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International Journal of Bioprinting 3D bioscaffolds with SR1 for vasculogenesis

± 1.56, and 112.08 ± 1.92 for the CT, NP, and SNP groups, 3.3. LC/MS release test of nanoparticles
respectively. These findings strongly indicate that SR1 The cumulative release tendency of SR1-laden nanoparticles
nanoparticles promote EPC proliferation, with the SNP is shown in Figure 3D. Percentage values of released SR1
group showing the most pronounced effect. were 0.6%, 2.7%, and 3.3% for the nanoparticles released
for 2, 4, and 6 days, respectively.
3.1.3. EdU cell proliferation assay
Cell proliferation in cultured EPCs was analyzed via 3.4. In vivo studies
EdU cell proliferation assay. Green signals indicate EdU- None of the experimental animals died before sacrifice, and
positive cells, while the nuclei were counterstained with no significant loss of body weight was observed among the
DAPI. Figure 1C shows the results of the EdU assay and experimental animals during the investigation. Schematic
the quantitative evaluation of the experimental groups. illustration of the surgery and expected results are shown
Remarkably, the SNP group exhibited a significantly higher in Figure 2B. Figure 4A shows the outline of the in vivo
percentage of EdU-positive cells compared to that of experimental design.
the CT (p < 0.05) and NP groups (p < 0.01). The specific
percentages of EdU-positive cells for all three groups are as 3.4.1. Bone formation analysis
follows: 32.17 ± 2.60, 28.40 ± 3.58, and 40.98 ± 1.76 for CT, Figure 4B shows MCT images of the defected sites in the
NP, and SNP groups, respectively. These results strongly rats 2 and 4 weeks after scaffold implantation. Quantitative
support the conclusion that SR1 nanoparticles exert a evaluations of the MCT results are presented in Figure
substantial stimulatory effect on EPC proliferation, with the 4C and D. MCT images indicated that new bone had
SNP group displaying the most pronounced enhancement. partially regenerated in the defect, and differences between
the groups were observed. The bone volume fraction
3.1.4. Wound healing assay (BV/TV%) and bone mineral density (BMD) of the CT,
A wound healing assay was performed to examine the NP@Sc, and SNP@Sc groups were analyzed. At 2 weeks
role of SR1 nanoparticles in cell migration, a fundamental postoperatively, the BV/TV% values were recorded as
process essential for vasculogenesis—the formation of new 13.01 ± 2.51%, 11.30 ± 1.23%, and 15.75 ± 2.61% area/
blood vessels. The scratched area was partially re-filled by ROI for the CT, NP@Sc, and SNP@Sc groups, respectively.
the migrating cells 8 h after taking out the insert that was Similarly, the BMD values were 102.53 ± 16.83%, 92.60 ±
previously placed to induce the scratching. Figure 1D shows 12.08%, and 125.27 ± 15.68%, showing trends of increasing
the migration area and the results of quantitative analysis bone area in the order of SNP@Sc > CT > NP@Sc. However,
on CT, NP, and SNP groups. The results demonstrated that these differences were not statistically significant (p > 0.05)
the migrated area of the SNP group was approximately at this early time point.
twice the size of the CT and NP groups, measuring at 0.61 Nevertheless, this trend persisted in the three groups,
± 0.11 mm (p < 0.01). These findings highlight the crucial and the differences became more pronounced (p < 0.01)
2
role of SR1 nanoparticles in enhancing the migration at 4 weeks after implantation, particularly for the SNP@Sc
capacity of EPCs, a key factor in facilitating vasculogenesis. group. At this later time point, the BV/TV% values were
3.2. Scaffold fabrication and morphology 17.00 ± 1.87%, 15.54 ± 2.83%, and 23.91 ± 4.57% area/ROI,
A schematic illustration of the scaffold manufacturing respectively, while the BMD values were 134.40 ±13.89%,
process is shown in Figure 2A. 128.51 ± 19.97%, and 189.75 ± 21.15%. In summary,
these findings strongly suggest that SR1 significantly
3.2.1. Scaffold morphology accelerated new bone formation within the rat calvarial
Figure 3A displays SEM images of the SNP@Sc and NP@Sc defects, with the most notable effects observed at 4 weeks
scaffolds. The appearance of the entire scaffold was revealed after implantation. This underscores the role of SR1 in
in the 30× magnification SEM images. A porous structure promoting bone regeneration, particularly in the later
was observed in the 500× magnification SEM images, and stages of healing.
a detailed one was observed in the 15,000× magnification
images. No difference in appearance was detected between 3.4.2. Histological analysis
NP and SNP groups. For additional evaluation, histological analysis was
performed using H&E and MT staining on sections of
3.2.2. Nanoparticle size distribution assay rat calvaria after sacrifice. Figure 5A and B shows the
SEM was used to analyze the size distribution of SR1- H&E and MT staining results of the rat calvarial samples
laden nanoparticles. Figure 3B shows an image of the obtained at 2 and 4 weeks after implantation. Figure 5C
nanoparticles. After measurement, their diameter was clearly illustrates that new bone formation in the SNP@Sc
found to be 189.34 ± 99.23 nm (Figure 3C). group significantly surpassed that of the CT and NP@Sc

Volume 10 Issue 3 (2024) 267 doi: 10.36922/ijb.1931