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Silva, et al.
tubular structures were then perfused through the 3 Results and discussion
core channel with warm sterile 1 × PBS to wash
away the sacrificial material. In silico simulations, varying the extrusion,
This bioprinting procedure was performed pressure was performed to investigate the impact
with the three designed nozzles varying the that the overall design of the coaxial nozzle
extrusion pressure of the printhead connected might have on cell viability. Since this nozzle
to channel b since the cell-laden bioink was is intended for the fabrication of single-layered
dispensed through this channel. The extrusion tubular structures, cell-laden hydrogels will only
pressure of the other two channels was adjusted be extruded through channel b; however, the
to achieve the same extrusion rate as that of simulations were performed for all channels.
channel b. Coaxial tubular structures were Fluid velocity and pressure distribution through
dispensed with three extrusion pressures: 26, 34, the entire channel geometry were collected from
and 40 kPa, through each of the designed nozzles. the simulations. The minimum and maximum
Each combination of bioprinting parameters was fluid velocities at the outlet were 13 and 43 mm/s,
performed in triplicates, resulting in a total of 27 respectively. Moreover, velocity appeared to
extruded tubular structures. remain constant through the entire geometry for
all extrusion pressures studied and, in each layer/
2.6 Cell viability assessment area, into the nozzle geometry. However, that
was not the case for pressure distribution since
For studying the effect of the inlet extrusion pressure it seemed to decrease as the fluid approached the
and the different coaxial nozzle geometries on cell air interface between the nozzle and the collecting
survival, a Live/Dead (Sigma-Aldrich, St. Louis, glass slide. According to the simulations, pressure
MO, USA) assay was performed on the bioprinted distribution values at the tip of the flow channels
tubular structures. Briefly, constructs were stained fell to between 2 and 10 kPa, while pressures
with calcein acetoxymethyl ester (calcein-AM) between 20 and 64 kPa could be experienced
and propidium iodide (PI) immediately after at the upper most regions of the in-silico flow
bioprinting and fluid perfusion through the lumen, channels. These values were then compared
to visualize live and dead cells, respectively. The to those validated experimentally by previous
staining solution was prepared according to the studies for ordinary [25,26] and coaxial nozzles.
[27]
manufacturer’s instructions. Bioprinted constructs Nair et al. reported that cell viability decreases
were subsequently submerged in it and incubated exponentially as a function of increasing shear
for 15 min at room temperature (~22ºC) protected stress, with cell viability above 60 % for pressures
from light. Samples were then washed with below 100 kPa and nozzle diameters between (150
1 × PBS and imaged using an epifluorescence and 400 µm) . Yu and colleagues investigated this
[26]
microscope (ZEISS Axio Observer, Carl Zeiss same relationship on coaxial nozzles and obtained
AG, Oberkochen, Germany). One image from a very similar results . Although these estimations
[27]
random location on each sample was captured and depend widely on the rheological properties of the
later analyzed using the ImageJ software. studied hydrogel and on the specific response of

2.7 Statistical analysis the cells utilized, we might be able to predict the
high viability of cells bioprinted with the present
Data for cell viability was statistically analyzed coaxial nozzle. The predicted values collected for
with the aid of the GraphPad Prism software pressures experienced by cells on the bioprinting
(GraphPad Software, La Jolla, CA, USA). The process fall within a safe range for cells according
statistical distribution of the data was first studied to these previous studies.
with the Shapiro-Wilk normality test and a two- To confirm these notions and to investigate
way ANOVA with Tukey’s multiple comparisons the effect of nozzle geometry, specifically the
tests was subsequently performed. flow channel Gauge, on cell viability, bioprinting

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