Page 186 - IJB-10-6

International Journal of Bioprinting 3D-printed EVs for nasal septal defects

2.4. Mechanical test using live/dead cell staining and cell counting kit 8
The tensile and compressive properties of composite stents (CCK-8) cytotoxicity assays.
were tested using a universal mechanical testing machine For live/dead staining, 1 × 10 cells were seeded on a 2 × 2
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(Sunstest, China) at a tensile and compressive velocity of cm composite scaffold and co-cultured with the composite
10 mm/min. The stress–strain curves were plotted using scaffold for 1, 3, and 7 days. The staining solutions (calcein
Origin 2021. AM and propidium iodide [PI]; Keygen, China) were

2.5. EV release test added to the cell-laden composite scaffold at 0.5 μL/ml each
The obtained EVs were incorporated into the composite using PBS solution until the working solution submerged
scaffold to assess the release of EVs from the scaffold. the sample. The samples were incubated at 37°C for 30 min
Briefly, the EV-doped composite scaffold was placed in a and subsequently observed using an inverted fluorescence
dialysis bag and incubated in PBS solution at 37°C. PBS microscope (Nikon, Japan).
samples were collected on days 1, 2, 3, 4, 7, and 10. After For CCK-8 cytotoxicity experiments, the GelMA
each EV collection, fresh PBS was added at the same hydrogel was soaked in DMEM for 24 h at room
volume. The EV concentration in the solution was detected temperature in advance to obtain a solution containing
using a BCA kit, and an EV-release curve was plotted. the hydrogel extract. PLGA-electrospun membranes of
2.6. In vitro degradation and swelling test the same size were cut and placed at the bottom of the
The swelling and degradation properties of composite 96-well plate. They were seeded on the biofilm with the
scaffolds were evaluated based on previous reports. Briefly, standard 2000 cells per well, and 100 μL of complete
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after lyophilizing the stent samples for 24 h, the initial weight medium containing the hydrogel extract was added. Cell
W was weighed. The samples were placed in PBS at 0.04 g/ viability was evaluated by measuring the absorbance at 450
0
mL, and the samples were freeze-dried on days 5, 10, 15, nm using a microplate reader (Agilent, USA) with CCK-8
20, 25, and 30, respectively. The corresponding mass W was solution (Beyotime, China) on days 1, 3, and 5.
t
weighed, and scaffold degradation is calculated as follows: 2.9. Cell proliferation and migration experiments
2.9.1. CCK-8 proliferation experiment
W − W Chondrocytes (P3–5) were seeded into 96-well plates at
Degradation rate (%) = 0 t × 100% (I) 2000 cells per well. After overnight cell culture, 100 μL
W 0 DMEM was added to the EVs/mL (5 × 10 , 10 × 10 , and
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20 × 10 EVs/mL) and control groups, respectively. After
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Swelling of the stent also employed a similar method. days 1, 3, and 5, 10 μL of CCK-8 reagent was added to
The weight of the freeze-dried stent is W , and the stent each well according to the manufacturer’s instructions
0
was soaked in PBS. The PBS on the surface of the stent was and incubated for 2 h in the cell culture incubator. Finally,
absorbed every 15 min using filter paper, and the weight the absorbance (optical density [OD]) of the samples at
W was recorded. The swelling weight of the stent was 450 nm was measured using a microplate reader to assess
t
obtained after 24 h, and stent swelling was calculated based cell proliferation.
on the following:
2.9.2. Wound-healing
Cell scratch assay was performed to evaluate the effect
W − W of different EV concentrations on the migration of nasal
Swelling ratio (%) = t 0 × 100% (II) septal chondrocytes. Briefly, 5 × 10 cells/mL were seeded
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W 0 in a six-well plate, and the medium was aspirated after
cell adhesion. A 200 μL pipette tip was used to create
2.7. Fourier-transform infrared spectroscopy three marks in the well plate to simulate a wound. Each
Fourier-transform infrared spectroscopy (FTIR) was group of cells (EV: 5 × 10 , 10 × 10 , and 20 × 10 EVs/
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employed to investigate the effect of EVs on the GelMA mL; and control) was photographed at 0, 12, 24, and 48
chemical functional groups. Freeze-dried GelMA hydrogel h, respectively. The migration area was quantified using
was ground into a powdered sample and completely mixed ImageJ, and statistical analysis of the migration area was
with potassium bromide. The processed samples were performed using Prism 9.5.
subsequently analyzed using FTIR at room temperature.
2.9.3. Transwell assay
2.8. Biocompatibility assessment An 8-μm transwell room was purchased from NEST
As previously described, the biocompatibility of nasal (China). A 24-well plate was grouped according to EV
septal chondrocytes and composite scaffolds was evaluated concentrations of 5 × 10 , 10 × 10 , and 20 × 10 EVs/mL
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Volume 10 Issue 6 (2024) 178 doi: 10.36922/ijb.4118