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

developed by Bonatti et al. and Paxton et al. 43,44 In the case It is noteworthy that the effect of induced shear stress
of the QEB, the recommended range of viscosity of the on the cells is independent of the extruder design. The
bioink would be similar to the typical range of viscosities mechanism by which the QES extrudes material can be
that are enabled by extrusion-based bioprinters ensuring categorized as a piston-driven mechanical extrusion.
48
printability and accuracy of bioprinted constructs. This extrusion mechanism was chosen given the lower
Typically, the viscosity ranges between 30 mPa·s and 6 × 10 associated cost compared to pneumatically controlled
7
mPa·s. As a caveat, the range of 3D-bioprintable materials ones due to the higher costs associated with pneumatic
45
with the QEB is currently limited to hydrogels that are controllers. The induced stresses are well studied in the
extrudable at temperatures between 20°C and 40°C. This literature computationally and analytically, and have
is due to the lack of an active temperature control unit. shown that these induced stresses that cells experience
However, a wide range of hydrogel materials can be utilized depend mainly on the nozzle type, the interchangeable
including, but not limited to, gelatin-based hydrogels, 3 mL syringe needle tips in the case of the QES developed
collagen-based hydrogel, Pluronic, polyethylene glycol herein, and the bioink material and its viscosity. 23,48-52
(PEG), as well as other materials such as ultrastretchable Hence, the developed QES has no risk of having additional
double-network hydrogel materials. 46,47 damaging effects on the extruded cells.

Without the frame for the QEH, it is difficult to 4.2. IAP and SBP outcomes
maintain a perfect alignment for the four nozzles along the IAP is a printing paradigm that enables the printing of basic
x–y directions. This is due to the non-uniformity within structures including grids and scaffolds. Minimal post-
the plastic syringes and the needle tips locked onto them processing is required, and high cell viability outcomes can
manually. The misalignment would be greater and would be achieved. IAP post-processing is considered minimal
cause more inaccuracies with longer needle tips that can compared to SBP post-processing, which requires at least
misalign more with a small initial deviation starting from two additional steps before retrieving the final printed
the needle fixture. To compensate for that and ensure construct, namely release and washing from the support
negligible misalignments, a needle frame was designed bath. However, scaling up the layer numbers invariably
and added to the QEH that forces all four syringes to have presents a challenge when working with hydrogels
an aligned tip position, as can be seen in Figure 1D. With specified at low material concentrations, i.e., ≤5% in the
the needle frame mounted, minimal nozzle alignment is case of GelMA, since low structural fidelity has been
required in the x–y directions and the different materials observed at these concentrations. 53-55 Notably, printed
printed align well side by side with minimal mixing at the bioinks will spread across the substrate and relinquish the
boundaries. The different structures shown in Figures 2 designed structure. Also, with multi-material printing,
and 3 with the different cases of IAP and SBP show that the mixing of different bioinks at the interfaces will occur.
the QEH renders multi-material structures with well- This will result in undefined multi-material structural
defined boundaries and interfaces between the different outcomes. Continuous crosslinking of the bioink during
bioinks. The nozzles Z-calibration process was done semi- printing is a way to minimize the spreading and loss of
automatically by moving the nozzles to a single point on the prescribed structural outcome. However, this would
the bed, where all the nozzles were calibrated to touch result in inconsistencies of mechanical properties within
and have the same zero level at that point. This was done the construct due to the earlier extruded layers being
by the help of the variable screw Z-limit switch designed. subjected to multi-fold crosslinking times compared to the
It is noteworthy that even with the same needle tips, the latter layers extruded. Alternatively, increasing the bioink
Z-level of each needle tip would be different since the tips concentration would help in maintaining a better structural
are manually attached to the Luer locks of the syringes. outcome and allow increased layer numbers when printing.
Thus, a perfect Z-level alignment cannot be attained However, other issues may arise like the printability
all the time due to human errors, causing the Z-home of the bioink and cell viability. At low temperatures,
level to vary every time with the change of syringes and high-concentration bioinks make material extrusion
needles. Thus, a Z-leveling solution needs to be devised. prohibitive owing to the decreased gelation temperature
For that reason, a variable limit switch was designed and increased viscosity. Moreover, the range of cell types
and added to the QEB to help calibrate the Z-home that can be incorporated within higher-concentration
level with every new set of syringes mounted, as can be bioinks would be reduced. This is because not all cells can
seen in Figure 1E. The added extension part above the survive and function natively in microenvironments that
limit switch can be extended or retracted using a screw are mechanically stiff and that have small micro-porous
mechanism to allow the accommodation of different structures. This may cause a drastic decrease in viability
needle sizes. with certain types of cells.

Volume 10 Issue 1 (2024) 305 https://doi.org/10.36922/ijb.0159