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International Journal of Bioprinting Bioprinted osteoarthritis scaffolds

37 °C for 2 h. Subsequently, the absorbance at 450 nm was constructing the 2D AC senescence model, and the optimal
measured using a microplate reader (Tecan, Switzerland). conditions were applied to construct the 3D-bioprinted
Ten samples from each group were included for cartilage senescence model. Briefly, the H O diluent was
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statistical analysis. removed, and the samples were rinsed with PBS. Then,
1 mL of β-galactosidase staining fixative (Beijing
2.13. Extracellular matrix secretion assay LABLEAD Inc, China) was added, and the cells were fixed
The secretion of extracellular matrix (ECM) (proteoglycans at room temperature for at least 15 min. Subsequently,
and glycosaminoglycans [GAGs]) by ACs in 3D-bioprinted the fixative was removed, and the samples were rinsed
cartilage models was detected using Safranin O and with PBS. Next, 2 mL of working staining solution
Toluidine Blue staining (Suzhou Haixing Biosciences, containing X-Gal substrate was added, and the samples
China) to evaluate the formation and function of cartilage were incubated overnight at 37°C. Finally, β-galactosidase
tissue. After printing, the constructs were cultured in activity was observed using a fluorescence microscope, and
10% FBS DMEM for 5 days. Prior to the experiment, the quantitative analysis was performed using ImageJ software.
3D-bioprinted cartilage models were rinsed with sterile
PBS to remove residual culture medium. Subsequently, 2.16. Construction of 3D-bioprinted cartilage
the samples were fixed with 4% PFA at room temperature senescence models
for 1 h and washed with sterile PBS to remove the fixative. We constructed the 3D-bioprinted cartilage senescence
The fixed samples were then immersed in Safranin O models using the optimal H O concentration and
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and Toluidine Blue staining solutions, respectively, and induction time (determined previously in Section 2.15).
incubated at room temperature for 40 min. After staining, Briefly, the complete medium was removed, and the
the samples were thoroughly rinsed with distilled water samples were rinsed with sterile PBS. Subsequently,
to remove excess dye. Dehydration was performed using 500 μM H O diluent was added, and the samples were
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a graded ethanol series (70, 90, and 100%). Finally, the cultured for 2 h. Finally, the H O diluent was removed, and
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stained samples were observed under an optical microscope the samples were rinsed with PBS for subsequent detection.
(Olympus, Japan).
2.17. Cell viability analysis of senescence models
2.14. Construction of 2D articular chondrocyte The cell viability of 2D AC senescence models and
senescence models 3D-bioprinted cartilage senescence models was evaluated
The effects of oxidative stress on 2D AC models and 2D AC using the CCK-8 assay to compare the proliferation of cells
senescence models were established using H O induction. in both models under senescence conditions. Briefly, the
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A 3% H O stock solution (Biosharp, China) was diluted 2D AC senescence models and 3D-bioprinted cartilage
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with a serum-free medium by volume to prepare H O senescence models were transferred into 24-well plates
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solutions at concentrations of 0 (control group), 100, 300, and cultured in a complete medium. After 24 h of culture,
500, and 700 μM. These were used for constructing the 2D the complete medium was removed, and the samples were
AC senescence models. The optimal H O concentration rinsed with sterile PBS. The medium was then replaced
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for constructing the senescence model was determined with fresh medium containing 10% CCK-8 (Biosharp,
through β-galactosidase staining (Biosharp, China), and China) and incubated at 37°C for 2 h. Subsequently, the
this concentration was subsequently used for constructing absorbance at 450 nm was measured using a microplate
the 3D-bioprinted cartilage senescence models. Briefly, reader (Tecan, Switzerland). Ten samples from each group
the 3% H O stock solution was sequentially diluted to the were included for statistical analysis.
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control group, 100, 300, 500, and 700 μM using a gradient
dilution method. Subsequently, the 2D AC models were 2.18. Western blot analysis
rinsed with sterile PBS to remove the residual culture The differences in senescence-related protein expression
medium on the surface. Finally, the 2D AC models were between 2D AC senescence models and 3D-bioprinted
cultured with different concentrations of H O for 2, 6, and cartilage senescence models were explored using Western
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12 h to determine the optimal concentration and time for blot. Briefly, total protein was extracted from the 2D
inducing the senescence model. After the induction of the AC senescence models and 3D-bioprinted cartilage
senescence model was completed, the H O diluent was senescence models using radioimmunoprecipitation lysis
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removed, and the models were rinsed with sterile PBS for buffer (Thermo Fisher, China) supplemented with 1%
subsequent detection. protease inhibitor and phosphatase inhibitor (Thermo
Fisher, China). The protein concentration was determined
2.15. β-galactosidase staining analysis using the bicinchoninic acid assay (Thermo Fisher, China).
The β-galactosidase staining was used to determine the Equal amounts of protein were electrophoresed by sodium
optimal H O concentration and induction time for dodecyl sulfate–polyacrylamide gel electrophoresis and
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Volume 11 Issue 4 (2025) 195 doi: 10.36922/IJB025150136