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International Journal of Bioprinting 3D-Printed scaffolds for diabetic bone defects

Then, the reaction termination solution was added and divided them into four groups of four animals each:
measured by an enzyme labeler (Thermo Fisher Scientific PCL, PCL@SS31, PCL@E7, and PCL@SS31@E7 groups
Co., Ltd., USA). The absorbance value at 405 nm was (n = 4). After the animals had been raised with a high-
detected, and the relative enzyme activity value was glucose and high-fat diet for 1 month, two subcutaneous
calculated. The experiment was repeated three times. In injections of streptozotocin (STZ) were administered,
addition, an Alizarin Red S staining kit (Solarbio, China) and blood glucose level of each rat was measured 2 weeks
was used to detect calcium nodule formation in BMSC after STZ injection. Rats were identified as diabetic if they
specimens after co-culturing with different scaffolds had blood glucose values ≥11.1 mmol/L. The diabetic rats
in high-glucose medium for 21 days. The BMSCs were were anesthetized using sodium pentobarbital (20 mg/
inoculated at 1 × 10 /well, and the medium was changed kg). The surgical area was dressed and disinfected; then,
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every 3 days. On day 21, the cells were fixed using 4% the femoral condyles were exposed along the medial side
paraformaldehyde for 30 min, stained with 1% Alizarin of the knee joint, and a 4-mm-diameter, 6-mm-deep bone
Red S solution for 30 min, and photographed and imaged defect area was created laterally in the femoral condyles
under a microscope. Quantitative analysis was performed using an electric drill and a 4.0 mm diameter metal
using ImageJ software, and the experiment was repeated needle. Four sterile porous scaffolds were implanted.
three times. After the surgery, the incision was closed layer by layer.
All rats were injected with penicillin (400,000 units/kg/
2.3.5. Regulation of mitochondrial function of BMSCs day) intramuscularly for 3 consecutive days after surgery.
BMSCs were inoculated in six-well plates and co-cultured In the third month after surgery, the rats were euthanized
with different scaffold systems (PCL, PCL@SS31, PCL@ by sodium pentobarbital overdose anesthesia, and the
E7, and PCL@SS31@E7) in a high-glucose environment femur, heart, liver, and kidneys were rapidly removed
for 24 h. The oxygen consumption rate (OCR) of the under aseptic conditions, fixed in 4% paraformaldehyde
cells was determined using an XF-24 flux analyzer solution, and prepared for examination.
(Agilent Technology). BMSCs were first treated with
2.0 μM oligomycin (Beyotime, China), and adenosine 2.4.2. Micro-computed tomography
triphosphate (ATP) production was measured in each All rat femoral condyle specimens were scanned with
group. Then, 1 μM FCCP (Beyotime, China) was added to micro-computed tomography (micro-CT; u-CT80,
the supernatant, and the increase in mitochondrial oxygen SCANCO, Switzerland). A cylindrical specimen, taken
consumption was detected as the maximum mitochondrial from the center of the implanted scaffolding site, with a
oxygen consumption. Finally, 0.5 μM antimycin A and diameter of 4 mm and a height of 6 mm was selected for 3D
0.5 μM rotenone (Beyotime, China) were added to the reconstruction. 3D reconstructed images of the new bone
supernatant to inhibit the respiratory chain and completely tissues within the scaffold were obtained. Bone mineral
prevent mitochondrial oxygen consumption. density, trabecular number, trabecular thickness, and
After culture for 24 h as described above, the cells trabecular separation were also quantitatively analyzed in
were scraped off and centrifuged at 3000 rpm for 5 min; each group of specimens.
afterward, the supernatant was discarded, and pre-cooled
glutaraldehyde fixative at 4°C was added slowly to the 2.4.3. Histological analysis
pellet. The dehydrated samples were sectioned in a LEICA All rat femoral condyle specimens were resin-embedded,
EM UC7 ultrathin microtome, and the slices were stained and heart, liver, and kidney specimens were paraffin-
with lead citrate solution and hydrogen peroxide acetate embedded. Then, 5 μm thick tissue sections were taken
50% ethanol saturated solution for 5–10 min each, and then from the specimens, and histomorphological analyses of
dried. The mitochondrial morphology was observed under bone defects and internal organs were performed using
a transmission electron microscope (Hitachi H-7650). hematoxylin and eosin (H&E) and von Kossa staining
(Servicebio, China). The volume of new bone tissue was
2.4. In vivo experiments analyzed using ImageJ software.

2.4.1. Construction of animal models 2.5. Statistical analysis
All animal experiments in this study were approved by All data are expressed as mean ± standard deviation.
the Ethics Committee of the Animal Experimentation Statistical analyses were performed using SPSS software
Centre of Chongqing Medical University (IACUC- (version 24) and GraphPad Prism 9 (GraphPad Software,
CQMU-2022-0022). Based on the literature related to United States), and the results were analyzed statistically
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sample size calculation for animal experimental models, by one-way analysis of variance (ANOVA). P < 0.05 was
we selected 16 two-month-old SD rats and randomly considered to indicate statistical significance.

Volume 10 Issue 4 (2024) 209 doi: 10.36922/ijb.2379