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Gantumur, et al.
the non-cross-linked Rho-Gel-Ph. Subsequently,
10T1/2 cells were seeded at 1 × 10 cells/cm and
4
2
incubated in DMEM at 37°C.
3 Results and discussion
3.1 Characterization and printability of inks
To determine the optimal printing condition of CNF
and alginate-based ink, the variable concentrations
of CNF (from 0 to 1.5 w/v%) and the fixed
concentration of Alg-Ph (0.5 w/v%) were mixed
to prepare four different inks. The concentrations
of HRP (100 units/mL) and glucose (44 mg/mL)
were determined based on the previous findings
in terms of gelation time [32,33] . Comparing to the
direct supply of H O as an aqueous solution ,
[38]
2
2
the gelation time of this method is much longer
and that makes it possible to mix all the ink
components at the same time. Figure 2A shows the
viscosity changes of the prepared inks at various
shear rates. The ink non-containing CNF (CNF 0)
has low zero-shear viscosity at the beginning of
cross-linking resulting in a poor shape fidelity
during the printing (Figure 2D). The viscosity
of Alg-Ph solution gradually increased with
increasing the concentration of incorporated CNF.
As already known , the shear-thinning property
[35]
of the ink containing CNF was confirmed by
showing high viscosity at low shear rates and low
viscosity at high shear rates. Dynamic viscoelastic
measurements in Figure 2B also show that the
storage modulus (G) and loss modulus (G )
ʹ
ʹʹ
substantially increased with a higher proportion
of CNF in the ink. These rheological responses
of the prepared inks were in agreement with the
printing resolution indicated by the widths of
printed lines (Figure 2C). The inks with higher
viscosities improved the printing resolution. Then,
the high printing resolution gave nicely printed
lattice-shaped hydrogel construct (Figure 2D).
Based on the results, the ink containing 1.5 w/v%
CNF was selected from the prepared inks as an
appropriate ink for printing. Incorporating more
high concentration of CNF into the ink caused
nozzle clogging.
Once an optimal concentration of CNF
incorporated in the ink was determined, a more
International Journal of Bioprinting (2020)–Volume 6, Issue 1
complex construct was printed. It was previously
noted that CNF can mimic the bulk collagen
matrix for cartilage tissue . Thus, the hydrogel
[40]
construct resembling human nose, which is one
of the cartilage tissues was successfully printed
using the selected ink. Even during up to 20 min
of printing procedure and slow cross-linking
process, the printed construct maintained its shape
without collapsing (Figure 3A). In addition, if it
is required to let the cross-linking more slowly
for longer printing procedure, the concentrations
of HRP and glucose can be decreased [32,33] .
After cross-linked through glucose-mediated
HRP-catalyzed reaction, the printed human nose
construct became mechanically stable and showed
elastic deformation after squeezing (Figure 3B).
Furthermore, the stability of hydrogel after
cross-linking in cell culture medium was
examined before evaluating the cell behavior
inside it. The diameters of disk-shaped hydrogels
obtained using the selected ink increased <15%
when compared with their initial sizes for the
first 2 days of soaking in medium. After that,
the changes in the size of hydrogels were barely
noticeable and stayed stable during the 8 days of
soaking (Figure 4). Taken together the results
from rheology, printability, and stability, it is
possible to print complex and stable hydrogel
constructs with good shape fidelity using the
proposed ink and the cross-linking method.
A B
Figure 3. Printed human nose construct based
on blueprint (A) before and (C) after postcross-
linking. Scale bars: 1 cm.
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