Enhanced Mechanical Properties, Corrosion Resistance, Cytocompatibility, Osteogenesis, and Antibacterial Performance of Biodegradable Mg–2Zn–0.5Ca–0.5Sr/Zr Alloys for Bone‐Implant Application

Magnesium (Mg) alloys are widely used in bone fixation and bone repair as biodegradable bone‐implant materials. However, their clinical application is limited due to their fast corrosion rate and poor mechanical stability. Here, the development of Mg–2Zn–0.5Ca–0.5Sr (MZCS) and Mg–2Zn–0.5Ca–0.5Zr (MZCZ) alloys with improved mechanical properties, corrosion resistance, cytocompatibility, osteogenesis performance, and antibacterial capability is reported. The hot‐extruded (HE) MZCZ sample exhibits the highest ultimate tensile strength of 255.8 ± 2.4 MPa and the highest yield strength of 208.4 ± 2.8 MPa and an elongation of 15.7 ± 0.5%. The HE MZCS sample shows the highest corrosion resistance, with the lowest corrosion current density of 0.2 ± 0.1 µA cm−2 and the lowest corrosion rate of 4 ± 2 µm per year obtained from electrochemical testing, and a degradation rate of 368 µm per year and hydrogen evolution rate of 0.83 ± 0.03 mL cm−2 per day obtained from immersion testing. The MZCZ sample shows the highest cell viability in relation to MC3T3‐E1 cells among all alloy extracts, indicating good cytocompatibility except at 25% concentration. Furthermore, the MZCZ alloy shows good antibacterial capability against Staphylococcus aureus.


Introduction
Nowadays, biodegradable metal boneimplant materials are expected to be used as biomedical materials to promote bone tissue repair due to their good degradability, biocompatibility, and no need for secondary surgery.Among these metal implant materials, pure magnesium (Mg) has a density of ≈1.79 g cm −3 and an elastic modulus of 45 GPa, similar to those of human cortical bone (1.75 g cm −3 , 5-30 GPa), which can alleviate the stress-shielding effect caused by elastic modulus mismatch. [1]Moreover, Mg is nonmagnetic and has a high specific strength, good casting and cutting properties, and high thermal conductivity. [2]g can be corroded and degraded in human body fluids containing chloride (Cl) ions and the degradation products can be absorbed or excreted from the body, which can effectively prevent the need for secondary surgical removal of implants after healing, thus reducing the pain and the economic pressure for patients. [3]In addition, Mg is abundant in human bones and cells as an essential nutrient element and participates in the majority of body metabolic processes. [4]Mg can induce the differentiation of bone-marrow mesenchymal cells into bone and cartilage tissue as a key regulator of osteogenesis and a low concentration of Mg 2+ s can promote bone healing without significantly changing local osmotic pressures. [5]Furthermore, Mg is involved in protein synthesis as a metabolism-essential enzyme activator, regulating the activities of the neuromuscular and central nervous system, activating various enzymes in the body, ensuring normal contraction in the myocardium, regulating body temperature, and stabilizing DNA and RNA structures. [6,7]Therefore, Mg alloys are favored by biomaterial researchers and medical professionals, and this has become a hot spot in the research area of biodegradable metals.At present, bone screws made of pure Mg and Mg alloys have been certified by Conformite Europeene and the Korea Food and Drug Administration. [8]owever, Mg has the lowest standard electrode potential compared to iron and zinc (Zn), indicating that Mg is chemically reactive and forms magnesia (MgO) products on its surface, which are soluble in water and thus ineffective in protecting its substrate. [9,10]Therefore, the complete degradation cycle of Mg alloy implants is generally shorter than the time required for bone healing at the damaged site, which is normally more than 18 weeks, thus showing inadequate mechanical stability. [11]In general, Mg alloy implants can only provide a stable mechanical environment at the initial stage of fracture healing and there is still no effective and sustained stress stimulation in the middle and late stages, resulting in local osteoporosis and refracturing. [12]At the same time, the excessive degradation rate of Mg alloy implants leads to high concentrations of metal ions, production of a large amount of hydrogen (H 2 ) gas, and alkalization of the surrounding site during the degradation process. [13]This will cause bone cysts and osteolysis lesions, and affect the bone healing and other physiological functions of the body, although low concentrations of Mg 2+ s and H 2 can be absorbed, utilized, and metabolized through the kidneys. [14]In addition, because implantation is associated with bacterial or microbial infection, bacteria can proliferate and form biofilms through the nutrients provided by the host in implant surgery and healing, which will cause a greater infection risk at the implantation site. [15]Therefore, infection is one of the main factors leading to the failure of implantation therapy.Currently, about 5% of implants need to be removed through a second operation due to aseptic loosening (≈18% of failures) or implant infection (≈20% of failures), resulting in implant failure. [16]At the same time, due to the inevitable wear and shedding of corrosion products, a large number of fine particles will be generated around the Mg alloy implant, resulting in osteolysis and aseptic loosening. [17]The abovementioned adverse factors seriously restrict the clinical application of Mg alloys.There-fore, improving the mechanical properties, corrosion resistance, biocompatibility, and antibacterial properties of degradable Mg alloys is a key issue to be urgently addressed.
The mechanical properties and corrosion resistance of Mg alloys are significantly affected by their chemical composition and microstructure. [18]As a very effective alloying element of Mg alloys, Zn has the dual effects of solid-solution and grain-refinement strengthening, which significantly improve the mechanical properties of pure Mg. [19] At the same time, the solid solution of Zn in Mg can increase the electrode potential of the -Mg matrix due to its higher standard electrode potential than that of pure Mg, which enhances the corrosion resistance of Mg-Zn alloys. [9]The addition of calcium (Ca) to Mg leads to the formation of a Mg 2 Ca intermetallic compound with a high melting point, which can refine the matrix phase size and reduce the precipitation and size of the second phase. [20]Simultaneously adding Zn and Ca to Mg leads to the formation of a stable Ca 2 Mg 6 Zn 3 intermetallic compound, which can significantly weaken the matrix texture and improve the mechanical properties. [21]The combination of Mg and Ca promotes the deposition of inorganic substance in bone, which has a positive effect on the prevention of osteoporosis and the promotion of bone healing and osteoblast growth. [22]Therefore, Mg-Zn-Ca alloys with low cost, good precipitation-hardening performance, and biocompatibility have broad prospects in biomedical applications.Zareian et al. [23] reported that an extruded Mg-2Zn-1Ca (ZX21) alloy showed an excellent combination of mechanical properties with an ultimate tensile strength ( uts ) of 283 MPa and a failure elongation of 29% due to recrystallization and grain refinement by Ca.Zhao et al. [24] reported that a rolled and annealed Mg-2Zn-0.2Cashowed  uts of 285 MPa, tensile yield strength ( ys ) of 204 MPa, and fracture elongation () of 24% due to an enhanced solid-solution strengthening effect.Still, it is challenging for Mg-Zn-Ca alloys to meet the required mechanical properties ( uts of ≥300 MPa,  ys of ≥200 MPa, and  of ≥10% [25] ) for bone implants.
A low dose of strontium (Sr) salt can reduce bone absorption, maintain a high bone formation rate, and promote bone synthesis, development, and osteoid formation as a crucial component of human bone and teeth. [26]Therefore, Sr-containing drugs such as strontium ranelate or alendronate are commonly used for osteoporosis treatment. [27]Gu et al. [28] reported that a Mg-2Sr alloy showed better corrosion resistance and mechanical properties than pure Mg, without cytotoxicity and with a good host response.In addition, adding Sr to Mg alloys can refine -Mg grains, improve the formability of Mg alloys, and increase their mechanical properties at room temperature (RT), along with high temperature and corrosion resistance. [29]Therefore, as a biomaterial for internal orthopedic fixation, an appropriate amount of Sr can be added to Mg alloys to improve mechanical properties and corrosion resistance, promoting bone growth and healing.Zirconium (Zr) was also reported to have good biocompatibility with no in vitro toxicity, mutagenicity, or carcinogenicity. [30]r helps improve the strength and elongation of Mg alloy due to its good grain-refinement effect. [31]Sun et al. [32] reported that adding Zr refined the grain size of -Mg and formed corrosion barriers, improving the corrosion resistance of a Mg-10Gd-3Y alloy.According to the Mg-Zn, Mg-Ca, Mg-Sr, and Mg-Zr phase diagrams, [33] the solid solubility of Zn, Ca, Sr, and Zr in Mg is Therefore, the Mg-2Zn-0.5Caalloy was used as the base alloy in this study, and Sr and Zr were added as the alloying elements.The mechanical properties, corrosion resistance, cytocompatibility, osteogenesis performance, and antibacterial properties of the as-cast (AC) and hot-extruded (HE) Mg-2Zn-0.5Ca-0.5Sr/Zralloys were comprehensively investigated for degradable bone-fixation implant applications.

