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Deng Y, et al.

2.3 Microstructure Characterization tests were performed to evaluate the hardness of the
composite by a hardness tester (Taiming Optical
The microstructure was analyzed by scanning electron Instrument Corporation, China). The applied load and
microscopy (SEM; JSM-5600LV, Japan). The sample loading time were 0.98 N and 15 seconds, respectively.
was prepared as follows: the ZK60/xβ-TCP composites Triplicate tests were conducted for each sample.
were successively grounded with abrasive papers (500,
1000 and 2000 grit), and polished with diamond grits. 2.6 In Vitro Cell Culture
The phase composition of the composites was identified MG-63 cells were used for the in vitro cell culture.
using an X-ray diffractometer (XRD, D8-Advance, Dulbecco’s modified eagle medium (DMEM) with
Germany). The condition was set using Cu Kα radiation 10% fetal bovine serum, 100 U∙mL penicillin and 100
−1
at 15 mA and 30 kV. Scans were performed with 2θ mg∙mL streptomycin were used as culture medium.
−1
−1
range of 5° to 80° at a scanning rate of 8°·min . ZK60/xβ-TCP samples were immersed in DMEM for 3
The relative density of the laser rapidly solidified com- days to prepare extracts (surface area to extracts volume
posites was studied using Image-Pro Plus 6.0 software. 1.25 cm ∙mL ) in humidified atmosphere (5% CO ,
−1
2
After grinded and polished, optical micrographs of each 37 °C). 2
sample were obtained. The obtained optical micrographs MG-63 cells were first cultured in DMEM in a 24-
were then turned into the gray mode. A proper threshold well plate. After 4 h, the cell culture media were
value of gray scale was determined as gray scale of the substituted by previously prepared extracts. Cells were
pores. Then, the area percentage of pores was obtained cultured in a humidified atmosphere for 1 day (5%
by calculating the area ratio of the marked regions to the CO , 37 °C). Subsequently, the cells were gently rinsed
2
whole micrograph. Triplicate tests were conducted for with phosphate buffered saline (PBS) and then stained
each sample. with Calcein-AM and Ethidium homodimer-1 reagents
2.4 Immersion Test (15 min, 37 °C). After gently rinsing with PBS, the
specimens were mounted onto glass slides and then
The immersion experiments were conducted to access observed by fluorescence microscopy (BX60, Olympus,
the corrosion behavior of the ZK60/xβ-TCP composites. Japan).
Simulated body fluid (SBF) served as the degradation Besides, CCK-8 assay was used to assess cell viability
medium. The pH variations of the SBF were recorded of MG-63 cells cultured in extracts of ZK60/xβ-TCP
during the immersion for 10 days. In addition, the composites. The MG-63 cells were seeded onto the 96-
corrosion surface of the composites was observed well plate (cells density 1×10 per mL) and cultured for
5
by SEM after immersion for 7 days. The chemical 1 day. Then, the cell culture media were substituted by
composition was analyzed by energy dispersive prepared extracts, with DMEM serving as control. After
spectroscopy (EDS, JSM-5910LV, Japan). Weight cultured for 1, 3 and 5 days, cells were incubated with
loss method was used to obtain the quantified data 10 μL CCK-8 (5 mg/mL, Sigma-Aldrich, St. Louis, MO,
on the degradation behavior of Mg-based composite. USA) for 2 h, then the absorbance was measured at 450
The weight of the samples after the corrosion test was nm by paradigm detection platform (BECK MAN, S.
measured after removal of the corrosion products in Kraemer Boulevard Brea, CA). The obtained optical
chromic acid. An average of three measurements was density (O.D.) values were proportional to the live cell
taken for each group. The in vitro corrosion rate was numbers.
calculated according to the equation: 2.7 Statistical Analysis

C= M /(qAT)
loss
Quantitative data were presented as mean ± standard
where C was the corrosion rate in mm/year, M was the deviation, and analyzed using the STATA (Data Analysis
loss
weight loss, q was the density of the material, A was the and Statistical Software). Statistical significance was
initial immersion surface area and T was the immersion defined when the p-value was <0.05.
time.
3. Results and Discussion
2.5 Mechanical Properties
3.1 Microstructure
The compressive strength was assessed by a universal
testing machine (WD-01, Shanghai Zhuoji instruments The typical microstructures of the laser rapidly solidified
Co. Ltd., China) at a loading rate of 0.5 mm·min . The ZK60/xβ-TCP composites were presented in Figure 2.
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test samples with the size of 3×3×6 mm were prepared For ZK60, only a small amount of second phases (MgZn
3
according to the ASTM-E9-09. Three identical samples intermetallic phase) distributed in Mg matrix (Figure 2A).
were used for the compressive tests. Besides, indentation After composited with 4 wt. % β-TCP, divorced slender
International Journal of Bioprinting (2018)–Volume 4, Issue 1 3