Impact of copper on corrosion behavior, biocompatibility and antibaterial activity of biodegradable Zn-3Mg-xCu alloys for orthopedic applications

Biodegradable zinc (Zn) based materials have been regarded as promising candidates for orthopedic applications owing to their suitable biodegradability. However, pure Zn exhibited poor mechanical performance and inadequate biofunctionality, which restricted its biomedical applications. Herein, biodegradable Zn-Mg-Cu alloys were developed to enhance the mechanical strength of the Zn matrix and endow the alloys with antibacterial activity. The effect of Cu addition on corrosion behavior, biocompatibility and antibacterial activity of biodegradable Zn-3Mg-xCu alloys was systematically investigated. In vitro immersion test revealed that Zn-3Mg-1Cu exhibited an increasing corrosion rate of 0.0504 mm y−1. The relative cell availability of MC3T3-E1 cells was over 70% after co-culture with 2-fold diluted extracts of the Zn-3Mg-xCu alloy for 3 d, indicating acceptable cytotoxicity. The Cu addition could enhance the antibacterial activity of the Zn-3Mg matrix, and Zn-3Mg-1Cu alloy exhibited the highest inhibition zone diameter (IZD) values of 10.4 mm and 6.0 mm against S. aureus and E. coli, respectively. Overall, the Zn-3Mg-1Cu could be recognized as a promising biodegradable orthopedic material owing to favorable degradation behavior, satisfying biocompatibility, and substantial antibacterial ability.


Introduction
Biodegradable implant materials, which could satisfy clinical requirements for mechanical properties, degradation rate, and requisite biocompatibility [1][2][3].In the past decades, there have been mainly three types of degradable metallic materials, including such as iron (Fe)-, magnesium (Mg)-, and Zn-based alloys [4,5].However, among these biodegradable, Fe-based materials exhibited an excessively low degradation rate, which can not meet the clinical requirement of total degradation in 2 years after implantation [6].Also, the degradation products of Fe-based materials were nonabsorbable and hard to excrete from the human body.On the other hand, Mg-based materials have proven effective in clinical applications with good biocompatibility and osteogenic capability, while the excessively high degradation speed restricted the application of Mg-based as biodegradable implants [7,8].Further, hydrogen generation during the degradation process of Mg-based implants could cause the hydrogen accumulation around the implant, inducing pain and inhibiting wound healing [9,10].
Zn, as a widely distributed essential trace element, plays a significant role in enzyme synthesis, DNA replication, transcription, and expression [11][12][13].More importantly, Zn can promote osseointegration and osteoinduction during the bone healing process, indicating the great potential of Zn-based materials as a biofunctional orthopedic implant [14,15].Moreover, elemental Mg, as a major essential dietary element participates in many biochemical reactions and enzyme reactions in the human body.From the perspective of biosafety, Mg should be an ideal alloying element for the Zn matrix to develop a biocompatible and biofunctional Zn-based alloy [16,17].Moreover, Mg is a critical factor for glucose metabolism and the cellular respiratory system and a stabilizing function of DNA and RNA [18,19].A previous study revealed that Zn-1Mg alloy lead to no side effect on cell multiplication and no cytotoxicity to MG63 osteoblasts, exhibiting improved cytocompatibility [20].Zhao et al revealed that Zn-3Mg implants trigger appropriate immune responses and appealing osteogenic reactions for guiding bone tissue regeneration [21].In another previous work, in vivo investigation proved that there is no gas evolution during degradation of a Zn-0.8Mg-0.2Sr orthopedic implant and no inflammatory reaction or bone resorption under service for 120 d [22].Furthermore, Cu was widely employed as an effective alloying element enabling metallic materials to inhibit bacterial infection and postoperative inflammation [23,24].Moreover, Cu showed high corrosion potential and high solid solubility in the Zn matrix [25].In other words, the substitutional solid solution of Cu in the Zn matrix could contribute to the homogeneously increased corrosion potential of the Zn matrix, thereby achieving the uniform and simultaneous release of Zn and Cu.Qu et al found that the Zn-Cu alloy suppressed wall synthesis, colonization, adhesion, and biofilm formation in methicillin-resistant staphylococcus aureus (MRSA) [26].In vivo investigation disclosed that Zn-Cu alloy eliminated MRSA infection and reduced inflammatory toxic side effects.Shi et al reported that alloying of 0.4 wt% Cu in the Zn-Mn alloys enhanced their strengths and upregulated the corrosion speed without the adverse effect on biocompatibility [27].Lin et al developed Zn-1Cu-0.1Tiand Zn-3Cu-0.2Tialloys with substantial antibacterial ability and good blood compatibility, which is attributed to the continuous release of Cu [28,29].Hence, the simultaneous addition of Mg and Cu in the Zn matrix could endow the Zn-based materials with adjustable degradation behavior, improved biocompatibility, enhanced osteogenic capability and substantial antibacterial activity.However, the comprehensive effect of Cu on the corrosion behavior, biocompatibility, and antibacterial activity was still obscure, and the feasibility of the newly designed Zn-Mg-Cu alloy for orthopedic applications indeed needs to be verified.
In the present study, we developed biodegradable Zn-Mg-Cu alloys with various Cu contents via gravity cast and hot extrusion process for orthopedic application.The synergetic addition of Cu and Mg could simultaneously enhance the biocompatibility and antibacterial activity of the Zn matrix.The microstructure, mechanical performance, physicochemical properties, in vitro cytocompatibility and antibacterial ability of alloys were systematically investigated to examine the feasibility of Zn-Mg-Cu alloys as implant materials.

