Page 361 - IJB-10-2

International Journal of Bioprinting 3D bioprinting for vascular regeneration

Experiments were performed on 8-week-old male bovine serum in PBS and stained with Pacific Blue anti-
BALB/c nude mice (Biogenomics, Seoul, South Korea) mouse CD86 (105022, BioLegend, USA), APC anti-mouse
maintained under a 12-h light/dark cycle in accordance CD206 (141408, BioLegend), and FITC anti-mouse F4/80
with the regulations of the Pusan National University. All (130-117-509, Miltenyi Biotec, USA) antibodies for 30
procedures were performed in accordance with the policies min at 4°C. Cells were analyzed with FACS Canto II (BD
of the Institutional Animal Care and Use Committee of the Biosciences, USA). Data were analyzed by FlowJo software
Pusan National University of Korea. In the murine hind (BD Biosciences, USA).
limb ischemia model, ischemia was induced by the ligation
of the proximal femoral artery and boundary vessels of 2.16. Statistical analysis
the mice. No later than 1 h following surgery, PBS (PBS Statistical analyses were performed using the GraphPad
injection), EPC (EPC injection), NP-R/V (artificial blood Prism software (La Jolla, CA, USA). To assess significance,
vessel loaded with NP-R), EPC@NP/V (artificial blood we performed unpaired two-sided Student’s t-tests. P-values
vessel loaded with NP and EPC), and EPC@NP-R/V <0.05 were considered statistically significant. Experimental
(artificial blood vessel loaded with NP-R and EPC) were data were presented as the mean ± standard deviation.
intramuscularly transplanted into the ischemic thigh area
of the experimental group. Blood perfusion was assessed 3. Results
by comparing the ratio of blood flow in the ischemic limb 3.1. Characterization of nanoparticles and
(left) to that in the nonischemic limb (right) using laser rapamycin-nanoparticles
Doppler perfusion imaging (LDPI; Moor Instruments Ltd, Before the fabrication of artificial blood vessels, NPs were
Devon, UK). prepared to increase the stability, solubility, and slow
down release of drugs to prevent stenosis. To safely load
2.13. Measurement of blood flow and tissue necrosis rapamycin into NPs in a manner that was harmless to the
Blood flow of the ischemic and nonischemic limbs was human body, the experiment was conducted as follows
measured using an LDPI analyzer on days 0, 3, 7, 14, 21, (Figure 1A): first, the fabricated NPs were prepared as a
and 28 after the induction of hind limb ischemia. Perfusion control without capturing any drug, followed by loading
of the ischemic and nonischemic limbs was calculated of NPs with rapamycin selected for restenosis prevention
based on colored histogram pixels; red and blue colors
indicated high and low perfusion, respectively. Blood (Figure 1A). The fabricated NPs were examined using
perfusion was expressed as the LDPI index, representing Bio-TEM, revealing similar sizes (Figure 1B and C).
the ratio of ischemic to nonischemic limb blood flow. The Additionally, the stability of the NPs was determined by
extent of necrosis in the ischemic hind limb was recorded measuring the zeta potential, confirming their stability
28 days postsurgery. (Figure 1D), and their slow release was verified (Figure
1E). Furthermore, when setting the ratio of rapamycin to
2.14. Histological and immunofluorescence analysis NPs at 1:2, 1:5, and 1:10, the measurement of drug loading
The ischemic thigh tissues were harvested and fixed with efficiency yielded results ranging from approximately 10%
4% paraformaldehyde (Biosesang, South Korea) 7 and 28 to 35% (Figure 1F).
days postsurgery. Each tissue sample was embedded in
paraffin. Immunofluorescence staining was performed 3.2. Enhancing drug delivery with nanoparticles:
using primary antibodies against CD31 (ab56299, Abcam, evaluating stability and efficacy for inhibiting
UK), alpha-smooth muscle actin (ab5694, Abcam), CD86 smooth muscle cell proliferation and migration
(19589S, Abcam), and CD206 (ab64693, Abcam), as well The CCK8 experiment was conducted to evaluate the
as secondary antibodies against Alexa-488 and Alexa-594 in vitro stability and effect of the fabricated NPs (Figure
(Invitrogen; USA). Nuclei were stained with DAPI using 2A). It was confirmed that the NPs containing rapamycin
ProLong Diamond Antifade Mountant with DAPI were able to inhibit the proliferation of smooth muscle
(Invitrogen), and stained samples were observed using the cells (Figure S1A in Supplementary File). Furthermore,
Lionheart FX automated microscope. compared with conventional drugs, the rapamycin
contained in NPs demonstrated a higher inhibition of
2.15. Flow cytometry analysis cell proliferation (Figure 2B and C; Figure S1B and C in
Raw 264.7 cells were pre-treated with 10 μg/mL of NP-R Supplementary File). In the case of smooth muscle cells,
for 24 h, followed by induction of polarization into M1 when a stent or artificial blood vessel was transplanted,
and M2 macrophages by treating with lipopolysaccharide they migrated to the graft and induced stenosis. To evaluate
(LPS; 100 ng/mL) and interleukin-4 (IL-4; 20 ng/mL) for the prevention of this migration, the migration ability of
24 h. Cells were harvested and centrifuged at 1,500 rpm smooth muscle cells was evaluated. Rapamycin contained
for 5 min at 4°C. The pellet was re-suspended in 2% fetal in NPs was found to induce a greater inhibition effect on

Volume 10 Issue 2 (2024) 353 doi: 10.36922/ijb.1465