Page 395 - IJB-10-5

International Journal of Bioprinting DEX-Loaded PLGA microspheres enhance cartilage regeneration

solution to create four types of mixed hydrogels. Finally, until 80% of maximum deformation was reached. The slope
the seeded cells (1 × 10 cells/mL) were mixed with the of the stress–strain curve was adjusted within the range
7
hydrogel to form the bioink. of 10–20% strain to calculate the Young’s modulus of the
To tissue-engineer cartilage in C57 mice, a silicone hydrogel. A humidifier was utilized to maintain ambient
rubber base (Sylgard™ 184, Dowsil, USA) was heated at air humidity throughout the entire testing process.
70°C for 3 h to prepare a circular mold with a diameter 2.9. Culture of 3D-bioprinted constructs
of 10 mm and a height of 4 mm. Subsequently, 180 μL of The 3D-bioprinted constructs were stained with the
bioink was aspirated and injected into the mold. The bioink Calcein-AM/PI dual staining kit (Dojindo, Japan) to
was then exposed to blue light with a wavelength of 450 nm assess cell viability within the hydrogel. Cell viability was
2
and an intensity of 30 mW/cm for 10 s to achieve complete evaluated using a confocal microscope (Leica TCS SP8
crosslinking. Following crosslinking, the bioink was CARS, USA).
crosslinked using blue light (450 nm) to form cylindrical
structures. These cylindrical structures were cultured in vitro 2.10. Scanning electron microscopy
for 24 h before being implanted subcutaneously into mice. After freeze-drying and gold sputter coating, the samples
After 24 h of in vitro culture of the cell-loaded constructs, were analyzed using a Quanta 2000 scanning electron
they were implanted subcutaneously into animals to microscope (FEI, the Netherlands), operating at an energy
observe cartilage formation. Specimens were collected at 3, level of 15 kV. The acquired SEM images were analyzed
7, and 14 days post-implantation for biomechanical testing, using ImageJ software.
determination of glycosaminoglycan (GAG) content, and
histological examination. 2.11. Fourier transform infrared spectroscopy
Fourier transform infrared spectroscopic (FTIR) analysis
Following the 3D bioprinting of the bioink, the was conducted using the iS10 FT-IR spectrometer
constructs were crosslinked and then cultured in vitro for (Thermo Fisher Scientific, USA) with a wavenumber range
24 h before being implanted subcutaneously in rabbits. of 400–4000 cm . The spectrometer featured a resolution
−1
After 24 h of in vitro culture of the 3D-bioprinted cell- of 4 cm and a signal-to-noise ratio of 50,000:1, with
−1
loaded constructs, they were implanted subcutaneously an average of 32 scans. During the testing process, the
into rabbit and the cartilage formation was observed Attenuated Total RTimes Perfactory RP slicing software
afterward. Specimens were collected at 7 and 14 days was utilized for slicing. The layer height was set to 320
post-implantation for histological examination and μm, and the sliced models were imported into the 3D
determination of GAG content. bioprinter’s visual machine. The internal structure of

2.7. Histological examination the scaffold was defined as a bordered lattice with a line
The tissue-engineered cartilage samples were fixed in spacing of 400 μm in the printing system. The extrusion
4% paraformaldehyde for 48 h, followed by dehydration, pressure for the composite hydrogels ranged from 0.2 to
embedding in paraffin, and sectioning using a microtome. 0.7 bar, with nozzle speeds ranging from 5.5 to 8.5 mm/s.
Subsequently, selected samples were stained using Images were captured using built-in cameras at the 2nd,
hematoxylin and eosin (H&E), Alcian blue (Solarbio, 4th, and 6th layers during the bioprinting process. After
China), and immunohistochemical staining to assess printing six layers, complete crosslinking was achieved
the deposition of extracellular matrix and histological under a blue light source with a wavelength of 450 nm
structure of the regenerated cartilage. Cells and tissues and intensity of 30 mW/cm² for 10 s.
30
were prepared according to the aforementioned methods. 2.12. Total RNA extraction, cDNA synthesis, and
Antibodies against CD86 (ab220188; Abcam, UK) and quantitative real-time PCR analysis
CD31 (ab9498; Abcam, UK) were used. The images were For every 100 mg tissue, 1 mL TRIzol (Invitrogen,
captured using Motic Images 2.0 (China), and the exported USA) was added. Tissue specimens were chopped on
images were analyzed for relative staining intensity around
cells by using ImageJ software (Rawak Software, Inc., ice and homogenized with sterile equipment. Following
Germany). Subsequently, the results were subjected to the addition of another 1 mL TRIzol, the mixture was
statistical analysis. incubated for 15 min at room temperature. Upon being
transferred to RNase-free tubes, the lysate was added with
2.8. Mechanical compression test 200 μL of chloroform, mixed, and incubated for 15 min.
Compression tests were performed utilizing the Instron The lysate was then centrifuged at 12,000 × g for 15 min
5967 biomechanical analyzer (Instron, USA), which at 4°C. Approximately 500 μL of the upper phase, after
was outfitted with a 100 N pressure sensor. A constant centrifugation, was transferred to new tubes, followed by
compression strain rate of 10 mm/min was maintained addition of 500 μL isopropanol, mixing, incubation for 15

Volume 10 Issue 5 (2024) 387 doi: 10.36922/ijb.3396