Materials and methods
2.1.Preparation of biodegradable Zn-3Mg-xCu (x = 0, 0.5, 1 wt%) alloys As-cast Zn-3Mg-xCu were cast by melting pure elemental Zn (99.995%),Mg (99.995%) and Cu(99.99%) in a graphite crucible at 873 K in an atmosphere of N 2 + SF 6 , followed by pouring into a cylindrical die which was preheated to 473 K. Homogenization treatment was executed on as-cast ingots at 633 K for 12 h followed by water quenching.After the homogenization process, the ingot were hot extruded at 473 K with an extrusion ratio of 56.25 and a crosshead speed of 2 mm/s.Finally, Zn-3Mg-xCu alloys bar with a diameter of 8 mm were obtained.The actual composition of the as-extruded Zn-3Mg-xCu alloys was determined by the x-ray fluorescence spectroscopy (XRF) and the results are summarized in table 1.

Phase identification of Zn-3Mg-xCu alloys
Phase identification of Zn-3Mg-xCu alloys were performed by x-ray diffractometry (Rigaku smartlab 9 kW) operating with the Cu Ka radiation, using a test range of 10°to 90°and a scanning speed of 5°min −1 .An annealed Si standard specimen was used for the calibration of the XRD device.

Microstructural investigation of Zn-3Mg-xCu alloys
Field-emission scanning electron microscopy (FE-SEM) (Quanta FEG 250) with energy-dispersive x-ray spectroscopy (EDS) detector was used for microstructure observation.Disc specimens for SEM investigation were cut from the cross-section of the bars.These specimens were ground with SiC papers and mechanically here k is a constant of 8.76 × 10 4 , M is weight loss after immersion test (g), A is contact area (cm 2 ), t is immersion period (h), andρ is relative density of Zn-3Mg-xCu specimens (g•cm 3 ).Three parallel samples were tested immersed for each period.

Cytotoxicity and cytocompatibility assay for Zn-3Mg-xCu alloys
The cytotoxicity of the Zn-3Mg-xCu specimens was evaluated using the extract method according to ISO 10993-12 standard [31].MC3T3-E1 pre-osteoblasts cell (ATCC CRL-2594) were purchased from Beijing Zhongyuan Ltd and selected as model cell.Zn-3Mg-xCu samples were set in alpha-minimum essential medium (α-MEM) for 24 h at 310 K. Thereafter, the extracts were collected.The cells were incubated with a medium consisting of 0.1 mM α-MEM with 10% fetal bovine serum (FBS), 100 units ml −1 penicillin, and 100 μg ml −1 streptomycin.Cells were seeded into a well plate by a density of 5000 cells per well.After culture for 24 h, the medium was replaced by extracts with various concentrations (6.5%, 12.5%, 25%, 50%, and 100%) and incubated for 1 d, 2 d and 3 d. 10 μl Cell Counting Kit-8 (CCK-8) reagent was added to every well and then the cells were cultured under the identical conditions for another 3 h.Absorbance for 450 nm irradiation was measured using a microplate absorbance reader (iMark, Bio-Rad, USA) and then the cytotoxicity were evaluated by relative absorbance.
Afterwards, live cell were stained by Hoechst 33342, while the dead cell was stained by Propidium Iodide (PI).Laser scanning confocal microscopy (LSCM) was used to observe the cell adhesion and cell distribution.

