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Rueda-Gensini, et al.
structure even after incubation at 37˚C. This is probably sharp edges was observed, which adequately maintained
because upon this thermal stimulus the formed covalent its shape during the bioprinting process and before
bonds have limited rotation for their 3D rearrangement. photocrosslinking (Figure 5B, left panel). Similarly, an
Thermal- and photo-induced crosslinking of SISMA- 8-layered cubic construct was successfully bioprinted
GO hydrogels was further compared by evaluating their (Figure 5B, right panel), which further demonstrates the
rheological behavior in the presence of both stimuli suitability of our bioink for maintaining deposited patterns
(Figure 4B and C). SISMA hydrogels were also assessed during the additive manufacturing of multilayered 3D
as a control to ensure that the presence of GO led to no constructs. Flow sweep experiments depicted in Figure 5C
detrimental impact on the crosslinking dynamics. Both show that SISMA and SISMA-GO hydrogels presented
crosslinking mechanisms effectively increased the storage the characteristic shear-thinning behavior of bioinks with
moduli (G’) in both hydrogels while the loss moduli (G’’) superior printability, in which a decrease in viscosity
remained unchanged. This confirms that both mechanisms occurs upon the application of a shearing force . After
[64]
increase the elastic response of the hydrogels and, fitting the data to a power law model (Figure S8), the
consequently, their structural stability. Moreover, there is no obtained n coefficients from Equation 2.9.1 were 0.208 and
crossover between G’ and G” curves during crosslinking, 0.223 for SISMA and SISMA-GO hydrogels, respectively,
which indicates that the hydrogels exhibit a predominantly indicating a pseudoplastic behavior with strong shear-
elastic behavior over time. This is particularly important in thinning (close to n = 2) . This is important since high
[24]
hydrogels intended for EBB, since it is crucial for assuring shear stress values predominate during the extrusion
filament formation during extrusion, and for maintaining process and shear thinning fluids attenuate the perceived
shape fidelity upon deposition . However, while the stress by cells by stress-relaxation mechanisms that
[24]
temperature stimulus increased G’ values of SISMA-GO ultimately help maintaining high cell viability . Again,
[65]
to 300 Pa after 5 min, blue-light irradiation led to a value of there was no observable difference between the shear-
about 400 Pa after 1 min, confirming that photo-stimulation thinning behavior of SISMA-GO and SISMA hydrogels,
yields faster and stronger crosslinking dynamics. meaning that GO shows no significant impact on the
Moreover, since the formed covalent bonds during rheological response of the produced bioinks.
photo-stimulation are expected to produce more stable hAD-MSCs were embedded and bioprinted in
structures than the weak hydrogen bonds formed during SISMA-GO hydrogels to evaluate the biocompatibility of
thermal-stimulation, we evaluated both the soluble and the materials and the suitability of the proposed bioprinting
gel fractions of thermally and photo-crosslinked SISMA- scheme. In addition, the proposed ascorbic acid-based
GO constructs as a function of mass loss over time, scheme for in situ reduction was also performed on day 3
such that their stability can be estimated. After a 24-h to assess whether any change was observable on construct
incubation period, their gel fractions approached 69.32 viability. Cell viability was assessed with nuclei (blue) and
± 3.41% and 90.39 ± 1.92%, respectively (Figure S6). dead cell (red) staining, where nuclei signal alone marked
These results suggest that photo-crosslinked SISMA-GO live cells and dual staining (seen as magenta) marked
constructs exhibit a much slower degradation rate and are, dead cells (Figure S9). Since propidium iodide has been
therefore, more structurally stable over time. Likewise, reported to interact with GO sheets , spots with only red
[66]
swelling tests confirmed that, upon incubation, irradiated staining were disregarded as they correspond to GO-PI
SISMA-GO constructs only swell up to 27.90 ± 4.88% complexes. The bioprinted constructs exhibit high cell
of their initial weight, and this value remains unchanged viability at all measured time points, with a slight decrease
after the first 8 h of incubation (Figure S7). This confirms on day 3 but a prompt recovery by day 7 (Figure 6D).
that the formed covalent bonds limit excessive hydrogel This small drop may be attributed to unreacted by-
swelling and maintain stable hydrogel structures over products from the photocrosslinking reaction, since
time. excess free radicals may induce the formation of reactive
oxygen species, which have shown to induce DNA
3.4. 3D bioprinting of cell-laden constructs damage and compromise cell viability . The ascorbic
[67]
Before incorporating hAD-MSCs into the composite acid supplementation on day 3 may have also contributed
hydrogel, we conducted several tests to ensure it presented to the observed drop. Despite this, a timely recovery
the required bioink behavior. Manual extrusion tests were was observed and high cell viability (96.9 ± 0.9%) was
carried out to select the adequate SISMA concentration achieved 1 week after bioprinting, confirming that neither
that allowed a stable filament formation during the process GO incorporation nor the proposed reduction scheme
(Figure 5A). Based on these results, a grid-like pattern compromises cellular survival. In addition, embedded
was deposited with a 3D bioprinter to evaluate the shape hAD-MSCs in both SISMA and SISMA-GO constructs
fidelity of the concentration that granted the best filament transitioned from rounded to elongated morphology
formation (20 mg/mL). A defined construct outline with after 6 days of incubation (Figure 5E), which suggests

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