Material Preparation
Mg-2Zn-0.5Ca-0.5Srand Mg-2Zn-0.5Ca-0.5Zr(wt%, hereafter denoted MZCS and MZCZ, respectively) alloy ingots were prepared by casting using a high-purity Mg ingot (99.99%,Suzhou Origin Medical Technology Co. Ltd., China), Zn ingot (99.99%;Huludao Zinc Industry Co. Ltd., China), Mg-20Ca, Mg-30Sr, and Mg-30Zr master alloys (wt%, Hunan Rare Earth Metal Material Research Institute, Co., Ltd., China).The raw materials were fully melted within a cast-steel crucible in a vacuum magnetic suspension furnace at 720 °C, gravity-cast into 250 °C preheated cast-steel molds after stirring and cooling to 660 °C, and then cooled down in air for solidification.The chemical compositions of the AC ingots determined using an X-ray fluorescence spectrometer (S4 Pioneer, Bruker, Germany) are listed in Table 1.The AC ingots were homogenized at 400 °C for 4 h under the protection of argon gas followed by quenching in air.Finally, HE MZCS and MZCZ alloy bars with a diameter of 16 mm were prepared by hot extrusion at an extrusion ratio of 25:1 of their billets with 80 mm diameter and 40 mm thickness after turning and a preheating treatment at 350 °C for 1 h.For comparison, commercially pure Mg (CP Mg) was prepared under the same conditions except for the alloying process.
For electrochemical corrosion, immersion, Kelvin probe force microscopy (KPFM), nanoindentation testing, cytotoxicity, alkaline phosphatase (ALP), and antibacterial assessments, disk samples with a diameter of 8 mm and a thickness of 1.5 mm were cut from the middle of the HE bars with the horizontal plane perpendicular to the extrusion direction using electrical discharge machining (EDM).The alloy disks were ground with 1000 grit silicon carbide (SiC) papers and polished with 1.0 μm diamond slurry.The disk samples were autoclaved at 121 °C for 15 min and then exposed to ultraviolet radiation for 30 min before cytotoxicity, ALP, and antibacterial assessments.

Microstructural Characterization
The phase constitutions of the AC and HE samples were determined using D/max 2500 X-ray diffraction (XRD; Rigaku, Japan) by Cu-K radiation.XRD patterns were employed over the 2 range of 20°-70°with a scan rate of 2°min −1 .Microstructural characterization of the AC and HE samples was carried out using Axio Imager 2 optical microscopy (OM; Zeiss, Germany).Subsequently, the microstructures and phase compositions of the AC and HE samples were further examined using Pro X FEI scanning electron microscopy (SEM; Phenom, Netherlands) and energy-dispersive X-ray spectroscopy (EDS; Oxford, UK) under the operating condition of 15 keV.Prior to microstructural observation, all samples were ground with SiC papers up to 1200 grit, polished with 0.5 μm diamond slurry, and etched with a picric acid solution (1.5 g picric acid, 25 mL ethanol, 5 mL acetic acid, and 10 mL distilled water).The grain size of AC samples was calculated from OM images using Image-Pro Plus software (v6.0,Media Cybernetics, USA).For analysis of the grain size and orientation distribution of the HE samples, electron backscattered diffraction (EBSD) was carried out using FEI Quanta 450 fieldemission SEM (Hillsboro, OR, USA) equipped with an EBSD system (EDAX-TSL, USA) under the operating condition of 20 keV.The samples for EBSD analysis were prepared by grinding with SiC paper up to 800 grit and electrolytically polishing using a mixed solution of 7% perchloric acid and 93% alcohol at 20 V for 1 min.EBSD data were interpreted using Channel 5 software.The longitudinal sections of HE samples were used for microstructural observation and EBSD analysis.

Tensile and Compression Testing
The tensile properties of the AC and HE samples were measured using an Instron-3369 universal testing machine (Instron, MA, USA) with a 1 mm min −1 deformation rate at RT. Tensile samples with a plate-type geometry and a gauge length of 25 mm were prepared by EDM following the guidelines provided in American Society for Testing Materials (ASTM) E8/E 8M-16 [34] and the HE samples were cut from the middle of the HE bars parallel to the extrusion direction.SEM combined with EDS was used to examine the fracture surfaces and phase compositions of the tensile samples after tensile testing.

Vickers Hardness and Nanoindentation Testing
The Vickers hardness (or microhardness) of the AC and HE samples was determined using a Micromet 6000 Vickers hardness tester (Buehler, USA) with an indentation load of 0.2 kgf and a holding time of 20 s.The nanohardness and elastic modulus of the HE MZCS and MZCZ samples were tested via nanoindentation testing using a Hysitron TI980 nanoindenter (Bruker, Germany).A Berkovich diamond indenter with ≈20 nm tip radius was used for the indentation under a 5 mN maximum load.

Electrochemical Corrosion and Immersion Testing
Potentiodynamic polarization (PDP) and electrochemical impedance spectroscopy (EIS) testing of the HE samples was carried out using an electrochemical workstation (ParStat 2273, Princeton Applied Research, USA) with a typical three-electrode  2. [35] Electrochemical samples with a 0.5 cm 2 exposure area were mounted using epoxy resin and then immersed in the Hanks' solution for 3, 10, and 21 days to simulate the degradation of different corrosion stages.The working electrode was set as a disk sample, while a saturated calomel electrode (SCE) and platinum sheet served as the reference and counter electrodes, respectively.The samples underwent PDP testing in a potential range of −0.3-0.8V relative to the steady open-circuit potential (OCP) with a scanning rate of 1 mV s −1 .The corrosion rate (V corr ) was calculated using the Tafel extrapolation method based on the corrosion current density (I corr ), following the guidelines outlined in ASTM G102-89. [36]For EIS testing, the samples were subjected to an applied frequency range of 10 5 -10 −2 Hz under OCP conditions with an amplitude of 10 mV.Samples that were not immersed were used as the control group.

Immersion Testing
For the immersion testing, the HE samples were immersed in Hanks' solution for 3, 10, and 21 days at 37 ± 0.5 °C with a 20 mL cm −2 surface-to-volume ratio.During immersion, the H 2 volume evolution was collected, and the pH of the Hanks' solution was monitored using a PB-21 pH meter (Sartorius, Germany).After 21 days of immersion, the concentrations of Mg, Zn, and Ca ions in the Hanks' solution were determined using an inductively coupled plasma optical emission spectrometer (ICP-OES; 720ES, Agilent, USA).The morphology and chemical composition of the corrosion products on the sample surfaces immersed at different times were evaluated using SEM and EDS.The phase constituents of the corrosion products on the sample surfaces were analyzed using XRD in the 10°-70°2 range.Fourier transform infrared spectroscopy (FTIR; Nicolet 6700, Thermo Fisher, USA) was employed to identify the functional groups of the corrosion products.The FTIR spectra were recorded with a resolution of 4 cm −1 within the 4000-400 cm −1 range.X-ray photoelectron spectroscopy (XPS; K-Alpha+, Thermo Fisher Scientific, USA) with an Al-K radiation source was utilized for chemical analysis of the corrosion products.High-resolution narrow spectra of Mg 2p, P 2p, C 1s, Ca 2p, and O 1s were collected to obtain detailed information about the chemical bonding.After immersion testing, the morphology and chemical composition of the corrosion products on cross-sections of the immersion samples were characterized using SEM-EDS.Subsequently, a 200 g L −1 CrO 3 solution was employed to eliminate the corrosion products from the sample surfaces.The weights of the samples were measured using an electronic balance before immersion and after the removal of corrosion products.The degradation rate (V w ) was determined by calculating the weight loss as per the guidelines provided in ASTM G31-12. [37]

KPFM Testing
Atomic force microscopy (Dimension Icon, Bruker, Germany) equipped with KPFM was utilized to measure the 3D surface topography and contact potential difference on the cross-section of the HE MZCS alloy after 21 days of immersion.KPFM testing was conducted in tapping mode with a scan frequency of 0.5 Hz at RT.The 3D images were captured with a resolution of 256 × 256 pixels over a 50 μm × 50 μm area.

