Introduction
As a single-layered carbon material, graphene exhibits high mechanical
strength, large surface area, superior chemical stability, and
extraordinary electrical and thermal conductivities, thus finding
application in energy storage, sensors, etc. (Y. Zhu et al., 2010).
Recently, graphene-based materials have shown promise as bio-scaffold
owing to their biocompatibility, conductivity, anti-inflammatory
properties, and differentiation effects during cell growth in multiple
cell types (Sahni et al., 2013). The ultra-low density of graphene leads
to easy biodegradation, and its high porosity enhances oxygen diffusion.
All these factors collectively help graphene mimic the physiological
environment better than many hydrogels and polymers (Loeblein et al.,
2016). However, most of the potential applications require a large
specific surface area for cells and chemical compounds to attach, along
with a macrostructure to operate, connect, and examine. In order to
circumvent the limitations of 2D materials and transfer the outstanding
properties into three dimensions, there is an emerging need to assemble
graphene into a 3D structure with specific dimensions and a
well-controlled micro-structure (C. Zhu et al., 2015). Since graphene is
hydrophobic, it can be modified with oxygen-containing groups, such as
hydroxyl and carboxyl groups, by a reversible oxidation process
(Hummers’ method), thereby forming graphene oxide (GO) (Hummers Jr &
Offeman, 1958), which has enhanced hydrophilicity, dispersity, and
forming property. GO retains most properties of graphene, except
conductivity, which can also be easily recovered by reduction reaction
with hydroiodic acid or ascorbic acid. Transformation of GO into a 3D
structure, based on aqueous solution or gel, can be easily controlled in
an inexpensive way, thus making it a preferred method for assembling
graphene-based materials toward multi-functional applications.
Conventional forming methods for graphene-based materials include
hydrothermal method and chemical vapor deposition (CVD)(Xu, Sheng, Li,
& Shi, 2010), among many others. The hydrothermal method, developed by
Xu et al, can fabricate self-assembled GO into 3D structure, with good
conductivity and mechanical strength; however, the shape is defined by
the container and pores are formed randomly (Xu et al., 2010). The
graphene foam (GF), developed by Chen et al through CVD, is widely used
as a bio-scaffold for hepatocytes, osteoblasts, chondrocytes, etc., and
shows good biocompatibility and biodegradability (Loeblein et al., 2016;
Xie et al., 2018; Yocham et al., 2018). Besides having similar
disadvantages as the product from hydrothermal method, this material
turns fragile under low pressure, and the method is not scalable.
Compared to the above-mentioned methods, 3D printing possesses the
ability to fabricate complicated and well-controlled 3D shapes through
additive manufacture process with the help of computer-assisted design
(CAD) (Wang, Jiang, Zhou, Gou, & Hui, 2017). Mainly, two kinds of 3D
printing technologies are used in GO printing: freeze-casting 3D
printing and extrusion-based 3D-printing technique. Zhang et al had
developed the freeze casting 3D printing method, which selectively
solidifies the droplets of GO suspension into the cold sink (-25 ℃)
while the water and low-viscous GO suspension are printed in a
drop-on-demand mode (Zhang et al., 2016). While the low concentration of
GO and drop-by-drop printing can achieve high precision, it can also
result in low mechanical strength of the structure, low speed of
printing, and large deformation in lyophilization process.
Extrusion-based 3D printing, on the other hand, directly accumulates the
ink into the 3D structure with higher efficiency and strength.
Nevertheless, this method requires high viscosity, high yield stress,
and a shear-thinning behavior of the ink material (Jiang et al., 2018).
Normally, GO aqueous solution is of low viscosity, and requires further
treatments. Jakus et al and Yao et al had achieved printable GO ink
property via concentration using evaporation and lyophilization,
respectively; however, both the methods were highly time-consuming
(Jakus et al., 2015; Yao et al., 2016). Centrifugation is a fast and
inexpensive way for concentrating, although the highest achievable
concentration depends on the speed limit of the centrifuge (Jiang et
al., 2018; C. Zhu et al., 2015). Based on centrifugation, Zhu et al had
used 10–20 wt% silica as the viscosifier to increase the storage
modulus of GO by an order of magnitude and achieve a highly compressible
3D structure (C. Zhu et al., 2015). However, silica is removed by
hydrofluoric acid, which can, in turn, induce cytotoxicity. Jiang et al
used 15 mmol/L CaCl2 as a crosslinker and achieved
high-resolution printing (Jiang et al., 2018). Almost all mentioned 3D
printing methods used lyophilization as the final treatment. However,
the diameter, distribution, and orientation of pores, which are crucial
parameters for cell attachment and proliferation, remain to be explored
in detail. Multiple organic compounds (PEO, HPC, etc.), including
divalent and trivalent ions (Ca2+,
Mg2+, Cu2+, Pb2+,
Cr3+, Fe3+), have been proven to
induce GO gelation; however, only seldom have been studied for
rheological properties, printability, or toxicity (Bai, Li, Wang, &
Shi, 2011). Therefore, 3D printing of GO is still in need of further
research and optimization, for designing a GO 3D structure with better
microstructure and stable mechanical strength, for advanced application
in tissue engineering. We herein proposed our work of improving the
mechanical strength of GO by adding trivalent ions with optimized
concentration as crosslinker. Fe3+ was selected due to
its low cytotoxicity and high coordination number, and was used as
crosslinker to fabricate the three-dimensional GO structure with both
macro and micro porosity.
In order to characterize and optimize GO hydrogel, rheological
properties of pure GO, following centrifugation, were tested first. As
shown in fig. 1a, oscillatory measurements proved GO to be in gel state
under low shear stress, with a storage modulus of 519 Pa and loss
modulus of 111 Pa. The sol-gel transformation point was 50.5 Pa.
Rotational measure proved the shear thinning behavior of GO hydrogel
(fig. 1b), and the shear stress-shear rate relation coincided with the
Herschel-Bulkley non-Newtonian fluid model (fig. 1c) (Truby & Lewis,
2016). A yield stress of 56.9 Pa was calculated by curve fitting.