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Zhou, et al.
kept at 4°C before use. LAP was added as an initiator photo-crosslinked, the hydrogel was then cut into
with a dosage of 0.003 wt.%. dumbbell-shaped specimens (gauge length, 12 mm;
width, 2 mm; and thickness, 1 mm). All the experiments
2.5. Vial-inverting test were conducted with a 50 N load cell and a stretch rate of
The sol-gel transition of the copolymer aqueous solutions 10 mm/min. At least three specimens were tested for each
was examined by the vial-inverting method. The vials sample to get reliable data.
containing 1 mL solution were immersed in a water bath. 2.8. Swelling ratio
The temperature was adjusted from 25 to 55°C with
an increment of 1°C/step. Samples were equilibrated The water absorption properties were evaluated using the
for 10 min at each temperature and then the state was conventional gravimetric method. For this purpose, the
confirmed. If no flow was noticed in 20 s after the vial inks were made into cylinder samples (4 mm height, 5 mm
was inverted, the state of the solution was regarded as diameter) by exposure to UV light at 37°C for 5 min. The
“gel,” otherwise a “sol” state would be recorded. hydrogels were then immersed in 5 mL deionized water at
37°C for 48 h. After that, samples were taken out and the
2.6. Rheological analysis surface water was wiped carefully with filter papers, the
(1) Sol-gel transition swollen hydrogels were weighted and noted as W . Then,
s
those samples were lyophilized and the dry weight (W) was
A rotational rheometer (TA, DHR) was used to investigate recorded. Similar studies were conducted in triplicates, and
r
the sol-gel-sol transition of the copolymer solutions and the the swelling ratio (s ) was calculated using the equation.
properties of the hydrogel. Temperature sweep experiments r
were performed to examine the phase transition phenomenon W −W
using a cone-plate geometry (1.985°, 40 mm diameters, s r = s d 100%×
0.05 mm gap). About 0.7 mL solution was added between W d
two plates at 25°C. Then, the margin of the plate was
covered with lowly viscous oil to prevent vaporization of
the solvent. The temperature was increased from 25 to 55°C 2.9. Degradation properties
at a rate of 2°C/min. The controlled stress and frequency To study the degradation properties of the dual-sensitive
were 1.0 Pa and 6.28 rad/s, respectively.
hydrogel, 0.5 mL solution was added to a vial and then
(2) Extrudability exposed to UV light for 5 min at 37°C. After gelation, 1 mL
phosphate buffer saline (PBS) containing 0.02 mg lipase
After adding to the plate, all the samples were then kept at was added. The degradation experiments were conducted
37°C for 5 min before measurement. To characterize the at 37°C and PBS was refreshed every day. At specific time
shear thinning nature of the hydrogel, the shear rate was intervals, the residual samples were taken out and freeze-
logarithmically increased in a range of 0.1 – 100 s and dried. The weight was recorded and the weight loss to the
−1
the viscosity was recorded. The elastic recovery behavior original weight of samples before incubation was seen as
was measured by alternating 1% and 100% strain for the degradation rate. All the experiments were performed
100 s at 6.28 rad/s angular frequency. The thixotropic in triplicates and the average value was used for analysis.
property of the ink was proved using a three-stage steady-
state flow test. At the first and the third stage, a shear rate 2.10. Printing experiments
of 0.1 s was applied for 100 s. The shear rate was set to
−1
100 s and lasted for 5 s during the second stage. (1) Preparations before printing
-1
(3) Mechanical properties The printing was performed in a commercial
microextrusion 3D bioprinter (Regenovo, Bio-Architect®-
To compare the physical strength of hydrogels after photo- Pro) with a temperature-controlled syringe and print bed.
crosslinking, the samples were made into disks with 20 mm The control of the printer was achieved through software
diameter and 1 mm thick by exposure to UV light (375 nm) (Bio-Architect v2.2) on a personal computer. First, a
for 300 s at 37°C. The disks were then tested on a parallel- printing model was designed in 3D computer-aided
plate (20 mm, 1.0 mm gap) fixture. A frequency sweep (1% design and saved as STL files. Those files were imported
strain, 0.1 – 50 rad/s angular frequency) was performed to to the software, and then the walking path was generated
examine the mechanical properties. automatically according to the printing parameters. To
print, inks were loaded into 10 mL syringes and store at
2.7. Tensile tests 4°C for 2 h to remove bubbles. After those syringes were
A commercial tensile tester (Instron 3343) was mounted onto the printing apparatus, equilibrating at 37°C
used to measure tensile modulus of samples. After for 15 min was required to make sure the inks stay at a
International Journal of Bioprinting (2021)–Volume 7, Issue 3 143