Cytotoxicity Evaluation
An indirect cytotoxicity and cell proliferation assay of HE samples was conducted using the mouse preosteoblast cell line (MC3T3-E1, CTCC GDC030) following the guidelines outlined in International Organization for Standardization (ISO) 10993-5 and the previously established protocols. [38]For the cytotoxicity evaluation, 3T3 cells were cultured in an -minimum essential medium (Cat.No.: C12571500BT) supplemented with 10% fetal bovine serum (Cat.No.: S711-001S) under a humidified atmosphere with 5% CO 2 at 37 °C.The HE samples were immersed in the culture media at a ratio of 1.25 cm 2 mL −1 for 2 days to prepare the extracts, conforming to the guidelines specified in ISO 10993-12. [39]The pH value and concentration of Mg 2+ ions in the undiluted extracts were determined using a pH meter and ICP-OES, respectively.
For the indirect cytotoxicity evaluation, 3T3 cells were seeded into 48-well plates at a density of 1 × 10 4 cells per well and cultured for 1 day.Subsequently, the cell-culturing media were replaced with the extracts at different concentrations of 100%, 75%, 50%, and 25% after incubating the extracts for 3 days under identical culturing conditions and gently washing them with phosphate-buffered saline (PBS).Next, 200 μL of new medium containing 10% 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) solution (5 mg mL −1 , Solarbio) was added to each well and incubated for an additional 4 h.The supernatants were then carefully removed and 150 μL dimethyl sulfoxide (Aladdin) was added to each well.The plate was shaken for 10 min to dissolve the purple formazan crystals.Subsequently, 100 μL of medium from each well was transferred into a new 96-well plate and the absorbance was measured using a Spark 10M automatic microplate reader (Tecan, Switzerland) at an absorbance wavelength of 570 nm.The control group consisted of cells cultured in a medium without any extract.
Cell morphology was assessed using fluorescence live/dead staining and 4′,6-diamidino-2-phenylindole (DAPI) staining for the cell-proliferation assay.A cell suspension of 300 μL, at a density of 3 × 10 4 cells mL −1 , was added to 48-well plates and cultured for 1 day.After the preincubation period, the original cell-culturing medium was replaced with 300 μL of extract at a concentration of 75%, and the cells were continuously cultured under consistent conditions for 3 days.The wells were filled with a 250 μL combination of Thermo Fisher Scientific calcein acetoxymethyl ester and propidium iodide, dissolved in PBS, and followed by incubation for 15 min for live/dead staining.The cells were fixed with 4% paraformaldehyde (PFA) before being successively stained with fluorescein-isothiocyanate-labeled phalloidin (green) and DAPI (blue) for DAPI staining.Finally, cell images were obtained using Axio Observer A1 fluorescence microscopy (Zeiss, Germany) at an excitation wavelength of 490 nm.

ALP Staining and Activity Assessment
MC3T3-E1 cells were seeded into 24-well plates at 3 × 10 4 cells per well density.After seeding, the cells were exposed to Thermo Fisher Scientific StemPro Osteogenesis Differentiation medium and 75% HE sample extract for a duration of 7 days, following the protocol outlined. [38]After 7 days of culturing, the cells were fixed in 4% PFA at 4 °C and subsequently stained using a 5-bromo-4chloro-3-indolyl phosphate/nitro blue tetrazolium ALP color development kit (Beyotime, China).Photomicroscopy images were taken using a SMZ800N stereoscopic microscope (Nikon, Japan).To quantify ALP activity, a commercial ALP assay kit (Beyotime, China) was utilized in accordance with the manufacturer's directions.The absorbance of the ALP activity was measured at a wavelength of 405 nm using an automatic microplate reader.

Antibacterial Assessment
To assess the antibacterial properties of the HE samples, both inhibition zone diameter (IZD) assay and colony-forming unit (CFU) enumeration were employed, following the protocols specified in ISO 20645 and ISO 6888-1, [40] and the procedures outlined in previous studies. [41]For the antibacterial assessment, S. aureus at ≈1 × 10 8 and 1 × 10 6 CFU mL −1 was used as the bacterial model for IZD and CFU assays, respectively.For the IZD assay, the IZD values were measured using a caliper after incubating the samples for 1 day at 37 °C.The control group consisted of commercially pure Ti (CP Ti) samples of the same size for comparison.
For the CFU assay, the bacterial suspension was added to a 24-well plate and the HE samples were then introduced into the wells.After 1 day of incubation under consistent conditions, 100 μL of the bacterial suspension was pipetted, sonicated for 20 min, and evenly plated onto a tryptic soy agar plate.The plates were then incubated for 1 day and the CFU number was determined using v2.0.0 ImageJ software.Following the 1 day incubation period under consistent conditions, the disk samples were washed 3 times with PBS after coculturing with S. aureus, prefixed with 2.5% glutaraldehyde fixative for 3 h, dehydrated using an ethanol gradient from 60% to 100%, and then air-dried.The surface morphologies were observed using JSM-7100F SEM (JEOL, Japan).

Statistical Analysis
The data were analyzed using one-way analysis of variance, followed by Tukey's multiple comparison test.Data were presented as mean values ± standard deviations (n = 3 independent samples).Statistical significance levels were defined as significant and highly significant for *p < 0.05 and **p < 0.01, respectively.

Microstructures of CP Mg, MZCS, and MZCZ Samples
Figure 1 shows the microstructural characteristics of the AC and HE samples.It can be seen that the -Mg matrix phase existed in all samples, while Mg 7 Zn 3 , Mg 2 Zn 11 , and Ca 2 Mg 6 Zn 3 phases were found in both the MZCS and MZCZ samples (Figure 1a).In addition, a Sr 2 Mg 17 phase was also found in the MZCS samples and there was no obvious diffraction peak of a Zr-containing second phase or pure Zr phase in the MZCZ samples.Figure 1b-g shows OM images of the AC and HE samples.In the AC condition, CP Mg showed a coarse and irregular -Mg grain with a grain size of 834.7 ± 106.4 μm.With the addition of Sr to MZCS and Zr to MZCZ, the -Mg grains of the AC MZCS and MZCZ samples became polygonal and equiaxed, with more continuous and spot-like second phases uniformly distributed at grain boundaries (GBs) and inside grains.Compared with CP Mg, the grain size was measured to be 163.2± 12.9 μm for the MZCS and 89.4 ± 7.8 μm for the MZCZ.After hot extrusion, the grains showed an equiaxed structure with the grain size significantly decreased compared to their AC counterparts, indicating that recrystallization occurred during hot extrusion (Figure 1e-g).In addition to the equiaxed grains, the HE MZCS and MZCZ samples also showed black-striped deformation bands parallel to the extrusion direction.
Figure 1h-k shows SEM images of the AC and HE MZCS and MZCZ samples, and the corresponding EDS analysis results of the marked Spots 1-11 are shown in Table 3. Spots 1 and 3 indicate the -Mg matrix phases, which contain primarily Mg, Zn, and Ca, with small amounts of Sr in the AC MZCS and Zr in the AC MZCZ.Spot 2 shows a reticulated second phase on GBs in the AC MZCS that contains Mg, Zn, Ca, and Sr with atomic content of 64.1 ± 2.7%, 20.4 ± 1.8%, 13.6 ± 1.0%, and 1.9 ± 0.4%, respectively, and the Zn/Ca atomic ratio is ≈3/2.Spot 4 is a reticulated second phase in the AC MZCZ containing mainly Mg, Zn, and Ca with atomic content of 67.6 ± 3.1%, 21.0 ± 0.9%, and 11.4 ± 2.2%, respectively, similar to the composition of Spot 2 except for no Sr.Spot 5 is a fine granular second phase in the AC MZCZ containing a large amount of Zr with an atomic content of 40.0 ± 1.8% and small amounts of Zn and Ca.Spots 6 and 9 show the -Mg matrices containing mainly Mg and Zn, with small amounts of Sr in the HE MZCS and Zr in the HE MZCZ, and the alloying element content is slightly higher than in Spots 1 and 3. Spot 7 shows a fine second phase at the deformation band in the HE MZCS containing 78.5 ± 0.7% Mg, 11.7 ± 0.2% Zn, 7.7 ± 0.7% Ca, and 2.1 ± 0.5% Sr, with a Zn/Ca atomic ratio of ≈3/2.Spot 8 shows a coarse granular second phase with a phase size of 7.2 ± 0.6 μm in the HE MZCS containing large amounts of Mg, Sr, and Zn and a small amount of Ca.Spot 10 shows a fine granular second phase distributed in the deformation bond along the extrusion direction in the HE MZCZ containing 85.8 ± 1.8% Mg, 9.9 ± 0.9% Zn, 4.2 ± 1.0% Ca, and 0.1 ± 0.1% Zr.Spot 11 shows a mixture of fine and coarse granular second phases distributed in the deformation bond along the extrusion direction in  Figure 2 shows SEM images and EBSD analysis results for the HE samples.All HE samples were composed of fine equiaxed recrystallized grains (Figure 2a-c).In addition, deformation bands and a coarse granular second phase were observed along the extrusion direction in the HE MZCS and MZCZ samples.Figure 2d-f shows EBSD inverse pole figure (IPF) maps of the HE samples.The CP Mg contained many twins within the coarse deformed grains (Figure 2d).There were almost no twins and the second phase was mainly composed of fine sub-micrometer particles in the HE MZCS and MZCZ samples (Figure 2e,f).In the [0001] texture of the -Mg phase, the preferred orientation of the samples was (0001) and the texture strength of the -Mg phase in descending order was: CP Mg > MZCZ > MZCS, with the CP Mg showing the highest basal texture with a maximum texture intensity of 10.9. Figure 2g-i shows GB maps of the HE samples and the corresponding grain-misorientation angle distributions are shown in Figure 2j-l.The red, green, and blue lines in the GB maps correspond to the low-angle grain boundaries (LAGBs), medial-angle grain boundaries (MAGBs), and high-angle grain boundaries (HAGBs) with misorientation angles of 1°-5°, 5°-15°, and >15°, respectively.The LAGBs of the HE CP Mg sample were larger than those of the HE MZCS and MZCZ samples.The larger LAGBs showed a high dislocation density, which makes them susceptible to becoming more tangled and entangled during dislocation movement, thereby storing more internal stress and distortion energy. [42]In addition, the grain-misorientation angle distributions of the HE CP Mg tended to be bimodal with a clear peak value at 87°.The grain size distribution of the HE samples is shown in Figure 2m-o.The grain size distribution of the HE samples tended to have a lognormal distribution and the average grain sizes of the HE CP Mg, MZCS, and MZCZ samples were 23.3 ± 4.6, 10.2 ± 2.1, and 4.6 ± 0.9 μm, respectively, all of which were significantly lower than those of their AC counterparts.In addition, the average grain size variation of the HE samples was consistent with that of the AC samples after alloying.

