Page 383 - IJB-10-3

International Journal of Bioprinting Multi-physical field control inkjet bioprinting

to demonstrate the ability to DOD-print thermosensitive was approximately 20 mm. The achievable aspect ratio with
materials; the ability of microdroplets to bond into curves GelMA during inkjet printing with the MFCPIB method
was also found to be good. was 4.0. The maximum aspect ratio in previous studies was
18
Inkjet printing has been widely used in industrial curved only 2.5. This demonstrated the promising fabrication
surface structure manufacturing due to its advantages of capability of the MFCPIB method, which could be utilized
noncontact printing. We printed an ear-shaped object with for the direct biofabrication of thermosensitive materials in
sharp and well-defined edges in 2.5 h (Figure 7C). This structures needed for patients.
demonstrated the promising fabrication capability of the For the bioprinting of living cells, the viability of cells
MFCPIB method, which can be utilized for manufacturing after printing is of great concern. SMCs were mixed in
tissue with curved surfaces. In the process of printing the GelMA to print the vascular structures. After printing the
complex curved surface, when a microdroplet combined cell-laden vessel-like structures, we sectioned and observed
with the microdroplet that had been printed previously, the bottom (I), middle (II), and top (III) of the vessel-like
the contact surface was not smooth but irregular in shape, structure, as shown in Figure 7D. We applied live-dead
such as a slope; the microdroplet easily collapsed and staining after culturing on day 1 (Figure 7E), and we also
rebounded due to the uneven contact surface, making it added dead and live staining test on day 7 (Figure 7F).
difficult to achieve ideal printing. However, this method Most stained cells showed green fluorescence, indicating
accurately controls the microdroplet bonding temperature, living cells, while only a few cells showed red fluorescence,
which ensures good printing resolution and achieves a good indicating dead cells. The test was repeated three times.
printing effect, even for the formation of complex curved The analyzed results, shown in Figure 7G, revealed that the
surfaces, and provides the possibility to manufacture cell viability at all three heights was about 90%, regardless
clinically complex organs. of whether the cells have been cultured for 1 day or 7 days.
To demonstrate the suitability of the MFCPIB method for According to the data presented in Figure 7G, the viability of
producing cell-laden constructs, large aspect ratio cell-laden SMCs in layer I on day 1 was 89.0 ± 1.1%, in layer II was 89.9
vessel-like structures (Figure 7D) with a diameter of 5.0 mm ± 0.4%, and in layer III was 88.0 ± 1.5%. Similarly, on day 7,
were fabricated using the MFCPIB method. Printing tubular the viability of SMCs in layer I was 89.7 ± 2.1%, in layer II was
structures with a large aspect ratio is beneficial because such 90.1 ± 0.5%, and in layer III was 89.8 ± 0.8%. We conducted
structures are needed for various biomedical applications, significance calculations on cells of different heights on the
such as vascular applications. Considering the temperature same day, yielding p-values of 0.52 for significance tests of
control and printing effect, we used a 150 μm nozzle. The layer I and layer II, 0.13 for significance tests of layer II and
voltage amplitude of the driving waveform was 160 V, and the layer III, and 0.52 for significance tests of layer I and layer
pulse width was 1 ms. The microdroplet diameter was 197 ± III. All p-values were greater than 0.05, indicating that there
15 μm, and the velocity was 0.17 ± 0.02 m/s. The MFCPIB was no significant difference in cell viability at each height
method helped control the microdroplet temperature layer. The same was true for the cell activity test on day 7,
throughout the printing process. Following the relationships with p-values of 0.85 for significance tests of layer I and
in Figure 6I, we set the top cover temperature to -5°C, the layer II, 0.98 for significance tests of layer II and layer III,
bottom plate temperature to 4°C, and the air temperature and 0.94 for significance tests of layer I and layer III. Again,
to 2.2°C. Finally, the microdroplet molding temperature was all p-values were greater than 0.05, indicating no significant
also approximately 16°C, which met printing requirements. difference in cell activity at each height layer on day 7. Our
While printing the first few layers that become the base of a experimental results demonstrate that the MFCPIB method
structure, it was necessary to deposit the material not only has low effect on cell viability. Furthermore, we conducted
on a surface that was homogeneous and smooth, but also on the CCK-8 assay to evaluate the proliferation of the SMCs.
one that ensured that the cells did not die from prolonged Based on the experimental findings, it was observed that
hypothermia. The gradually rising vessel-like structure is the cells cultured in the three gel layers for a period of 7
also needed to ensure the same molding effect as the first days exhibited a remarkable improvement in absorbance as
few layers while printing successive layers. Otherwise, the determined by the CCK-8 assay. This also correlated with an
printing of the structure with a large aspect ratio could not increase in cell area and fluorescence intensity as observed in
be realized because of the accumulation of errors. In this our fluorescence staining. These results further validate that
case, the MFCPIB method satisfied these requirements by the MFCPIB method is safe for cells. Finally, hydrogels were
using the uniform temperature field formed by the dual- fixed and stained for cell nuclei (DAPI, blue) and F-actin
machine temperature control and thus realized the printing (phalloidin, red) on day 10. As shown in Figure 7I, most cells
of structures with large aspect ratios. The maximum height encapsulated in the gel showed excellent spreading, which
of a vascular structure obtained using the MFCPIB method is similar to mature SMCs in vivo. These results indicated

Volume 10 Issue 3 (2024) 375 doi: 10.36922/ijb.2120