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

tracheal defect of rabbits. The TETCs were intermittently (Videoclip S2 in Supplementary File). These findings
sutured using 5-0 monofilament COVIDIEN sutures, and demonstrated that the synthesized hydrogel is suitable for
the survival rate of experimental rabbits in both groups bioprinting cell-laden constructs.
was recorded after 8 weeks. At the end of the 3-week
transplantation, the experimental rabbits in both groups 3.2. In vitro anti-inflammatory and anti-bacterial
were sacrificed for gross and histological examinations. activities of GelMA, ICA/GelMA, CS/GelMA, and ICA/
Immunofluorescence fluorescence in situ hybridization CS/GelMA hydrogels
(FISH) staining was performed to evaluate bacterial To evaluate the anti-inflammatory effect of the hydrogels
distribution, and the obtained images were used to quantify in vitro, RAW264.7 macrophages stimulated with LPS were
the bacterial intensity per field. Immunofluorescence incubated with GelMA, ICA/GelMA, CS/GelMA, and ICA/
staining of COL II was used to evaluate cartilage-related CS/GelMA hydrogels for 24 h. Compared to the GelMA
characteristics. COL II contents were quantified using and CS/GelMA groups, the ICA/GelMA and ICA/CS/
ELISA, and Young’s modulus was analyzed using a dynamic GelMA groups exhibited significantly reduced expression
mechanical analyzer, as described in section 2.3.2. of TNF-α and IL-6, the inflammatory cytokines, as
confirmed by immunofluorescence staining (Figure 3a–b).
2.11. Statistical analysis Western blot results also showed a significant decrease in
Statistical analysis was performed using GraphPad Prism relative protein expression of TNF-α and IL-6 in the ICA/
8 software, with a minimum of three samples per group. GelMA and ICA/CS/GelMA groups compared to the
Differences between treatments were assessed using one- GelMA and CS/GelMA groups (Figure 3c). Additionally,
way ANOVA or unpaired Student’s t-test, as appropriate. qPCR examination confirmed that the relative gene
Results are expressed as mean ± standard deviation (SD), expression of TNF-α and IL-6 was significantly suppressed
and statistical significance was defined as P < 0.05. in the ICA/GelMA and ICA/CS/GelMA groups compared
to the GelMA and CS/GelMA groups (Figure 3d). These
3. Results results indicate that the addition of ICA enhances the anti-
3.1. Synthesis and characterizations of GelMA, ICA/ inflammatory effect of GelMA-based hydrogels.
GelMA, CS/GelMA, and ICA/CS/GelMA hydrogels To determine the anti-bacterial activity of the hydrogels
To create tissue-specific bioinks, GelMA was used as a in vitro, both gram-positive (S. aureus) and gram-negative
substrate, and a photocrosslinking approach was employed (E. coli) bacteria were cultured in Petri dishes and treated
to ensure stability for 3D bioprinting. After exposure to UV with different hydrogels. The GelMA and ICA/GelMA
18
light (365 nm, 20 mW/cm ) for 30 s, GelMA, ICA/GelMA, groups exhibited good bacterial viability for both S. aureus
2
CS/GelMA, and ICA/CS/GelMA hydrogel precursors and E. coli compared with the control group, while the
were rapidly crosslinked (Figure 2a). Additionally, the CS/GelMA and ICA/CS/GelMA groups demonstrated
encapsulation of chondrocytes did not interfere with a significant reduction in bacterial viability for both S.
gelation (Figure 2b). GelMA, ICA/GelMA, CS/GelMA, aureus and E. coli (Figure 4a–b). The quantitative analysis
and ICA/CS/GelMA hydrogels exhibited shear-thinning showed that the bacterial survival rate for S. aureus and E.
behavior (Figure 2c), which is critical for an injectable coli in control, GelMA, and ICA/GelMA groups was higher
hydrogel to flow continuously. Dynamic modulus (G’ than that in the CS/GelMA and ICA/CS/GelMA groups,
and G”) of various hydrogels under UV light at different indicating that the addition of CS significantly enhances
times (Figure 2d) showed that gelation was achieved the anti-bacterial activity.
after photo-triggered crosslinking, and the gelation of the In conclusion, these results suggest that ICA endows
hydrogel could be controlled by adjusting the irradiation ICA/GelMA and ICA/CS/GelMA hydrogels with
time. Mechanical analysis (Figure 2e–f) revealed that all significant anti-inflammatory ability, while CS endows CS/
GelMA, ICA/GelMA, CS/GelMA, and ICA/CS/GelMA GelMA and ICA/CS/GelMA hydrogels with credible anti-
groups exhibited no significant differences, indicating bacterial ability.
that the addition of ICA and CS did not affect the
mechanical properties of GelMA hydrogel. We designed 3.3. Cytocompatibility of GelMA, ICA/GelMA, CS/
a customized 3D model for a C-shaped ring (Figure 2g), GelMA, and ICA/CS/GelMA hydrogels
and our results showed that chondrocyte-laden hydrogels To evaluate the viability, spreading, and proliferation of
were successfully printed into the C-shaped rings in all chondrocytes in the hydrogels, the chondrocyte-laden
GelMA, ICA/GelMA, CS/GelMA, and ICA/CS/GelMA hydrogels in GelMA, ICA/GelMA, CS/GelMA, and ICA/
groups (Videoclip S1 in Supplementary File and Figure CS/GelMA groups were cultured in vitro. Live/dead
2h). Moreover, the printed C-shaped rings could maintain staining (Figure 5a) showed that chondrocytes in all groups
their original shape after being immersed in PBS solution exhibited high viability with minimal cell death. Notably,

Volume 10 Issue 1 (2024) 165 https://doi.org/10.36922/ijb.0146