Mechanical Properties of CP Mg, MZCS, and MZCZ Samples
Figure 3 shows the mechanical properties and fracture morphologies of the AC and HE samples.The tensile stress-strain curves of the AC and HE samples are shown in Figure 3a and their corresponding tensile properties are shown in Figure 3b.For comparison, the mechanical properties of these alloys and those of other Mg-Zn-based alloys published in the literature, as well as the target values for orthopedic implant materials, are listed in Table 4.All of the AC samples showed poor mechanical properties, especially the elongation, making it challenging for them to meet the orthopedic implant material requirements.After hot extrusion, the mechanical properties of the HE samples were significantly improved compared with those of the AC samples.The strength values of both the AC and HE alloy samples and the elongation of the HE alloy samples are higher than those of their CP Mg counterparts.The HE MZCZ sample showed the best mechanical properties with  ys of 208.4 ± 2.8 MPa,  uts of 255.8 ± 2.4 MPa, and  of 15.7 ing for them to meet the orthrthopedic implant material requirements [25] except for  uts .
Figure 3c-h 1 shows the tensile fracture surfaces of the AC and HE samples.The AC CP Mg showed a smooth fracture surface, many dissociation facets, and a small number of small and shallow dimples, indicating a mixed mode of cleavage and ductile fracturing.The AC MZCS and MZCZ samples showed typical brittle fracture characteristics with an intergranular fracture morphology, many torn edges, and almost no cleavage fractures or dimples.In addition, large amounts of the second phases were deposited at the GBs and many second phases were separated from the b-Mg matrix and became sources of cracking.However, the fracture surfaces of the HE samples became notably finer than those of the AC samples.The HE CP Mg still had a large number of cleavage planes and a small number of dimples, with clearer plastic deformation, smaller cleavage planes, and deeper, wider, and more uniform dimples than its AC counterpart, showing better plastic deformation ability.A large number of dimples and a small number of cleavage planes were distributed on the HE MZCS sample with almost no intergranular fracture morphology, showing a mixed fracture mode of large ductile fracturing and small cleavage fracturing.In addition, the second phase with some cracks was uniformly embedded in the bottom of the dimples.The HE MZCZ samples had the deepest and widest dimples among all HE samples, showing the best plastic deformation ability, consistent with their elongation results.In addition, the HE MZCZ samples had tiny nanoscale second phases at the bottom of the dimples, showing a typical dimple-microporous aggregation fracture mode.
Figure 3i shows the microhardness values of AC and HE samples.The hardness values of the AC and HE samples in descending order were MZCS > MZCZ > CP Mg and the HE samples showed higher hardness values than their AC counterparts.The HE MZCS sample showed the highest hardness value of 62.2 ± 1.5 HV among all AC and HE samples.Figure 3j-o  In the solidification process of Mg alloys, Zn and Ca mainly cause -Mg GB segregation through the solute effect, forming a strong component of undercooling due to solute redistribution at the solid-liquid interface front, thus hindering -Mg grain growth. [53]At the same time, the aggregation of Zn atoms at the -Mg crystallization interface front can also hinder the growth of -Mg grains because the binding energy between Zn and Mg atoms is lower than that of the Mg solid-liquid interface. [54]Therefore, the two factors together have a notable grain-refining effect.According to the Mg-Sr phase diagram, [33] Sr can be enriched at the solid-liquid growth interface, hindering the continuous growth    of -Mg grains. [55]In this work, the addition of Sr to the MZCS alloy exceeded the solid solubility limit of -Mg, so a eutectic reaction occurred at ≈585 °C to form the Sr 2 Mg 17 eutectic phase.In the solidification process, the second phase particles were gradually pushed to the GBs and induced grain refinement through the mechanism of grain growth inhibition. [56]According to the Mg-Zr phase diagram, [33] when the melting temperature of the MZCZ alloy was lower than 653.6 °C, a peritectic reaction occurred and the excessively added Zr precipitated out of the melt and formed -Zr particles.The lattice constants of Zr and Mg are similar due to their hexagonal close-packed (hcp) crystals. [57]At the same time, the Mg/Zr interfacial energy is much lower than that of the solid and liquid Mg, resulting in Mg undergoing less growth in nucleation energy with -Zr particles than the uniform nucleation growth energy of Mg itself. [58]Therefore, the fine -Zr particles can act as heteronucleation sites for Mg alloys, thus inhibiting the grain growth of -Mg and playing a role in grain refinement. [58,59]Compared with the MZCS alloy, the MZCZ alloy exhibited significantly finer grain size.This may be because Zr has the highest growth restriction factor (38.29) among the metals Zn (5.31), Ca (11.94), and Sr (3.51). [60]After extrusion deformation, the Mg alloy samples underwent recovery and dynamic recrystallization, which refined the grains and the second phases, and redistributed the second phases along the extrusion direction. [61]During the recrystallization process, Sr and Zr significantly inhibited the recrystallization process of the Mg alloys, thus giving them a fine recrystallized structure and preserving the high-density dislocation and fibrous structure generated during the deformation process. [62]he CP Mg sample showed the largest LAGBs with the largest distortion energy, which caused strength improvement and elongation deterioration. [63]However, as pure Mg is a hcp structure with few slip surfaces and few slip systems, the AC pure Mg showed a poor plastic deformation capacity and low mechanical strength. [64]However, as the grain size of the MZCS and MZCZ alloys became smaller, the number of GBs increased and the hindrance effect of limiting dislocation movement was strengthened.Dislocation accumulating at GBs forms a stress field and activates more slip systems, thus improving the yield strength and plastic deformation capacity, leading to grainrefining strengthening. [65]However, although the AC MZCS alloy exhibited a smaller grain size than the CP Mg, its corresponding elongation also decreased compared with the CP Mg.This may have been due to the presence of a large amount of the reticular and spot-like hard and brittle Sr 2 Mg 17 second phase on the GBs of -Mg, resulting in the poor plasticity of the AC MZCS alloy.Second, due to the differences in atomic radii between Zn, Ca, Sr, Zr, and Mg, lattice distortion will occur when these metal elements are solidly dissolved in the -Mg matrix, [66] which can hinder dislocation movement, thus achieving solid-solution strengthening.In addition, the high hardness and elastic modulus values of the Ca 2 Mg 6 Zn 3 , Sr 2 Mg 17 , and -Zr second phases formed in the MZCS and MZCZ alloys pinned GBs, hindered GB migration, and played a role in second phase strengthening. [67]he refinement of grains and second phases caused by dynamic recrystallization after hot extrusion contributed to the improvement in the mechanical properties and hardness of the MZCS and MZCZ alloys. [68]In addition, a modest increase in the solid solution of the alloying elements in the -Mg phase of the HE MZCS and MZCZ alloys also played a role in solution strengthening, promoting strength and hardness enhancements.Therefore, the improvement in the mechanical properties of the HE MZCS and MZCZ alloys compared with the CP Mg was mainly due to the finer grains and second phases, and a small amount of solid-solution strengthening.

