Page 303 - IJB-9-2

International Journal of Bioprinting Zn-doped coatings with osteogenic and antibacterial properties

CHA-M, CHA-M (with 3-layer coating), and CHA-M (with kit (Beyotime, China) was used to measure alkaline
9-layer coating) were soaked in Tris-HCl buffer solution phosphatase (ALP) activity, and the optical density (OD)
(pH 7.4; Scientific Phygene, China) with a scaffold mass to value was measured with a microplate reader at 405 nm.
solution volume was 5 g/L at 37°C for 1, 2, 3, and 4 weeks. Cellular samples were stained with BCIP/NBT Alkaline
The solution was replaced with fresh tris-HCl solution every Phosphatase Color Development Kit (Beyotime, China),
7 days. After a predetermined soaking period, each sample after being fixed with 4% paraformaldehyde for 20 min.
was completely dried and weighed (W ). The relative mass After 21 days of incubation, the samples were stained
b
loss at time b was calculated as: Weight loss = (W -W )/W using the Alizarin Red S Staining Kit for Osteogenesis
a
b
a
× 100%. W was the initial mass of the scaffold. A pH meter (Beyotime, China) for 30 min, followed by a rinse with
a
was used to measure the pH value in solution. distilled water. Then, an inverted optical microscope
2.5. Bioactivity in vitro assessment (CKX53, Olympus, Japan) was used to observe the stained
cells. Afterwards, the 10% (w/v) cetylpyridinium chloride
Mouse calvarial pre-osteoblast cell line MC3T3-E1 (diluted with PBS; Aladdin, China) was used to quantify the
(MC3T3-E1; Institute of Life Science Cell Culture Center, Alizarin red staining (ARS). The absorbance was measured
Shanghai, China) was used in the experiment. The cell at 562 nm. In addition, the amount of Zn released from
2+
growth medium was AMEM (Wisent, Canada) containing Zn-doped samples in the collected medium was detected
10% fetal bovine serum (Gibco, USA) and 1% penicillin/ using commercial kits (Zinc, China).
streptomycin (Wisent, Canada). After being resuscitated
from frozen storage, the cells were cultured in incubator 2.6. Antibacterial assay
at 37°C with 5% CO . The medium was replaced every The antibacterial properties of HA, CHA-0, CHA-L, CHA-
2
2 – 3 days.
M, and CHA-H were assessed against Gram-negative
2.5.1. Cytotoxicity test by live/dead staining Escherichia coli (E. coli, ATCC 25922) and Gram-positive
Staphylococcus aureus (S. aureus, ATCC 29213). Before
Before the test, all samples were sterilized by autoclave the test, the bacteria on logarithmic phase were dispersed
boiler (Shenan, Shanghai, China). Then, pre-osteoblasts into PBS, and the concentration of bacterial suspension
(5 × 10 cells/mL) were inoculated directly on the scaffolds. was adjusted using a microplate reader (10 CFU/mL;
4
8
After 4 days of incubation, the scaffolds were stained with OD =0.1).
Live/Dead Double Staining Kit (Beyotime, China), and 600
then, the staining results were observed under a confocal Disk diffusion method was used to qualitatively
laser scanning microscope (LSM710, Zeiss, Germany). evaluate antibacterial properties of samples. 100 μL of the
bacterial suspension (10 CFU/mL) were equably spread
7
2.5.2. Cell proliferation and attachment over a 90 mm agar plate, followed by dispersedly placing
Cells (5 × 10 cells/well) were seeded onto scaffolds in 24-well the discoid samples onto the plate and culturing in an
4
plates. After culturing for 1, 4, and 7 days, the samples were incubator with a humidity of about 90% at 37°C for 24 h.
washed three times with phosphate-buffered saline (PBS; Afterward, the zone of inhibition was photographed.
Wisent, Canada), followed by 10% Cell Counting Kit-8 Plate colony-counting method was applied to
solution (CCK-8, diluted with medium; Beyotime, China), quantificationally evaluate the antibacterial activities. The
which was used for replacing the original medium and samples were exposed to bacterial suspension (10 CFU/mL)
6
incubation for another 2 h. The cells grown in the blank and then incubated on a shaker for 2 h and 8 h at 37°C. The
well plate were defined as the control. Then cell viability was group without scaffold was considered a control. After the
measured on a microplate reader (Multiskan FC, Thermo appropriate proportion of dilution, the bacterial suspension
Fisher Scientific, USA) at 450 nm. To examine the cell was inoculated onto solid medium and then cultured until
morphology on scaffolds, the scaffolds were disposed by colonies were formed. The antibacterial efficiency was
DAPI (4′,6-diamidino-2-phenylindole, Beyotime, China) computed based on following equation:
and Actin-Tracker Green (Beyotime, China) after 4 days
of co-culture. Fluorescent images of cells on scaffolds were Antibacterial efficiency (%) = (A – A )/A × 100%
s
c
c
observed under a confocal laser scanning microscope. Where A and A are the bacterial colony numbers of
s
c
control and sample groups, respectively.
2.5.3. Cell differentiation
4
The cells (10 cells/mL) were cultured with HA, CHA-0, 2.7. Statistical analysis
CHA-L, CHA-M, CHA-H, and CHA-G in 12-well plate. The data were processed using Origin 2018 software
After 14 days of culture, the alkaline phosphatase assay (Originlab, USA). Values were expressed as means ±
Volume 9 Issue 2 (2023) 295 https://doi.org/10.18063/ijb.v9i2.668