9 Issue 5 (2023) 421 https://doi.org/10.18063/ijb.770