Corrosion Behavior of CP Mg, MZCS, and MZCZ Samples
Figure 4 shows the corrosion and degradation behaviors of the AC and HE samples in Hanksn the -Mg phase of the HE MZCS and MZCZ alloys also played a role in solution strengthening, promoting strength and hardFigure 4a).The OCP values in descending order were CP Mg > MZCZ > MZCS at the same immersion time, except for the MZCZ-3d and MZCZ-10d samples.Furthermore, MZCZ showed a significantly lower OCP value relative to the CP Mg and MZCS samples after 10 days of immersion.
Figure 4b displays PDP curves of the AC and HE samples in Hanks different immersion times; (h) degradation rates after different immersion times; and (i) mTafel region are given in Table 5.The changing trend of the E corr is consistent with the OCP values.With the prolongation of immersion time, both the I corr and V corr of the HE samples showed decreasing trends, except after immersion for 3meays.Both the I corr and V corr in descending order were: CP Mg > MZCZ > MZCS, indicating that the MZCS exhibited the best corrosion resistance.The MZCS showed the lowest I corr of 0.2 ± 0.1 μ. cm −2 and the lowest V corr of 4 ± 2 μm per year among all the corrosion samples after 21 Mays of immersion.Figure 4c displays the Nyquist plots of the HE samples.There are two capacitive loops at high and medium frequency, and inductive arcing at low frequency in the Nyquist plots of the unimmersed, 3 days immersion, and MZCZ-10d samples.After 10 and 21 days of immersion, the samples had capacitive loops at high, medium, and low frequency, respectively (Figure 4c).Two equivalent circuit models are shown in Figure 4d.R s , R 2 , and R 3 in the two circuit diagrams represent the solution resistance, the electric double layer resistance linked to charge transfer and electrochemical double-layer/oxide film effects between the electrode surface and solution, and the resistance that is related to mass transport relaxation due to the diffusion of Mg 2+ ions through the corrosion layer, respectively.The CPE 1 and CPE 2 correspond to the capacitance of R 1 and R 2 , respectively. [69]L is related to coverage relaxation due to the adsorption of Mg 2+ intermediates in the equivalent circuit models of the unimmersed, 3mmays immersion, and MZCZ-10 d samples. [69,70]In the equivalent circuit models of the HE samples after 10 and 21 days of immersion, R 3 and CPE 3 are the resistance and associated constant phase elements of the corrosion products, respectively. [71]The EIS parameters are given in Table 6 after being fitted according to the equivalent circuit models.The R total showed a gradually increasing trend with prolonged immersion time and the R total of the samples in descending order was: MZCS > MZCZ > CP Mg for the same immersion time.This indicates that the corrosion resistance of the HE samples gradually increased with prolonged immersion time and the MZCS samples showed the best corrosion resistance among these HE samples.This is consistent with the corrosion rate results measured by PDP curves except after immersion for 3 days.Figure 4e,f shows the Bode impedance modulus and phase-angle diagrams of the AC and HE samples.At low frequency (10 −2 -10 0 Hz), there was a positive correlation between the impedance modulus and the R total of the HE samples.The phase-angle curves of the HE samples after 10 and 21 days of immersion showed three peaks, except for the CP Mg-10d samples, denoting three time constants in the equivalent circuits related to the two reactance arcs in the Nyquist curves.
Figure 4g displays the H 2 volume and pH value evolutions of Hanks' solution immersed for different times.The pH value evolution showed a rapidly increasing trend at first and then tended to become stable with the prolongation of immersion time.The H 2 volume and pH values of the samples in descending order were: CP Mg > MZCZ > MZCS for the same immersion time.In addition, the evolution trends of the H 2 volume and pH value were consistent with the corrosion rate measured by electrochemical testing; a greater corrosion rate corresponded with greater H 2 volume and higher pH value.The minimum and maximum H 2 volumes of the MZCS alloy were 1.66 ± 0.03 mL cm −2   per day after 21 days, both of which are lower than the hydrogen absorption level of 2.25 mL cm −2 per day for the human body, [72] indicating that the body could tolerate this level of H 2 evolution.Figure 4h displays the degradation rates of the AC and HE samples with different immersion times and the V w values of the samples are listed in Table 5.The V w values of the samples were consistent with the change law of V corr measured by electrochemical corrosion testing except after 3 days of immersion; that is, the V w of the HE samples showed a decreasing trend with the prolongation of immersion time and their V w values in descending order were still: CP Mg > MZCZ > MZCS for the same immersion time.After 21 days of immersion, the MZCS showed the smallest V w of 368 ± 38 μm per year among these samples, which meets the degradation rate requirement of less than 0.5 mm per year for degradable implants. [73]The metal ion concentrations in the Hanks' solution after 21 days of immersion are displayed in Figure 4i.The Mg 2+ concentration in Hanks' solution was positively correlated with the V w of the metal samples; a higher V w of the metal samples corresponded with a higher Mg 2+ concentra-tion.The variation trends of the Zn and Ca ion concentrations of the MZCZ and MZCS samples were also consistent with the V w of these two samples.The MZCS showed the lowest metal ion concentration with Mg, Zn, and Ca ion concentrations of 247.5 ± 4.4, 4.7 ± 0.3, and 1.1 ± 0.1 μg mL −1 , respectively.
Figure 5 shows the surface morphologies and chemistry analyses of the corrosion products on the HE samples after immersion in Hanks' solution for 3, 10, and 21 days.After immersion for 3 days, the SEM images show gray spherical and white fine granular corrosion products on all sample surfaces, with the corrosion products not completely covering the substrate surface, and the MZCS sample surface exhibited the fewest corrosion products (Figure 5a-i), which is consistent with the corrosion rate of the HE samples.In addition, there were many cracks on the corroded surfaces.With immersion time extended to 10 days, the corrosion products on the sample surfaces continued to increase and the crack length and width gradually increased, with local areas showing protrusions.With immersion time extended to 21 days, the sample surfaces were mainly composed of large gray flocculent corrosion products, white granular and gray short rod corrosion products embedded in the surfaces, and the gray spherical corrosion products disappeared.The corrosion product structures on the sample surfaces were rough, loose, and cracked.
Figure 5j shows EDS profiles of the corrosion products marked on the MZCZ alloy surface after 21 days of immersion in Figure 5i.Spot 1 is a white granular corrosion product that contains large amounts of O, C, Mg, and Ca, and the atomic ratio of Ca to P is close to 1.6:1.Spot 2 is a short gray bar corrosion product with more C and Mg, less Ca, P, and Cl, and almost no Ca or P compared to Spot 1. Spot 3 is a gray flocculent corrosion product that contains the most Mg and Cl and the least O among the three corrosion products and almost no Ca or P.
Figure 5k shows XRD patterns of the corrosion products on the sample surfaces after immersion in Hanks' solution for different times.The corrosion products on the sample surfaces were mainly composed of -Mg, Mg(OH) 2 , MgCl 2 , Mg 5 (CO 3 ) 4 (OH) 2 •8H 2 O, and Ca 3 (PO 4 ) 2 (HA) phases.At the same time, the diffraction peak intensity of the corrosion products gradually increased with the extension of immersion time, indicating that the amount of corrosion products also gradually increased with the extension of immersion time.
Figure 5l shows FTIR spectra of corrosion products on the sample surfaces after immersion in Hanks' solution for different times.The absorption peaks at wave numbers ≈410, 582, and 877 cm −1 correspond to the bending vibration absorption peak of metallic oxide (M─O) groups.Additionally, the absorption peaks at ≈793 and 1076 cm −1 are associated with P─O groups.Notably, the absorption peaks at 582 cm −1 may also be attributed to P─O groups.The absorption peaks at ≈850, 1415, and 1475 cm −1 are attributed to C─O groups.In addition, the absorption peaks at 3695 cm −1 represent O─H groups and the absorption peaks at 1655 cm −1 signify the O═H groups. [74,75]The normalized transmittance of the samples gradually increased with increasing immersion time, indicating that the amounts of the above functional groups on the sample surfaces gradually increased.
Figure 5m shows XPS spectra of corrosion products on the sample surfaces after immersion in Hanks' solution for different times.The corresponding high-resolution XPS spectra after 21 days of immersion are shown in Figure 5n.The corrosion products contained C, O, P, Ca, Na, and Mg in the survey spectra, which aligns with the EDS analysis results (Figure 5j).[77][78] The P 2p peak at 132.8 eV can be attributed to the P chemical bond in HA. [75,79,80] C 1s peaks at 285.0, 285.4,and 288.9 eV can be attributed to the C chemical bonds in C─C, C─OR, and CO 2− 3 structures. [75,76]Ca 2p observed at 347.4 and 350.9 eV may be attributed to the Ca chemical bond in the Ca 3 (PO 4 ) 2 structure. [80][77] Based on the findings obtained from EDS, XRD, FTIR, and XPS analyses, it can be inferred that the corrosion products on the sample surfaces after immersion in Hanks' solution primarily consisted of -Mg, Mg(OH) 2 , MgCl 2 , Mg 5 (CO 3 ) 4 (OH) 2 •8H 2 O, and HA phases, and Spots 1-3 are in-ferred to be HA, Mg 5 (CO 3 ) 4 (OH) 2 •8H 2 O, and MgCl 2 , phases, respectively.These corrosion products demonstrate the potential for absorption through in vivo metabolism, as reported in previous studies. [81]igure 6a-i shows SEM images of the corrosion products on cross-sections of the corroded samples after immersion in Hanks' solution for 3, 10, and 21 days.With the extension of immersion time, the thickness of the corrosion product layers on all sample surfaces gradually increased and the cracks on the corrosion product layers gradually increased.At the same immersion time, the corrosion product layer thickness in descending order was: CP Mg > MZCZ > MZCS, except CP Mg, which is positively correlated with degradation rate and surface corrosion product content after immersion.After immersion for 21 days, the MZCS showed the smallest corrosion product thickness of 69.2 ± 7.4 μm, the least cracking, and the most uniform and dense corrosion layer, indicating the best corrosion resistance.In addition, there were more second phases around the corrosion front at the bottom of the corrosion layers of the MZCS and MZCZ alloys.Figure 6j-l illustrates the corresponding EDS mapping results for the corrosion products on cross-sections of the samples after 21 days of immersion.Large amounts of O, Mg, C, and small amounts of Zn, Ca, P, Cl, and/or Sr/Zr were uniformly distributed on the corrosion product layers.Figure 6m-o shows the 3D surface morphology, 3D Volta potential map, the corresponding surface height, and Volta potential line profiles of KPFM on cross-sections of the MZCS sample after 21 days of immersion.The phase height and Volta potential of the MZCS sample in descending order were: Sr 2 Mg 17 > -Mg > CP layer and the Sr 2 Mg 17 showed a greater height of 0.86 ± 0.15 μm and higher Volta potential of 45.3 ± 3.9 mV than those of the -Mg phase.
The corrosion behavior of pure Mg and its alloys is related to their microstructure, the content and distribution of second phases, texture, and defects. [82]The standard electrode potentials of Mg, Zn, Ca, Sr, and Zr are −2.372,−0.762, −2.868, −2.899, and −1.529 V, respectively. [22]Therefore, Zn with high solid solubility and standard electrode potential can significantly increase the H 2 evolution potential of the -Mg matrix, thus improving the corrosion resistance.Second, the addition of Zn contributes to forming a dense passivation film on the sample surface, [80] which causes a good corrosion protection effect on the matrix.Third, the fine grain interaction of Sr and Zr can significantly increase the GB area and chemical uniformity of Mg alloys and act as a physical corrosion barrier in the corrosion process. [81]Therefore, significant grain refinement of the MZCS and MZCZ alloys effectively improved the corrosion resistance of the Mg matrix.In addition, the CP Mg had significantly more LAGBs than the MZCS and MZCZ alloys, resulting in higher distortion energy and deterioration of corrosion performance.Since the amounts of Sr and Zr in the MZCS and MZCZ alloys exceeded their maximum solid solubility in -Mg, the excess Sr and Zr was precipitated in the second phase.However, Sr 2 Mg 17 , -Zr, and Ca 2 MgZn 3 showed higher corrosion potential than the -Mg matrix. [83]Therefore, the microgalvanic corrosion between the second phase as the cathode and -Mg occurred in the corrosion environment containing Cl ions due to the potential difference. [84]This can promote the corrosion of the contact interface between the second phase and -Mg matrix, forming the corrosion fronts and spots, thus accelerating the corrosion of Mg alloys.
Although the MZCZ alloy had a smaller grain size than the MZCS alloy, the corrosion rate of the MZCZ alloy was higher than that of the MZCS alloy, which may be because the potential difference between Sr 2 Mg 17 and -Mg is smaller than that between -Zr and -Mg.This can result in a tendency toward lower galvanic corrosion of the MZCS alloy than that of the MZCZ alloy.Second, the corrosion product layer had a lower corrosion potential than the -Mg matrix (Figure 6o).When the corrosion layer was not dense and part of the metal matrix was exposed to the presence of Cl ions, the corrosion product as an anode formed galvanic corrosion with -Mg, thus accelerating the corrosion process.Therefore, the V corr and V w of the HE samples were inconsistent after immersion for 3 days.In the degradation process, the MZCS alloy had a denser, more continuous, and finer corrosion layer with finer cracks, which effectively prevented the corrosion caused by contact between the corrosive medium and the matrix, and weakened the galvanic corrosion between the corrosion layer and the matrix.Finally, the MZCS alloy had slightly fewer LAGBs, which also contributed to improving the corrosion resistance.After the degradation process, a violent H 2 evolution reaction occurred on the sample surfaces.The hydroxide ions formed during the degradation process and the carbonate and phosphate ions in the solution reacted with Mg and Ca metal ions to form the corrosion products covering the sample surfaces, including magnesium hydroxide, magnesium phosphate, HA, and magnesium carbonate.This makes the corrosion partly passivated, increasing the exposed area of matrix material and reducing the corrosion rate.At the same time, with the gradual increase in the passivation area and the continuous thickening of the corrosion product layer in the passivation area, the corrosion of the sample was further inhibited and the degradation rate further reduced.However, the Cl ions in Hanks' solution can easily penetrate the crevices of the corrosion layer and enter the substrate surface to corrode the metal sample further.At the same time, Cl ions can dissolve the corrosion product layer to form soluble MgO, which destroys the dense structure of the corrosion layer. [85,86]ith the prolongation of corrosion time, the corrosion product layers formed on the sample surfaces exhibited sharp increases at first and then stable tendencies for both the pH value and Mg 2+ concentration of Hanks' solution and the degradation rate, mainly due to the H 2 evolution reaction tending to become stable when the formation and dissolution of corrosion products on the sample surface layers reached equilibrium.Figure 7b shows the cell viability of MC3T3-E1 cells after culturing with different concentration extracts.All concentrations of the metal extracts showed high cell viability greater than 95%, demonstrating grade 0 to 1 cytotoxicity according to ISO 10993-5. [87]At 100% and 75% concentrations, the MZCS and MZCZ alloys showed higher cell viability than the CP Mg, indicating that both samples had better cytocompatibility.When the extracts were diluted to 50%, all extracts showed a significant cell proliferation effect with a cell viability of >100%.When the extracts were diluted to 25%, the cell viability of the CP Mg was higher than those of the MZCS and MZCZ alloys.The MZCZ alloy showed the highest cell viability among all the metal extracts, exhibiting the highest cytocompatibility except at 25% concentration.

