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Wang, et al.
then samples were kept at 37°C. PBS was replaced at (1) Cytotoxicity analysis
weekly intervals. At predefined time points, samples To collect the extracts of different groups, bare PCL
were weighed to record the residual mass after rinsing stents, stents coated with PDLLA nanofibers, and stents
and vacuum drying. SEM observation of nanofibers after coated with PDLLA/DP nanofibers were immersed in a
degradation was performed to analyze fiber morphology culture medium of RASMCs and HUVECs at 37°C for
and integrity. Three replicates of each group were used at 24 h, respectively. The cell suspensions of RASMCs and
each time point.
HUVECs were added to 96-well plates at densities of 5.0
3
2.7. In vitro hemocompatibility evaluation × 10 /100 μL in each well. The plates were incubated
at 37°C with 5% CO . After 24 h of incubation, the cell
2
In this study, the in vitro hemocompatibility evaluation culture medium was replaced by extracts of different
was performed following the International Standard (ISO groups. After culturing in extracts for 48 h, 10 μL of cell
10993-4) and National Standard of the People’s Republic of counting kit-8 (CCK-8, Dojindo Laboratories, Japan)
China (GB/T 16886.4). To evaluate the hemocompatibility per 100 μL of culture medium was slowly instilled into
of different stents, male Sprague-Dawley rats (8 weeks each well, and then the plates were incubated for 3 h at
old) were applied to obtain fresh whole blood. 37°C with 5% CO . Subsequently, the absorbance value
2
for each well was recorded at 450 nm using a microplate
(1) Platelet adhesion reader (Multiskan, Thermo, USA). Cell viability was
[11]
To collect platelet-rich plasma (PRP), fresh whole blood calculated in detail elsewhere .
was centrifuged at 1500 rpm for 10 min. As a previous
study reported , 3D-printed bare PCL stents, stents (2) Cell viability and proliferation
[24]
coated with PDLLA nanofibers, and stents coated with RASMCs and HUVECs were seeded in different stents to
PDLLA/DP nanofibers were incubated in PRP for assess biocompatibility via cell viability and proliferation
30 min at 37°C and washed 3 times in PBS. The samples experiments, respectively. Before seeding the cells,
were fixed, dehydrated, air-dried in sequence, and then the specimens were sterilized in 75% ethanol for 5 h,
observed by SEM. washed 3 times in PBS, irradiated with UV for 1 h, and
then incubated in culture medium overnight. Stents were
(2) Hemolysis analysis transferred into a new 24-well plate, and then RASMCs

As reported in the previous studies [24,25] , the hemolysis rate and HUVECs were seeded onto the inside surface of
was measured by detecting hemoglobin release. Briefly, stents at densities of 5.0 × 10 and 1.0 × 10 cells per well,
5
4
3D-printed bare PCL stents, stents coated with PDLLA respectively. All samples were incubated at 37°C with 5%
nanofibers, and stents coated with PDLLA/DP nanofibers CO . The cell culture medium was changed every 2 days.
2
were soaked in 10 mL of normal saline at 37°C for 60 min. For cell-seeded stents, cell viability was analyzed by a
Fresh whole blood (4 mL) was diluted by adding 5 mL of cell live/dead staining assay kit (Dojindo, Japan) on days
normal saline, and then 0.2 mL of diluted blood was added 1 and 7, according to the manufacturer’s instructions.
to these centrifuge tubes. Similarly, 10 mL of deionized Samples were observed using a laser confocal microscope
water and normal saline solution were set as positive and (Nikon A1, Japan). In addition, CCK-8 was applied to
negative controls, respectively. After incubation at 37°C evaluate cell proliferation on days 1, 4, and 7.
for 60 min, all samples were centrifuged for 5 min at
3000 rpm to collect the supernatant. The optical density (3) Cell morphological analysis
(OD) of the supernatant was recorded at 545 nm by a To further observe the cell morphology of the RASMCs
spectrophotometer (NanoDrop 2000, Thermo, USA). and HUVECs on different stents, cytoskeletal staining
and SEM analysis were conducted after culture for 7 days.
2.8. In vitro biocompatibility assessment Samples for immunostaining and SEM observation were

In this study, rat aortic SMC (RASMCs) were purchased treated with the same process as reported in our previous
[23]
from Shanghai Zhong Qiao Xin Zhou Biotechnology work .
Co., Ltd. (Shanghai, China), and human umbilical 2.9. In vivo evaluation of stent implantation
vein endothelial cells (HUVECs) were obtained from
American Type Culture Collection (ATCC, USA). Six healthy white minipigs weighing 30 ± 5 kg were used
RASMCs were cultured in Dulbecco’s modified eagle in this study. All experimental procedures were carried
medium (DMEM) supplemented with 10% fetal bovine out with approval by the Animal Ethics Committee
serum and 1% penicillin-streptomycin. HUVECs were of Nongnong Life Science and Technology Company
cultured in endothelial cell growth medium-2 (EGM-2, (Beijing, China). 3D-printed bare PCL stents and stents
Lonza, Switzerland). coated with PDLLA/DP nanofibers (inner diameter:

International Journal of Bioprinting (2022)–Volume 8, Issue 2 83