Page 342 - IJB-10-2

International Journal of Bioprinting 3D printing with drug for vascular repair

CU-50, Electron Microscopy Sciences, USA), and the grid Institutional Review Board of Pusan National University
was dried at room temperature at 24 h. All images were Yangsan Hospital, Republic of Korea (Approval No.
recorded using a Gatan 4k × 4k Thermo Scientific Ceta PNUYH-05-2017-053). Total mononuclear cells were
CMOS. The dimensions of the images acquired within each isolated with Ficoll (GE Healthcare, Buckinghamshire, UK)
grid were quantified using the ImageJ analysis program gradient density centrifugation of human umbilical cord
and visually represented through a size distribution blood. Freshly isolated cells were cultured in endothelial
graph. While preparing sample for transmission electron growth medium-2 (EGM-2) supplemented with 5% fetal
microscope-energy dispersive spectrometer (TEM- bovine serum (FBS), human vascular endothelial growth
EDS; Talos F200X, Thermo Fisher Scientific, Carlsbad, factor (VEGF), human basic fibroblast growth factor,
CA, USA), 100 μL of the particles sample in methanol human epidermal growth factor, human insulin-like growth
was dropped on copper grid (FCF200-CU-50, Electron factor 1, ascorbic acid, and GA-1000 (Lonza, Walkersville,
Microscopy Sciences, USA) and washed with deionized MD, USA). The cultures were maintained at 37°C in a 5%
water twice. To observe the sample, the grid was dried at CO₂ humidified atmosphere. After 4 days of culture, non-
room temperature for 24 h. The composition of rapamycin, adherent cells were discarded, and the attached cells were
NP, and NPR was analyzed with Fourier transform infrared further cultured. The cells were subjected to long-term
(FTIR) spectroscopy. The FTIR spectra of the samples were culture to allow the formation of spindle-shaped colonies,
generated by Nicolet 560 FTIR spectroscope (Nicolet Co., and the medium was replenished every 14–21 days. Flow
Madison, WI, USA) with 4.0 cm resolution and 16 scans cytometric analysis performed to characterize several
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ranging from 4000 to 750 cm . surface and pivotal functional markers confirmed that the
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2.3. Zeta potential analysis of NP, NPS, and NPC EPCs were positive for endothelial lineage markers (CD31
The zeta potential in NP, NPS, and NPC was measured and VEGFR2) and hematopoietic stem cell markers (CD34,
using Zetasizer Nano ZS (Malvern Instruments). The CXCR4, and c-Kit), and negative for hematopoietic markers
evaluation of zeta potential was performed based on the such as CD11b, CD14, and CD45.
principle of electrophotoretic light scattering. A dip cell 2.6. Cell viability assay
(zen1002, Malvern Instruments, Malvern, UK) with a The viability assay for EPCs was conducted using a cell
pair of parallel palladium electrodes was used to provide counting kit-8 (CCK-8) (Dongin, CCK-3000, Seoul,
electrical trigger on charged particles. The experiments Republic of Korea), according to the manufacturer’s
were performed in triplicate, and the data were analyzed instructions. For the analysis of cell toxicity, 5000 cells/
using Zetasizer Software. well were seeded into each 96-well plate, the medium was

2.4. Determination of drug release replaced with fresh medium containing NP, NPS, and NPC
We prepared calibration standard solutions by diluting at various concentrations, and the cells were incubated for
NPS and NPC in distilled water, yielding the first standard 24, 48, and 72 h. After incubation, the medium was replaced
solution of 10 mg/mL, which was further diluted with with 100 µL of 1:10 diluted CCK-8 solution. The plates
methanol to final concentrations of 10, 50, and 100 ppb. were then incubated for an additional 1 h. Absorbance was
The samples were prepared by diluting and soaking measured at 450 nm using a spectrophotometer (TECAN,
NPS and NPC in distilled water, yielding solutions at a Grodig, Austria). Each experiment was repeated at least
concentration of 1 mg/mL, for 7 days prior to releasing. three times.
The released samples were filtered with a pore filter (pore 2.7. Proliferation assay
size: 0.22 μm). The filtered solution was centrifuged at To evaluate the proliferation of EPCs, Click-iT® Plus EdU
10,000 × g for 10 min, and the supernatant was diluted Alexa Fluor® 488 Imaging Kit (C10637; Thermo Fisher
with methanol to a final concentration of 0.01 mg/mL. Scientific) that uses modified 5-ethynyl-2’-deoxyuridine (a
QTRAP 6500 + low-mass liquid chromatography/mass nucleoside analog of thymidine), which can be efficiently
spectrometry (LC/MS) system (SCIEX, Canada) was used
to analyze the cumulative release of NPS and NPC. incorporated into newly synthesized DNA, was used. For
the analysis of cell proliferation, 10,000 cells/well were
2.5. Isolation of endothelial progenitor cells and cell seeded into each 8-well chamber (154534, Thermo Fisher
culture Scientific, Carlsbad, CA, USA) and pre-conditioned with
Human endothelial progenitor cells (EPCs) were NP, NPS, and NPC in EGM-2 media for 24 h. The samples
isolated from human umbilical cord blood as previously were incubated for 2 h in EdU solution and processed
described. Briefly, human umbilical cord blood was according to the manufacturer’s instructions. The labeling
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collected from healthy volunteers after obtaining informed index was evaluated as EdU-labeled nuclei relative to
consent according to the protocol approved by the 4’,6-diamidino-2-phenylindole (DAPI)-stained nuclei.

Volume 10 Issue 2 (2024) 334 doi: 10.36922/ijb.1857