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Chen, et al.
where W is the remaining weight and W is the microparticles with a cell density of 6 × 10 cells/ml
6
r
initial weight. i were prepared as previously described Pan et al.
[21]
The cell-laden microparticles were mixed with the
2.8 In vitro cytotoxicity test HA-Alg ink at a mass ratio of 1:3. The cell-laden
HAc-Alg and HAc-Alg/CaP hydrogel pieces cast inks were printed in the gelatin slurry support bath
in plastic molds were used for the cell viability at a feed rate of 5 mm/s and air pressure of 0.5 bar
tests. All of the gel pieces were prepared in 12-well using the 3D printer with a 0.51 mm nozzle. The
plates. The fibroblast cell line L929 (a derivative printed scaffolds were then exposed to UV light for
of Mus musculus strain L) was used to assess the 2.5 min and incubated at 37°C to melt and remove
cellular responses to the hydrogels. Before seeding, the support bath. All of the cell-laden scaffolds
the samples were washed with Dulbecco’s PBS and were the washed 3 times with PBS to remove
then sterilized under UV irradiation for 1 h. The any remaining gelatin and were then incubated
cells were seeded at a density of 5 × 10 cells/mL. with live/dead staining solution containing 2 μM
4
They were cultured in minimum essential medium calcein-AM and 4 μM ethidium homodimer-I
(MEM)-alpha medium with 10% fetal bovine stains for 30 min at 37 °C. The stained cells
serum (FBS) and 1% penicillin-streptomycin in an were observed using CLSM (LSM 800, ZEISS,
incubator with 5% CO at 37°C. Cell viability was Germany).
2
tested via the AlamarBlue assay after 3 and 5 days 2.9 In vitro differentiation test
of culturing. Cellular morphology on the surfaces
of both hydrogel specimens was observed using Osteogenic differentiation was evaluated using
FE-SEM (FE-SEM; Quanta 200F, FEI Company, MC3T3-E1 pre-osteoblast cells cultured in MEM-
USA) after 5 days of culturing. All of the samples alpha medium (no ascorbic acid, Gibco, Thermo
were washed 3 times using PBS. They were then Fisher Scientific, USA) with 10% FBS and 1%
fixed in 2.5% glutaraldehyde solution and were penicillin and streptomycin in an incubator with
gradually dehydrated with a series of ethanol 5% CO at 37°C. The cells were seeded at a
2
solutions with the following concentrations: 30%, density of 2 × 10 cells/well on a 12-well plate
5
50%, 70%, 80%, 90%, 95%, and 100%. for the control and 1 × 10 cells/scaffold for the
6
For cell attachment tests of 3D-printed composite HAc-Alg/CaP composite hydrogel scaffolds.
hydrogels, fluorescence microparticles (FluoSpheres They were cultured in either cell maintenance
carboxylate-modified microspheres, 1.0 µm, blue medium or osteogenic medium (cell maintenance
fluorescent [350/440], Invitrogen, Thermo Fisher medium supplemented with 0.05 mg/mL ascorbic
Scientific, USA) were incorporated into the inks acid and 10 mM β-glycerophosphate) for 2 weeks.
to stain the printed scaffolds for imaging. The cells Gene expression was evaluated by quantitative
were seeded at a density of 5 × 10 cells/ml and real-time polymerase chain reaction (qPCR) after
5
cultured for 7 days. The cells were stained with 14 days of culturing. RNA was extracted from
nucleic acid stain (Hoechst 33342, trihydrochloride, the cells by TRIzol (Invitrogen, Thermo Fisher
trihydrate, Invitrogen, Thermo Fisher Scientific, Scientific, USA) and then converted to cDNA
USA) and plasma membrane stain (CellMask, deep through reverse transcription by Moloney Murine
red plasma membrane stain, Invitrogen, Thermo Leukemia Virus reverse transcriptase (Promega,
Fisher Scientific, USA). Cell adhesion was observed USA). To test in vitro cell differentiation, the
using a confocal laser scanning microscope (CLSM, following four markers, Runx2, Collagen type 1
LSM 800, ZEISS, Germany). (COL1), osteopontin (OPN), and osteocalcin
For the cell viability test of cell-laden HA-Alg (OCN), were chosen, while actin was used as a
porous scaffolds, pure hydrogel inks consisting of reference gene [ref]. The sequences of each gene
4% w/v GMHA, 0.5% w/v Alg, 0.5% v/v NVP, are shown in Table 1. The qPCR was conducted
0.9% w/v NaCl, and 0.5% w/v Irgacure 2959 using a CFX Connect real-time system (Bio-Rad,
were prepared for 3D printing. Cell-laden Alg USA) with SsoAdvanced Universal SYBR Green

International Journal of Bioprinting (2020)–Volume 6, Issue 2 33