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Shuai C, et al.

adhesion and proliferation level, with PHBV scaffolds to exclude the effects of possible self-decomposition of
serving as control. The bacterial suspensions were NBT.
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diluted to a concentration of 5×10 CFU/mL as this
is the clinically relevant concentration in orthopedic 2.5 Degradation Properties
infections [27] . Before seeding, the scaffold specimens The degradation properties of the PHBV/nMgO scaf-
(diameter 8 mm, thickness 4 mm) were sterilized in folds were evaluated by immersing them in PBS (pH
XFS-260 autoclave at 120 °C for 20 min, followed by = 7.4). Prior to immersion, the initial weights of the
immersing in phosphate buffer solution (PBS) overnight specimens were recorded. Approximately 1 g specimen
to prewet. Afterwards, the specimens were seeded with was added into a cap-sealed tube containing 10 mL
the diluted bacterial suspensions and incubated in low PBS and incubated in an electronic thermostat water
glucose Dulbecco’s Modified Eagle Medium (DMEM) bath at 37 °C. After the predetermined immersion time
at 37 °C in 5% CO /95% air atmosphere. After 24 h (7, 14, 21, 28 and 35 days), the specimens were taken
2
of incubation, the bacterium/scaffold constructs were out and the PBS was collected. The pH values of the
gently washed using PBS, and then were fixed with 2.5% collected PBS were measured using a digital pH meter
glutaraldehyde. Subsequently, they were dehydrated with a resolution of 0.01 (PHS-3C, Shanghai Xiaosheng
with a graded ethanol series, and dried in vacuum drying Instrument Manufacturing Co., Ltd., Shanghai, China).
oven. Afterwards, the dried specimens were installed The specimens were dried in a vacuum drying oven
on copper stubs, sputtering with platinum. Finally, until their weights were constant. The weight loss of the
the adhesion and proliferation level of E. coli were specimens was calculated by the equation (2.2):
characterized using a SEM (Phenom ProX, Phenom- Weight loss (%) = (W −W t )/W ×100 (2.2)
World BV, Netherlands) installed with EDS (INCA, 0 0
Oxford Instruments, UK) under backscattering mode. where W and W represents the initial weights and
t
0
Reactive oxygen species (ROS) was reported to play the residual weights of the specimens after t days of
a significant role in exerting the antibacterial activity immersion, respectively. The pH and weight loss tests
of some metallic oxide including nMgO [12,13] . Hence, were carried out in quintuplicate.
an oxidation-reduction method [28,29] based on reducing After the weight loss was determined, the specimens
nitroblue tetrazolium (NBT) by ROS was employed were used to characterize the degradation morphologies.
to detect the production of ROS in the suspensions Before SEM characterization, the specimens were
containing PHBV/5%nMgO scaffolds, with PHBV installed on copper stubs and sputtered with gold. The
scaffolds serving as control. Firstly, approximately surface morphologies of the specimens were observed
50 mg PHBV/5%nMgO scaffold specimens were by Phenom ProX SEM using backscattering mode under
added into a cap-sealed tube filling with 50 mL PBS 15 kV acceleration voltage.
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containing 2.5×10 M NBT, followed by incubating at 2.6 Cytocompatibility
37 °C in a water bath shaker (SHA-C, Hunan Lichen
Instrument Technology Co., Ltd., Changsha, China). The cytocompatibility of PHBV/5% nMgO scaffold
After incubating for 1 min, 1 mL of the suspension was was evaluated by seeding with MG63 cells (American
aspirated and filtered in order to determine the initial Type Culture Collection, Manassas, VA, USA) and
absorbance. After that, another 1 mL of the suspension assessing the cellular responses. The MG63 cells were
3
was regularly aspirated and filtered at a fixed time harvested using trypsin/EDTA, centrifuged at 1×10
interval of 10 min until 60 min. The absorbance of the rpm for 3 min and resuspended in DMEM. The scaffold
initial filtrates and the filtrates taken out at the fixed time specimens (diameter 8 mm, thickness 4 mm) were
interval was measured with an ultraviolet-visible (UV- sterilized in an autoclave (XFS-260, Zhejiang Xinfeng
vis) spectrophotometer at 259 nm where NBT showed a Medical Devices Co., Ltd., Shaoxing, China) at 120 °C
maximum absorbance. The amount of the produced ROS for 20 min, followed by immersing in PBS overnight
was proportional to the reduction percentage of NBT, to prewet. Afterwards, the specimens were seeded with
3
5
which was calculated by the equation (2.1): MG63 cells (at a density of 2×10 /well, 1×10 /well

5
Reduction percentage of NBT (%) = and 5×10 /well for SEM observation, Cell Counting
Kit-8 (CCK-8) assay and alkaline phosphatase (ALP)
(A 0 −A t )/A 0 ×100 (2.1)
staining, respectively) and incubated in low glucose
where A and A represent the absorbance of the initial DMEM supplemented with 10% fetal bovine serum
0
t
filtrates and the filtrates taken out at t min, respectively. and 1% antibiotic-antimycotic solution at 37 °C in 5%
The ROS detection tests were performed in quintuplicate. CO /95% air atmosphere. After the selected incubation
2
Besides, a blank control was also set, where no scaffold time, the cell-scaffold constructs were sacrificed to
specimens were added into PBS/NBT solution, in order assess the cellular adhesion, proliferation and osteogenic
International Journal of Bioprinting (2018)–Volume 4, Issue 1 3