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Xie, et al.
with angiogenesis. The encapsulated HUVECs showed diameter. When the gelatin microdroplets were finally
obvious 3D sprout and the vascularization protein (VE- received in silicon oil that was precooling to 4°C, the
cadherin) was tested to be positive. We believe that receiving Petri dish was transferred to refrigerator at 4°C
this bioink system would become a powerful tool in with a thermo-crosslinking time of 400 s. Finally, the
corresponding biomedical applications in future. totally crosslinked TSMs were transferred to centrifugal
tubes and centrifugated for 3 times at 1000 rpm to remove
2. Materials and methods the silicon oil.

2.1. Rheological testing of electrospraying ink 2.3. Rheological testing of GelMA precursor
The non-modified gelatin was mixed in the phosphate solution
buffered saline (PBS) at the concentration of 15% w/v and Frozen-dried GelMA and photoinitiator LAP were mixed
fully dissolved for 1 h with a water bath with magnetic
mixer at 37°C. It was then filtered through a 0.22 μm in the PBS at the concentration of 5% w/v and 0.5 w/v,
filter for sterilization and kept at the temperature of 4°C. respectively, and fully dissolved for 1 h in a water bath
In the flow step measurement of gelatin electrospraying at 37°C. It was then filtered through a 0.22 μm filter for
sterilization and kept at the temperature of 4°C. A parallel
ink, a parallel plate rotor with a diameter of 50 mm was
selected and the testing clearance was set to 0.2 mm. The plate rotor with a diameter of 25 mm was selected and
shear rate was selected as 10 s , with a sampling period the testing clearance was set to 1 mm. In flow step
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of 6 s and a total duration of 1200 s. The electrospraying measurement, GelMA precursor solution was preheated in
ink was preheated in a 37°C water bath for 30 min, then a 37°C water bath for 30 min and transferred to rheometer
the rheometer temperature was set to 30°C and 37°C at initial temperature of 37°C and kept for another 5 min.
respectively and the preheated electrospraying ink was The cooling process was set at 4°C, 5 min followed by
warmed up again on the rheometer for 5 min. In the flow stabilization process at 24°C, 10 min. Sampling period
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sweep, the rheometer temperature was maintained at was set to 10 s and the shear rate was 10 s . In flow
37°C and the shear rate range was set to 1 s ~100 s . sweep, GelMA precursor solution was scanned at a
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In the low amplification oscillation time sweep of temperature of 24°C and the shear rate range was set to
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thermo-crosslinking process, a parallel plate rotor 0.1 s ~100 s . In low amplification oscillation frequency
with a diameter of 25 mm was chosen, and the testing sweep, the amplification was set to 1% and the oscillation
clearance was set to 1 mm. The electrospraying ink was angular frequency was set to 0.1 rad/s~1000 rad/s.
preheated in a 37°C water bath for 30 min and added 2.4. Preparation and rheological properties of
to rheometer platform. Then, the testing temperature TSM-B with different recipes
was set to 4°C and the testing was started at once. The
oscillation angular frequency was set to 1 rad/s and the After TSMs were generated, GelMA precursor solution
oscillation amplification was set to 0.01%. The sampling was loaded in 10 mL syringe and preheated at 37°C and
period was set to 12 s, and the total duration was set to was then transferred to 4°C refrigerator for 5 min. TSMs
720 s. In the low amplification oscillation time sweep were added to the GelMA precursor solution at 120 s during
of extruding bioprinting process, the testing sample in cooling process at certain volume proportion according to
the one of thermo-crosslinking process with stable G’ the syringe scale and the syringe was constantly flipped.
and G’’ was directly tested. The oscillation angular Finally, the syringe was transferred to bioprinter at 24°C
frequency was set to 10 rad/s and the other parameters and rest for more than 560 s. In the rheological testing
remained unchanged. of TSM-B with different recipes, the applied voltage in
electrospraying was set as 0 kV, 2 kV, 3 kV, and 3.5 kV
2.2. Electrospraying of TSMs (35%) and the volume proportion was set as 50%, 35%,
The TSMs were prepared with nozzle-ring electric field. and 20% (3.5 kV), respectively. The testing temperature
The environment temperature was set at 37°C and the was set as 24°C. A parallel plate rotor with a diameter
humidity was 50%. The nozzle was selected as 27G of 25 mm was selected. It is worth to be mentioned that
(outer diameter 0.4 mm and inner diameter 0.2 mm), 28G because TSM-B was a kind of two-phase bioink with both
(outer diameter 0.35 mm, and inner diameter 0.19 mm), solid and liquid phase, the testing clearance was set to
30G (outer diameter 0.3 mm and inner diameter 5 times the diameter of the selected TSMs. In flow sweep,
0.15 mm), and initially hydrophobically treated with GelMA precursor solution was scanned at a temperature
nano waterproof sprays in order to ensure the surface of 24°C and the shear rate range was set to 0.1 s ~100 s .
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hydrophobicity. The flow rate of the electrospraying In low amplification oscillation frequency sweep, the
ink was set to 50 μL/min and 100 μL/min. The applied amplification was set to 1% and the oscillation angular
voltage was selected according to the desired TSM frequency was set to 0.1 rad/s~1000 rad/s.
18 International Journal of Bioprinting (2022)–Volume 8, Issue 4