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Liang H, et al.

We firstly compared the width of the printed filaments microstructures were observed with scanning electron
with or without applied voltage under different nozzle microscope (SEM, SU8010, Hitachi, Japan).
diameter when alginate feeing rate and stage moving
speed were fixed at 600 μL/h and 15 mm/s, respectively. 2.7 Electrohydrodynamic Printing of 3D Cell-
Three kinds of coaxial nozzles were used with the core/ Laden Constructs
sheath diameter of 160/500 μm (30G/21G), 260/840 To demonstrate the capability of the presented strategy
μm (25G/18G) and 410/1010 μm (22G/17G). The for cell printing, 3D cell-laden constructs with a layer
morphology and width of the printed filaments were number of 30 were electrohydrodynamically printed.
characterized with an inverted fluorescence microscope To evaluate cell viability, Live/Dead assay (Thermo
(ECLIPSE Ti, Nikon, Japan). For each condition, three Fisher Scientific, USA) was performed according to the
samples were separately printed with nine locations manufacture’s specifications. The 3D fluorescent images
totally measured. of the constructs were reconstructed with a confocal

2.4 Effect of Process Parameters on the Width microscopy (Nikon, Japan). Cell number and cell
of Electrohydrodynamically Printed Filaments viability at specific layer of 5, 15 and 25 were quantified.
The quantified data is expressed as mean ± standard
The effect of alginate feeding rate and stage moving deviation. Statistical analysis was performed using
speeding on the width of the electrohydrodynamically analysis of variance in Microsoft Excel software. Values
printed filaments was studied when the applied voltage of p < 0.05 was considered to be statistically significant.
and nozzle-to-substrate distance were fixed at 4.5 kV 3. Results and Discussion
and 200 μm. Alginate feeding rate of gradually increased
from 200 μL/h to 1000 μL/h when the stage moving Figure 2A–F show the morphology of alginate filaments
speed was fixed at 30 mm/s. The moving speed changed electrohydrodynamically printed by different nozzle
from 15 mm/s to 35 mm/s when alginate feeding rate diameter without/with applied voltage. When the voltage
was fixed at 400 μL/h. The morphology of the printed was not applied, the width of the printed filaments
filaments was characterized and the filament width was gradually increased from 166.15 ± 2.67 μm to 196.78
expressed as mean ± standard deviation. ± 4.87 μm as the core nozzle diameter changed from
160 μm to 410 μm. When the voltage of 4.5 kV was
2.5 Effect of CaCl Feeding Rate on the applied, the width of the electrohydrodynamically
2
Electrohydrodynamic Printing of 3D Constructs printed filaments increased from 144.24 ± 4.82 μm to
167.33 ± 7.40 μm as the nozzle diameter increased.
To fabricate 3D hydrogel constructs using the presented In all cases, the width of the electrohydrodynamically
electrohydrodynamic printing method, it is necessary to printed filaments was obviously smaller than that of
simultaneously feed alginate and CaCl solutions using extrusion-based printing filaments as shown in Figure
2
the coaxial nozzle to ensure instant crosslinking when 2G. This indicated that applied voltage could decrease
the layer number is over 3. The effect of CaCl feeding the width of the printed filaments. Previous studies also
2
rate on the maximum layer number of the printed indicated that a thinner Taylor cone could be achieved
constructs was investigated. CaCl feeding rate varied in under a higher voltage, which can decrease line width
2
the range of 0–300 μL/h and the maximum layer number during the printing process [19,20] . Therefore, in the
was recorded when the electrohydrodynamic printing following experiment, applied voltage of 4.5 kV and the
process became unstable.
coaxial nozzle with core diameter of 160 μm and sheath
2.6 Characterization of the Elec tro hy dro dy- diameter of 500 μm were used to achieve relatively
nam ically Printed 3D Hydrogel Constructs smaller filaments.
Figure 3A shows the filament morphology as well
3D hydrogel constructs with different layer number as the measured width of the electrohydrodynamically
of 10, 30, 50 and 70 were electrohydrodynamically printed filaments under fixed stage moving speed of
printed. The macro/microscopic images of the resultant 30 mm/s and different alginate feeding rate. When the
constructs were viewed with a digital camera (Nikon, alginate feeding rate was lower than 400 μL/h, the
Japan) or optical microscope. The 3D profiles of the printed filaments were discontinuous. As the alginate
printed constructs were reconstructed using a confocal feeding rate increased from 400 μL/h to 1000 μL/h, the
laser scanning microscope (OLS4000, Olympus, USA), filament width significantly increased from 92.53 ± 2.75
based on which the construct height was quantified. μm to 137.70 ± 2.99 μm. When alginate feeding rate
The electrohydrodynamically printed constructs with was fixed at 400 μL/h, the printed filament was straight
50 layers were further freeze-dried in a lyophilizer (FD- and continuous and the filament width significantly
1A-50, Biocool, Beijing, China) for three days. The decreased from 122.24 ± 4.42 μm to 92.53 ± 2.75 μm as

International Journal of Bioprinting (2018)–Volume 4, Issue 1 3