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Gu, et al.
2.2.4. Perfusion chip establishment and perfusion system at a displacement rate of 1 mm/min. Stresses at the strain
assembly of 50% and 60% were inspected particularly.
After 5–7 days of culture, the cell-laden tube was inserted 2.3.3. Perfusion performance
with a PCL stent to be cast into bulk. First of all, tube with
stent was placed into a PLA frame, which was printed by Perfusion performance was measured by inserting an 18G
an FDM printer (CR-10 MAX, Creality 3D Technology needle into a 3-cm long tube with/without stent under the
Co., Ltd., China). Secondly, 75 μl of GelMA solution flow rate of 6 μl/min, 60 μl/min, 600 μl/min, and 6000 μl/
containing VEGF (PeproTech, US) at the concentration min from a peristaltic pump (BT300-2J, Longer Precision
of 200 ng/ml was pipetted into the frame. Photocuring Pump Co., Ltd.).
for 25 s was applied afterward. Subsequently, the PLA
frame with hydrogel bulk inside was fitted onto a PLA 2.3.4. Swelling
subplate. Next, it was mounted in a PDMS (Sylgard 184, Hydrogel tubes with/without stent reached equilibrium
Dow Corning, US) wall and surrounded by two PET swelling after being immersed in culture medium for 24 h.
films; two cover plates of stainless steel were pressed on After that, bright field images were taken by microscope
both sides and fixed with bolts and nuts subsequently. (WMF-3690, Shanghai Wumo Optical Instrument Co.,
After that, needles of inlet and outlet were inserted into Ltd., China) to measure the inner diameters of tubes.
the tube through PDMS. Finally, a medium container, a
peristaltic pump (BT300-2J, Longer Precision Pump Co., 2.3.5. Diffusion
Ltd., China), a bubble remover (FluidicLab, China), and To examine the barrier function of the endothelialized
the perfusion chip were connected by silicone tube to hydrogel tube, moistened tube was placed on the
form a perfusion circulation. platform of fluorescent microscope (WMF-3690,

2.3. Property characterization Shanghai Wumo Optical Instrument Co., Ltd.) at first,
and 6 μl fluorescein isothiocyanate (FITC)-dextran with
2.3.1. Rheology a molecular weight of 40 kDa at the concentration of 500

Rheological properties of GelMA and gelatin were μg/ml was injected into the tube. Fluorescent images
measured by a rheometer (MCR 102, Anton Paar, were captured every 15 min under the same parameters.
Austria) equipped with a 50 mm-diameter plate-plate Images were transformed into grayscale images and
in all measurements. All hydrogel samples were placed analyzed with ImageJ software.
on the plate at the beginning to completely fill the gap 2.3.6. Scanning electron microscopy (SEM) analysis
(1 mm) between the two plates. The measure of storage/
loss modulus and temperature was performed by varying Hydrogel bulks with/without stent were treated by graded
temperature from 37 to 10°C, or from 10 to 37°C at ethanol dehydration. Afterward, the constructs were
the rate of 2°C/min, while the hydrogel samples were coated with platinum in a sputter coater (Ion Sputter
equilibrated at 37°C/10°C, respectively. For the measure E-1045, Hitachi, Japan), and then imaged by an SEM
of viscosity as a function of shear rate and storage/loss system (SU-6600, Hitachi, Japan).
modulus as a function of amplitude sweep, the initial
temperature of hydrogels samples was 10°C, and then, 2.4. Bioactivity characterization
the samples were warmed to 20°C before the two tests. 2.4.1. Cell culture
The measure of viscosity and shear rate was performed
by changing shear rate from 0.1 to 1000. The measure HUVECs were cultured in ECM with 10% fetal bovine
of storage/loss modulus toward periodic amplitude sweep serum (Gibco, US), 1% penicillin (100 units/ml),
was performed by setting the amplitude of shear strain and streptomycin (100 μg/ml) (Qizhenhu Biological
as 1% and 200%, which alternated every 30 s for three Technology Co., Ltd.) at 37°C and 5% CO Cells were
2.
loops. passaged every 4 days and culture medium was changed
every 2 days.
2.3.2. Mechanical properties
2.4.2. Cell morphological analysis
The mechanical properties of hydrogel bulks with/without
stent were characterized by compression tests using a Morphologies of HUVECs were visualized by cell
dynamic mechanical analysis instrument (ElectroForce, cytoskeleton staining, including F-actin and nucleus
TA Instruments, US) at 25°C. Each hydrogel sample was staining utilizing HUVECs-laden hydrogel tube with
cast as the same size as the one in the perfusion chip inner/outer diameters of 500 μm/1200 μm. F-actin
(4.5 × 4.5 × 4 mm ), enveloping the same hydrogel tube. staining was applied using TRITC phalloidin (Yeasen
3
Samples were placed between two plates and compressed Biological Technology Co., Ltd., China), and nucleus
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