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International Journal of Bioprinting Low-cost quad-extrusion 3D bioprinting system

In the case of SBP, many of the aforementioned (based on the nozzle diameter) for each of the different
difficulties with scaling up IAP outcomes can be overcome. cases printed at 25°C vs. 30°C. The significant differences
By leveraging the thixotropic properties of the support between the IAP and SBP measurements are caused by
baths, the bioinks are well supported in their extruded the bioink extruded into the support bath being withheld
locations and no mixing between different bioinks will by the effect of the thixotropic properties of the SBM that
occur. More well-defined boundaries and edges can be helps maintain the extruded shape. In comparison, in IAP,
attained, even with bioinks of very low concentrations. the absence of any support medium around the extruded
The one-time crosslinking of the printed outcomes in SBP bioink on a substrate in the air leaves the bioink free to
helps also to produce consistent mechanical properties spread around the substrate, leading to a deterioration in its
across the whole construct. Although the food dye can be designed shape. This is more clearly realized when printing
seen diffusing into the support bath, the printed material at GelMA bioink concentrations of 5% or less. Regarding
remains in its printed location. After crosslinking, the dye the effect of UV crosslinking, the difference between the
is photo-bleached and the crosslinked GelMA is what is measurements before and after UV crosslinking still exists,
finally left without being affected by the diffusion, as can although it is not very significant. This may be due to the
be seen in Figure 3E-ii. Further images and a video of bonds formed, as well as the slight dehydration that may
the bioprinted structures using SBP can be accessed via happen within the GelMA, causing the volume/width to
the GitHub link, under the folder “Support Bath Printing shrink compared to that before UV crosslinking. It can be
Outcomes,” mentioned in the Availability of data section. deduced that the structures printed using SBP techniques,
Some difficulties, however, may also arise with SBP. One at 25°C, and that are UV-crosslinked show the closest
issue would be the difficulty of extracting thin structures compliance to the designed CAD model. Geometric design
from the supporting bath without causing any damage to fidelity is a multi-faceted issue that is affected by multiple
it. Post-processing damage is more likely to happen with parameters at once. In addition to the tested parameters
constructs printed with low-concentration bioinks and above, it is observed that the structural fidelity is highly
thin structures. Moreover, within small hollow structures, affected by material and process parameters, including the
the removal of the SBM would be a challenge without temperature and concentration of the bioink, along with
causing any damage to the construct. Interlayer adhesion the SBM and the printing speed. Thus, further parametric
between the different bioinks may be problematic in the studies that identify the optimal values for the different
case of multi-material printing if the bioinks have very printing techniques would be valuable in creating a guide
different properties and compositions that do not allow for multi-material printing with reliable outcomes. The
them to adhere to each other at the boundaries. This makes bioink properties are among the parameters that affect the
SBP a less favorable candidate for biological assays for thin shape fidelity of the bioprinted construct. Relevant work
multi-material structures, such as grids or scaffolds. The includes a study by Ouyang et al. regarding the effect of
additional post-processing requirements may add more bioink properties on printability and cell viability for
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strains on cell viability due to the extended times when 3D bioplotting of embryonic stem cells.
cells reside outside the physiological incubator conditions. 4.4. Biological characterization
It is noteworthy that the images presented in Figures 2 The HTR-8 cells used to formulate the cell-laden bioinks
and 3 are qualitative and serve as evidence of functionality are invasive cells and are capable of breaking down
of the QEB developed. However, more quantitative the hydrogel they are embedded in and invading in
studies are being conducted in conjunction with material certain directions where a better supply of nutrients and
parameters, which are needed to understand and optimize chemoattracting factors would exist. Figures 5B and D
the bioprinting outcome of multi-material constructs. clearly show the proper expected invasive behavior of the
HTR-8 trophoblasts embedded in the GelMA constructs
4.3. Structural characterization throughout the 3 days of incubation. The cells invaded
In order to characterize the structural fidelity of the printed the periphery of the GelMA boundaries, where they
outcomes using the QEB, measurements of printed grid started to proliferate more extensively and easily with the
structures, composed of 5% GelMA bioink, were carried abundant availability of cell media. This is also proven
out under different conditions as previously mentioned. It by the fact that the core of the printed strands carried a
is apparent that at 25°C, the bioink exhibits a slightly higher minimal number of cells, mainly dead cells, after 80 h of
viscosity that allows it to maintain a structure close to that incubation. The samples imaged over time show a clear
of the designed one compared to the samples printed at development of the number of cells, which is realized
30°C. This can also be visualized by the average percent by the increased green fluorescence in the bioprinted
increase in strut width from the designed width of 0.45 mm constructs over time. Also, the shape extensions of cells

Volume 10 Issue 1 (2024) 306 https://doi.org/10.36922/ijb.0159