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Gantumur, et al.
cross-linking, a dispersion of cellulose nanofibers 2.2 Ink preparation and rheological
(CNF) was incorporated as a supporting material. characterization
CNF has already made an impact in the field of
bioprinting due to its outstanding shear thinning Phosphate-buffered saline (PBS, pH 7.4) containing
and mechanical properties . The combination Alg-Ph (0.5 w/v%), HRP (100 units/mL), and
[34]
of alginate and CNF has been previously used for D-glucose (44 mg/mL) was prepared. As a
bioink and separately treated with calcium ions supporting material, CNF (autoclaved at 121°C for
for postcross-linking [35-37] . In this study, the cross- 20 min, 0.5~1.5 w/v%) was added. The rheological
linker (i.e., enzyme and glucose) mixed in the properties of the prepared bioinks were analyzed
bioink led to a slow hydrogelation during and the using a rheometer (HAAKE MARS III, Thermo
following printing step while CNF supported well Fisher Scientific, MA, USA) with a parallel plate
the stability of complex construct. In addition to of 20 mm radius at 25°C, immediately after
the printability, the cell behavior inside or on the mixing. Dynamic viscoelasticity measurement
was performed at a constant shear strain of 1% and
surface of gelatin-coated hydrogel was investigated the gap between the parallel plate and the stage
to ensure the cell compatibility of the proposed was set to 1 mm.
method.
2.3 Bioprinting procedure
2 Materials and methods
The prepared inks were printed using a software-
2.1 Materials assisted 3D printer (Reprap Prusa i3, HIC

Sodium alginate (Kimica I-1G, high content of Technology Co. Ltd., Hong Kong, China) modified
guluronic acid and molecular weight, 70 kDa), to have one syringe connected to a syringe pump.
gelatin (type B from bovine skin), and CNF The inks were extruded from a 27-gauge stainless
suspension (Rheocrysta I-2SX, dry CNF needle at 22 mm/s onto the stage moving at
2
content, 2 w/v%) were purchased from Kimica 22 mm/s to build lattice- (20 × 21 mm , thickness
(Tokyo, Japan), Kewpie (Tokyo, Japan), and 1 mm, 10 layers) and a human nose-shaped (12 ×
2
DKS Co. Ltd. (Kyoto, Japan). HRP (200 units/ 15 mm , thickness 7 mm, 70 layers at maximum)
mg), D-glucose, and N-hydroxysulfosuccinimide 3D hydrogel constructs. The thickness of a layer
(NHS) were obtained from FUJIFILM Wako was set at 0.1 mm. The bioprinting process was
Pure Chemical Corporation (Osaka, Japan). carried out at room temperature and the obtained
Water-soluble carbodiimide (WSCD) and constructs were rested at room temperature for 2 h
tyramine hydrochloride were purchased from after printing to let the postcross-linking.
Peptide Institute (Osaka, Japan) and Chem- 2.4 Swelling of hydrogel in medium
Impex International (Wood Dale, IL, USA),
respectively. 3-(4-hydroxyphenyl) propionic acid PBS containing Alg-Ph (0.5 w/v%), HRP
and rhodamine (Rho) were purchased from Tokyo (100 units/mL), D-glucose (44 mg/mL), and CNF
Chemical Industry (Tokyo, Japan). Alginate (Alg- (1.5 w/v%) was poured into PDMS mold and
Ph, 1.4 × 10 mol-Ph/g) and gelatin (Rho-Gel-Ph, rested at room temperature for 2 h to obtain disk-
−4
1.5 × 10 mol-Ph/g) derivatives possessing Ph shaped hydrogel with 15 mm in diameter. The
−4
groups were synthesized by conjugating tyramine resultant hydrogels were then soaked in DMEM.
hydrochloride using NHS and WSCD, as The medium was changed every day. The diameter
previously reported [38,39] . Mouse 10T1/2 fibroblast of the specimen was measured using the software
cells were provided by Riken Cell Bank (Ibaraki, Image J (National Institutes of Health, USA).
Japan) and cultured in Dulbecco’s Modified 2.5 Cell behavior in hydrogel
Eagle’s Medium (DMEM, Nissui, Tokyo, Japan)
supplemented with 10% fetal bovine serum at 10T1/2 cells were suspended in PBS containing
37°C in 5% CO . Alg-Ph (0.5 w/v%), HRP (100 units/mL),
2
International Journal of Bioprinting (2020)–Volume 6, Issue 1 45