. antibacterial evaluation for Zn-3Mg-xCu alloys
A agar disk diffusion test was used to estimate the antibacterial ability of the Zn-3Mg-xCu alloys.The sterilized Zn-3Mg-xCu alloy discs were set in the center of the agar plates.Thereafter, 500 μl the bacterial suspension was uniformly painted on the plates.The inhibition zone were determined after culture at 310 K for 24 h, and IZD values were calculated by the following formula [28]: where D is the diameter of the inhibition circle and d is the discs' diameter.

Results and discussion
3.1.Phase identification, microstructure investigation of Zn-3Mg-xCu alloys Figure 1 shows x-ray profiles of as-extruded Zn-3Mg-xCu alloys, suggesting that the main constitution in these alloys was α-Zn.Despite different Cu contents, the existence of Mg 2 Zn 11 and MgZn 2 secondary phases in addition to the α-Zn matrix were confirmed in all Zn-3Mg-xCu alloys.Mg 2 Zn 11 and MgZn 2 secondary phases were familiar in Zn-Mg alloy system, which could strengthen the materials and facilitate the degradation process.No peak related to CuZn 5 secondary phase was confirmed in the experimental specimens, which could attribute to the low amount of Cu and high solid solubility of Cu in the Zn matrix.Figure 2 exhibits the SEM micrographs of the cross-section of the as-extruded Zn-3Mg-xCu alloys bars.For Zn-3Mg and Zn-3Mg-0.5Cuspecimens, there are some equiaxed α-Zn grains with relatively large size, which incorporate with many eutectic area.This kind of eutectic area is very familiar in Zn-Mg alloys system, which is consisting of α-Zn and Mg 2 Zn 11 dendrites.On the contrary, the boundary between α-Zn and eutectic area is ambiguous in Zn-3Mg-1Cu alloy, and the size of intermetallic dendrites obviously increased.Figure 3 provides the EDS point analysis results for Zn-3Mg-0.5Cuand Zn-3Mg-1Cu alloys.Two kinds of Zn-Mg intermetallics were confirmed in Zn-3Mg-0.5Cuspecimens.Combined with the XRD results, the Mg-rich intermetallic possessed a Zn to Mg ratio of about 4 : 1 referring to MgZn 2 phase, while the Mg-lean intermetallic exhibiting a Zn to Mg ratio of about 9 : 1 should be to Mg 2 Zn 11 secondary phase.It is worth mentioning that as compared with the theoretical ratio of these secondary phase, relatively low Zn to Mg ratio were detected by EDS.Small grain size of these intermetallics and the receive of the signal from the adjacent Zn matrix could be responsible for the higher deviation of the detected Zn to Mg ratio.On the other hand, Cu signal (0.8 at%) was confirmed in the α-Zn grains in Zn-3Mg-1Cu alloy, suggesting the solid solution of Cu in the Zn matrix.Interestingly, the 3.1  at% Cu was detected in the dendrites, which is higher than the solid solubility of Cu in the Zn matrix.Moreover, the ratio of Cu + Mg concentration to Zn content is close to 1 : 9, indicating that substitution solid solution in Mg 2 Zn 11 secondary phase occurred and resulted in a (Cu, Mg) 2 Zn 11 secondary phase.The disparate microstructure between Zn-3Mg and Zn-3Mg-1Cu alloys could attribute to high solid solubility of Cu in Mg 2 Zn 11 phase.The solid solution of Cu lead to the release of Mg and then facilitate the precipitation of more Mg 2 Zn 11 phase.Eventually, a whole eutectic structure consisted of α-Zn matrix and (Cu, Mg) 2 Zn 11 dendrites was garnered.It is worth mentioning that the the Cu(Zn) 5 secondary phase can not be confirmed in Zn-3Mg-0.5Cuand Zn-3Mg-1Cu alloy.The Cu only existed in the α-Zn matrix and (Cu, Mg) 2 Zn 11 as solid solution atom.According to the EDS analysis (figures 3), 3.1 at% Cu, which is much higher than the designed Cu concentration in Zn-3Mg-1Cu alloy, were detected in the (Cu, Mg) 2 Zn 11 dendrites.On the contrary, the Cu concentration of Cu(Zn) 5 secondary phase was 16.7 at% higher than that of (Cu, Mg) 2 Zn 11 secondary phase.It is reasonable to assume that higher Cu concentration may induce severe Cu enrichment and result in the precipitation of Cu(Zn) 5 in Zn-Mg-Cu alloy system, but in this study the addition of 1 wt% Cu can not lead to the precipitation of Cu(Zn) 5 secondary phase.

