Page 370 - IJB-10-3

International Journal of Bioprinting Multi-physical field control inkjet bioprinting

2.3. Rheological test The core component of the printhead was a piezoelectric
To comprehend the physical properties of the GelMA ceramic tube (C82, FUJI, Japan). According to the inverse
hydrogel, we analyzed the rheological properties of GelMA piezoelectric effect of ceramic, when an external electric
before the printing test with a rheometer (MARS 60, field was applied to the tube, it would deform along the
HAAKE, Germany). The parameters for our experiment radial direction, and Equation I describes the relationship
involve using an oscillation frequency of 1 Hz and a between its displacement and the electric field: 35
cooling rate of 2 Temp/min. We monitored the results (I)
closely to ensure accuracy and consistency of the findings. ∆d = d U
33
The rheological study mainly included energy storage and
loss modulus measurements. where ∆d, d , and U are shape changes of the
33
2.4. Cell viability piezoelectric ceramic, piezoelectric constant, and applied
After printing the cell-laden vessel-like structures, in voltage, respectively. The d of our used ceramic is 600 ×
33
-12
order to analyze the cell viability, LIVE/DEAD Viability/ 10 m/V. The equation expresses that the shape change is
Cytotoxicity Kit for mammalian cells (Thermo Fisher proportional to the voltage. We controlled the deformation
Scientific, Waltham, USA) was used to examine the post- of a piezoelectric ceramic by controlling the driving voltage
fabrication cell viability after 24 h. The vessel-like structure to adjust the pressure field in the printhead and regulate
was sliced at the top and bottom and mid-height of 10 mm. microdroplet formation.
The slices were washed with PBS twice. Subsequently, a The proposed printhead was actuated by a signal
diluted LIVE/DEAD staining solution was added to cover generator (FY3200S-20M, FeelTech, China) and high-
the hydrogel for 60 min. Finally, the gel was cleaned with voltage amplifiers (Model 2350, TEGAM, USA), and we
PBS for 10 min. A fluorescent microscope (DMi8, LEICA, optimized the actuating waveform online by adjusting
Germany) was used to image the stained hydrogels, and various parameters. As shown in Figure 1A, we selected the
ImageJ (National Institutes of Health, USA) was used to square wave as the actuation waveform. The rectangular
quantify the live and dead cells. The test was repeated three wave made the pressure field change rapidly and encouraged
times to obtain the survival rate of cells. the formation of microdroplets. A square wave is defined

To examine cell proliferation in various layers, the upper, by voltage amplitude, pulse width, and frequency. In the
middle, and lower layers of the blood vessel model were waveform optimization process, the voltage amplitude
separated into evenly weighted sheets. The cell counting and pulse width affected the strength and action time of
kit-8 assay (CCK-8) was used to measure cell proliferation. the pressure field. Therefore, we chose them as the critical
Next, hydrogels were incubated in 200 μL of cell culture parameters for optimization. This study set the voltage
media mixed with 10% CCK-8 solution at 37°C for 30 min amplitude in the range from 0 to 300 V and pulse width in
in a 48-well plate. Afterward, 100 μL of the resulting liquid the range from 0 to 2 ms. To ensure that the second droplet
was transferred to a 96-well plate, and absorbance for each was printed only after the first droplet had completely
group was read at the wavelength of 450 nm. solidified and to avoid the high temperature of the
second droplet from melting the first droplet, the printing
For actin staining, cell-laden hydrogels were fixed frequency was set at a low rate. This provides enough time
with 4% paraformaldehyde for 2 h at room temperature. for the first droplet to cure, which is usually set at 1–5 Hz.
Subsequently, hydrogels were washed twice with Dulbecco’s To maintain cell activity, the temperature of the bottom
PBS (D-PBS) for 10 min and permeabilized with 0.5% of plate of the temperature-controlled chamber had to be set
Triton X-100 in D-PBS for 2 h followed by washing twice at a minimum of 4°C. Although the gelling speed of GelMA
with D-PBS for 10 min. Hydrogels were incubated in 100 is not very fast at this temperature, reducing the printing
μL of TRITC phalloidin diluted by D-PBS containing 1% speed ensures that the microdroplet on the bottom plate can
bovine serum albumin (BSA) overnight. Hydrogels were fully cool the gel, allowing the next microdroplet of GelMA
washed twice with D-PBS for 10 min, and cell nuclei were with a higher temperature not to affect the formation of
stained with DAPI for 10 min and washed twice with D-PBS. the previous microdroplet after it falls. By adopting a lower
Hydrogels were imaged with a fluorescent microscope. frequency, better control of the temperature field shaping
was achieved. Printing the vascular structure took about 2
2.5. MFCPIB method h, and the low-frequency printing ensured good structural
The MFCPIB method was realized by controlling the
pressure field and temperature field. The pressure field shaping without affecting cell activity.
controlled the formation of microdroplets, and the Figure 1B shows the process of printing a tubular
temperature field controlled the assembly of microdroplets. piezoelectric ceramic. According to the inverse piezoelectric

Volume 10 Issue 3 (2024) 362 doi: 10.36922/ijb.2120