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Coaxial Electrohydrodynamic Bioprinting of Pre-Vascularized Tissues
voltage generator. The sheath and core layer of the coaxial and distribution after different days in culture were
nozzle is fed with alginate solution and collagen/CaCl characterized with an inverted fluorescence microscope
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solution, respectively, by high-resolution syringes. and confocal microscope. The endothelialized filaments
after 14 days in culture were captured using the digital
2.3. EHD bioprinting process camera (Nikon, Japan).
The process parameters of the applied voltage and nozzle- 2.7. Fabrication of 3D hydrogel constructs
to-collector distance were fixed at 4.5 kV, 3.5 mm, 300 μm
according to our previous studies . When the high voltage The EHD printing strategy was also employed to
[30]
syringe pumps run, alginate and collagen/CaCl solution fabricate 3D complex lattice hydrogel by precisely
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was simultaneously fed into the coaxial nozzle. Alginate stacking the filaments in a layer-by-layer manner. In this
was immediately cross-linked by the CaCl solution when study, the macro/microscopic images of the 3D hydrogel
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they met in the nozzle head. During the EHD bioprinting constructs were captured with a digital camera and
process, the printing room temperature should be lower optical microscope. A confocal laser scanning microscope
than 16℃ to avoid collagen solution gelling. After printing, (OLS4000, Olympus, USA) was used to rebuild the 3D
the constructs were put into the cell culture incubator set at profiles of the printed constructs, which were used to
37℃ for 10 min for collagen solution gelling. quantify the height of constructs with different layers. The
printed constructs with 5 layers were further freeze-dried
2.4. Optimization of EHD printing process in a lyophilizer (FD-1A-50, Biocool, Beijing, China) for
parameters 3 days. The microstructures were observed with a scanning
The effects of the feeding rate of alginate and collagen electron microscope (SEM, SU8010, Hitachi, Japan).
solution and the moving speed of stage on the width of the 2.8. Fabrication and characterization of cell-
printed core-sheath filaments were investigated when the
applied voltage, the distance between nozzle and collecting laden 3D constructs
substrate were fixed at 4.5 kV and 300 μm, respectively. To fabricate pre-vascularized 3D lattice constructs, the
Green fluorescent particles (Lumisphere, BaseLine, China) inner-layer bioink was prepared with GFP-HUVECs,
were added to the collagen solution to distinguish the core collagen, and CaCl at the final concentration of 2×10 /mL,
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line from the sheath line in the hydrogel filaments. The 0.3% (w/v) and 3% (w/v). The outer-layer bioink was 0.3%
bright-field and fluorescent images of the printed filaments (w/v) pure alginate solution or 0.3% (w/v) alginate solution
were captured with an inverted fluorescence microscope laden with red H9C2 cells at the final concentration of
(ECLIPSE Ti, Nikon, Japan), which were utilized to 2×10 /mL. The bioprinted lattice constructs were cultured
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measure the size of the core line and sheath line. statically for different days. The proliferation of the
printed cells was quantified using CCK-8 assay (Dojindo
2.5. Characterization of the lattice hydrogel with Molecular Technologies) on the culturing day of 1, 4, and
core-sheath filaments 7. The cell viability was quantified by performing Live/

A single layer of complex lattice hydrogel with core-sheath Dead assay (Thermo Fisher Scientific).
filaments was printed. To show the distribution of collagen
and alginate hydrogel in the electrohydrodynamically printed 2.9. Statistical analysis
core-sheath filaments, red and green fluorescent particles were All quantitative results were presented as mean
added into the alginate and collagen solution, respectively. ±standard deviation. Statistical significance was
Hollow filaments were printed by replacing collagen solution determined using one-way analysis of variance
with 3% (w/v) CaCl solution. The fluorescent cross-section (ANOVA) followed by Tukey post-hoc test for multiple
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images of the printed hollow filaments were reconstructed comparisons using SPSS statistical software. The
using confocal microscope (Nikon, Japan). To evaluate the differences were considered statistically significant if
perfusability of the lattice hydrogel with hollow filaments, a the p-value was <0.05 (*).
syringe was connected to the opening of the hollow filament
and a blue dye solution was injected; the process was recorded 3. Results and discussion
by a commercial camera.
3.1. EHD bioprinting of core-sheath hydrogel
2.6. Cell culture within the core-sheath filaments filaments

The inner-layer bioink was prepared with GFP-HUVECs, Figure 1 shows the EHD printing strategy integrated with
collagen, and CaCl at the final concentration of 2×10 /mL, a coaxial nozzle for fabricating the thick vascularized
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0.3% (w/v) and 3% (w/v). The outer-layer bioink was construct, assembled by the cross-linked core-sheath
0.3% (w/v) pure alginate solution. Cellular morphology filaments. As illustrated in our previous study, an insulating
88 International Journal of Bioprinting (2021)–Volume 7, Issue 3