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International Journal of Bioprinting 3D-bioprinted peripheral nerve scaffold

50 mM CaCl solution was subsequently used for crosslinking PBS three times. A 1:500 dilution of secondary goat anti-
2
for 5 min. Following this, the scaffold was introduced into the rabbit antibody (ab150077; Abcam, UK) was prepared in
culture medium. The linear structure would be rolled up to the same antibody dilution buffer. The cells and scaffolds
eventually form the scaffold before subsequent experiments were then incubated with the secondary antibody solution
or use (Figure 3K and L). for 1 h. Following three rinses with PBS, 4’,6-diamidino-
2-phenylindole (DAPI) solution (ZLI-9557; ZSGB-BIO,
2.4. In vitro experiments China) was added to the cells and scaffolds for 10 min,
2.4.1. Cell proliferation assay before visualization with a laser confocal microscope
A Cell Counting Kit-8 (CCK-8) (AQ308-500T; Aoqing (LMS710, Zeiss, Germany).
Biotech, China) was used to assess the proliferation 2.4.4. Cytoskeleton staining
of scSHEDs in two-dimensional (2D) cultures and To visualize the cytoskeleton topography of the scSHEDs
3D-bioprinted scaffolds. Briefly, a working solution was within the scaffold, we stained the 3D-bioprinted scaffolds with
prepared by combining the CCK8 solution and culture 1× Phalloidin-iFluor 594. The scaffold samples were stained on
medium in a 1:9 ratio. Subsequently, 2 ml working the fourth day after 3D printing. Before staining, the scaffolds
solution was added to the samples (n = 3) and incubated were crosslinked for 5 min by immersing them in a solution
for 1 h. Thereafter, 110 μl supernatant of each sample was
transferred into a 96-well plate. The microplate reader containing 100 mM CaCl . The samples were then treated with
2
(BioTek ELX800, BioTek, USA) was utilized to measure 4% paraformaldehyde for fixation for 10 s and subsequently
the optical density (OD) of the samples in the 96-well plate washed with PBS two to three times. The scaffolds were then
at 450 nm. The assay was performed on days 1, 3, 5, and 7. treated with 0.1% Triton X-100 for 3–5 min and subsequently
washed with PBD two to three times with PBS. A working
2.4.2. Cell viability analysis solution was produced by mixing 1% BSA and 1 μl 1000×
We evaluated scSHEDs viability within the 3D-printed phalloidin in 1 ml PBS. Thereafter, the scaffolds were stained
hydrogel with the Live/Dead fluorescence stain (KGAF001; with the working solution for 30 min and subsequently with
KeyGen Biotech, China). Briefly, a working solution was the DAPI staining solution before visualization using a laser
prepared by dissolving 4 μl propidium iodide (PI) and 1 μl confocal microscope (LMS710, Zeiss, Germany).
calcein AM concentrates in 5 ml PBS. After incubating the
scaffolds in the working solution for 30 min, we washed 2.4.5. Quantitative real-time polymerase
the scaffolds with PBS buffer three times. A fluorescence chain reaction
microscope was used for observation. Viable cells To conduct a gene expression analysis of SHEDs, scSHEDs,
fluoresced green (calcein AM) at 490 nm, while dead cells and scSHEDs on scaffolds for 7 days, qRT-PCR was used.
fluoresced red (PI) at 535 nm. The scaffolds were immersed in 100 mM sodium citrate for
5 min to facilitate de-crosslinking of the hydrogels through
2.4.3. Immunofluorescence staining gentle stirring, and cells were regained after centrifuging
The protein expression levels of S-100β, GFAP, and P0 at 1000 rpm for 5 min and PBS washing. We used TRIzol
(specific to Schwann cells) were evaluated for both SHEDs reagent (Invitrogen, USA) to perform total RNA extraction
and scSHEDs. Immunostaining of S-100β was performed following the manufacturer’s protocol. To achieve reverse
on scSHEDs within the printed hydrogel to determine transcription, the PrimeScript RT reagent kit (RR036A;
TM
its expression. The scaffold samples were stained on Takara, Japan) was utilized. FS Universal SYBR Green
the fourth day after 3D printing. Before staining, the Master Mix (Rox) (4913914001; Roche, Germany) was
scaffolds were immersed in a solution containing 100 mM employed for qRT-PCR. Sangon Biotech synthesized the
CaCl for 5 min. Briefly, the samples were treated with primers targeting nerve growth factor (NGF), platelet-
2
4% paraformaldehyde for fixation for 30 min, followed derived growth factor (PDGF), brain-derived neurotrophic
by blocking in a solution containing 5% bovine serum factor (BDNF), protein S-100β (S-100β), glial-derived
albumin (BSA) and 0.3% Triton X-100 in PBS for another neurotrophic factor (GDNF), and myelin protein zero (P0)
30 min. Rabbit anti-human S-100β antibody (ab52642; (Table 2). The cycle threshold value of each target gene was
Abcam, United Kingdom [UK]), rabbit anti-human GFAP normalized to that of GAPDH, and the fold difference in
antibody (ab68428; Abcam, UK), and rabbit anti-human expression change was calculated by the Ct (2 −ΔΔCt ) method.
P0 antibody (#10572-1-AP; Proteintech, USA) were
respectively diluted (1:100) in antibody dilution buffer 2.4.6. Nerve growth factor release analysis
(1% PBS/1% BSA/0.3% Triton X-100). Thereafter, the The release of NGF from SHEDs or scSHEDs in the 2D culture
samples were cultivated overnight at 4℃ in the antibody and scSHEDs within 3D-printed scaffolds was quantitatively
solutions. The following day, the samples were rinsed with determined using the human NGF (hNGF) ELISA kit (ELH-

Volume 10 Issue 4 (2024) 463 doi: 10.36922/ijb.2908