Page 380 - IJB-9-5

International Journal of Bioprinting Vascularized bone regeneration

and the resulting slurry was continuously stirred for 2.7. Biocompatibility of the scaffold
homogenization. The 3D-printed PMBG/TCP scaffolds BMSCs were seeded onto a 24-well tissue culture plate at
were then vacuum dried at 80°C for 1 week before being a density of 1 × 10 cells per well and co-cultured with the
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calcined in a muffle furnace at 850°C, with a heating rate of double-phase scaffold. Cytotoxicity was evaluated using
0.5°C per minute, for a duration of 6 h. the CCK-8 reagent (Biomake, USA). On day 1, day 3, and
day 5, 600 μL of CCK-8 reagent (CCK-8: α-MEM = 10:90)
2.4. Characterization of the scaffold was incorporated to each well. After 2 h of incubation,
The surface morphology, chemical content, and elemental 0.1 mL of the incubation supernatant was collected and
distribution of the PMBG/TCP scaffolds were examined analyzed using an enzyme immunoassay (EIA) reader to
using scanning electron microscopy (SEM; GAIA3, measure the absorbance at 450 nm. To assess cell viability
Czechoslovakia) and energy dispersive spectroscopy after 48 h of co-culturing with the scaffold, a live/dead cell
(EDS; GAIA3, Czechoslovakia). Mechanical strength was kit (YEASEN, China) was used. A fluorescence lens was
measured using a universal material experiment device used to see the stained cells after they had been stained
(AG-2000A, Japan) at a constant loading rate of 0.1 cm/ with a mixed dye for 40 min (600× magnification).
min. Compressive strength was measured using 1 × 1 ×
1 cm cubes, and the ultimate compressive strength was 2.8. Alkaline phosphatase staining and Alizarin red
3
determined as the stress at which the specimens shattered. S staining
Three test specimens were averaged to determine the BMSCs were seeded onto the scaffold at a density of 1 ×
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findings for each group. To detect the possible presence 10 cells per well in 24-well plates and allowed to adhere
of functional groups in the PMBG gels, Fourier-transform for 24 h. After this initial period, the cells were induced
infrared (FTIR) spectroscopy and H nuclear magnetic to differentiate into osteoblasts by the addition of a
1
resonance (NMR) spectroscopy were conducted. specific medium. The cells were stained with an alkaline
phosphatase (ALP) kit (YEASEN, China) after 7 days
2.5. In vitro degradation and mineralization of the of culture, and the optical density (OD) values were
scaffold determined by enzyme labeling at 405 nm.
The scaffolds were immersed in Tris-HCl buffer solution To observe the mineralized nodules formed by the
(200 μL/mg, pH = 7.4) and subjected to in vitro degradation BMSCs on the scaffolds, Alizarin red S (ARS) staining
experiments on a constant-temperature shaker at 37°C. was performed. The same cell culture process was used
The pH value was measured at specific time intervals, as for the ALP staining, except that the Alizarin Red kit
and the soaking solution was then collected. The calcium (Beyotime, C0138) was used for staining after 21 days of
ion and silicate ion concentrations were measured using cell growth. After staining, the culture plates were treated
inductively coupled plasma emission spectrometry (ICP). with 10% acetic acid and incubated overnight to dissolve
Following a 35-day period, the scaffolds from each group the dye. After 15 min of centrifugation, the supernatant
were retrieved and analyzed for their surface mineralization was combined with a 10% ammonium hydroxide solution,
morphology via SEM and EDS. and the OD value was measured using the same method as
for the ALP assay.
2.6. BMSC isolation and culture
Three male Sprague-Dawley rats, aged 6 weeks, were 2.9. Tube formation investigation of scaffolds
euthanized with an excessive dose of anesthesia (3% To prepare the substrate, Matrigel was applied to the
pentobarbital sodium) and subsequently submerged in surface of a 24-well plate and incubated in fresh Dulbecco’s
75% ethanol for 15 min. Bone marrow was extracted Modified Eagle Medium (DMEM) at 37°C for 1 h. A
from the femurs and tibias of the rats using disinfected substrate was inoculated with human umbilical vein
tools, and bone marrow-derived mesenchymal stem cells endothelial cells (HUVECs) at a density of 1 × 10 cells per
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(BMSCs) were suspended in a culture medium consisting well and co-cultured with the extracted fluid from each
of α-MEM (Gibco, USA) with 10% fetal bovine serum group of scaffolds for 3 and 6 h in a 5% CO incubator
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(FBS; Gibco, USA) and 1% penicillin–streptomycin (PS) at 37°C. The results were observed using an inverted
(Biosharp, China), and cells were incubated at 37°C in a microscope. The total tube length, number of connections,
carbon dioxide (CO ) incubator for the duration of the number of meshes, and mesh area within each group were
2
culture period. The culture medium was replenished every further analyzed using ImageJ software.
2–3 days, and the BMSCs were sub-cultured when they
reached 80% confluence in the culture dish. Only cells 2.10. Scratch migration assay
between passages three and five were used in subsequent Scratch assay was used to further explore the impact of
experiments. different scaffolds on the migration of HUVECs. HUVECs

Volume 9 Issue 5 (2023) 372 https://doi.org/10.18063/ijb.767