Vickers hardness of biodegradable Zn-3Mg-xCu alloys
Table 3 exhibits the Vickers hardness of Zn-3Mg-xCu alloys determined using an applied load of 0.05 kg and a holding time of 15 s.The According to table 3, the Vickers hardness value of Zn-3Mg-xCu arise with Cu addition.The HV value was increased from 170.8 ± 1.4 to up to 191.0 ± 2.7 by 1 wt% Cu addition.In other words, the Vickers hardness increased by 11.8% after 1 wt% Cu addition.It is supposed that the solid solution of Cu in Zn matrix and Mg 2 Zn 11 secondary phase contributed to the elevated HV value.

In vitro degradation behavior of biodegradable Zn-3Mg-xCu alloys
The degradation behaviour of Zn-3Mg-xCu alloys was evaluated by immersion test in SBF solution at 310 K for 3 to 30 d. Figure 4 exhibits the corrosion morphology of Zn-3Mg-xCu alloys after different immersion times.Obviously, the amount of corrosion products increased with the extended immersion time.In spite of various immersion period, scratches caused by mechanical grinding were confirmed on all the specimens' surface, suggesting the uniform and mild corrosion process with a quite thin corrosion product layer.Hence, the original morphology (scratches) could be remained after 30 d immersion.Some corrosion product particles were observed after 30 d immersion and the distribution of these particles was relatively homogeneous, indicating that the degradation process of Zn-3Mg-xCu were mild and homogeneous.In the perspective of corrosion morphology, no apparent difference was confirmed among Zn-3Mg-xCu during the first 7 d immersion.Contrarily, when the immersion period was longer than 14 d, higher content of corrosion products were confirmed on the surface of Zn-3Mg-1Cu alloy than those on Zn-3Mg and Zn-3Mg-0.5Cualloys, illustrating a more severe corrosion process of Zn-3Mg-1Cu alloy.Furthermore, SEM-EDS investigation was performed to characterize the constitution of corrosion product layer.Figure 5. shows SEM images of Zn-3Mg-xCu alloys and corresponding EDS analysis results after 30 d immersion test.The signal of Zn, Mg, Ca, P and O were confirmed  on these corroded surface, possibly indicating the formation of ZnO, MgO and calcium-phosphate compounds.
It is widely believed that ZnO have a substantial antibacterial activity against various bacterial [32].The Ca/P ratio of the corrosion products varied from 0.44 to 0.49, which is lower than the Ca/P ratio of human bone.
Although the Ca/P ratio is not sufficient to build bone, a small amount of Ca and P can facilitate osteogenic differentiation and adhesion of osteoblast.Due to the rapid materials exchange in the human body, sufficient Ca and P could be transported to implant/bone interface for the bone regeneration by osteoblast.Hence, the deposition of Ca and P on the Zn-3Mg-xCu alloys mainly accelerate bone healing process by facilitating the osteogenic differentiation and osteoblast adhesion rather than providing the raw materials for bone regeneration.Even more to point, the signal of Cu can not be detected on the corroded surface.One possible reason for the absence of Cu signal was the very low content of Cu in Zn-3Mg-xCu.Another reason is the Cu ion could react with Zn matrix and product elemental Cu due to the negative Gibbs free energy (−173.8kJ mol −1 at room temperature) [33].Hence, the Cu could be detached from the corroded area as elementary Cu particles, resulting in the absence of Cu signal detected by EDS.More importantly, Cu particles not only could provide a broad-spectrum antibacterial effect by themself but also could contribute to a synergistic antibacterial effect with Zn ions. Figure 6 shows surface morphology without corrosion products of Zn-3Mg-xCu alloys after immersion test in SBF solution for 30 days.According to these SEM images, some localized corrosion pits and others uniformly corroded area were confirmed on these corroded surface.The number of corrosion pits increased with the increasing Cu content, probably suggesting the increased tendency of the localized corrosion.Moreover, as compared with Zn-3Mg alloy, the corroded surface of uniformly corroded domain became more rough and fluctuate in Zn-3Mg-1Cu alloy.Hence, the addition of Cu could facilitate both uniform corrosion and localized corrosion, suggesting a shortened degradation period of Zn-3Mg-1Cu alloy.The relatively high degradation rates may attribute to the lack or discontinuity of the protective corrosion product layer.Accompany with the increasing immersion time (7-30 days immersion), thickness, continuity and stability of protective corrosion product layer increased, contribute to the continuous declination of degradation rates.After 30 d immersion in SBF solution, Zn-3Mg, Zn-3Mg-0.5Cuand Zn-3Mg-1Cu alloys possessed degradation rates of 0.032 mm y −1 , 0.036 mm y −1 and 0.050 mm y −1 , respectively.Thus, the addition of Cu barely impact degradation progression of the initial stage of corrosion, while Cu addition can slightly accelerate the corrosion process after 21 d immersion.This phenomenon may attribute to the prevalence of (Cu, Mg) 2 Zn 11 secondary phase.According to SEM investigation, the 1% Cu addition facilitate the precipitation of (Cu, Mg) 2 Zn 11 secondary phase.As is known to all, (Cu, Mg) 2 Zn 11 possessed lower surface Volta potential than that of Zn matrix, which means that the Zn matrix as potential anode and (Cu, Mg) 2 Zn 11 secondary phase as possible cathode could form a galvanic coupling, thereby accelerating the Zn matrix dissolution and corrosion progression.The a large amount of (Cu, Mg) 2 Zn 11 secondary phase could induce the massive galvanic corrosion, and could deteriorate the integrity of protective layer.Eventually, the Cu addition up-regulate the degradation rate during the 30 d immersion test in  Zn-3Mg-1Cu alloy.Generally, Zn-based materials exhibited a longer degradation period than the ideal one (total degradation in two years after implanation) [6].For the sake of well-matched degradation and bone regeneration periods, a shortened degradation process is preferable to extended one for biodegradable Zn-based alloy.Hence, Zn-3Mg-1Cu alloy possessed a more favorable degradation period than Zn-3Mg alloy as a potential orthopedic material.Above all, in vitro immersion test revealed that Zn-3Mg-1Cu alloy possessed an accelerated corrosion rates and can prompt the deposition of biofunctional ZnO and calcium-phosphate compound, thus exhibiting a favorable degradation behavior as a potential orthopedic material.

