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Multiresponsive Graphene-Oxide Embedded ECM Hydrogel for 3D Bioprinting
disorder ratio (I /I ) is <1, which is typical of oxidized This integrates two of the most important aspects for
G
D
graphene . The observed D peak intensity in the rGO recapitulating the environment of native tissues and, in
[73]
spectrum surpasses that of the G peak, which indicates that turn, for bioink formulation. In addition, GO incorporation
incubation with ascorbic acid increased structural disorder. followed by its in situ reduction harnessed the bioactivity
This structural disorder ratio above 1 has been reported for and hydrophilicity of GO to improve its colloidal stability
rGO because previously oxidized structures fail to recover within the bioink, and once it was stably dispersed,
the original carbon backbone of graphene . Moreover, improved its electroconductive properties by two orders
[74]
the process permanently destroys some functional groups, of magnitude. As such, its bioactive, mechanically stable,
thereby affecting the carbon backbone even further . and electroconductive properties are a clear advantage
[74]
To further characterize GO’s reduction, the above previous bioink formulations.
conductivity of GO before and after incubation with The enhanced electrical conductivity of rGO sheets
ascorbic acid was assessed with the four-point probe increases the potential of SISMA-GO for the biofabrication
method. As shown in Figure 6C, ascorbic acid-based of tissues that require electrostimulation while avoiding
reduction significantly increased the electroconductivity current limitations of electroconductive hydrogels, such
of GO sheets by nearly two orders of magnitude (i.e., as cytotoxicity and poor dispersibility . As such, it
[82]
from 0.49±0.04 mS/m to 30.59±1.27 mS/m). We decided could contribute significantly to the development of
to measure electroconductivity on the rGO sheets directly, electrosensitive tissues (e.g., skeletal muscle, cardiac
instead of the composite hydrogel, to isolate the effect of the muscle, and neural tissue), directing differentiation
reduction from that of electrolytes in the culture medium profiles (e.g., chondrogenesis , and osteogenesis ),
[84]
[83]
and avoid measurement variabilities due to the localized and tissue regeneration . Future work will be focused
[85]
electrical percolation networks . This is a promising on optimizing the in situ reduction procedure to further
[75]
result for the intended electrostimulation applications improve the observed electroconductivity, and assessing
since increased electroconductivity within the sheets may its direct contribution to different electro stimulation
help direct electrical currents through the bioink. However, applications. This work paves the way for exploiting the
despite the large increase in conductivity with respect to potential of SISMA-GO within the field of electrosensitive
GO, rGO sheets still exhibit a lower conductivity than tissue engineering and regenerative medicine.
those reported for rGO by other reduction methods [76-78] .
This was an expected result due to the mild temperature Author contributions
and acid concentration used during the process to
maintain viability unaffected. Nonetheless, considering L.R. and J.A.S. performed most of the experiments
electrostimulation studies have been performed in and J.C performed experiments related to nanomaterial
hydrogels without conductive materials due to the characterization. L.R. analyzed the data and wrote the
conductivity of cell culture medium (1,700 mS/m ) and manuscript with contributions from J.A.S., review and
[79]
that of cell surfaces (10 – 10 mS/m [80,81] ), the presence of editing, L.R., J.C.C., and C.M.-C.; supervision, J.C.C.
-5
-2
rGO sheets could enhance previous efficiencies by aiding and C.M.-C.; project administration, J.C.C. and C.M.-C.;
the electrical connectivity and directing currents within funding acquisition, J:C:C ans C.M.-C.
the hydrogel. This is specifically advantageous if currents Funding sources
are applied during early maturation stages, since cells are
yet to form interconnected networks themselves. This work was funded by the Department of Biomedical
Engineering and by the Vice-Chancellor for Research
4. Conclusions and Creation at Universidad de los Andes. Publishing
Tailoring the mechanical and biological properties of expenses was funded by Convocatoria CI-001 Publica
biomaterials for the biofabrication of tissues with specific tus conocimientos o expone tus nuevas creaciones by the
maturation profiles has been an ongoing research focus Vice-Chancellor for Research and Creation at Universidad
for their translation to medical scenarios. The herein de los Andes.
developed photocrosslinkable nanocomposite bioink Conflict of interest
showed great promise for 3D bioprinting applications,
considering that it can be directly printed with little The authors declare no conflicts of interest.
composition modifications and without the need of other
excipient materials. The limited modification to the overall References
ECM composition enabled by methacryloyl modification
maximizes its bioactive and biomimetic advantages, 1. Kim B, Das S, Jang J, et al., 2020, Decellularized Extracellular
while also enhancing its mechanical properties upon the Matrix-based Bioinks for Engineering Tissue and Organ-
formation of stable interconnected crosslinked networks. specific Microenvironments. Chem Rev, 120:10608–61.

136 International Journal of Bioprinting (2021)–Volume 7, Issue 3