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International Journal of Bioprinting Bioprinted osteoarthritis scaffolds

2.5. Mechanical strength analysis 2.7. Porosity test
To evaluate the mechanical properties of the 3D-bioprinted The porosity of the 3D-bioprinted Gel–Alg hydrogel
Gel–Alg hydrogel scaffolds, a universal testing machine scaffolds was measured using the ethanol displacement
(Instron, USA) was used. Briefly, the scaffolds were method. Briefly, the scaffolds were freeze-dried to remove
fabricated into 3D-bioprinted rectangular columns with internal moisture. The dried scaffolds were accurately
dimensions of 8 × 8 × 8 mm and subjected to uniaxial weighed (W0). Subsequently, the dimensions of the freeze-
compression at a rate of 1 mm/min until 50% strain was dried scaffolds were measured using a vernier caliper to
reached. The compressive Young’s modulus was calculated calculate their theoretical volume (V0). The dried scaffolds
from the linear region of the initial 10% of the stress–strain were then placed in a test tube pre-filled with ethanol,
curve, while toughness was determined by measuring the ensuring complete immersion of the scaffolds. The tube
area under the curve. All measurements were performed was sealed and placed under a vacuum for 2 h to allow
in triplicate. ethanol to fully infiltrate the pores of the scaffolds. The
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process was monitored continuously until no bubbles
2.6. Water absorption and degradation behavior were observed, indicating complete displacement. After
The swelling and water absorption properties of the removal, the scaffolds were gently blotted with filter paper
3D-bioprinted hydrogel scaffolds were evaluated using to remove excess ethanol and immediately weighed (W1).
the conventional weighing method. Briefly, the scaffolds The experiment was repeated three times independently
were freeze-dried to obtain their initial dry weight (W0) to ensure data reliability. The porosity of the scaffolds was
and then immersed in 2 mL of PBS. The wet samples calculated using Equation III:
were maintained in a 37°C incubator. At specific time
intervals (100, 200, 300, 400, and 500 min), the scaffolds
were removed, and excess water was blotted with filter W1 W0 × 100% (III)
paper. The wet weight (Wt) of each scaffold was recorded. ρV0
The percentage of swelling rate and water content was
calculated using Equation I: where W0 is the initial weight, W1 is the measured
weight at each time period, ρ is the density of ethanol, and
Wt W0 V0 is the scaffold volume.
Water content (%) × 100% (I)
W0 2.8. Isolation and amplification of
articular chondrocytes
where W0 is the initial dry weight, and Wt is the wet All four-week-old male Sprague-Dawley (SD) rats were
weight of each sample at a different time point. Each provided by the Department of Laboratory Animals of
sample was independently run in triplicate. the Zhejiang Province Experimental Animal Center. The
The in vitro degradation behavior of the 3D-bioprinted study strictly adhered to the guidelines of the Institutional
hydrogel scaffolds was evaluated using a gravimetric Animal Care and Use Committee of Hangzhou Institute
analysis method. Briefly, the scaffolds were freeze-dried, of Medicine Chinese Academy of Sciences and was in
and their initial dry weight (W0) was recorded. The accordance with the principles of the Declaration of
scaffolds were immersed in PBS (pH 7.4) containing type Helsinki for the care and use of animals. After anesthesia,
II collagenase (COL2) and maintained in a shaker at 37°C four-week-old male SD rats were euthanized, and the
with a shaking speed of 40 rpm. At specific time points carcasses were disinfected by immersion in 75% ethanol for
(0, 2, 4, 6, 8, and 10 h), the samples were removed, dried, 20 min. The skin was removed, and the lumbar spine was
and weighed again (Wt). Each experiment was performed isolated, discarding the tail and ankles while preserving the
in triplicate to ensure the reliability of the results. The hip and knee joints. The joints were then rinsed with 75%
degradation rate of the scaffolds was calculated using ethanol. Under aseptic conditions, the joint capsule was
Equation II: opened, and the surrounding muscle tissue was carefully
removed. The meniscus was excised to expose the articular
cartilage. Cartilage from the knee joint and femoral
W0 Wt
Degradation rate (%) × 100% (II) head was harvested and rinsed with PBS. The tissue was
W0 transferred into a trypsin–EDTA solution (Sigma, USA)
and digested on a shaker at 37°C and 85 rpm for 30 min.
where W0 is the initial weight, and Wt is the measured Following digestion, the samples were washed with PBS
weight at each time period. Each sample was independently and transferred to a centrifuge tube containing 0.2% COL2
run in triplicate. (Sigma, USA) for further digestion at 37°C for 4 h under

Volume 11 Issue 4 (2025) 193 doi: 10.36922/IJB025150136