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International Journal of Bioprinting 3D-Bioprinted human lipoaspirate-derived cell-laden skin constructs

2.8. Scanning electron microscopy Scaffolds carrying ADSCs were placed in 24-well plates,
To remove moisture from the hydrogel, acellular samples and then DMEM-F12 medium was added for culture
were vacuum-freeze-dried, followed by sputtering, under standard conditions at 37°C. On days 0, 3, and 7,
and coated with iridium to avoid charge accumulation. scaffolds were collected, rinsed with PBS three times, and
Lyophilized samples were cut with a razor to expose the stained in 2-mL solution containing 2 μmol/L calcein-AM
internal structure and placed on the sample stub, with and 4.5 μmol/L PI at 37°C for 15 min. Fluorescent images
tangent facing up. A scanning electron microscope (SEM, of stained cells within scaffolds were obtained using a
S-4800, HITACHI, Japan) was used to examine sample pore confocal laser scanning microscope (Leica TCS SP8, Leica,
structures, and images were obtained with an accelerating Germany). To evaluate ADSC viability at different time
voltage of 15 kV at a working distance of 10 mm. points, the numbers of live and dead cells were counted in
the fluorescent confocal microscopy images using Image-
2.9. Cytocompatibility of photocrosslinked bioinks Pro-Plus 6.0 software.
After crosslinking with 405-nm UV light, 200 μL of
acellular bioink samples were transferred to a 24-well plate 2.12. In vivo wound healing
and immersed in 2 mL Dulbecco’s modified Eagle medium All animal experiments were approved by the ethics
(DMEM)-F12 medium containing 10% FBS in a humidified committee of the Fourth Medical Center of the PLA General
atmosphere of 5% CO at 37°C for 3 days. The samples Hospital (No. 2022-X18-03). Thirty female BALB/c nude
2
were then removed, and the solution containing the eluent mice, aged 4–5 weeks old and weighing 19.13–22.47 g, were
was collected. The eluent solution was supplemented with purchased from Beijing Vital River Laboratory Animal
10% FBS and 1% penicillin–streptomycin–amphotericin Technology (Beijing, China). After anesthesia, two full-
and stored at 4°C for later use. ADSCs (5 × 10 cells/well) thickness excisional skin wounds with 8-mm diameters
3
were cultured in 96-well plates with 100 μL of the above- were created using a trephine on the backs of nude mice.
mentioned eluent solution per well for 1, 3, and 7 days. The A rubber ring with an 8-mm inner diameter was sutured
blank control group was cultured in DMEM-F12 medium around the wound with 6-0 nylon to minimize wound
containing 10% FBS and 1% penicillin–streptomycin– contraction. Mice were randomly divided into five groups
amphotericin. After washing with PBS, ADSCs were treated (n = 6 per group) where wounds were filled with no
with a mixture of DMEM-F12 medium (90 μL) and CCK-8 treatment, ADSC-laden adECM–GelMA–HAMA, acellular
reagent (10 μL) for 2 h. ADSC proliferation was determined adECM–GelMA–HAMA, ADSC-laden GelMA–HAMA,
using a CCK-8 assay (Dojindo Molecular Technologies, or acellular GelMA–HAMA. After scaffold placement, the
Japan), and the absorbance value of the sample solutions at wound was covered with a semipermeable membrane and
450 nm was measured using a microplate reader (ELX 800; bandaged, and mice were housed and fed in separate cages.
BioTek Instruments, USA). Images of the wounds were taken at 0, 7, and 14 days, and
the wound residual rate was calculated using Equation III:
2.10. Fabrication of 3D-bioprinted scaffolds
Four-layer circular scaffolds were printed layer-by-layer Wound residual rate = (Residual area/Original area) × 100%
using a commercial 3D printer (Envision TEC, Germany) (III)
by extruding bioinks with a thermo-controlled chamber Mice were euthanized at either day 7 or 14 after
at 16°C–18°C, a 27G nozzle with an inner diameter of implantation for the harvesting of wound tissues, which
200 μm, a pneumatic pressure of 0.8–1.2 bar, and a moving were then processed for analysis.
speed of 3.2–5.6 mm/s. Scaffolds had an 8-mm diameter
with an 800-μm strand spacing and 180-μm layer height. 2.13. Histological and immunohistochemical
Photocrosslinking was performed by irradiating the staining of wound tissues
scaffolds for 3–5 s with 405-nm UV light immediately after Wound tissues harvested after 7 or 14 days were fixed in 4%
the printing process for each layer was completed. Scaffolds paraformaldehyde, dehydrated in a graded alcohol series,
were temporarily immersed in DMEM-F12 medium and embedded in paraffin. The tissues were then sliced
containing 10% FBS and maintained under normal culture into 4-μm-thick sections perpendicular to the wound
conditions for later use. surface for further staining. Each section was stained
with H&E and Masson’s trichrome stain. Picrosirius red
2.11. Biocompatibility of adECM–GelMA–HAMA staining was performed to visualize collagen fibers using
scaffold a Picrosirius Red Staining Kit (Yeasen Biotechnology,
A calcein-AM/propidium iodide (PI) double staining kit China). Images were taken under polarized light with a
(Dojindo Molecular Technologies, Kumamoto, Japan) Nikon Eclipse Ci microscope (Nikon, Tokyo, Japan). For
was used to assess the biocompatibility of prepared immunohistochemical staining, deparaffinized sections
scaffolds according to the manufacturer’s instructions. were evaluated for CD31 expression using a staining kit

Volume 9 Issue 4 (2023) 33 https://doi.org/10.18063/ijb.718