Mira4 lmh

Electric discharge machining (EDM) was used to cut all the coating samples to ensure no extra stress was created during the process. Then, the samples were ground and polished using abrasive papers ranging from 400 to 1200 grit and SiC suspension. Then, the samples were etched in an etchant (2.7 g Fe (NO₃)₃, 10 mL C₂H₅OH, and 10 mL deionized water) for 15 s before being measured using a scanning electron microscopic (SEM, MIRA4 LMH, TESCAN, Czech) that was equipped with energy-dispersive spectroscopy (EDS) to observe the microstructure (cross-sectional) and elemental analysis. To calculate the porosity of the coating, image analysis software employing the SEM micrographs of cross-sectional samples after polishing was used. At the same time, an optical profiler (WYKO NT9100, Veeco Metrology Inc., Plainview, NY, USA) was used to measure the surface roughness of the coating as well as its 3D morphology. In addition, the phase composition of coatings and powders were detected using X-ray diffraction (XRD, Empyren, PANalytical, The Netherlands) employing Cu-Kα radiation of 1.5418 Å with an operating voltage of 45 KV, operating current of 40 mA, 2θ range of 20–80°, and scan rate of 0.02 °/s.

Pure zinc discs (purity 99.99%, 10 mm in diameter, 1 mm in thickness) were used (denoted as Zn). The entire surface of the specimen was wet ground until reaching 1200 grit with silicon carbide abrasive paper. Next, specimens were cleaned ultrasonically in absolute ethanol for 10 min. Pure Zn was further pre-treated with a complete cell culture medium under standard cell culture conditions (denoted as P-Zn). The formation of the initial degradation layer can partially mimic the degradation interface changes occurring in the early post-implantation stage, as previously reported [27 (link),28 (link),29 (link)]. Specifically, Zn discs were immersed in 2 mL Dulbecco’s modified Eagle medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, ExCell Bio, Shanghai, China) under standard cell culture conditions for 7 days. Surface morphology and its elemental composition were characterized by a scanning electron microscope with an energy dispersive X-ray spectroscopy instrument (SEM-EDS, MIRA4 LMH, TESCAN, Brno, Czech Republic). According to ISO 10993-12: 2012 [30 ], titanium alloy (Ti-6Al-4V) and pure Cu were used as a negative control (N.C.) and positive control (P.C.), respectively. Prior to cell culture experiments, all samples were disinfected with ultraviolet radiation for 1 h.