Page 471 - IJB-9-5

International Journal of Bioprinting 3D printed hydrogel for infected wound healing via PDT

photo-crosslinked hydrogels were formed at a distance of prepared as the CON group. The live/dead staining was
20 mm using a 3 M Epilar FreeLight 2 LED dental curing implemented according to the manufacturer’s instructions.
lamp with 1200 mW/cm at the source for 30 s. The stained cells were observed using CLSM (Leica,
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Wetzlar, Germany). Dead cells appeared red whereas living
2.7. Scanning electron microscopy examination cells appeared green.
The samples were freeze-dried in a vacuum for 24 h so that
the surface microstructures and cross-sectional shapes 2.12. Cell migration assay
of the hydrogels could be seen. The samples were then The scratch wound healing assay was performed to evaluate
sprayed with gold and examined with a scanning electron the effects of MB@UiO-66(Ce)/PH on cell migration .
[44]
microscope (S4800, Japan) at an acceleration voltage of 5 kV. L929 cells were seeded in 12-well plates, and when the
cells reached 90%–100% confluence, a line was scraped in
2.8. Mechanical properties of MB@UiO-66(Ce)/PH each well using the tip of a sterile plastic pipette and the
The mechanical properties of the hydrogels were evaluated cells were then treated with MB@UiO-66(Ce)/PH extracts.
both via compression tests and tensile tests. In compression DMEM containing 10% FBS was prepared as the CON
tests, hydrogels were prepared using a cylindrical model group. All plates were fixed with 4% polyformaldehyde
with height of 10 mm and a diameter of 10 mm. Then, at predetermined time points (0, 6, 12, and 24 h). Finally,
samples were testified at room temperature with speed at they were visualized using a microscope (Ti-U, Nikon,
2 mm/min on a universal mechanical tester (HY-0230, Japan) and calculated using ImageJ software.
China). In tensile tests, hydrogels were prepared using a
dumbbell model (dowel: 30 mm in length, 3 mm in width, 2.13. Antibacterial activity of MB@UiO-66(Ce)/PH
2 mm in thickness; bell: 10 mm in length, 15 mm in width, Gram-positive S. aureus (ATCC 25923) and gram-negative
2 mm in thickness) and stretched using a clamp attachment E. coli (ATCC 11775) were selected for the detection
at a strain rate of 10 mm/min (HY-0230, China). of antibacterial activity in the MB@UiO-66(Ce)/PH .
[45]
MB@UiO-66(Ce)/PH were blended with bacteria in a
2.9. Morphological observation of L929 cells on the logarithmic phase (10 CFU/mL), with or without a 20-min
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surface of MB@UiO-66(Ce)/PH treatment of laser irradiation at 660 nm, 0.5 W/cm . The
2
Cells of the 1.5 × 10 mouse fibroblast L929 cell line (CCL-1, conventional plate counting method was adopted to value
4
ATCC) were cultured on different 3D-bioprinted MB@ the changes in the number of colonies due to the MB@
UiO-66(Ce)/PH for 24 h. Samples were then fixed with UiO-66(Ce)/PH treatment. The colonies of surviving
4% polyformaldehyde; cytoskeletons were stained with bacteria after 24 h were calculated using ImageJ software.
FITC-phalloidin; and nuclei were stained with DAPI.
The morphology of the L929 cells was observed using a 2.14. Animal experiments
confocal laser scanning microscope (CLSM; Leica, Wetzlar, All animal experiments in this study were conducted
Germany). in accordance with the guidelines for the protection of
animal life and protocols approved by the Institutional
2.10. CCK-8 assay Animal Care and Use Committee (SH9H-2021-A32-1)
The CCK-8 assay was used to test the viability and and following the Animal Experimental Ethical Inspection
proliferation of L929 cells after exposure to the procedure of the Ninth People’s Hospital, which is affiliated
3D-bioprinted hydrogels. Extracts of 100 μL/well of MB@ with the Shanghai Jiao Tong University School of Medicine.
UiO-66(Ce)/PH were put into 96-well plates, and then 2 ×
10 L929 cells were seeded and cultured. Cells were seeded A full-thickness skin defect mouse model was adopted
3
directly in the well plates as a control (CON) group. After to investigate the effects of MB@UiO-66(Ce)/PH treatment
cell culture for 1, 3, 5, and 7 days, the CCK-8 working on skin wound repair in vivo. Briefly, the full-thickness
solution was added. The absorbance was measured at defect model was established using 8-week-old Kunming
450 nm (Safire, Tecan, Switzerland) after incubation at mice. Wounds were made using a sterile, 5-mm biopsy
37°C for 1.5 h. punch outlining two circular wound patterns on each side
of the mouse midline. The skin in the middle of the outline
2.11. Live/dead assay was lifted with serrated forceps; a full-thickness wound was
The live/dead assay was conducted to evaluate the activity created with iris scissors that extend into the subcutaneous
of L929 cells cultured in the MB@UiO-66(Ce)/PH extracts. tissue; and this circular tissue was excised. S. aureus (50 μL,
In this study, the extracts of MB@UiO-66(Ce)/PH were 1 × 10 CFU/mL) was injected at the wound sites on the
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prepared according to the ISO-10993 standard. Then, 1.5 × next day to simulate infection. Day 0 was designated as
10 L929 cells were seeded on glass-bottom culture dishes the first day of infection. All mice were randomly divided
4
and cultured for 3 days. DMEM containing 10% FBS was into five groups (PH-0, PH-0.1, PH-0.5, PH-1, and CON

Volume 9 Issue 5 (2023) 463 https://doi.org/10.18063/ijb.773