Page 69 - IJB-9-6

International Journal of Bioprinting 3D Aerosol Jet® printing for microstructuring

Table 2. AJ®P print parameters and postprocess used for the print investigation for 3D microstructures, divided by ink type and
print strategy
Print parameters (23°C, 50% rh)
Printer and offset, z [mm] Optomec AJ300s (USA), 3 mm
Ink AgNPs-based or water-diluted PEDOT:PSS-based (standard and Collagen-based ink
(1:4) ink, 1 mL own-formulated) ink, 850 µL
Substrate VWR Superfrost Glass Slides VWR Cover Glasses
Printing strategy CJD LBL CJD or LBL CJD, LBL or PW
Nozzle, Ø [μm] 300 100 150 150
Focusing Ratio, R = SGF/CGF 40:10 30:18 35:18 40:19
f
Platen temperature, T [°C] 40 100 80 37
Printing speed, s (mm/s) 0 0.4 0.4 0 (CJD)
0.4 (LBL)
0.01 (PW)
Printed patterns CJD-not required / LBL: 6×4 array, Ø = 50 µm Pillars (Ø = 50 µm) and pyramids
Number of layers, n (#) 1 50 50 50 (LBL)
Postprocessing Thermal sintering (200°C, 1h) Thermal annealing (140°C, 1h) Chemical crosslinking (EDC-NHS)

Table 2 discloses the print parameters and print in terms of height (h, μm), width base-tip (w, μm), AR,
strategy used for each ink (ambient conditions 22°C, internal structure (bulk, hollow), process reproducibility
55%rh). Every parameter was selected after a trial-and- (low, medium, high), and printing time (t, s). Geometrical
error screening for 3D printing, with as output of interest: dimensions were detected via the software ImageJ.
(i) the plate temperature, T (°C), which facilitates drying
out and presintering, and (ii) the focusing ratio, R (#), 2.4 In vitro biocompatibility assessment
f
that is the ratio between SGF and CGF, which regulates The exploitation of 3D AJ®P can be foreseen in several
the jet focusing during in-flight and evaporation rate application domains for customized multimaterial and
upon deposition. R also avoids a common phenomenon multifunctional 3D microstructuring, among which
f
in AJ®P: the overspray (OS), that is undesired material life sciences, particularly the fields of tissue engineering
deposited in the form of porous drops or streams along and bioelectronics, is impendent. In this context, in
the printed pattern. For each ink, a homogenous aerosol vitro biocompatibility is a crucial requirement for the
mist was obtained in at least 15 min after starting the use of such 3D AJ®-printed microstructures. Therefore,
U-AJ®P atomization. After all, 10 min were used to reach potential cytotoxicity effects were evaluated for all three
the deposition of a stable ink before starting printing. In inks. Specifically, cell viability assays were performed with
case of using a heated platen, the substrates were allowed respect to specific applications for each ink.
to reach thermal equilibrium by placing them on the 2.4.1. AgNPs-based inks
platform for 10 min. After printing, a postprocess was Commercial AgNPs-based inks have already been
executed based on the selected ink. In detail, a thermal proven by the same authors to have serious cytotoxicity
sintering (200°C, 1 h) and a thermal annealing (140°C, 1 h) effects (dose- and time-dependent) on different cellular
process were pursued on AgNPs- and PEDOT:PSS-based lineages . To confirm the results obtained from previous
[37]
inks (Heraeus, DE), respectively, to ensure full evaporation data, a direct rATP assay (CellTiter-Glo® 3D Cell Viability,
of the co-solvents and particle aggregation. The collagen Promega cat. no. G9681) was here performed on human-
samples normally also undergo crosslinking postprinting induced pluripotent stem cells (h-iPSCs)-derived neural
using a chemical crosslinking approach reported by Gibney stem cells (NSCs) after 48 h of culture on a commercial
et al. . However, the crosslinking of collagen was out of AgNPs-based ink (SI-AJ 20X, Agfa NV, BE). This PE
[36]
the scope of this work. dispersion has a silver content of 19 wt%, a Ø
avg,particle size
Each experiment was repeated at least three times. ~ 100 nm, and a viscosity of 12–18 mPas. Compared to
Visual inspection and geometrical analyses were performed the AgNPs-based ink proposed for the printing of 3D
using an optical microscope (Hirox KH8700) or a scanning microstructure, this ink also contains glycol-type solvents,
electron microscope (SEM) (Tescan Vega 3, Czech but a lower Ag content (~ 60% lower) and a larger Ø avg,particle size
Republic). Particularly, the final shapes were inspected (100 nm vs 35 nm). Since the cytotoxicity effects increase

Volume 9 Issue 6 (2023) 61 https://doi.org/10.36922/ijb.0257