Page 567 - IJB-10-6

International Journal of Bioprinting Internally-crosslinked ADA/Alg/Gel bioinks

Gel containing hydrogel was significantly higher than that in vitro models of hepatic tissue. 31,32 In these reports, the
for ADA/Alg/Gel_50/50/0 at all the evaluated time points. authors demonstrated the hydrogel ability to support mid/
At longer times, well-defined 3D structures could not be long-term cultures of human liver cancer cells (HepG2). 31-33
printed, and both compositions exhibited spreading ratios In this work, we investigated the bioprinting of ADA/
above 3 (4.3 ± 0.5 for ADA/Alg/Gel_50/40/10 after 10 min; Alg/Gel hydrogels, exploiting an internal crosslinking
8 and 5.8 ± 1.3 for ADA/Alg/Gel_50/25/25 after 10 and 20 mechanism to fabricate biodegradable, biocompatible,
min, respectively). For ADA/Alg/Gel_50/40/10 hydrogel, and self-standing ADA-based 3D-printed scaffolds for
the average spreading ratio decreased from 3.1 ± 0.3 to prospective cardiac TE. Considering the time-dependent
1.8 ± 0.2 between 20 and 45 min (i.e., 3.1 ± 0.3, 2.5 ± 0.2, nature of the internal crosslinking mechanism, an in-
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and 1.8 ± 0.2 after 20, 30, and 45 min); while the spreading depth investigation of ADA/Alg/Gel printability was
ratio for ADA/Alg/Gel_50/25/25 decreased from 2.5 to 1.5 performed, evaluating viscosity and shape fidelity (i.e.,
between 30 and 60 min (2.5 ± 0.4, 2.1 ± 0.5, and 1.5 ± 0.2 spreading ratio and Pr) over time. Specifically, printability
after 30, 45, and 60 min). was optimized within short time intervals (i.e., below 90
Finally, the Pr was measured to evaluate possible under- min), as long printing times can severely decrease the
or over-gelation phenomena of the inks (Figure 5H). At viability of cells embedded in the bioink. 83,84 ADA/Alg/
conditions allowing the printing of continuous filaments Gel_50/50/0, ADA/Alg/Gel_50/40/10, and ADA/Alg/
and well-defined grid structures (10–30 min for ADA/ Gel_50/25/25 hydrogels demonstrated suitable printability
Alg/Gel_50/50/0; 10–45 min for ADA/Alg/Gel_50/40/10; times of 10, 20, and 30 min, respectively. Given the internal
and 20–60 min for ADA/Alg/Gel_50/25/25), the three gelation mechanism exploited in the study, the printability
compositions exhibited a Pr between 0.9 and 1.1, indicating of the hydrogels is attributed to the gradual pH-triggered
the formation of square-shaped pores (Pr = 1, indicates release of calcium ions, leading to time-dependent
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perfectly square-shaped pores), with no statistically crosslinking, as evidenced by printability studies. The
significant differences among the samples with/without delay in printability observed for the samples with higher
Gel (Figure 5H). Gel content was probably related to the steric hindrance of
uncrosslinked Gel within the semi-IPN network, slowing
Microextrusion printing of Alg-based hydrogel was 39
previously investigated using external ionic crosslinking down the crosslinking process. Indeed, the presence of
additives in internally crosslinked Alg-based hydrogels
with calcium ions and supporting baths. The process significantly affected their rheological behavior, including
requires optimization of the hydrogel ink and supporting gelation. 33,85,86 However, it is not possible to exclude other
bath to print complex 3D structures. Moreover, external
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gelation is inherently associated with the formation of factors that might concur to determine such delay.
non-homogeneous filaments with a highly crosslinked Finally, as a proof-of-concept, Figure 5I and J features
surface, causing potential detrimental effects on nutrient 3D structures based on ADA/Alg/Gel_50/25/25, printed
exchange. 24,25 Additionally, for drug delivery applications, after 60 min from hydrogel components, having a grid
the use of a crosslinking solution can lead to a fast release (1.25 mm height, five layers; Figure 5I) and cylindrical
of the encapsulated molecules during the gelation phase. geometry (2.25 mm height, 10 layers; Figure 5J). Hence,
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To overcome these drawbacks, internal ionic crosslinking ADA/Alg/Gel hydrogels can be used for printing self-
represents a promising approach for the development standing 3D structures with various shapes by internal
of Alg-based ink. Only a few studies have investigated crosslinking with no need for a supporting bath. Unlike
internal gelation for Alg-based inks, highlighting its previous works, multilayer structures were developed
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ability to generate homogeneous scaffolds without the without the need for curing or post-processing of
need for post-crosslinking strategies or supporting baths. the bioink.
Specifically, Remaggi et al. investigated the application
of internally crosslinked Alg inks for drug delivery for 3.3.4. Bioink in vitro cytocompatibility
breast cancer treatment, using printing conditions (i.e., and printability
curing time of three days at 2°C) not suitable for cell In vitro indirect cell cytocompatibility tests were carried
bioprinting. Sardelli et al. explored Alg ink printability out to investigate the potential cytotoxic effect of eluates
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via internal gelation, evaluating material properties from ADA/Alg/Gel_50/50/0, ADA/Alg/Gel_50/40/10,
and fiber printability over 24 h, but without biological and ADA/Alg/Gel_50/25/25 hydrogels by in vitro culture
characterization. To our knowledge, only three studies of AHCFs for 24 h in the presence of eluates, following
by the same group developed a bioink for cell bioprinting ISO 10993. Results indicated increasing cell viability for
based on an internally crosslinked Alg hydrogel added hydrogels with increasing Gel content (Figure 6A). Indeed,
with porcine liver-derived decellularized ECM to design AHCFs cultured with eluates from ADA/Alg/Gel_50/50/0,

Volume 10 Issue 6 (2024) 559 doi: 10.36922/ijb.4014