Page 273 - IJB-10-3

International Journal of Bioprinting 3D bioscaffolds with SR1 for vasculogenesis

heart was exposed by vertically incising along their bodies’ at 25°C. Moreover, the nuclei were counterstained with
midline. An incision (<1 cm) was made in the left ventricle, DAPI. Finally, the slides were observed using a motorized
and a feeding tube was inserted into their aorta. Afterward, fluorescence microscope (Eclipse Ti-E, Nikon, Minato
the following procedures were conducted: First, the blood City, Tokyo, Japan). In this study, the regions of interest
31
vessels were flushed by cardiac perfusion using 200 mL of (ROIs) were defined as areas within the tissue sections that
heparin (1 IU/mL)-containing normal saline, and the right exhibited distinct staining patterns indicative of specific
atrium was incised to allow the perfused normal saline markers or characteristics. These areas were identified
to flow out. Next, 20 mL of Microfil solution (prepared based on staining intensity, color, and patterns observed
by mixing 10.5 mL of Microfil compound, 13.2 mL of in the histological sections, particularly those stained with
specific diluent, and 1.3 mL of specific curing agent) was MT and H&E. The positive areas were quantified using
used to perfuse the blood vessels in the same manner with image analysis software, which allowed us to measure
an initial pressure of 120 mmHg and a perfusion rate of 2 the extent of marker expression or tissue characteristics
mL/min. Following this, the animals were stored overnight within the defined ROIs. This approach facilitated the
at 4°C to facilitate the primary setting of visualizing the evaluation of various parameters, including angiogenesis,
vasculature system of whole cranial region by MCT tissue regeneration, and cellular responses within the
scanning Eventually, the animals were fixed in 10% buffered treated samples. 32-34
formaldehyde solution for a week and were soaked in 14%
EDTA for decalcification for 3 weeks before scanning. 2.8. Statistical analysis
Quantum FX (PerkinElmer, Waltham, Massachusetts, All experimental data were processed and plotted using
USA) was used to perform MCT scanning of the Microfil GraphPad Prism version 9.3 (GraphPad Software, Inc.,
compound-perfused and decalcified rat calvarial samples. Boston, MA, US). Data are expressed as mean ± standard
deviation (SD). Analysis of variance (ANOVA) was used to
2.6. Histological analysis test whether the obtained data had a statistical difference,
All animals were sacrificed immediately after MCT and significance levels were set at *p < 0.05, **p < 0.01, and
scanning, and cranial bone samples were extracted from ***p < 0.001.
the sacrificed animals. Harvested samples were fixed in
10% buffered formaldehyde solution for a week and were 3. Results
also decalcified with 14% EDTA for 3 weeks. Next, the 3.1. In vitro studies
samples were cut horizontally, dehydrated, embedded in
paraffin, and trimmed into sections of 4 µm thickness. The 3.1.1. Flow cytometry analysis
sections were stained with hematoxylin–eosin (H&E) and Figure 1A shows the result of flow cytometry analysis
Masson’s trichrome (MT). Quantitative evaluation of the using biomarkers (CD34, c-kit, CXCR4, VEGFR2, and VE-
percentage area of new bone to the total defective area was cadherin) on the differently treated groups. The histogram
conducted according to the results of the H&E- and MT- for CD34 revealed that 26.6%, 24.6%, and 35.3% of CD34
+
stained slides at 2 and 4 weeks after implantation. cells were present in CT, NP, and SNP groups, respectively.
Among the CD34 cells of each group, the percentage values
+
2.7. Immunohistochemistry and of expressing c-kit (CD117), CXCR4 (CD184), VEGFR2,
immunofluorescence and VE-cadherin (CD144) were 13.6%, 21.4%, 23%, and
Immunohistochemical analysis was conducted on the 23.9% for the CT group; 12.2%, 20.6%, 18.5%, and 24.8%
sectioned slides to detect the expression of CD31 and for the NP group; and 17.9%, 27.2%, 32.4%, and 34.6%
α-smooth muscle actin (α-SMA) using the following for the SNP group, respectively. In summary, SR-1-laden
procedures: The sectioned slides were deparaffinized and nanoparticles significantly boosted CD34 cell presence
+
rehydrated using a series of ethanol and xylene solutions. and enhanced EPC marker expressions, suggesting their
The slides were then labeled with elastin and biotin- potential to promote angiogenesis and tissue regeneration.
conjugated secondary antibodies. Labeled slides were
visualized using the Vector Nova RED substrate kit after 3.1.2. CCK-8 assay
incubation with an ABC complex kit. The nuclei were A CCK-8 assay was conducted to evaluate the effect of
counterstained with hematoxylin. The slides were probed the SR1 nanoparticle on EPC proliferation. The results
overnight at 4°C with the following antibodies: anti-CD31 (Figure 1B) clearly demonstrate a substantial difference in
(#ab182981, Abcam, Cambridge, England) and anti-α- the relative optical intensity among the three groups. The
SMA (#ab5694, Abcam, Cambridge, England). Afterward, SNP group revealed a significantly higher proliferation
the slides were washed with PBS and incubated for an hour rate compared to that of the CT (p < 0.001) and NP groups
with proper Alexa Fluor-coupled secondary antibodies (p < 0.05); the proliferation rates were 100.00 ± 5.04, 105.23

Volume 10 Issue 3 (2024) 265 doi: 10.36922/ijb.1931