Page 210 - IJB-9-3

International Journal of Bioprinting Dual ions mixed GelMA for hair follicle regeneration

2.4. Tube formation assay 2.9. Compression test
The effects of ions on angiogenesis were evaluated by A universal testing machine (Instron 3365, UK) with a
tube formation assay. Briefly, Matrigel (356234, Corning, 100-N load was employed to perform the compression
USA) was added into 48-well plates and was solidified. tests. Samples were shaped into cylinders (height =
Then, HUVECs (2 × 10 per well) were seeded into the 10 mm and diameter = 20 mm) and were compressed at
4
wells and treated with ions in different concentrations. a strain velocity of 1 mm/min until fracture at 25°C. The
After incubation for 8 h, an inverted optical microscope displacement in the hydrogel height and the increasing
(Leica DMI 4000 B, Germany) was employed to capture load were recorded. Then, typical curves were obtained
the images, and the length of the tube and the number of and the first 15%–25% of the curve was used to calculate
branch points were counted by ImageJ . Young’s modulus .
[15]
[17]
2.5. Preparation of hydrogels integrated with 2.10. In vitro degradation
silicon/zinc ions The initial dry weight of the hydrogels was recorded (W ).
0
Briefly, 1 g of GelMA was dissolved in 5 mL phosphate- Then, the samples were shaped into cylinders (height =
buffered saline (PBS) containing lithium phenyl 10 mm and diameter = 20 mm) and were incubated in
(2,4,6-trimethylbenzoyl) photoinitiator (EFL-LAP, EFL, PBS at 37°C. The PBS was replaced by the fresh solution
China) and then, the solution was put into a water bath at at determined time intervals, and the dry weight of the
37°C for 1 h. About 312.5 μL Zn/Si dual ions solution was remaining hydrogels was recorded (W ) at different time
d
added to 5 mL GelMA hydrogel solution and then, PBS points. The degeneration rate was calculated according to
was added to 10 mL, which means the final concentration Equation V .
[19]
of ions reached the level of 1/32 Zn/Si ions solution (Zn W
0.625 μg/mL, Si 3.75 μg/mL). The hydrogel with Zn/Si dual Remaining mass of the hydrogel d 100% (V)
ions was named GelMA-Zn/Si, and the hydrogel without W 0
ions was named GelMA for control.
2.11. Releasing profile of ions from hydrogel
2.6. Structural characterization 1 mL solidified GelMA-Zn/Si hydrogel was immersed
A scanning electron microscope (SEM; S-4800, Hitachi, in PBS (pH = 7.4, 9 mL) at 37°C. At predetermined time
Japan) was employed to characterize the structure of points (1, 3, 5, and 7 days), samples were collected, and
hydrogels . Briefly, the hydrogels were dehydrated by the concentration of ions was measured by inductively
[16]
freeze-drying and were coated with gold-palladium in a coupled plasma emission spectrometer (ICP).
Hitachi ion sputter. Then, the images were captured, and 2.12. Mouse excisional model and hydrogel
the pore size and the porosity were quantified via ImageJ.
treatment
Pore area Female C57 mice aged 4 weeks were obtained from SPF
Porosity 100% (III)
Total area Biotechnology Company (China). GelMA were adequately
exposed to ultraviolet light for sterilization, and Zn/Si dual
2.7. Swelling property ions solution was sterilized through 0.22-micrometer-
Samples were shaped into cylinders (height = 10 mm and pore-size filters (SLGVR33RB, Millipore, Germany).
diameter = 20 mm) whose values of initial weight were The PBS was sterilized at 124°C for 30 min, and then,
recorded as W . Then, the hydrogels were put into PBS the sterile GelMA-Zn/Si hydrogel was prepared. The
0
solution (pH = 7.4) for complete swelling at 37°C, and the mouse excisional wound model was established after
values of constant mass were recorded as W . The swelling being anaesthetized with pentobarbital sodium solution
t
[20]
ratio was calculated according to Equation IV . (100 mg/kg) . A wound with a diameter of 10 mm was
[17]
created. Then, the GelMA-Zn/Si hydrogel was loaded into
W W a syringe. One milliliter hydrogel was in situ printed and
Swelling ratio t 0 (IV)
W 0 fully covered the wounds. White light was employed to
crosslink the hydrogel for 60 s. Gauze and bandage were
2.8. Rheological test employed to dress the wound surface. A new hydrogel
A rheometer (TA-ARES G2, USA) with a 40 mm-diameter dressing was replaced every 3 days. In addition, we set
parallel plate was applied to assess the rheological another three groups using saline, hydrocolloid and pure
properties of the hydrogels. Frequency sweep tests were GelMA, respectively, for comparison with GelMA-Zn/Si
conducted from 0.1 to 100 rad/s at 1% strain amplitude at group. Gross images of the wound area were captured on
25°C. The elastic modulus (G′) and viscous modulus (G′) days 0, 7, and 14 postoperation, and 1-cm diameter rubber
were investigated as a function of frequency . rings were used as a size reference.
[18]

Volume 9 Issue 3 (2023) 202 https://doi.org/10.18063/ijb.703