Page 122 - IJB-8-3

3D Scaffold for Combined Antibacterial and Antitumor Therapy
2.6. Photothermal effect of scaffold to MG63 and 2.8. Statistical analysis
bone marrow mesenchymal stem cell (BMSC) In this study, multiple replicate tests were performed
cells for each group of samples and the final experimental
The composite scaffolds were sterilized with 75% alcohol results were expressed as mean ± standard deviation. All
for 1 h and then ultraviolet for 2 h. human osteosarcoma experimental data were statistically analyzed by SPSS
cells (MG63) and mouse BMSC were cultured with software (ver. 23.0; IBM Corporation, NY, USA). The
DMEM (10% fetal bovine serum, 1% penicillin, and results were regarded as statistically significant only
streptomycin) in a cell incubator (5% CO , 37℃) for 48 h. when P < 0.05.
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The cell suspensions were then placed into a composite 3. Results and discussion
scaffold and irradiated with or without 808 nm NIR laser
for 10 min. Cell Counting Kit-8 reagent was added to 3.1. Micromorphology of MoS and Ag@PMoS
each hole and incubate at 37°C for 2 h. Cell survival was 2 2
evaluated by measuring the absorbance of the supernatant Micromorphology and elemental compositions of the
at 450 nm (Beckman, USA). Propidium iodide (PI) and MoS and Ag@PMoS NSs were observed by TEM
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calcein-AM were added, respectively, and the cells live/ and SEM equipped with EDS. As shown in Figure 2A,
dead assay was evaluated using a fluorescence microscope the unmodified MoS NSs presented a relative smooth
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(BX60, Olympus, Japan). Apoptosis rate was evaluated surface. Figure 2B shows the TEM images of Ag@
according to the instructions of the VFITC/PI Apoptosis PMoS NSs. It can be seen that the size of Ag particles
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Detection Kit (BIOBOX, China). For Western blotting ranges from 5 nm to 20 nm and is evenly distributed on
detection of Bcl-2 and Bax expression, the MG63 cells the surface of PMoS NSs. This is because MoS is coated
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were cocultured with the composite scaffold, then by PDA and has abundant phenols and nitrogenous
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irradiated with or without 808 nm NIR laser (1 W/cm ) for groups on its surface, which can absorb Ag and help
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[34]
20 min and then incubated for 2 h. Then, the protein was the formation of Ag NPs . The high-resolution TEM
collected and the protein concentration was measured. image of Ag@PMoS nanosheets shows crystal structure
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Finally, the expression of Bcl-2 and Bax was examined with lattice spacing about 0.27 nm for the (100) plane
by Western blotting. of MoS NSs and 0.237 nm for the (111) plane of
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metallic Ag (Figure 2C-E) [35,36] . In the selected area of
2.7. Antibacterial function of scaffolds electron diffraction images (Figure 2F), the lattices
The antibacterial performance of scaffolds was of (200), (111), (220), and (311) were observed, which
evaluated with Gram-negative bacteria E. coli. The further confirmed the formation of Ag NPs. To check
composite scaffolds were sterilized with 75% alcohol for the distribution of silver in PMoS NSs, EDS assay was
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1 h and then ultraviolet for 2 h. E. coli suspension (1 × performed. As shown in Figure 2G, the silver element
10 CFU/mL) was cultured with sterilized scaffolds and is uniformly distributed on the surface of PMoS NSs.
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irradiated with or without 808 nm NIR for 10 min at 4 h Above results indicate that the Ag had been reduced
intervals. After 24 h, the absorbance of the suspension to Ag NPs and distributed uniformly on the surface of
at 600 nm was measured to evaluate the antibacterial PMoS NSs, which could help with the dispersion on
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activity of the scaffold (Beckman, USA). At the same each other. On the one hand, the accumulation of MoS
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time, the suspension was imaged by digital camera. NSs may be inhibited by the sandwiched Ag NPs. On the
Finally, the scaffolds attached with bacteria were fixed other hand, the aggregation of Ag NPs may be hindered
with 2.5% glutaraldehyde for 1 h after washed with by the MoS nanosheets.
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PBS, then dehydrated in a gradient concentration of 3.2. Characterization of Ag@PMoS
ethanol (10, 30, 50, 75, 95, and 100% v/v) for 10 min. 2
The number and morphology of bacteria on the scaffold XRD and XPS were performed to characterize the crystal
were observed by SEM. 2’,7’-dichlorofluorescin structure and chemical component of MoS NSs, PMoS
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diacetate (DCFH-DA) was used to evaluate the ROS in NSs, and Ag@PMoS NSs. As shown in Figure 3A, the
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E. coli. In details, E. coli suspension (1 × 10 CFU/mL) pattern of the Ag@PMoS exhibited all diffraction peaks
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was cultured with sterilized scaffolds and irradiated associated to Ag and MoS . The diffraction peak near 14°
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with or without 808 nm NIR for 10 min at 4 h intervals. reduces in PMoS NSs and Ag@PMoS NSs due to the
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After 24 h, E. coli was collected, 500 μL phosphate- stack of MoS layers is disrupted by the PDA coating .
[35]
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buffered PBS with 10 μM of DCFH-DA was added Compared with PMoS and MoS , it can be observed
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and then incubated at 37°C for 30 min in the dark. The that four new diffraction peaks at 38.13°, 44.35°, 64.52°,
images were captured with fluorescence microscope and 77.44° in Ag@PMoS , which belong to the (111),
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(BX60, Olympus, Japan). (200), (220), and (311) reflection planes of metallic Ag,
114 International Journal of Bioprinting (2022)–Volume 8, Issue 3
Please cite this article as: Zheng L, Zhong Y, He T, et al., 2022, A Codispersed Nanosystem of Silver-anchored MoS Enhances Antibacterial
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and Antitumor Properties of Selective Laser Sintered Scaffolds, Int J Bioprint, 8(3):0025. http://doi.org/10.18063/ijb.v8i3.0025