In vitro cytocompatibility of biodegradable Zn-3Mg-xCu alloys
Figure 8 shows the cell availability of mouse osteoblasts MC3T3-E1 cell after 1, 2, and 3 days cultured with 6.25%, 12.5%, 25%, 50% and 100% extracts.When the extract was diluted, the cell viabilities of all experimental groups with various extract concentrations were over 70%, indicating 0-I grade cytotoxicity and no obvious cytotoxicity of diluted extracts to MC3T3-E1 cell.However, the indirect contact assay indicated that 100% extracts could deteriorate the cell availability to lower than 60%, which means a confirmed cytotoxicity.Moreover, the addition of Cu resulted in decreasing viability of MC3T3-E1 cell of the groups using 50% and 100% extracts.On the contrary, the groups cultured by 6.25%, 12.5% and 25% extracts exhibit similar cell viability, suggesting the Cu addition have limited effect in these groups.Due to the rapid materials exchange caused by metabolism in the human body, the diluted extracts was more suitable to evaluate the cytocompatibility of biodegradable materials than the original one.A 6 to 8 fold dilution of extract was appropriate to simulate the actual situation in the human body [34].Therefore, although the Cu addition could induce the slightly decreased of cell viability in the groups using 50% and 100% extracts, the negative effect of Cu addition in biodegradable Zn-3Mg-xCu implant should be limited under service conditions.
Figure 9 exhibits the fluorescent images of MC3T3-E1 cell after directly co-culture with Zn-3Mg-xCu alloys for 24 h.(MC3T3-E1 cell were stained by Hoechst 33342 (blue) and PI (red)).Blue fluorescent with uniform dispersion and high intensity were observed in the Zn-3Mg group, illustrating the excellent viability of MC3T3-E1 cell on the surface of Zn-3Mg alloy.With the increasing Cu content, the declined intensity of blue fluorescent were confirmed on the surface of Zn-3Mg-0.5Cuand Zn-3Mg-1Cu, possibly indicating the lower cell adhesion ability or substantial cytotoxicity of these surface.Furthermore, a very few dot with the red fluorescent were confirmed on the experimental surface, suggesting non cytotoxicity of these surface.Hence, the decreased blue fluorescent intensity was derived from the interior cell adhesion rather than substantial cytotoxicity.More importantly, the blue intensity were uniformly detected on all the experimental surface, which means MC3T3-E1 cell homogeneously adhered on the Zn-3Mg-xCu alloys, thereby probably resulting in a uniform bone regeneration effect.To sum up, acceptable biocompatibility to MC3T3-E1 cells was testified in Zn-3Mg-xCu alloys, suggesting that the Zn-3Mg-xCu alloy could be potential candidates as the biodegradable implant materials.

