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International Journal of Bioprinting 3D-printed Mg scaffolds promote bone defect repair

2. Materials and methods to calculate the degradation rate of the scaffolds, and the
formula is as follows :
[13]
2.1. Sample preparation and characterization
2.1.1. Preparation of scaffolds and coating samples Degradationrate Weight before soaking Weight after soakingg (I)
The 3D-printed porous Mg alloy scaffolds were fabricated Soak time
using a selective laser melting 3D printer as previously
described [13] . The design porosity of 3D-printed Mg alloy 2.1.4. Drug release in vitro
scaffolds was 80%, and the design pore diameter was The 3D-printed Mg alloy scaffolds with ZA-loaded
600 μm. The main component of the Mg alloy powder ceramic coating were placed in 15 mL of Dulbecco’s
used for printing was Mg-3Nd-0.2Zn-0.4Zr, abbreviated phosphate-buffered saline (DPBS, pH = 7.4). Oscillate
as JDBM. The printed scaffolds were electrochemically at a predetermined interval of 120 rpm in a constant
polished. Square scaffolds with 10 × 10 × 10 mm temperature oscillator (ZD-85A, Langyue, China) at
diameters were fabricated for in vitro experiments, and 37°C. Each time, 3 mL DPBS was taken out for analysis
cylindrical scaffolds with 3 × 4 mm diameters were and replaced with the same amount of fresh DPBS. The
used for animal experiments. In addition, 5 × 5 × 3 mm concentration of ZA released into DPBS was measured
cast Mg alloy bulk samples were prepared for coating using an ultraviolet-visible spectrophotometer (Evolution
characterization. 201, Thermo Fisher Scientific, USA) at 210 nm wavelength.
Further, 15 g butyl acetate and 5 g polysilazane were 2.2. In vitro cell experiments
stirred under nitrogen for 1 h. Subsequently, 2 mg of ZA 2.2.1. Preparation of scaffold extracts
was added and stirred for 2 h to obtain polysilane coating According to ISO10993-12, the scaffolds sterilized with
materials containing ZA (332.6 μM). The JDBM Mg alloy ethylene oxide were placed in Alpha-Modified Eagle
samples were immersed into the coating material for Medium (α-MEM, Hyclone, USA) containing 10% fetal
3 min. The ZA drug load of each sample was about 40 μg. bovine serum (FBS, Hyclone, USA) and 1% penicillin–
The prepared drug-loaded coating samples were heated in streptomycin (Hyclone, USA) with a concentration of
the oven at 150°C for 4 h; the ceramic-coated JDBM Mg 0.1 g/mL. Subsequently, the culture medium was incubated
alloy samples containing ZA are referred to as Mg/Sc/ in 37°C and 5% CO cell incubator for 72 h. The extracts
2
ZA hereafter. Pure ceramic coating samples without ZA were stored in refrigerator at 4°C for standby.
were prepared using the same method and are referred to
as Mg/Sc, and the uncoated JDBM Mg alloy samples are 2.2.2. Cell viability
represented by Mg as the control group. Bilateral ovariectomy was performed in 3-month-
old female Sprague-Dawley rats. Rat bone marrow
2.1.2. Sample characterization mesenchymal stem cells (rBMSCs) were isolated and
The surface morphology, cross-section, and elemental expanded 3 months after the operation, according to
composition of the samples were examined using scanning previously described methods . The rBMSCs were
[19]
electron microscopy (SEM, HITACHI S4800, Japan) seeded in 96-well plates at a density of 2 × 10 cells/well.
3
combined with an energy dispersive X-ray spectrometer Twenty-four hours after inoculation, the original culture
(EDS, OXFORD, UK). SEM images were analyzed and medium was replaced with scaffold extracts. The cells were
measured using the ImageJ 1.53e software (National incubated in 37°C, saturated humidity, and 5% CO for 1,
2
Institutes of Health, USA) to obtain the pore diameter of 3, 5, and 7 days. Further, 100 µL α-MEM and 10 µL Cell
scaffolds and the coating thickness of samples. The static Counting Kit-8 (Dojindo Molecular Technology, Japan)
contact angle (CA) and sliding angle (SA) of ultrapure solution were added to each well and incubated at 37°C
water and hexadecane on the coating were measured for 2 h. The optical density (OD) at 450 nm was measured
using an optical contact angle tester (Attention Theta Flex, using a microplate reader (Bio-Tek, USA) to determine cell
Switzerland). The coating adhesion of the samples was viability.
determined according to ASTM D3359-22.
2.2.3. Osteogenic differentiation
2.1.3. In vitro degradation The osteogenic differentiation of rBMSCs was evaluated
The 3D-printed Mg alloy scaffolds were immersed in using alkaline phosphatase (ALP) and Alizarin Red (AR)
simulated body fluid (SBF) for 7 days at 37°C. The pH, Mg staining. The rBMSCs were seeded into 48-well plates at a
ion concentration, and hydrogen release were measured density of 1 × 10 cells/well. After the cells reached about
4
every 24 h. After soaking for 7 days, the corrosion products 80% confluence, the medium was replaced with osteogenic
were removed, and the surface morphology of the scaffolds differentiation medium (Sigma-Aldrich, USA) containing
was observed by SEM. The weight loss method is used different scaffold extracts and supplemented with 10 mM

Volume 9 Issue 5 (2023) 403 https://doi.org/10.18063/ijb.769