Page 443 - IJB-10-1

International Journal of Bioprinting Bioactive scaffold for necrosis bone repair

bought from Beyotime Biotechnology (Shanghai, China). After placing the scaffolds on the device platform, the
Dexamethasone and penicillin solution were purchased probe could move in three dimensions according to the
from Sinopharm Chemical Reagent Co. (Beijing, China). design of the software (Regenovo Co, China), and the focus
BMP-2, Runx2, and β-actin antibodies were purchased position of the light source could be adjusted for automatic
from Santa Cruz Biotechnology, USA. Hematoxylin and quantification of parameters.
eosin (H&E), Masson, and BMP-2 staining kits were
purchased from Pinuofei Bio Co. (Wuhan, China). Cell 2.3.4. Mechanical properties of scaffolds
culture dishes and cell culture plates were acquired from The standard mechanical properties of the scaffold were
NEST Biotechnology Co. Ltd (Wuxi, China). tested using a universal testing machine (Shenzhen SUNS,
China), based on an experimental load of 250 N and a
2.2. Preparation of scaffolds speed of 0.1 mm/s. The compressive strength values of the
To prepare bioactive bioink, 7.2 g of PLGA granules and 1.8 scaffolds at breakage were recorded.
g of β-TCP were added to 30 mL of 1,4-dioxane solution,
followed by vortex shaking for 1 min and magnetic stirring 2.3.5. Biotin release curve of HBPT
until the mixture was homogenous. Then, various biotin The HBPT was immersed in PBS, the supernatant was
mass ratios (0%, 0.5%, 1%, and 2%) were added to the collected weekly, and PBS was added to maintain the
liquid level. The biotin content of the supernatant was
bioink and thoroughly mixed. The bioink containing then studied using a UV-Vis spectrophotometer (Thermo
biotin was transferred to the print cartridge, which was Fisher Scientific, USA) to plot the release curve of biotin
equipped with a piston and needle of the appropriate size from the scaffold. The UV-Vis wavelength range was 190–
(Φ 0.25 mm), and then loaded onto the Bio 3D printer 1100 nm, where the maximum absorption peak for biotin
(Regenovo Co, China). The layer height was 0.15 mm, and was detected at 225 nm.
the print pitch was 1 mm. The scaffolds were then placed
in a -80°C refrigerator for 2 h, freeze-dried for 24 h, and 2.4. In vitro cytotoxicity analysis
stored at -20°C until use. The scaffolds were designated
β-TCP scaffold (PT), low-biotin β-TCP scaffold (LBPT), 2.4.1. Cell culture
medium-biotin β-TCP scaffold (MBPT), and high-biotin Rat bone marrow mesenchymal stem cells (rBMSCs) were
β-TCP scaffold (HBPT) based on the quantity of biotin purchased from the Shanghai Cell Bank, Chinese Academy
they contained (0, 0.5, 1, and 2%) respectively. of Sciences, and cultured in α-MEM complete medium
containing 10% FBS and 1% penicillin mixture at 37°C, 5%
2.3. Characterization of scaffolds CO , and saturated humidity. The medium was changed
2
every 2 days. rBMSCs were passaged under an inverted
2.3.1. Morphology observation and microscope (Olympus, Japan) at 80–90% confluence, and
elemental analysis cells at passage 3 were taken for subsequent experiments.
Scanning electron microscopy (SEM; Thermo Fisher
Scientific, USA) was used to analyze the surface 2.4.2. Cytotoxicity
micromorphology of the scaffolds after gold spraying. The leaching solution was prepared according to the
Then, the main elemental composition of the surface of method reported in the literature. Six scaffolds were
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the scaffolds (without gold spraying) was analyzed using randomly selected for each group, washed in PBS,
an energy dispersive spectrometer (EDS; Thermo Fisher sterilized with ethylene oxide, and placed in an extraction
Scientific, USA). vessel. The scaffolds were weighed and subsequently placed
into a centrifuge tube, and complete medium was added
2.3.2. Elemental composition and chemical bonding at a proportion of 0.2 g/mL. Subsequently, the tubes were
of the scaffolds incubated in a cell culture incubator at 37°C for 72 h. The
The structure and composition of the material of the leaching solution was then filtered and stored at 4°C.
scaffold were analyzed using Fourier infrared spectroscopy
(FTIR; Thermo Fisher Scientific, USA) with the following rBMSCs were cultured in 96-well plates. Each well was
parameter settings: spectral resolution = 4 cm , number of seeded with 3000 cells and 100 µL of leaching solution of
-1
-1
scans = 32, wave number range = 300–4000 cm . a different concentration; six replicates were used for each
concentration of leaching solution, which was changed
2.3.3. Porosity, pore size, filament diameter every 24 h. The 96-well plates were removed on days 1
of scaffolds and 3 for testing. Each well was incubated with 10 µL
Measurements were carried out according to previous of CCK-8 solution for 2 h. The absorbance at 450 nm
methods. In brief, the above parameters were automatically (A450) was measured with the use of an enzyme marker
4
quantified by optical coherence chromatography imaging. (BioTek, USA), and the standard deviation was calculated

Volume 10 Issue 1 (2024) 435 https://doi.org/10.36922/ijb.1152