Page 444 - IJB-10-1

International Journal of Bioprinting Bioactive scaffold for necrosis bone repair

to account for differences between groups. The experiment 2.5.3. Western blotting
was repeated 3 times. The scaffolds were co-cultured with cells for 6 days. The
expression of osteogenesis-related proteins in rBMSCs
Live/Dead staining was conducted according to the
manufacturer’s instructions after the cells were cultured was analyzed. Lysis solution at a volume of 200 μL was
added to the culture dish, and the cells on the scaffold and
as described above. Finally, dead cells were stained red, in the dish were lysed for 20 min. Then, the precipitate
and live cells were stained green. Fluorescence images was discarded after centrifugation at 12,000 r/min for 20
were captured using an inverted fluorescence microscope min, and the protein concentration in the supernatant was
(Olympus, Japan).
measured using a BCA protein kit. The target proteins
2.4.3. Co-culture of cells with scaffolds were then separated by gel electrophoresis of the protein
rBMSCs were grown on the scaffold and co-cultured for samples using SDS-PAGE. After that, the proteins were
24 h and 48 h before removing the medium. Then, the cells transferred from the gel onto PVDF membranes, which
were fixed in paraformaldehyde for 5 min and rinsed in were then blocked with 5% skimmed milk powder for 2
PBS for 10 min. 0.5% TritonX-100 was added to the cells, h. The PVDF membranes were incubated with BMP-2,
which were then incubated for 20 min. The cytoskeleton Runx2, and β-actin overnight at 4°C and the next day with
was stained with phalloidin (red), and the nuclei were secondary antibodies for 1 h at room temperature. Finally,
stained with DAPI (blue) according to the instructions. detection and quantification of target protein fragments
Images of stained cells were captured using an inverted by means of a chemiluminescent image analyzer were
fluorescence microscope (Olympus, Japan). performed (Bio-Rad, USA).

2.5. In vitro osteogenic analysis 2.6. Animal housing and modeling
2.5.1. ALP staining 2.6.1. Animal housing
rBMSCs were inoculated in 12-well plates at a density of This study was approved by the Ethics Committee of The
4
3 × 10 cells/well and placed in an incubator. When the Affiliated Hospital of Nanjing University (approval number:
cells were 70–80% confluent, the scaffolds were placed 2021 DW-12-02). Thirty male rabbits (2500 ± 100 g) were
in the 12-well plates and osteogenic induction medium housed in a room at 24–26°C. All rabbits were humanely
(complete medium with 50 mg/L ascorbic acid, 10 mmol/L cared for and housed in conditions that conformed to the
β-glycerol sodium phosphate, 100 nmol/L dexamethasone) Guide for the Care and Use of Laboratory Animals.
and incubated for 6 days and 9 days, with fluid changes 2.6.2. ONFH rabbit modeling
every 2 days. Staining was performed at predetermined ONFH-type bone defect was induced in rabbits following
time points according to the manufacturer’s instructions. previously reported protocols. Lipopolysaccharide was
4,27
After staining for 30 min, the staining results were administered intramuscularly at 20 µg/kg once daily for 3 days,
observed under an inverted microscope (Olympus, Japan), and dexamethasone solution at 5 mg/kg was administered
and six randomly selected regions of interest were used for intramuscularly twice weekly for 6 weeks beginning on day
quantitative analysis of the positively stained areas using 4. The ONFH diagnostic criteria were applied in radiological
ImageJ software. or histopathological diagnosis. Therefore, all rabbits were
subjected to a 3.0 T magnetic resonance imaging (MRI;
2.5.2. Fluorescent staining of BMP-2 Siemens, Germany) after the modeling procedure was
rBMSCs were cultured for 6 days in leaching solution complete. MRI scans demonstrating edema at the femoral
medium; then, the medium was discarded, and the cells head or craniocervical junction and the typical double
were washed with PBS for 5 min. The cells were then fixed sign were used as the criterion for determining successful
in 4% paraformaldehyde for 15 min and washed again in modeling. Then, we stained the femoral head of MRI-
PBS 3 times for 5 min each. The cells were permeabilized confirmed ONFH rabbits and normal rabbits using H&E
with 2 mL of 0.5% Triton X-100 for 10 min, and then staining in order to compare the ONFH rabbit and normal
washed with PBS. Two percent goat serum was added rabbits in terms of bone trabeculae.
for 30 min. Fluorescent primary antibody (BMP-2) was
added. Afterward, the cells were incubated for 1 h at room 2.6.3. Bone repair surgery
temperature and washed with PBS. Then, fluorescent For the dead bone removal and scaffold implantation
secondary antibody was added, followed by incubation procedure, ONFH rabbits were selected and randomly
for 40 min and washing with PBS for 3 times. The staining divided into PT and HBPT groups according to the
was then observed by inverted fluorescence microscopy type of scaffolds. The rabbits that were subjected to this
(Olympus, Japan). procedure had been sedated with isoflurane using a gas

Volume 10 Issue 1 (2024) 436 https://doi.org/10.36922/ijb.1152