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International Journal of Bioprinting Cell viability in printing structured inks

in the outer ink layer, while the other contained cells in a novel 3D printing method based on structured inks,
both the outer and intermediary ink layers. The HUVECs which was employed for constructing tissue structures
concentration was set at 1 × 10 cells/mL. The distribution (Figure 1A). This method is assumed to reduce both types
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of cells within the structured inks was regulated by the of fluid forces since, in this approach, the nozzle size is
geometric dimensions of the molds. Fiber structures larger compared to conventional printing when printing
were constructed through post-extrusion crosslinking, patterns with the same resolution. However, conventional
employing both types of structured ink-based printing printing methods usually have lower inlet flow velocity to
and 18G needles. Conventional printing of cell-loaded ensure printing accuracy. Therefore, the fluid forces in the
bioinks with a 27G needle, followed by crosslinking, was printing of structured inks were tested. If the fluid pressure
also utilized for constructing fiber structures, facilitating and shear stress at different cross-sectional positions
a comparison of cell viability post-printing. Subsequently, of the nozzle are smaller in this method compared to
cell viability was assessed using the live/dead assay with conventional methods, this approach can be preliminarily
calcein AM and propidium iodide (PI), according to considered advantageous for cell viability. Both the average
the manufacturer’s instructions. Fiber structures were and maximum values of pressure and shear stress were
visualized in longitudinal and cross-sectional views, with analyzed, as the average fluid forces reflect overall cell
the latter being created after cutting the structures with a viability, influenced by fluid forces, and the maximum
surgical blade. Live and dead cells were imaged using an values directly determine cell viability. To achieve this goal,
inverted fluorescence microscope (Olympus IX73). simple 2-symmetric inks and 4-symmetric inks (Figure S2
in Supplementary File) were selected as research targets to
2.11. Data analysis and statistics examine the advantages of fluid forces using this printing
The fluid forces were analyzed using ANSYS Workbench method. For 2-symmetric inks, two different biomaterial
CFD-Post package (version 2020R2). For each set of inks were considered. As for 4-symmetric inks, only cases
results concerning the geometric distribution of material involving the same biomaterial inks and varying cells loaded
phases, quantitative data analysis with three measurements with corresponding material phases were considered, in
was performed using the open-source software ImageJ. order to simplify the calculations due to the complexity
Quantitative data from biological experiments were of phase interfaces formed by different biomaterial inks.
obtained from three independent experiments and are Specifically, pressure at different cross-sections, wall shear
presented as mean ± standard deviation (SD). For the stress, and shear stress at the phase interfaces were analyzed
comparison of cell viability, an unpaired two-tailed (Figure S3 in Supplementary File). Notably, the maximum
Student’s t-test was utilized. A p-value of less than 0.05 was shear stress at the phase interfaces was not calculated, as
considered to indicate a statistically significant difference. its maximum falls on the corresponding wall shear stress.
Biological statistical analyses were conducted using
GraphPad Prism software (version 8.0). The fluid forces of 2-symmetric and 4-symmetric ink-
based printing were systematically investigated for fluid
3. Results and discussion average pressure, maximum pressure, average shear stress,
In this section, the advantages of structured inks were and maximum shear stress within the fluid domain of
primarily confirmed through a comparison of fluid nozzles, in comparison to conventional printing processes.
pressure, wall shear force, and shear stress at material phase Figure 3A–H displays the contours of pressure and wall shear
interfaces using simple 2-symmetric and 4-symmetric stress for 2-symmetric ink-based printing, 4-symmetric ink-
inks. Furthermore, these benefits were explored through based printing, and corresponding conventional printing.
the evaluation of vascular-like inks and hepatic lobule Figure S4A (Supplementary File) displays the contours
analogue-like inks in tissue engineering. Additionally, of shear stress at interfaces for 2-symmetric ink-based
methods for enhancing cell viability were investigated printing. The results revealed difference in pressure and
through equivalent analysis of fluid forces experienced shear stress distribution. In 2-symmetric ink-based printing,
by cells, drawing from symmetric and core–shell inks. for the domains created, an increase in height (relative to
Finally, a workflow for designing structured inks with the nozzle outlet) correlated with a gradual rise in pressure.
consideration of cell viability was proposed. For instance, as the cross-sectional height increased from
3.6 mm to 21.6 mm, the average pressure rose from 8.36e+2
3.1. Examination of fluid forces with 2-symmetric Pa to 1.12e+3 Pa, and the maximum pressure increased
and 4-symmetric inks from 8.56e+2 Pa to 1.12e+3 Pa, as depicted in Figure 4A
In the context of bioprinting, the fluid forces exerted and B. Similarly, in 4-symmetric ink-based printing, the
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on cells, including pressure and shear stress, are crucial average pressure for the domain increased from 7.87e+2
factors influencing cell viability. Recently, a team reported Pa to 1.05e+3 Pa, and the maximum pressure increased

Volume 10 Issue 4 (2024) 245 doi: 10.36922/ijb.2362