Cytotoxicity of HE CP Mg, MZCS, and MZCZ Samples
Figure 7c-f shows fluorescent live/dead staining images of MC3T3-E1 cells after culturing with control and metal extracts at 75% concentration.The live (green) MC3T3-E1 cells in the control group and all extracts were triangular or short spindleshaped, with normal morphology and good growth, and almost no dead cells (red), showing a typical fibroblast-like morphology.The cell density of the MZCS and MZCZ alloys was higher than that of the control group and the CP Mg, showing better cellproliferation ability, consistent with the cell viability.Figure 7g-i shows DAPI staining images of MC3T3-E1 cells after culturing with the same culture medium.Many blue oval nuclei and purple cytoskeletons were observed on MC3T3-E1 cells in the control group and all metal extracts.The cells exhibited a fusiform or elongated morphology, without any notable presence of abnormal cells.This strongly suggests that the osteoblasts on the sample displayed robust and prolific growth, with a high cellular density.
In the high-concentration extracts of 100% and 75%, the CP Mg showed lower cytocompatibility than the MZCS and MZCZ alloys.This was mainly caused by the higher pH value and metal ion concentration, as a high pH strongly inhibits cell proliferation. [88]The alkalosis microenvironment caused by pH increase is not conducive to the proliferation and differentiation of murine embryonic stem cells. [89]Most osteoblasts can maintain active growth and structural integrity when the pH value ranges from 7.5 to 7.8. [90]In addition, there were Zn, Ca, Sr, and Zr ions in the MZCS and MZCZ extracts which were beneficial to cell proliferation. [43]Zn ions are essential in promoting osteoblast proliferation and differentiation. [91]hang et al. [92] reported that a Mg-Zn alloy was beneficial to the adhesion and mineralization of MC3T3-E1 osteoblasts and increased the mRNA expressions of collagen I 1 (COL1) 1 and osteocalcin (OC) proteins.Sr ions can stimulate osteoblast activity, promote bone formation, and inhibit bone resorption. [93]amamoto et al. [94] reported that Zr ions did not induce toxicity (>50%) for L-929 and MC3T3-E1 cells when Zr ions were lower than 10 −3 mmol L −1 .Zr can promote osteoblast proliferation at low concentrations. [95]Zr-containing absorbable metal stents showed good biocompatibility in vivo after implantation. [96]With the dilution of the extract concentration, the cell activity of the CP Mg gradually increased and became even higher than those of the MZCS and MZCZ alloys when the concentration was 25%.This may be because the concentration of metal ions in the alloy extract at 25% concentration was lower than that of the CP Mg, resulting in a less significant proliferation effect on MC3T3-E1 cells.

