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

New Zealand white rabbits were randomly divided into 2A). Furthermore, western blotting confirmed the
four groups: sham group, control group, Gel-PLGA group, presence of EV-specific markers CD63 and TSG101 on
and EVs-Gel-PLGA group (2.0–2.5 kg; The rabbits were the surface of the extracted EVs (Figure 2C). Subsequent
divided equally into the 6-week group and the 12-week cell uptake experiments demonstrated the successful
group,n = 3 in each group) . After weighing the rabbit internalization of EVs by nasal septal chondrocytes,
before surgery, 2% sodium pentobarbital was injected with fluorescence microscopy revealing endocytic entry
into the rabbit’s ear margin intravenously at a dose of 2 into the cytoplasm and enrichment outside the nucleus
mL/kg of anesthesia. Next, an incision about 4 cm long (Figure 2E). These findings collectively demonstrate the
was exposed in the center of the nasal bone of the rabbit, successful generation of 3D EVs using coaxial printing
and a grinding drill was used to create a gap (2 × 1 cm) at techniques.
the nasal bone in the center of the incision to ensure nasal
septal cartilage exposure. Defects (length: 5 mm; depth: 3.2. Physicochemical properties of
5 mm; width: 1 mm) were made on the septal cartilage composite scaffolds
using ophthalmic scissors and a ruler, ensuring the same Observation of electrospun biofilms and GelMA-PLGA
defect size for all experimental animals. Electrospun composite scaffolds by SEM (Figure 3A) revealed that
biofilm of the same size was cut and fitted to the defect most fibers in the electrospun biofilms had a diameter
site. The surrounding area was filled with 10% GelMA of approximately 1 μm (Figure 3B). The GelMA-PLGA
solution and light-cured to fix the scaffold. An adequate composite scaffold exhibited significant mechanical
amount of EVs was mixed with GelMA in the EVs-Gel- strength, as evidenced by the test results for tensile
PLGA group, before injecting the hydrogel. Light-curing modulus (Figure 3E) and compression modulus (Figure
was performed in all groups for 30 s. The rabbits were 3F). Calculations indicated that the tensile modulus
continuously injected with penicillin for three days after of the Gel-PLGA composite scaffold was 5.4249 MPa;
surgery. Thereafter, the rabbits were over-anesthetized 3.4271 MPa for the PLGA scaffold; and much lower for
at week 6 and euthanized at week 12. The nasal septum the GelMA hydrogel at only 0.00154 MPa. Additionally,
samples were collected accordingly. the compression modulus of the PLGA scaffold was
measured at 0.1661 MPa, but with the incorporation of
2.14. Histomorphology evaluation hydrogel, this value decreased to 0.08995 MPa for the
After fixation, decalcification, dehydration, and paraffin Gel-PLGA composite scaffold. The compression modulus
embedding of the nasal septum, the sample was sectioned of GelMA is 0.001497 MPa. The swelling experiment
at 3 μm using a cryomicrotome. Sections were stained (Figure 3C) revealed that the swelling rate of the
with hematoxylin and eosin (H&E; Beyotime, China) composite stent reached 750%. To prevent nasal cavity
and toluidine blue (Solarbio, China) according to the blockage from the high swelling rates post-implantation,
manufacturer’s instructions. Immunohistochemical we opted for the composite stent with non-freeze-dried
evaluation was performed using Col II according to the treatment for defect implantation. The degradation
results of previous studies. 24 experiment (Figure 3D) demonstrated that the composite
2.15. Statistical analysis scaffold can undergo continuous and stable degradation
Data was statistically analyzed using Prism 9.5 software in PBS at 37°C, with hydrogel experiencing partial
(GraphPad, USA) and ImageJ, and all experiments were degradation over approximately 30 days, while the
repeated at least three times. Data is presented as mean electrospinning biofilm exhibited a slower degradation
± standard deviation. For multiple comparisons, one- rate. The PLGA component in the composite bracket was
way analysis of variance (ANOVA) was performed with largely retained at day 30, with approximately 7% of its
Tukey’s post-hoc test, and p-values < 0.05 were considered total weight remaining.
statistically significant. The extended-release EVs experiment (Figure 2D)
demonstrated that the EV release rate was faster in the
3. Results first three days of the experiment; from the fourth day
3.1. Characterization of ADSCs-EVs onwards, the EV release rate began to slow down; by day
Coaxially printed ADSC-derived EVs were characterized 14, the total number of EVs released was close to 65%. The
using particle size analysis, TEM, and western blotting. experimental results suggest that the composite scaffold
The particle size analysis revealed that the diameter of could effectively achieve the sustained release of EVs. The
EVs ranged from approximately 120–150 nm (Figure FTIR spectra(Figure 3G) indicated that the functional
2B), and TEM observation indicated that the EVs groups of the GelMA hydrogel did not change after loading
exhibited a circular or oval membrane structure (Figure the EVs.

Volume 10 Issue 6 (2024) 180 doi: 10.36922/ijb.4118