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International Journal of Bioprinting Sr-doped printed scaffolds for bone repair

further washing, the slices were drained and directly stained ± standard deviation, and comparisons between groups
in 1% hydrochloric acid in alcohol for 2–30 s. After rinsing, were statistically analyzed using one-way analysis of
the sections were further differentiated with 1% acetic acid variance (ANOVA) and independent samples t-tests.
and dehydrated in two tanks of anhydrous ethanol (75% Graphs were plotted using GraphPad Prism (GraphPad
and 95%). For sealing, the sections were immersed in a Software, America) and Origin software (Beijing Tianyan
third vat of anhydrous ethanol (99%) for 5 min, xylene Rongzhi Software Co., Ltd, China). Statistically significant
for 5 min for transparency, and sealed with neutral gum. differences are indicated by *p < 0.05, **p < 0.01, and
Collagen fibers appeared blue, while myofibrils, fibrin, and ***p < 0.001.
erythrocytes were stained red under a light microscope.
3. Results
2.5.6. Micro-computed tomography
Micro-computed tomography (micro-CT) analysis was 3.1. Scaffold preparation and characterization
performed using a ZKKS-MCT-Sharp scanner (Zhong 3.1.1. Characterization of SrBG
Ke Kai Sheng, China). Cranial specimens from each The microscopic morphology of SrBG was characterized
group at 1-, 2-, and 3-months post-operation were fixed using SEM. The SrBG prepared consisted of many
in the scanner along the long axis. Scanning parameters regular stacks of small particles with well-dispersed glass
were set as follows: voltage at 70 kV, power at 7 W, four- particles arranged in clusters (Figure 3A). DLS analysis
frame superimposition, an angular gain of 0.72°, and an (Figure 3B) revealed that the SrBG particle size distribution
exposure time of 100 ms. Each sample was rotated 360° was 350–700 nm, with the most prevalent size being 493.9
to complete the scan. The scanned images were denoised nm, accounting for about 38% of the total distribution.
and reconstructed to obtain 3D images. Bone repair and
bone growth in the cranial defects of rats were observed; 3.1.2. Characterization and physicochemical
the bone volume fraction of the newborn bone in the properties of P, SBP, and PSBP scaffolds
cranial defects of rats was calculated using a Bruker high- The surface morphology of the composite scaffolds
resolution micro CT (CTAn). Finally, the cranial micro- (P, SBP, and PSBP) is presented in Figure 4A. Compared
CT data of rats from different subgroups were statistically to the P scaffolds, the pore size of the SBP scaffolds did not
analyzed to compare the differences. change significantly, and the surface became roughened
by the addition of SrBG. The surface of the SBP scaffolds
2.5.7. Immunofluorescence staining
The SD rats were euthanized at postoperative months 1, 2, had SrBG particles attached, but the pores on the surface
and 3, and the cranial tissues were removed and fixed in became smaller, and the number of pores decreased. After
4% paraformaldehyde for 48 h. The cranial specimens were the addition of PDA to the SBP scaffolds, there was no
then placed in a 10% EDTA demineralization solution for significant change in the pore size of the scaffolds, but
decalcification. To determine the new bone formation, the number of pores was further reduced, mainly because
hematoxylin and eosin (HE) staining, Masson staining, the PDA coating covered the original pores. At higher
and immunofluorescence staining were performed on magnification (× 2000), the surface of PSBP scaffolds was
the sections of each sample. Sections with 5 μm thickness smoother compared to the SBP scaffolds, and SrBG particle
were cut from paraffin-embedded tissues for histological attachment was observed on the surface of PSBP scaffolds.
assessment. These sections were immersed in 3% (w/v) The porosity of the P, SBP, and PSBP scaffolds was 68.29
H O and blocked with 3% (v/v) Bull serum albumin (BSA) ± 5.62%, 65.00 ± 2.62%, and 60.53 ± 2.87%, respectively
2
2
solution. After enzymatic antigen repair, the sections were (Figure 4C). The porosity of the PSBP scaffold was
again incubated with the following primary antibodies: slightly lower than that of the P and SBP scaffolds, but the
BMP-2 (rat, 1:100 dilution; Abcam, USA); VEGF (rat, difference was not significant, consistent with the results of
1:200 dilution; Abcam, USA); CD163 (rat, 1:100 dilution; SEM. The contact angles of the scaffolds in each group were
Abcam, USA); and iNOS (rat, 1:2000 dilution; Abcam, examined by the static contact angle method (Figure 4B1);
USA). After rinsing twice with PBS, the samples were the contact angles of the P, SBP, and PSBP scaffolds were
incubated with the corresponding horseradish peroxidase- 99.01 ± 2.62°, 72.04 ± 2.27°, and 0°, respectively (Figure 4B2).
labeled secondary antibodies and then visualized by Generally, a smaller contact angle would indicate a more
staining with 4’,6-diamidino-2-phenylindole (DAPI). Cell hydrophilic material. Overall, the addition of PDA and
nuclei were stained with hematoxylin. SrBG significantly improved the hydrophilicity of PCL
materials, and the PSBP scaffolds had better hydrophilicity
2.6. Statistical analysis compared to the P and SBP scaffolds. The SBP and PSBP
Data were processed using SPSS 29.0 software (IBM, scaffolds were immersed in PBS, and the release of Sr² was
+
China). The experimental data were expressed as mean measured on days 1 and 3. The SBP and PSBP scaffolds

Volume 11 Issue 4 (2025) 357 doi: 10.36922/IJB025210211