Osteogenesis Performance of HE CP Mg, MZCS, and MZCZ Samples
Figure 8 compares the ALP activity performance of the HE samples to that of the control group.The HE samples demonstrated deeper and denser purple-black areas on the walls compared to the control group (Figure 8a-d), suggesting that the HE samples had better capacity to promote mineralization.Furthermore, the HE MZCS and MZCZ samples exhibited significantly deeper and denser purple-black areas compared to the CP Mg sample, indicating that the incorporation of alloying elements contributed to the enhancement of mineralization capacity. Figure 8e illustrates the ALP activity of the control group and HE samples.The change law for ALP activity is consistent with the ALP staining results; the deeper and denser purple-black areas showed higher ALP activity.Notably, the HE MZCS showed the most ALP activity among the HE samples, more than 1.86 ± 0.26-fold that of the control group.
According to the ALP staining and ALP activity results, it can be concluded that the HE MZCS and MZCZ samples showed better osteogenic performance than the CP Mg.This is mainly because Zn and Ca ions promote osteoblast differentiation.Zn ions can activate ALP, carbonic anhydrase, and collagenase in bone metabolism and bone formation. [97]Ca ions can activate fracture callus, calcify cartilage, and form ALP. [98] In addition, Sr and Zr can stimulate osteoblasts to secrete new bone-tissue matrix, inhibit osteoclasts, and facilitate bone mineralization. [99]

Antibacterial Property of HE CP Mg, MZCS, and MZCZ Samples
Figure 9 shows the antibacterial effects of the HE samples versus CP Ti after 1 day of coculturing with S. aureus.There was no visible bacteriostatic ring around the CP Ti sample (Figure 9a), indicating that Ti had no antibacterial effect.By contrast, semitransparent circular bacteriostatic rings of different sizes existed around the Mg samples, indicating that the Mg samples showed good antibacterial effects (Figure 9b-d).This may be due to the rapid increase in pH value of the bacterial suspension of the Mg samples, showing a strong antibacterial effect against S. aureus. [100]The IZD values of the HE samples were 0.36 ± 0.05 mm for the CP Mg, 1.06 ± 0.03 mm for the MZCS, and 1.79 ± 0.05 mm for the MZCZ (Figure 9i).According to ISO 20645, [101] an IZD equal to or larger than 1 mm indicates a significant antibacterial capacity.It can be concluded that the CP Mg showed some antibacterial effect, the MZCS showed a significant antibacterial effect, and the MZCZ showed the best antibacterial effect among all the metal samples.
The numbers of adherent CFUs and CFUs of the CP Ti were significantly higher than those of the Mg samples (Figure 9e-h,j).At the same time, the change rule for the CFU was negatively correlated with the IZD; a higher IZD value showed a smaller CFU.The MZCZ alloy showed the lowest IZD among all the metal samples, confirming its best antibacterial effect.Figure 9k-n shows SEM images of bacterial adhesion on the sample surfaces.Some white spherical colonies were attached to the sample surfaces (red arrow) and the numbers of bacteria were consistent with the numbers of CFUs.
The MZCS and MZCZ alloys showed a higher antibacterial effect than the CP Mg, mainly due to the release of Zn 2+ with a good antibacterial effect during the degradation process.Zn ions have antibacterial effects on both S. aureus and Escherichia coli. [102]Zn 2+ ions have been shown to potentially interact with negatively charged bacterial cell membranes, causing changes in membrane permeability and reducing membrane integrity. [103]his interaction can result in leakage and lead to bacterial death.Additionally, Zn ions have the ability to bind to and inactivate bacterial proteins, and these Zn ions also interact with bacterial nucleic acids to hinder bacterial migration. [104]mpared with the MZCS alloy, the MZCZ alloy had a better antibacterial effect.This may be due to the higher concentration of metal ions in the bacterial culture medium of the MZCZ alloy based on the corrosion rate, especially the Zn 2+ concentration, which improved antibacterial performance.At the same time, Zr ions can destroy the integrity of the cell membrane after contact with it, resulting in leakage of bacterial contents and thus promoting improvement in antibacterial effect. [105]

Conclusions
In this study, MZCS and MZCZ alloys have been developed by alloying and hot extrusion as biodegradable metals for boneimplant applications.Their mechanical properties, corrosion be-havior, cytocompatibility, osteogenesis performance, and antibacterial ability have been systematically assessed.The main conclusions are as follows.Overall, the MZCZ sample is a promising candidate material for bone-fracture fixation applications such as biodegradable bone-plate implants due to its unique combination of good mechanical properties, suitable degradation rate, and good cytocompatibility, osteogenesis performance, and antibacterial ability.