In vitro antibacterial behavior of biodegradable Zn-3Mg-xCu alloys
Figure 10 depicts the antibacterial behavior of Zn-3Mg-xCu alloys against E. coli and S. aureus.Obvious inhibition zone around Zn-3Mg-xCu alloys were observed by the agar disk diffusion assay against S. aureus, proving the substantial antibacterial capability of Zn-3Mg-xCu.Moreover, the Cu addition lead to the expansion of the inhibition zone, which means the Cu addition enhance the antibacterial effect of Zn-3Mg alloy.Interestingly, the only very small inhibition area around Zn-3Mg alloy were confirmed against E. coli.Although the inhibition zone enlarged with the 1% Cu addition, the inhibition area of Zn-3Mg-1Cu against E. coli was lower than that against S. aureus.That may be due to the sensitivities of E. coli to Zn ion and Cu particles were lower than those of S. aureus.The IZD value determined by the agar disk diffusion assay were summarized in figure 11.Zn-3Mg alloy exhibits very limited antibacterial activity against E. coli, whose IZD value is 2.0 ± 0.2 mm.The IZD value against E. coli significantly increased with the increasing Cu content and up to 6.0 ± 0.5 mm at 1% Cu content.Similar to the case using E. coli, as model bacterial, the IZD value against S. aureus also increased from 8.5 ± 0.8 mm in Zn-3Mg group to 10.4 ± 1.1 mm in Zn-3Mg-1Cu group.The previous study revealed that Zn ions was an effective ion against various bacterial.Notably, the Zn ions released from Zn-3Mg-xCu during the corrosion process could suppress the bacterial and result in antibacterial ability.However, the limited antibacterial ability of Zn-3Mg against E. coli indicated that only released Zn ion can not endow the Zn-  based materials with significant antibacterial activity.On the other hand, many kinds of Cu-containing or Agcontaining biomaterials were extensively researched to develop the metallic materials with antibacterial activity [35,36].Elementary Cu particles and Cu ions can effective destroy the bacterial membrane and eliminate the various bacterial [37].Dai Y et al verified substantial antimicrobial ability of Zn-4Ag-1Cu alloy, and the synergistic effect of Zn, Ag and Cu considerably suppressed the formation of bacterial colony and inhibited bacterial reproduction [38].More importantly, the Cu ions and particles exhibits more destructive effect on the double membrane structure of Gram-positive bacterial (E. coli in these study) [37].The Cu addition significantly enhance the antibacterial activity against E. coli, while the IZD value to S. aureus only slightly increased after alloying with 1% Cu.Hence, synergy effect of released Zn ions, Cu ions and Cu particles could be responsible for the ameliorated antibacterial property of Zn-3Mg-1Cu biomaterial.
To sum up, Zn-3Mg-1Cu possessed slightly accelerated corrosion rate, permissible biocompatibility, and ameliorated antibacterial performance can be a promising biomaterials for orthopedic applications.An in vivo model will be well-designed and established in the future to systematically investigate the in vivo biocompability and degradation behavior of Zn-3Mg-1Cu alloy.

