Page 381 - IJB-9-5

International Journal of Bioprinting Vascularized bone regeneration

(1 × 10 ) were seeded in a 12-well plate. When the cell (LSCM; LSM800, ZEISS, Germany). To determine the
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density reaches 90%, a linear scratch was created using fluorescence intensity, three random fields of view were
the tip of a 200-μL pipette, and the cells were washed with chosen, and the fluorescence intensity was calculated using
phosphate-buffered saline (PBS) thrice to remove dead ImageJ, a tool for image analysis.
cells and cell debris. Then, the extract of each scaffold
containing 2% FBS was added; and photos of the cells were 2.13. Surgical procedure of animal studies in vivo
taken at 0, 12, and 24 h after scratching. In order to investigate the effects of scaffold treatment on
bone repair and resorption, a rat cranial defect model was
2.11. Real-time quantitative polymerase chain used. Thirty male Sprague-Dawley rats, aged 6 weeks and
reaction weighing 250 g, were divided into three groups: control,
The expression levels of osteogenic genes (RUNX2, COL1, PMBG, and PMBG/TCP. In the control group, cranial defects
OPN, and β-actin) and angiogenic genes (ANG, FGF, were created without the use of any scaffold materials.
and CD31) were evaluated using real-time quantitative A 2-cm incision was made along the sagittal suture after
polymerase chain reaction (RT-qPCR), with GAPDH the cranium had been cleaned, disinfected, and shaved.
serving as the internal reference gene. Sterile scaffolds were After that, the subcutaneous muscle was divided. On both
co-cultured with BMSCs and HUVECs. sides of the parietal bone, bony defects measuring 5 mm in
At a density of 10 cells per well, BMSCs were seeded in diameter were made using a trephine. The scaffolds were
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6-well dishes and induced for osteogenic differentiation by then implanted into the bone defects at a depth of 1 mm
changing the medium to an osteogenic induction medium and a diameter of 5 mm. After that, 4-0 silk sutures were
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containing 10 M dexamethasone, 50 μg/mL ascorbic used to seal the skin incision. The animals were euthanized
acid, and 10 mM β-glycerophosphate, following overnight at 6 and 12 weeks following the operation, and samples
culture in α-MEM. Genomic analysis was performed from the calvarium were obtained and fixed in formalin for
on day 7. On day 2, HUVECs were seeded in DMEM 72 h. The present study was performed in compliance with
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at a density of 4 × 10 cells per well in 6-well plates and the Animal Care and Experiment Committee guidelines
subjected to genetic analysis. of Shanghai Ninth People’s Hospital, Shanghai Jiao Tong
University School of Medicine.
Total RNA was isolated from the cells using TRIzol
reagent (Sangon Biotech, China), and cDNA was 2.14. Bone repair assessment in vivo
synthesized from the mRNA using the Prime Script RT kit To evaluate the efficacy of the scaffold, the animals were
from Takara, Japan, and the SYBR Green RT-PCR kit from sacrificed at 6 and 12 weeks after the operation, and their
Biomake, according to the manufacturer’s protocol. Real- skulls were removed and fixed with 4% paraformaldehyde
time PCR was conducted on a Thermo PCR instrument. for 24 h. Micro-computed tomography (micro-CT) scans
The primer sequences for each gene are provided in were then performed on the skull defect areas, and 3D
Table S1 (Supplementary File).
reconstructions were created for analysis. Bone volume/
total volume (BV/TV) and bone mineral density (BMD)
2.12. Immunofluorescence data were obtained to assess the bone repair effect of each
At a density of 2 × 10 cells/cm , BMSCs were inoculated scaffold group.
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onto the various scaffold groups in α-MEM, and they
were co-cultured for 7 days. Similarly, HUVECs were co- After fixation in 4% paraformaldehyde for 7 days, the
cultured with the scaffold groups in DMEM at a density tissue samples were treated with tissue fixative two more
of 10 cells/cm . Then, each sample was fixed with 4% times before being decalcified in 10% Ethylene Diamine
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paraformaldehyde for 15 min and washed three times Tetraacetie Acid (EDTA) for approximately 30 days to
with PBS. The samples were treated with 0.1% Triton prepare for future histopathological analysis. Hematoxylin
X-100 in PBS for 5 min after being blocked in a PBS and eosin (H&E) as well as Masson’s trichrome staining
solution containing 5% bovine serum albumin (BSA) were carried out following the instructions provided
for an hour. Next, the primary antibodies anti-COL1 by the manufacturer. The samples were air-dried, then
(1:400; Proteintech) and anti-CD31 (1:400; Abcam) were permeabilized with 0.1% Triton X-100 for 10 min prior
incubated with the samples overnight at 4°C. Then, the to immunostaining. To prevent non-specific binding,
secondary antibodies were incubated for 2 h at room the samples were treated with 5% BSA for 1 h at room
temperature. Finally, F-actin and 4,6-Diamino-2-Phenyl temperature. Following this, they were blocked with anti-
Indole (DAPI) were used to stain the cytoskeletons CD31 (1:200) and anti-OCN (1:200) at 4°C. After washing
and nuclei, respectively. The images were observed and with PBS, appropriate Alexa fluorescence-conjugated
collected using a laser scanning confocal microscope secondary antibodies were applied for 2 h at room

Volume 9 Issue 5 (2023) 373 https://doi.org/10.18063/ijb.767