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Li, et al.
31°C, respectively. A steady extrudate temperature (HK) (ScienCell, CA, USA) cells. Cells were cultured in
was simulated. The results were verified, and the T25 flasks with Dulbecco’s modified Eagle’s medium/F12
film coefficient was fitted by experiments conducted (Life Technologies, USA) supplemented with 10% fetal
under identical conditions. bovine serum (Fisher Scientific, USA) and 1% penicillin-
3. The outer surface temperature of the syringe was set streptomycin (Life Technologies, USA) under a 5% CO
2
to a constant temperature of 27°C, while the AT was concentration. The culture media were replaced daily. The
set to 20°C, 25°C, 30°C, 35°C, and 40°C, respectively. HKs were detached using Trypsin-EDTA (0.25%, Life
Then, a steady extrudate temperature was simulated. Technologies, USA) and were centrifuged at 1,000 r/min
4. The outer surface temperature of the syringe was set to for collection at a cell density of ca. 1.5 × 10 cells/mL.
7
a constant temperature of 27°C, which was identical The cells were suspended in growth media (ca.
to the initial temperature of the material. The AT was 1.5 × 10 cells/mL), centrifuged, and gently mixed
7
set to 20°C (lower than the nozzle temperature), 27°C into the sodium alginate–gelatin composite hydrogel at
(equal to the nozzle temperature), and 32°C (higher 37°C before being drawn into a sterilized commercial
than the nozzle temperature), respectively. Then, the 30-cc syringe. The typical final concentrations of each
transient temperature change of the extrudate at the component were as follows: 2% sodium alginate, 10%
tip was simulated. gelatin, and ca. 3 × 10 cells/mL.
6
2.5. Printing parameters 2.7. Cell-laden scaffolds printing
This study’s main focus was the linewidth as a printed For higher cell viability, the 23-G nozzle was used in
outcome, which was measured using fluorescence the printing of cell-laden scaffolds . The pressure and
[23]
microscopy (DM IL LED, Leica, Germany) and velocity were set to 130 kPa and 7 mm/s, respectively. To
industrial microscopy (JC-2001G, Jingtuo, China). Two investigate the effect of temperature on cell viability, the
different types of nozzles were used; the 23-G nozzle was extrudate temperature was regulated to 24°C, 27°C, and
cylindrical with a 13-mm cylinder length and an ID of 30°C, respectively.
0.34 mm, and the 32-G nozzle was cylindrical with a 13-
mm cylinder length and an ID of 0.11 mm. The distance 2.8. Cell viability assay
between the tip of the nozzle and the platform was set
as the nozzle diameter’s value to exclude its effect and A Live/Dead cell viability/cytotoxicity assay kit (Thermo,
produce continuous fibers . USA) was used to determine cells’ viability before and
[3]
1. The study conducted two sets of orthogonal after printing. The assay solution was prepared by mixing
experiments to determine the most suitable zone 1-μL/mL Cal-AM and 2-μL/mL propidium iodide in
for well-defined printing lines. In these tests, the PBS. Then, the mixture was added to the samples and
extrudate temperature, pressure, and velocity were was incubated (Thermo, USA) at 37°C for 20 min. After
varied separately. three 3-min washes with PBS, the samples were evaluated
2. To validate the physical model and to determine the using fluorescence microscopy (Leica, Germany). For
coefficients, additional fiber extrusion experiments each sample, three fluorescent images were obtained from
were conducted to investigate the effects of different areas. Live cells were shown in green, while dead
temperature, extrusion pressure, and nozzle velocity cells were shown in red. The number of live and dead cells
on the linewidth. Identical conditions were used was counted using ImageJ (NIH, USA) software under
except for the primary variable under investigation. the same thresholds. Cell viability was calculated as the
3. The observed results were used to determine the number of living cells divided by the total number of cells.
parameters for guiding the printing with a fixed
linewidth step. The values at which the extrudate 2.9. Statistical analysis
temperature, pressure, and velocity were set were The linewidth data were expressed as mean ± standard
calculated from the established physical model to deviation. The inter-group variations were analyzed via
acquire lines with a 50-μm linewidth step, respectively. a one-way ANOVA test in conjunction with Tukey’s test
2.6. Cell culture and bioink preparation using SPSS (IBM, USA) software.

According to the authors’ previous research, sodium 3. Results and discussion
alginate–gelatin composite hydrogels are suitable for the 3.1. Rheological measurements
in vitro fabrication of corneal substitutes [37-39] ; this finding
has received increasing attention over recent years. To test The rheological properties of the sodium alginate–
the effect of the printing process on cell viability, the cell- gelatin composite hydrogel (Figure 2) were measured
laden scaffold printing experiment used human keratocyte in a temperature sweep test, in which the temperature

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