Figure 1 .
Figure 1.Microstructural characteristics of AC and HE samples: a) XRD patterns; b) OM image of AC CP Mg; c) OM image of AC MZCS; d) OM image of AC MZCZ; e) OM image of HE CP Mg; f) OM image of HE MZCS; g) OM image of HE MZCZ; h) SEM image of AC MZCS; i) SEM image of AC MZCZ; j) SEM image of AC MZCS; k) SEM image of HE MZCZ; l) EDS mapping of HE MZCS; and m) EDS mapping of MZCZ.
Figure 3c-h 1 shows the tensile fracture surfaces of the AC and HE samples.The AC CP Mg showed a smooth fracture surface, many dissociation facets, and a small number of small and shallow dimples, indicating a mixed mode of cleavage and ductile fracturing.The AC MZCS and MZCZ samples showed typical brittle fracture characteristics with an intergranular fracture morphology, many torn edges, and almost no cleavage fractures or dimples.In addition, large amounts of the second phases were deposited at the GBs and many second phases were separated from the b-Mg matrix and became sources of cracking.However, the fracture surfaces of the HE samples became notably finer than those of the AC samples.The HE CP Mg still had a large number of cleavage planes and a small number of dimples, with clearer plastic deformation, smaller cleavage planes, and deeper, wider, and more uniform dimples than its AC counterpart, showing better plastic deformation ability.A large number of dimples and a small number of cleavage planes were distributed on the HE MZCS sample with almost no intergranular fracture morphology, showing a mixed fracture mode of large ductile fracturing and small cleavage fracturing.In addition, the second phase with some cracks was uniformly embedded in the bottom of the dimples.The HE MZCZ samples had the deepest and widest dimples among all HE samples, showing the best plastic deformation ability, consistent with their elongation results.In addition, the HE MZCZ samples had tiny nanoscale second phases at the bottom of the dimples, showing a typical dimple-microporous aggregation fracture mode.Figure3ishows the microhardness values of AC and HE samples.The hardness values of the AC and HE samples in descending order were MZCS > MZCZ > CP Mg and the HE samples showed higher hardness values than their AC counterparts.The HE MZCS sample showed the highest hardness value of 62.2 ± 1.5 HV among all AC and HE samples.Figure3j-oshows OM images of the testing area, load-depth variation graphs, nanohardness, and reduced elastic modulus of different phases of the HE MZCS and MZCZ samples after nanoindentation.The nanohardness values of the different phases in the HE MZCS alloy in descending order were Sr 2 Mg 17 (2.75 ± 0.50 GPa) > deformation band (1.35 ± 0.10 GPa) > -Mg (0.87 ± 0.09 GPa) and their corresponding reduced elastic modulus values in descending order were Sr 2 Mg 17 (55.5 ± 5.3 GPa) > deformation band (41.6 ± 0.9 GPa) > -Mg (39.3 ± 1.0 GPa).It can be inferred that the Sr 2 Mg 17 second phase had greater nanohardness and lower elastic modulus than those of the -Mg matrix and deformation band.The nanohardness and reduced elastic modulus of the HE MZCZ alloy in the deformation band (0.86 ± 0.08 and 43.1 ± 1.5 GPa) were higher than those of the -Mg (0.72 ± 0.07 and 36.7 ± 1.3 GPa).In the solidification process of Mg alloys, Zn and Ca mainly cause -Mg GB segregation through the solute effect, forming a strong component of undercooling due to solute redistribution at the solid-liquid interface front, thus hindering -Mg grain growth.[53]At the same time, the aggregation of Zn atoms at the -Mg crystallization interface front can also hinder the growth of -Mg grains because the binding energy between Zn and Mg atoms is lower than that of the Mg solid-liquid interface.[54]Therefore, the two factors together have a notable grain-refining effect.According to the Mg-Sr phase diagram,[33] Sr can be enriched at the solid-liquid growth interface, hindering the continuous growth

Figure 2 .
Figure 2. SEM images and EBSD analysis results for HE samples: a-c) SEM images; d-f) EBSD IPF maps; g-i) the corresponding GB maps; j-l) orientation distribution; and m-o) grain size distribution.

Figure 3 .
Figure 3. Mechanical properties and fracture morphologies of AC and HE samples: a) tensile stress-strain curves; b) tensile property bars; c-h 1 ) tensile fracture surfaces; i) microhardness values of AC and HE samples; OM image of nanoindentation area in j) HE MZCS; and m) HE MZCZ; load-depth variation graph of k) HE MZCS; and n) HE MZCZ; nanohardness and reduced elastic modulus of l) HE MZCS; and o) HE MZCZ.

Table 4 .
Mechanical properties and microhardness of AC and HE samples compared to Mg-Zn-based biomaterials and target values for orthopedic implant materials.HT: heat-treated; HR: hot-rolled; ST: solution treated.

Figure 4 .
Figure 4. Corrosion and degradation behaviors of AC and HE samples in Hanks' solution: a) OCP curves; b) PDP curves; c) Nyquist plots; d) equivalent circuit model; e) Bode impedance diagrams; and f) Bode phase-angle diagrams; g) H 2 volume and pH value evolution of Hanks' solution with different immersion times; h) degradation rates after different immersion times; and i) metal ion concentrations of Hanks' solution after 21 days of immersion.

Figure 5 .
Figure 5. Surface morphologies and chemistry analyses of HE CP Mg, MZCS, and MZCS samples after immersion in Hanks' solution for 3, 10, and 21 days: a-i) SEM images; j) EDS spectra; k) XRD patterns; l) FTIR spectra; m) XPS spectra of corrosion products; and n) the corresponding highresolution XPS spectra of corrosion products after immersion for 21 days.

Figure 6 .
Figure 6.a-i) SEM images of corrosion products on cross-sections of samples after immersion in Hanks' solution for 3, 10, and 21 days; j-l) the corresponding EDS mapping results for corrosion products on cross-sections of HE samples after 21 days of immersion; m) 3D surface morphology; n) Volta potential map; o) the corresponding surface height and Volta potential line profiles of KPFM on cross-sections of MZCS sample after 21 days of immersion.

Figure 7 .
Figure 7. a) Mg 2+ concentration and pH of extracts; b) cell viability; c-f) fluorescent live/dead staining images; and g-j) DAPI staining images of MC3T3-E1 cells after 3 days of culturing with HE sample extracts.

Figure 7a shows
Figure 7a shows the Mg 2+ concentration and pH values of the HE sample extracts.The changing trends of Mg 2+ concentration and pH values of the extracts of the alloy samples were consistent with those of the Hanks' solution after the immersion testing, and the Mg 2+ concentration and pH of the extracts were lower than those of the Hanks' solution after immersion.The MZCS alloy showed the lowest Mg 2+ concentration of 15.7 ± 0.4 μg mL −1 and the lowest pH of 8.4 ± 0.1 among all samples.Figure7bshows the cell viability of MC3T3-E1 cells after culturing with different concentration extracts.All concentrations of the metal extracts showed high cell viability greater than 95%, demonstrating grade 0 to 1 cytotoxicity according to ISO 10993-5.[87]At 100% and 75% concentrations, the MZCS and MZCZ alloys showed higher cell viability than the CP Mg, indicating that both samples had better cytocompatibility.When the extracts were diluted to 50%, all extracts showed a significant cell

Figure 8 .
Figure 8. ALP activity of HE samples: a) ALP staining of control; b) ALP staining of CP Mg; c) ALP staining of MZCS; d) ALP staining of MZCZ; and e) ALP activity of HE samples and control.

Figure 9 .
Figure 9. Antibacterial effect of HE samples after 1 day of coculturing with S. aureus: a-d) IZD images; e-h) adherent CFU images; i) IZD values; j) CFU numbers; and k-n) SEM of bacterial adhesion on sample surfaces.

1 )
The microstructure of the AC and HE MZCS and MZCZ samples was composed of -Mg matrix phase and intermetallic phases of Mg 7 Zn 3 , Mg 2 Zn 11 , and Ca 2 Mg 6 Zn 3 , as well as a Sr 2 Mg 17 intermetallic compound in the MZCS.Most fine in-termetallic compounds were attached to the black-striped deformation bands that ran parallel to the extrusion direction.The HE MZCZ sample showed the finest grain size among all the AC and HE samples due to recrystallization and the grain-refining effect of Zr. 2) The HE MZCZ alloy exhibited the highest tensile strength among all other AC and HE alloys, with  ys of 208.4 MPa,  uts of 255.8 MPa, and  of 15.7%, while the HE MZCS alloy showed the highest hardness of 62.2 HV among all other alloys.3) In PDP testing, the HE samples showed increased corrosion resistance with increasing immersion time in Hanks' solution except for the 3 days immersion samples.The HE MZCS sample showed the highest corrosion resistance among all the samples, with the lowest V corr of 4 μm per year obtained from electrochemical testing, the lowest V w of 368 μm per year, and the lowest H 2 volume of 0.83 mL cm −2 per day obtained from immersion testing after 21 days of immersion.4) The HE samples showed grade 0-1 cytotoxicity toward MC3T3-E1 cells at all concentrations of metal extracts.The MZCZ sample showed the highest cell viability among all the metal extracts, exhibiting the highest cytocompatibility except at 25% concentration.Further, the MZCZ alloy showed better osteogenesis performance and antibacterial effect against S. aureus than the CP Mg.

Table 3 .
EDS analysis results for Spots 1-11 marked in Figure1h-k of SEM images of AC and HE MZCS and MZCZ.

Table 5 .
Electrochemical performance parameters and degradation rates of AC and HE samples in Hanks' solution.

Table 6 .
Fitted data of EIS spectra for AC and HE samples in Hanks' solution.