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International Journal of Bioprinting Bioprinted osteoarthritis scaffolds

However, printing smaller models with a diameter of significantly lower than that of 7 Gel–3 Alg (39.2 ± 3.22%).
less than 10 mm was challenging due to the limitations This difference is attributed to the denser structure of
of extruded 3D printing technology. Although laser- 7 Gel–3 Alg, as supported by SEM analysis (Figure 2H).
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assisted bioprinting has higher precision and can be used To further determine the water content of Gel–Alg
to construct Gel–Alg hydrogel scaffolds, it is challenging to hydrogel scaffolds at swelling equilibrium in deionized
maintain their bioactivity. This study prioritized physical water, gravimetric analysis was conducted. The results
crosslinking for improved biocompatibility, avoiding revealed similar equilibrium water contents of 7 Gel–3
cytotoxicity associated with UV-initiated chemical Alg and 8 Gel–2Alg scaffolds at approximately 30%,
crosslinking. To eliminate potential mineral deposits reflecting similar hydrophilic properties (Figure 2I). This
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from residual calcium ions on the scaffold surface, hydrophilicity is likely due to hydrophilic groups (–NH2,
constructs were rinsed with sterile PBS. Figure 2C displays –COOH, and –OH) within the molecular chains of Gel
the structural profiles of Gel–Alg hydrogel scaffolds at three and Alg components. Water absorption and swelling
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different concentration ratios. Furthermore, SEM images are critical for retaining structural integrity and cellular
reveal microstructures of the scaffolds in both crosslinked microenvironments. Thus, achieving adequate swelling
and non-crosslinked states. Figure 2C also indicates that capacity is essential for optimal scaffold performance.
the 3D-bioprinted Gel–Alg scaffolds maintained a stable
multi-layered grid structure with tightly stacked layers and The degradation behavior was evaluated in the
an interconnected porous network suitable for AC growth. COL2 solution to assess scaffold stability. Following
The pore sizes of the 7 Gel–3 Alg, 8 Gel–2 Alg, and 9 Gel–1 2 h of incubation, 8 Gel–2 Alg and 9 Gel–1 Alg scaffolds
Alg scaffolds are 5.47 ± 0.87 µm, 2.29 ± 0.048 µm, and 1.18 exhibited significant mass loss, with degradation rates of
± 0.061 µm, respectively. High-magnification observations 67.2 ± 2.90% and 89.2 ± 4.76% after 10 h, respectively. This
revealed dense pore walls formed by Alg crosslinking observation suggests that increased Gel content accelerates
intertwined with Gel-derived microfiber structures, degradation. In contrast, the 7 Gel–3 Alg scaffold exhibited
providing rough surfaces that enhance cell adhesion and a degradation rate of 41.4 ± 2.86% after 10 h, indicating
increase specific surface area. The uniformity of printed that Alg enhances degradation resistance proportionally
fiber diameters and seamless interlayer fusion suggest that with its concentration in the composite (Figure 2J).
structural integrity was maintained through optimized The porosity of the scaffolds was further evaluated,
printing parameters. and the porosity of the 7 Gel–3 Alg, 8 Gel–2 Alg, and 9
The mechanical strength was evaluated to confirm Gel–1 Alg scaffolds was 88.9 ± 3.67%, 77.91 ± 4.66%, and
the stability of the polymer network following physical 64.63 ± 2.48%, respectively. SEM comparisons attributed
crosslinking, and the stress–strain curves were plotted the superior porosity of 7 Gel–3 Alg to its structured pore
(Figure 2F). Based on previous research, a higher network with multi-scale pore distribution, fostering
concentration of Gel–Alg hydrogels ionic-crosslinked efficient nutrient and fluid transport to create an optimal
with CaCl significantly enhanced the mechanical strength environment for cellular adhesion and proliferation
2
of the scaffolds. Therefore, according to Figure 2G, (Figure 2K).
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Young’s modulus of the 7 Gel–3 Alg scaffold is 11.7 ± 3.2. In vitro biocompatibility and cartilage
1.66 kPa, significantly higher than the values for 8 Gel–2 extracellular matrix secretion by gelatin and sodium
Alg and 9 Gel–1 Alg (8.52 ± 6.67 kPa and 5.56 ± 2.23 alginate hydrogel scaffolds
kPa, respectively). This suggests superior stiffness under ACs isolated from the knee joints of four-week-old male
load, crucial for maintaining structural integrity and SD rats were cultured for integration with 3D-bioprinted
performance in physiological conditions.
Gel–Alg hydrogel scaffolds (Figure 3A). After isolation
The swelling behavior was assessed to evaluate the and expansion, primary ACs were stained with Safranin O,
hydrophilicity of the Gel–Alg scaffolds, which is crucial Alcian Blue (pH 1.0), and Toluidine Blue to confirm ECM
for maintaining a hydrated microenvironment conducive synthesis capacity. Studies have demonstrated that the
to nutrient diffusion, cellular viability, and growth. All Gel component provides a scaffold that mimics the ECM
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scaffolds demonstrated rapid water absorption in the first of ACs, while the Alg component imparts mechanical
120 min, followed by a deceleration in absorption from properties similar to those of cartilage. Gel–Alg promotes
120 to 240 min, with equilibrium swelling achieved after the secretion of AC ECM, manifesting as increased cell
240 min. The equilibrium swelling ratios of 8 Gel–2 Alg viability, higher levels of collagen II and proteoglycan
and 9 Gel–1 Alg were 34.4 ± 1.48% and 16.8 ± 1.48%, expression, and elevated GAG content. 58,59 Safranin O

Volume 11 Issue 4 (2025) 198 doi: 10.36922/IJB025150136