Conclusions
In the current work, biodegradable Zn-3Mg-xCu alloys were developed via gravity casting and hot extrusion process.The microstructure, degradation performance, cytocompatibility and antibacterial ability of the experimental alloys were systematically investigated.The main findings can be summarized as follows: 1. Zn-3Mg exhibited a microstructure consisted of α-Zn matrix, Mg 2 Zn 11 secondary phase and MgZn 2 secondary phase.The Cu atom not only can dissolve in α-Zn matrix as solid solution atom but also could substitute the Mg atom in Mg 2 Zn 11 secondary phase, thereby facilitating the precipitation of (Cu, Mg) 2 Zn 11 secondary phase in Zn-3Mg-1Cu alloy.
2. The Cu addition could facilitate the degradation process of Zn-3Mg-xCu alloy.Zn-3Mg-1Cu exhibited a increasing corrosion rate of 0.0504 mm•y −1 after 30 d immersion test.The corrosion products layer mainly consisted of ZnO, MgO and calcium-phosphate compounds, possibly indicating antibacterial ability and osteogenic capability.
3. The cytotoxicity assay revealed that 2-fold diluted extracts of the Zn-3Mg-xCu alloy showed limited cytotoxicity.The relative cell availability of MC3T3-E1 cells were over 70% after co-culture with 2-fold diluted extracts of the Zn-3Mg-xCu alloy for 3 d.
4. The Cu addition also could enhance the antibacterial activity of Zn-3Mg-xCu alloy.Zn-3Mg-1Cu alloy possessed the highest antibacterial ability, and exhibited the IZD values of 10.4 mm and 6.0 mm against S. aureus and E. coli, respectively.
Collectively, Zn-3Mg-1Cu alloy is a promising biodegradable materials for orthopedic applications, and the in vivo degradation behavior and biocompatibility of the alloy will be investigated in the near future.

Figure 4 .
Figure 4. Corrosion morphology of Zn-3Mg-xCu alloys after immersion test in SBF solution for different periods.

Figure 7
Figure7displays degradation rate of the Zn-3Mg-xCu in SBF solution at 310 K with various immersion periods.For all the experimental alloys, the degradation rate declined with increasing immersion period.During the first 7 days of immersion tests, the relatively high degradation rates around 0.1 mm•y −1 were determined in Zn-3Mg-xCu alloys despite different Cu content.The relatively high degradation rates may attribute to the lack or discontinuity of the protective corrosion product layer.Accompany with the increasing immersion time (7-30 days immersion), thickness, continuity and stability of protective corrosion product layer increased, contribute to the continuous declination of degradation rates.After 30 d immersion in SBF solution, Zn-3Mg, Zn-3Mg-0.5Cuand Zn-3Mg-1Cu alloys possessed degradation rates of 0.032 mm y −1 , 0.036 mm y −1 and 0.050 mm y −1 , respectively.Thus, the addition of Cu barely impact degradation progression of the initial stage of corrosion, while Cu addition can slightly accelerate the corrosion process after 21 d immersion.This phenomenon may attribute to the prevalence of (Cu, Mg) 2 Zn 11 secondary phase.According to SEM investigation, the 1% Cu addition facilitate the precipitation of (Cu, Mg) 2 Zn 11 secondary phase.As is known to all, (Cu, Mg) 2 Zn 11 possessed lower surface Volta potential than that of Zn matrix, which means that the Zn matrix as potential anode and (Cu, Mg) 2 Zn 11 secondary phase as possible cathode could form a galvanic coupling, thereby accelerating the Zn matrix dissolution and corrosion progression.The a large amount of (Cu, Mg) 2 Zn 11 secondary phase could induce the massive galvanic corrosion, and could deteriorate the integrity of protective layer.Eventually, the Cu addition up-regulate the degradation rate during the 30 d immersion test in

Figure 7 .
Figure 7. Degradation rates derived from immersion test in SBF for various periods.

Figure 11 .
Figure 11.The inhibition zone diameter of Zn-3Mg-xCu alloys against (a) S. aureus and (b) E. coli after co-culture for 1 day.
were removed.Thereafter, corrosion products were removed by chromic acid solution (200 g/l).The weight loss before and after immersion tests were measured by a digital balance.Finally, the degradation rate (DR)was calculated by the following equation:

Table 2 .
The components of SBF solution.

Table 3 .
Vickers hardness of Zn-3Mg-xCu alloys and the hardness improvement caused by the Cu addition.