Where C, L, and A are the circularity, perimeter, and area of the grids
respectively. For 3D printing of hydrogel, the ideal gelation condition
should demonstrate square shape of grids (Pr = 1), with higher and lower
Pr values representing over-gelation and under-gelation states,
respectively. In fig. 1f & 1g, the measured printability coincided with
rheology results, in which 8 mmol/L samples showed Pr values closest to
1 (proper gelation). Fe3+ ion, at the concentration of
8 mmol/L, exhibited the best results in both rheology and printability,
which was, therefore, selected as the optimal concentration for further
printing.
Then, structures with different filament distances (1 mm, 1.5 mm, 2 mm,
2.5 mm, and 3 mm) were printed in order to test the forming property of
Fe3+-crosslinked GO hydrogel under different printing
conditions. During the printing process, parameters including the nozzle
diameter, extrusion speed, scanning speed, and nozzle height were
adjusted to achieve an optimized printed structure. The grid within the
printed network became a macroporous structure. Ultimately, under the
conditions of all filament distances, an intact structure with three-way
pores (along x, y, and z axes in fig. 2b) was obtained (fig. 2c).
Based on the stable and well-controlled structure with the help of
Fe3+ ion, lyophilization properties could be further
studied. There are three steps in lyophilization: freezing, main drying,
and final drying. Printed structures were frozen under -80 ℃ and -20 ℃
to explore the relationship between freezing temperature and pore size.
Pore structure with a mean diameter of 11.19 ± 5.46 μm (n = 107) was
produced by freeze-drying at a uniform temperature field of -80 ℃ (fig.
3a, 3b). For -20 ℃ field, the diameter of pores was 22.41 ± 11.55 μm (n
= 93) (fig. 3d, 3e). The distribution of pore size, as shown in fig. 3c
& 3f, can be roughly fitted into Gaussian distribution. Additionally,
samples were frozen at the temperature gradient field to test their
feasibility to form oriented porous structures (fig. 3g, 3h, and 3i).
Oriented pores with a mean width of 86.7 ± 19.0 μm were observed
therein. During the freeze drying process, ice crystals were formed in
the freezing stage that sublimed in the main drying stage. As a result,
the space that used to be occupied by ice crystals became pores after
lyophilization, shape, and size of the pores being decided by the
crystallization process, and ultimately controlled by the freezing
parameters. Compared to those obtained by freezing at -80 ℃, we obtained
pores of approximately double the diameter (22.41 μm vs. 11.19 μm) at a
higher freezing temperature (-20 ℃), which is in line with the mechanism
of crystalline growth. The oriented pores in fig. 3g, 3h, and 3i proved
the temperature gradient field to successfully guide the growth
direction of ice crystal.
Cell attachment experiment was then conducted to test the performance of
GO structures as a bio-scaffold using Human hepatoma cell line HepaRG,
which is able to be differentiated and applied to build liver tissue
model. According to the live/dead assay (fig. 4), we found HepaRG cells
to have good viability (of 91.0 ± 7.2%) in the GO scaffold, as evident
from the assistance of enhanced oxygen and nutrient transportation
through the porous structures. More attached cells were found near the
grid area, both on the surface and interior, which proved that the macro
pores increased the specific surface area for cell attachment. According
to DNA content measurement, the immobilized cells were approximately
3.06 × 106 cells/cm3 in the GO
scaffold. However, during z-axis scanning, few cells were detected in
the interior, which could be attributed to either the opaqueness of
graphene oxide or less cell distribution within. Therefore, the current
thickness of 4 mm of the GO scaffold might be too far for the cell to
travel through, even with the help of macro and micro pores; further
improvement in design is expected in the future.
Appreciable cell viability and attachment behavior proved the
biocompatibility of Fe3+-crosslinked GO structures and
showed that Fe3+ at the concentration ≤ 8 mmol/L does
not induce cytotoxicity. Consequently, such GO structures can be applied
as promising bio-scaffold in tissue engineering. The remaining problem
was the immobilized cell density, which was not comparable in magnitude
to that seen in vivo (108 to
109 cells/cm3) due to the thick
structure along the z-axis. Hence, further geometric optimization would
be helpful for higher cell density. Two-dimensional (2D) graphene
materials had demonstrated the capability of pre-concentrating the
differentiation inducers, thus accelerating the differentiation of human
mesenchymal stem cells (MSCs) (Lee et al., 2011). Therefore, we expect
the differentiation of HepaRG into hepatocytes and bile duct cells could
also be enhanced with the effect of concentrating dimethylsulfoxide
(DMSO) in the 3D GO structure (Guillouzo et al., 2007). Moreover, 3D
graphene showed anti-inflammatory ability in microglial cells while 2D
graphene failed to do so (Song et al., 2014). Our GO scaffold may also
be used to culture neural stem cells owing to the same property. All
these novel properties are beneficial to future applications of GO
scaffold in multiple cell types.
In summary, we improved the rheological behavior and printability of
graphene oxide hydrogel by inducing ferric ion as the crosslinker and
using micro extrusion-based 3D printing technology to 3D-print the
hydrogel, at room temperature, with a wide range of filament distances
and diameters. Additionally, controllable porous structure inside the
graphene structures was achieved by varying the freezing parameters in
lyophilization process. The Fe3+-crosslinked GO
structure is of low cytotoxicity, on which HepaRG cells showed good
viability and attachment behavior. Considering the excellent properties
of graphene materials in cell culture and their anti-inflammatory
effects, the current stable, well-controlled, porous, and easily
fabricated GO structure would be a promising bio-scaffold for developingin-vitro liver tissue models.