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International Journal of Bioprinting Dual tuning of 3D-printed SilMA hydrogel

by keeping the shear strain constant at 1% and varying the at an excitation wavelength of 360 nm and an emission
frequency range from 0.158 to 25.1 rad/s. wavelength of 460 nm. GAG content in the supernatant was
quantified using the dimethylmethylene blue colorimetric
2.6. Isolation and cultivation of rat method (n = 3).
articulator chondrocytes
The cartilage from the knee joints of 24-h old male Sprague 2.7.3. Histological analyses
Dawley rats (n = 4) (Laboratory Animal Center of Anhui To assess the viability and distribution of ACs within
Medical University (Hefei, Anhui Province, China) was the hydrogel during in vitro culture, hydrogel samples
extracted under sterile conditions. The cartilage slices containing cells were cultured for 21 days. The samples
were washed with PBS and then digested with 0.25% were then fixed with 4% paraformaldehyde, embedded in
trypsin (BioFroxx, Germany) for 30 min, followed by paraffin, and sectioned at 5 μm. Sections were stained with
three additional PBS washes. Subsequently, the slices HE, Safranin O, and Alcian Blue to evaluate the survival of
were digested with 0.3% type II collagenase (MCE, United ACs after 21 days of culture.
States) at 37°C for 4 h. The digested solution was filtered
through a 70 µm cell strainer (BioSharp, China) to remove 2.7.4. Real-time quantitative polymerase
collagen impurities and then centrifuged at 1000 rpm for chain reaction
5 min to collect articulator chondrocytes (ACs). The cells The expression levels of MKI67, collagen II (COL2A1),
were cultured and expanded in Dulbecco’s Modified Eagle aggrecan (ACAN), and SRY-box transcription factor 9
Medium/F-12 complete medium at 37°C, 95% humidity, (SOX9) genes in ACs were evaluated using real-time
and 5% carbon dioxide. ACs from passages 2–3 were used quantitative polymerase chain reaction (RT-qPCR) across
for experiments. For the cell-laden 3D printing, the ACs different hydrogel groups. Total RNA was isolated from the
were mixed into the bio-ink before printing. The cell- cells using Trizol reagent, followed by reverse transcription
loaded hydrogels were immersed in a culture medium to synthesize complementary DNA. RT-qPCR was
immediately following the 3D printing process. conducted using TB Green Premix Ex Taq™ II FAST qPCR
kit (Takara Bio, United States). Data was analyzed using
2.7 In vitro cytocompatibility and the 2 −∆∆CT method to determine means and standard
cartilage formation deviations. The primer sequences utilized in this study are
detailed in Table S1. This experiment was repeated thrice,
2.7.1. Live/dead cell staining with each group having three replicate wells per repetition.
Hydrogels loaded with ACs (1 × 10 cells/mL) were
6
prepared and incubated for 1 and 7 days to evaluate cell 2.8. Formation of in vivo cartilage
viability. Cell viability was assessed using a live/dead
staining kit (Beyotime, China). The staining solution was 2.8.1. Subcutaneous implantation of articulator
prepared by diluting the dyes to a final concentration of 2.5 chondrocyte-laden hydrogels in non-obese diabetic/
µM calcein acetoxymethyl ester and 1.25 µM propidium severe combined immunodeficiency mice
iodide. The AC-laden hydrogels were immersed in 400 Animal experiments were approved by the Animal Welfare
µL of the diluted staining solution and incubated at Ethics Committee of the Anhui Provincial Center for
37°C for 30 min in the dark. The growth of ACs within Disease Control and Prevention (No.: 2024024). Porous
the hydrogels was observed using a confocal microscope and non-porous hydrogel precursors were prepared as
(ZEISS, Germany). described previously. ACs were mixed into the prepared
hydrogel precursors at a concentration of 1 × 10 cells/
8
2.7.2. Quantification of DNA and glycosaminoglycan mL, serving as the bio-ink. The bioprinting parameters
For the quantitative measurement of DNA and are detailed in Table 1. During the bioprinting process, the
glycosaminoglycan (GAG) content in hydrogels, AC- photocrosslinkable bio-ink was cured under continuous
laden hydrogel samples were incubated for 7 and 14 days UV light irradiation. After bioprinting, the cell-laden
in Eppendorf tubes containing 1 mL of PBS, followed constructs were cultured in a complete medium for 7 days
by homogenization using a homogenizer. Then, 2 µL of and then transplanted into both sides of the back of male
proteinase K solution (Ron, China) was added to the non-obese diabetic/severe combined immunodeficiency
homogenized solution, and the mixture was incubated mice (n = 4) to observe in vivo cartilage formation.
in a water bath at 56°C for 6 h. After centrifugation, the
supernatant was collected. DNA content was determined 2.8.2. Histological and immunohistochemical staining
using Hoechst 33258 dye (MCE, United States). The The specimens were collected and subjected to histological
fluorescence intensity of each sample was measured and immunohistochemical analyses. Briefly, the samples
using a microplate reader (PerkinElmer, United States) were fixed with 4% paraformaldehyde, embedded in

Volume 11 Issue 4 (2025) 282 doi: 10.36922/IJB025140118