Page 470 - IJB-10-4

International Journal of Bioprinting 3D-bioprinted peripheral nerve scaffold

2.1.3. Shear rate-viscosity and (M8211; Solarbio, China) and 1% penicillin/streptomycin
temperature-viscosity tests (15140-122; Gibco, USA), without fetal bovine serum
The Discovery HR2 rotational rheometer was also utilized (FBS) (10099–141; Gibco, USA) for a duration of 24 h.
to assess the viscosity of the hydrogel. By subjecting For the following 72 h, the medium was changed to the
the bioink to a 1% strain for 200 s at 20℃, we obtained standard culture medium (αMEM containing 20% FBS
composite viscosity–shear rate curves. Subsequently, and 1% penicillin/streptomycin) supplemented with 35
a shear rate of 0.1–1000 s was used to establish the ng/ml all-trans-retinoic acid (A8539; ApexBio, USA).
-1
correlation between the shear rate and viscosity of the After 3 days, the medium was switched to standard culture
hydrogel. To obtain the temperature–viscosity curve, 1 Hz medium supplemented with 5 μM forskolin (B1421;
frequency and 1% strain were applied to the hydrogel for ApexBio, USA), 5 ng/ml platelet-derived growth factor AA
245 s at varying temperatures (10–42℃). (HZ-1215; Proteintech, USA), 10 ng/ml basic fibroblast
growth factor (HZ-1285; Proteintech, USA), and 200 ng/ml
2.1.4. Tensile modulus test and maximum tensile test human neuregulin-β1 (ab50227; Abcam, USA). Thereafter,
A tensile meter (SH-10; WD, China) was used to measure the the medium was renewed every 3 days, and the SHEDs
maximum tensile force of the hydrogel (after crosslinking), were maintained at least 2 weeks before examination or
PCL, and composite scaffolds. The constructed scaffolds
(from section 2.3. 3D bioprinting) would be hooked to bioprinting. Flow cytometry analysis was performed to
both ends of the tensile meter and stretched at a rate of evaluate the expression levels of the surface antigens CD29,
0.5 mm/s until fracture. The instrument would record the CD34, CD45, CD73, CD90, and CD105.
maximum tensile force endured during the stretch. 2.3. 3D bioprinting
2.1.5. Scanning electron microscopy Briefly, a cuboid (with 10 mm length, 10 mm width, and
The hydrogels were freeze-dried and gold-coated prior to 0.3 mm height) was designed using a 3D bioprinter (BMP-
observing the micromorphology using a scanning electron C300-T300-IN3; Medprin, China). The cuboid was designed
microscope (SEM) (SU8020; Hitachi, Japan). The Image in two types: linear or reticulated cuboid. Thereafter, PCL
J software was used for porosity measurements, and the was placed in the high-temperature melting cylinder
average porosity was obtained. (Cylinder 1), while 1 ml injector was placed into Cylinder 2
after mixing the scSHEDs with the hydrogel. A 3D-printing
2.1.6. Fourier infrared analysis pinhead, with an internal diameter of 100 μm, was selected
We used a NICOLET IS10 Fourier transform infrared for Cylinder 1, while another pinhead, with a diameter of
(FTIR) spectrometer (Thermo Fisher Scientific Inc., USA) 160 μm, was selected for Cylinder 2. Table 1 displays the
to investigate the chemical composition of the hydrogels. 3D-bioprinting parameters. For the linear cuboid (Figure 3I),
To clarify the stability of the hydrogel components after the Cylinder 1 was used to print the first layer, while Cylinder 2
introduction of the RGD peptide, we performed assays on printed the second layer. As for the reticulated cuboid, both
two hydrogels: 6% RGD-Alg/5% GelMA and 6% Alg/5% layers were printed by Cylinder 2, and the cuboid was used
GelMA. The lyophilized hydrogel was meticulously for subsequent cell Live/Dead, immunofluorescence, and
pulverized in an agate mortar with 200 mg of KBr at a ratio cytoskeleton staining purposes. After printing the construct,
of roughly 1:20 and compacted into a slender section. The it was solidified via 430 nm blue light exposure for 10 s, and
sample was then positioned within an automated sample
enclosure for approximately 100 s for identification, while Table 1. 3D-bioprinting parameters
the spectral range was 400–4000 cm and the resolution
-1
was 4 cm . The FTIR spectra were analyzed after baseline Parameter Value/ranges
-1
and background corrections. Temperature (℃)
2.2. Cell culture and induction Cylinder 1 90
SHEDs were offered by the Oral Stem Cell Bank of Cylinder 2 20–25
Beijing, Tason Biotech Co., Ltd. The cells were cultured Nozzle 1 100
in a mesenchymal stem cell medium (MSCM) (#7501; Ambient 20
ScienCell, USA) within a humidified environment at 37℃, Fill density (%) 60
and the cells were subcultured every 3 days. Passages 4–7 XY plotting speed (mm/s) 50
were collected for experimental purposes.
Inner diameter (μm)
Cell induction was initiated when the cells reached Nozzle 1 100
a confluency of ≥ 80%, and the medium was changed to Nozzle 2 220
αMEM supplemented with 1 mM β-mercaptoethanol

Volume 10 Issue 4 (2024) 462 doi: 10.36922/ijb.2908