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International Journal of Bioprinting Bioprinted osteoarthritis scaffolds

agitation. When visible cartilage fragments were reduced, and rinsed three times with distilled water for 1 min each.
43
an equal volume of Dulbecco’s Modified Eagle Medium Dehydration was performed using a graded ethanol series
(DMEM) (Gibco, USA), supplemented with 10% fetal (70, 90, and 100%) for 2 min each. Finally, the stained
bovine serum (FBS) (Bio Channel, China), was added samples were observed under an optical microscope.
to terminate the enzymatic reaction. The mixture was
centrifuged at 1200 rpm for 10 min, the supernatant was 2.10. Construction of 3D-bioprinted
discarded, and the cell pellet was collected. The cells were cartilage models
seeded in DMEM containing 10% FBS and 1% penicillin– The primary ACs of SD rats were selected as seed cells,
streptomycin (Gibco, China). AC growth was monitored and three different concentration ratios of Gel–Alg natural
under a microscope, and the culture medium was replaced polymer hydrogels were used as scaffolds to construct
every 2–3 days. When the primary ACs reached 80–90% cartilage models using 3D bioprinting technology. Based
confluence, they were used for subsequent staining on preliminary research, the optimal Gel–Alg ratios
identification and 3D bioprinting of cartilage models. were determined to be 7:3, 8:2, and 9:1. The bioinks were
thoroughly mixed on a shaker at 60°C. For each formulation,
2.9. Identification of articular chondrocytes 500 µL of bioink was transferred to a printing cartridge,
and 50 µL of ACs suspension was immediately added to
2.9.1. Safranin O staining the cartridge and mixed with the hydrogel. Printing was
The ACs were rinsed three times with PBS to remove performed on a 4°C 3D printer platform, with the filament
the residual culture medium. Subsequently, 4% diameter set to 200 µm, the number of layers set to four, and
paraformaldehyde (PFA) was added, and the cells were fixed the printing spacing set to 0.22 mm. Printing parameters,
at room temperature for 20 min. After fixation, the samples including extrusion pressure (0.25, 0.3, 0.35, and 0.4 Mpa),
were washed three times with PBS for 5 min each to remove printing speed (5, 8, 10, and 12 mm/s), and printing nozzle
the fixative. The fixed samples were then immersed in temperature (16, 18, and 20°С), were dynamically adjusted
Safranin O staining solution (Suzhou Haixing Biosciences, during the process to optimize the printing outcome. After
China) and incubated at room temperature for 10 min. printing, the cartilage models were crosslinked in a sterile
Following staining, the samples were thoroughly rinsed 3% CaCl solution for 3 min and rinsed with sterile PBS to
2
with distilled water to remove excess dye. Dehydration remove residual crosslinking agents. The cartilage model
was performed using a graded ethanol series (70, 90, and was then transferred to DMEM culture and placed in a cell
100%) for 2 min each. Finally, the stained samples were culture chamber at 37 °C and 5% CO to support cartilage
observed under an optical microscope. tissue growth. 2

2.9.2. Alcian Blue staining 2.11. Live/dead cell assay
The ACs were rinsed three times with PBS to remove The potential damage to AC viability during the 3D
the residual culture medium. Subsequently, 4% PFA was bioprinting process was assessed using live/dead staining
added, and the cells were fixed at room temperature for 20 assay. After printing, the constructs were cultured in 10%
min. The fixed samples were then immersed in Alcian Blue FBS DMEM for 1 day. Prior to the experiment, the culture
staining solution (Suzhou Haixing Biosciences, China) medium was removed, and the constructs were rinsed
and incubated at room temperature for 30 min. Following with sterile PBS. The constructs were then incubated
staining, the samples were thoroughly rinsed with distilled for 15 min in an assay buffer solution containing 4 μM
water to remove excess dye. Dehydration was performed calcein AM and 4.5 μM propidium iodide (Biosharp,
using a graded ethanol series (70, 90, and 100%) for 2 min China). After staining, the constructs were rinsed again
each. Finally, the stained samples were observed under an with sterile PBS. A fluorescent microscope (Nikon, Japan)
optical microscope. was used for observation at a wavelength of 490 ± 10 nm.
Cell viability was quantified using ImageJ software (Media
2.9.3. Toluidine Blue staining Cybernetics, USA).
The ACs were rinsed three times with PBS to remove the
residual culture medium. Subsequently, the cells were fixed 2.12. Cell counting kit-8 assay
with 4% PFA at room temperature for 20 min. A 0.1% The proliferation activity of ACs within the hydrogel
Toluidine Blue staining solution (pH 2.5–3.0) (Suzhou scaffolds was evaluated using the cell counting kit-8 (CCK-
Haixing Biosciences, China) was added to cover the 8) assay. After printing, the constructs were cultured
samples, followed by incubation at room temperature for in 10% FBS DMEM for 1 day. On days 1, 4, and 7, the
10–15 min. The samples were then quickly immersed in medium was replaced with fresh medium containing 10%
a 0.1% acetic acid solution for 5–10 s for differentiation CCK-8 (Shenzhen JHHS Biotech, China) and incubated at

Volume 11 Issue 4 (2025) 194 doi: 10.36922/IJB025150136