Page 172 - IJB-10-1

International Journal of Bioprinting Bioprinted cell-laden hydrogel for tracheal application

(Escherichia coli) bacteria, we carried out the following following the manufacturer’s instructions and examined
procedures. We mixed 80 mg of different hydrogel samples them using a fluorescence microscope (Olympus). We also
(GelMA, ICA/GelMA, CS/GelMA, and ICA/CS/GelMA) assessed cell proliferation via the DNA quantification and
with 200 μL of bacterial solution (1 × 10 cfu/mL) in sterile Cell Counting Kit-8 (CCK-8) assay to further evaluate the
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Eppendorf tubes. Eppendorf tubes containing 200 μL viability of chondrocytes in hydrogels. DNA was quantified
of bacterial solution (blank) only were used as a control. using the PicoGreen dsDNA assay kit (Invitrogen, USA)
After incubation, we diluted the samples 100-fold and then according to the manufacturer’s instructions. The CCK-
plated them to observe colony growth. We measured the 8 assay was performed according to the manufacturer’s
bacterial-occupied area using the following formula: instructions, and the absorbance was measured at 450 nm
100% using a spectrophotometer.
To evaluate the chondrogenic capacity of the various
where α is the area of colonies with the treatment of the hydrogels, we cultured the printed cell-laden hydrogels
hydrogel and β is the total area of agar plates. in the culture medium (DMEM supplemented with
10% FBS) for 3 weeks. Then, we analyzed the expression
2.6. Chondrocytes preparation level of collagen type II (COL II) in the samples by
To obtain chondrocytes for our experiments, we collected immunofluorescence staining, as described in section 2.4.
auricular cartilage samples from rabbits through surgery. We We also quantified the COL II contents using an enzyme-
then digested the samples using 0.15% type II collagenase linked immunosorbent assay (ELISA).
and cultured the resultant chondrocytes in DMEM
containing 10% FBS and 1% penicillin–streptomycin at 2.9. In vivo TETC regeneration
37°C in a 5% CO incubator. We used chondrocytes at the To facilitate the in vivo regeneration of TETCs, a total of
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second passage (P2) for further experiments. 24 rabbits were randomly assigned to four groups: GelMA,
ICA/GelMA, CS/GelMA, and ICA/CS/GelMA, with six
2.7. Bioprinting of C-shaped rings using cell-laden rabbits per group. The 3D-printed C-shaped rings in all
hydrogels the cell-laden hydrogel groups were interrupt-stacked
For 3D bioprinting, we homogeneously mixed onto a silicone tube and implanted into the platysma
chondrocytes in the different hydrogels at a concentration of anesthetized autologous rabbits. The C-shaped rings
of 1 × 10 cells/mL. The chondrocyte-loaded hydrogels underwent 3- or 6-week implantation to generate TETC
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were then loaded into 5 mL syringes equipped with tissues. The retrieved TETC samples were photographed
0.21 mm diameter needles and mounted into the syringe with an SLR camera for gross observation and sectioned for
pump extruder on a 3D BioArchitect workstation histological evaluation, including hematoxylin and eosin
(Regenovo). The temperatures of the syringes and platform (H&E), safranin-O, and immunohistochemical staining for
were maintained at 16 ± 1°C. To fabricate a single layer COL II. Glycosaminoglycan (GAG) and COL II contents
of ring-shaped constructs (9.0 mm external diameter, were quantified using the dimethylmethylene blue assay
6.0 mm internal diameter, and 1.5 mm height for C rings), (DMMB, Sigma-Aldrich) and ELISA, respectively.
we printed four layers of each bioink and photocrosslinked
them upon light irradiation (365 nm, 20 mW/cm ) within Moreover, the expression levels of TNF-α and IL-6,
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30 s. The printing parameters were set as follows: line gap, as well as an apoptosis-related marker (TUNEL) in the
400 μm; layer thickness, 400 μm; photocrosslinking time, samples, were analyzed by immunofluorescence staining,
30 s per ring; pneumatic pressure, 0.2 MPa; extrusion as described in section 2.4. A TUNEL apoptosis detection
speed, 1.5 mm /s. After printing, we incubated the kit (C1086; Beyotime, Shanghai, China) was used to detect
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bioprinted constructs in DMEM supplemented with 10% apoptotic cells by immunofluorescence staining. The
FBS and 1% penicillin/streptomycin at 37°C and 5% CO . acquired images were analyzed using ImageJ software to
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We conducted each step under sterile conditions to ensure calculate the relative intensity. Specifically, we used the
the integrity of the bioprinted constructs. formula α/β × 100%, where α represents the occupied area
of the positive expressed marker, and β represents the area
2.8. Cell viability, proliferation, and chondrogenesis of the total image.
evaluations
After printing, the cell-laden hydrogels were incubated at 2.10. Orthotopically transplantation of TETC in
37°C in a 5% CO incubator for 9 days. The culture medium autologous rabbits
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(DMEM supplemented with 10% FBS) was changed every The TETCs generated in the ICA/GelMA and ICA/
other day. On days 1, 4, and 9, we evaluated the cell viability CS/GelMA groups (as described in section 2.9) were
using the Live/Dead Cell Viability Assay (Invitrogen) orthotopically transplanted into the 0.6-cm-long native

Volume 10 Issue 1 (2024) 164 https://doi.org/10.36922/ijb.0146