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Rueda-Gensini, et al.
of the bioactive molecules described above can promote biofabrication of tissues and its advantages over other 3D
regeneration processes and restore tissue homeostasis [5-7] , bioprinting techniques have been extensively described
underscoring the potential of dECM hydrogels for cellular elsewhere [25,26] .
scaffolding and advanced biomanufacturing applications However, for the particular case of biomanufacturing
above other natural materials. tissues that require electrical stimulation to acquire their
Despite their great composition-wise resemblance final biological function, dECM hydrogels face relevant
to native tissues, solubilization procedures during dECM limitations as building blocks, mainly because of their
hydrogel preparation have shown to detrimentally alter insulating properties . Moreover, electrostimulation
[27]
the native structure of its fibrous proteins, mainly because approaches have been reported to be beneficial in wound
of the use of proteolytic enzymes to aid matrix digestion . healing [28,29] , bone regeneration [30,31] , cell differentiation [32-34] ,
[8]
This results in diminished biomechanical stiffness and and tissue maturation [35,36] . The incorporation of
higher biodegradability rate, which limits their prolonged electroconductive nanostructured materials into hydrogels,
functionality [9,10] . Specifically, temperature-dependent such as graphene derivatives, gold nanoparticles, and
gelation, the principal crosslinking mechanism in dECM- carbon nanotubes, has shown great promise to alleviate
and collagen-based hydrogels, is often not enough for these shortcomings . Graphene, in particular, is a
[37]
maintaining structural stability within three-dimensional flexible nanomaterial made of sp hybridized carbon atoms
2
structures due to the weak forces (e.g., hydrogen bonds) organized in a single two-dimensional layer, which generate
holding together the digested components in the solubilized a π-electron cloud responsible for electroconductivity .
[38]
matrix . As a result, dECM hydrogels usually need to be However, due to its hydrophobic surface, the colloidal
[10]
combined with other natural materials, such as hyaluronic stability of graphene in hydrophilic media is highly limited.
acid, alginate, gelatin, chitosan, and silk fibroin, to facilitate Consequently, its oxidized derivative, graphene oxide
alternative crosslinking mechanisms and improve their (GO), has attracted significant attention due to its superior
mechanical rigidity [11-13] . In this regard, the addition of hydrophilicity. This can be attributed to the presence of
crosslinking agents, such as glutaraldehyde and 1-ethyl- functional groups, such as hydroxyl and carboxyl moieties,
3-(3-dimethylaminopropyl) carbodiimide hydrochloride which facilitate its dispersion in aqueous media. Moreover,
(EDC)/N-hydroxysulfosuccinimide (NHS), allows the they can promote protein adsorption from either culture
formation of dense hydrogel networks [10,14] . However, media or cellular secretions, which improves overall cell-
unreacted reagents and reaction by-products from these hydrogel interactions by increasing available cell anchoring
approaches usually reduce the viability of the obtained sites [39,40] . However, the introduction of functional groups
hydrogel-based cellular constructs [15,16] . The biochemical on GO disrupts the highly ordered carbon structures and
modification of natural materials with methacryloyl results in reduced electroconductivity when compared to
groups has also emerged as a suitable alternative for pristine graphene or reduced GO (rGO) . Leveraging
[41]
photocrosslinking schemes , a method where material the bioactive and electroconductive properties of GO
[17]
excipients are largely avoided for formulating the nanostructures is, therefore, a major challenge for the
hydrogels and that favors crosslinking agents of low development of electroconductive hydrogels for tissue
cytotoxicity [18-20] . In particular, improved mechanical engineering applications.
stability has been reported in kidney- and bone-derived Accordingly, the aim of this study is to develop an
dECM bioinks upon methacryloyl modification, allowing extrudable bioink based on methacryloyl-modified dECM
tunable degrees of crosslinking with moderate ultraviolet and fully exfoliated GO nanosheets to render the potential
or blue-light irradiation dosages [13,21] . Moreover, numerous for the electrostimulation of 3D bioprinted tissue constructs.
biocompatible photoinitiators are commercially available We propose a fabrication scheme for the incorporation of
to mediate this reaction (e.g., LAP, eosin Y, and riboflavin GO that exploits both its bioactive and hydrophilic properties
[RF]) [13,20,22,23] . Therefore, methacryloyl modification of during the initial maturation stage of extruded constructs
dECMs represents one of the most valuable avenues for and simultaneously improves GO’s electroconductivity
the development of biomimetic hydrogels that closely upon in situ reduction. In particular, we harness the protein
recapitulate the biological and structural environment adsorption capacity of GO to induce homogeneous nanosheet
of native tissues and this, in turn, makes them suitable dispersion within the dECM hydrogel, thus facilitating
candidates for 3D bioprinting applications. In particular, the formation of interconnected electrical networks and
these hydrogels can be precisely tuned for extrusion- aiding initial cellular anchoring. The electro- and photo-
based bioprinting (EBB) since their pseudoplastic addressable hydrogel obtained here, and the corresponding
rheological behavior facilitates their extrusion at high bioprinting scheme, hold much promise in the biofabrication
viscosity values, which are needed to achieve adequate of electrosensitive tissue constructs. Moreover, it may enable
shape fidelity and stability upon deposition . EBB is cell differentiation and tissue maturation processes by highly
[24]
one of the most adopted bioprinting technologies for the controllable electrostimulation strategies.
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