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International Journal of Bioprinting Bioprinted osteoarthritis scaffolds

7.37%, and 29.67 ± 2.52%, respectively (Figure 4D and model enriched with approximately 3 × 10 ACs,
6
E). Compared to the control group, the 300 μM H₂O₂ sterile PBS was used to rinse away residual H O . The
2
2
group exhibited significant senescence (p < 0.001), with 3D bioprinting technology enabled the construction
apoptosis observed at higher concentrations. At 12 h, the of aging models that more accurately simulate the
SA-β-galactosidase-positive cell rates were 7.67 ± 2.08%, intracellular environment, increase cell interaction with
69.67 ± 10.79%, 27.67 ± 5.69%, 18.33 ± 2.52%, and 11.0 biogenic ECM, and improve the stability and accuracy
± 4.58%, respectively (Figure 4F and G). Compared to of senescence phenotype. This study assessed p21
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the control group, the 100 μM H₂O₂ group reached peak and p16 expression levels through Western blot and
senescence (p < 0.001), while apoptosis was evident at immunofluorescence staining, with semi-quantitative
higher concentrations. evaluation using ImageJ software. As displayed in
Figure 6A, Western blot and semi-quantitative
These findings suggest that high-concentration oxidative
stress overwhelms cellular regulatory mechanisms, causing analysis revealed dose-dependent upregulation of
p21 and p16 in the 500 μM group versus controls
macromolecular lesions and ultimately disrupting redox (Figure 6B and C). These results were further validated
homeostasis. 65–67 The optimal conditions for inducing AC by immunofluorescence staining, and semi-quantitative
senescence were identified as a 2-h exposure to 500 μM analysis revealed markedly elevated expression levels of
H₂O₂, resulting in 85.0 ± 3.61% SA-β-gal-positive cells senescence-associated proteins p21 and p16 in the 500
without significant apoptosis (p < 0.001 vs. control); and μM H₂O₂ group compared to the control, with enhanced
the 500 μM H₂O₂ concentration was applied to induce green fluorescence intensity indicating substantial
senescence in 3D-bioprinted cartilage models. Prolonged protein accumulation (Figure 6D–G). These results
exposure (6 and 12 h) induced significant apoptosis suggest that H O accelerates senescence in cartilage
(50.33–29.67% viable cells), making these conditions models through oxidative stress responses.
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2
unsuitable for further studies.
To further evaluate alterations in COL2 content within
The expression levels of senescence-associated the 3D-bioprinted cartilage models of the control and
proteins p21 and p16 were assessed through Western 500 μM groups, semi-quantitative analysis was performed
blot analysis and immunofluorescence, followed by using immunofluorescence techniques. As illustrated
semi-quantitative analysis using ImageJ software. in Figure 7A and B, immunofluorescence staining and
The senescence phenotype is not limited to cell cycle semi-quantitative evaluation demonstrated a statistically
arrest; senescent cells are metabolically active and significant reduction in COL2 expression in the 500 μM
exhibit changes in protein expression and secretion, group compared to the control, indicative of oxidative stress-
leading to Senescence-Associated Secretory Phenotype induced degradation of the cartilage matrix. Collectively,
development. Results indicated significant H₂O₂- the semi-quantitative assessment of COL2 highlights the
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induced effects on AC senescence. As illustrated dual impact of oxidative stress on both cellular senescence
in Figure 5A, Western blot and semi-quantitative and ECM disruption in the 3D-bioprinted cartilage model,
analysis revealed dose-dependent upregulation of p21 thereby advancing our mechanistic understanding of
and p16 in the 500 μM group versus controls (Figure pathological cartilage aging processes.
5B and C). These results were further validated by
immunofluorescence staining, and semi-quantitative 4. Conclusion
analysis revealed markedly elevated expression levels
of senescence-associated proteins p21 and p16 in the In this study, three different concentration ratios of
500 μM H₂O₂ group compared to the control, with Gel–Alg natural polymer hydrogels (7 Gel–3 Alg, 8
enhanced green fluorescence intensity indicating robust Gel–2 Alg, and 9 Gel–1 Alg) were used as tissue
protein accumulation (Figure 5D–G). Collectively, engineering scaffolds. Primary ACs served as seed cells
H₂O₂ treatment significantly promotes p21 and p16 and were combined with 3D bioprinting technology
expression in a concentration-dependent manner, to construct cartilage models. Among the tested
accelerating AC senescence. concentrations, the optimal hydrogel concentration ratio
was identified as 7 Gel–3 Alg. Compared to the 8 Gel–2
3.4. Construction and functional characterization of Alg and 9 Gel–1 Alg hydrogel scaffolds, the 7 Gel–3 Alg
a 3D-bioprinted cartilage senescence model hydrogel scaffold exhibited excellent biocompatibility,
The protocol for inducing AC senescence was optimized mechanical properties, and biological functions,
to 500 μM H₂O₂ for 2 h, as previously validated. After such as promoting ECM secretion. Furthermore, 2D
inducing senescence in the 3D-bioprinted cartilage AC senescence models and 3D-bioprinted cartilage

Volume 11 Issue 4 (2025) 201 doi: 10.36922/IJB025150136