Hongyn Wng ,Mingyun Bi ,Honn Yun ,Ychen Hou ,Yiho Liu ,Zhe Fng ,Yufeng Sun,Jinfeng Wng,Shijie Zhu,Shokng Gun,b,?

a School of Materials Science and Engineering,Zhengzhou University,Zhengzhou 450001,China

b Henan Key Laboratory of Advanced Magnesium Alloys,Zhengzhou 450002,China

Abstract The protein adsorption has an immense influence on the biocompatibility of biodegradable Mg alloy.In this work,the effect of Zn content on the fibrinogen(Fg)adsorption behavior in Mg-Zn binary alloy was systematically investigated.Experimental results showed that the Fg adsorption amount increased at first and then decreased with the increase of Zn content.The adsorption mechanism was investigated by molecular dynamic and density functional theory simulations.The simulations results showed that Zn with low content existed in the inner layer of Mg alloys due to the lower system energy,which promoted Fg adsorption and the promotion effect was more obvious with the increase of Zn content.When Zn content increased to a higher concentration,parts of Zn atoms started to precipitate in the surface,and the Fg-surface interaction energy started to increase.Moreover,the Zn sites favored the formation of ordered water molecules layers,which inhibit the stable adsorptions of Fg.The inhibition effects of Fg adsorption was enhanced with the Zn content increase.In short,the simulation results explain the experimental phenomena and reveal the microscopic mechanism.This study would provide a significant guidance on the design of biodegradable Mg-Zn alloys.

Keywords: Molecular dynamics;Protein adsorption;Magnesium alloy;Zn content;DFT simulations.

1.Introduction

Biodegradable Mg and its alloys have been turned out to be a promising candidate for cardiovascular stents and orthopedic applications [1–3].However,the rapid degradation rate and the insufficient mechanical strength of pure Mg impede its clinical application[2–6].Addition of proper elements into Mg matrix is a convenient and main-stream method to make up these deficiencies [7,8].Until now,many biodegradable Mg alloy systems have been developed [1–5,7,9–13].Especially,the addition of Zn element has received extensive attention.Zn is one of the most abundant nutritionally essential elements for human beings[14].Moreover,Zn is also beneficial to elevate the corrosion potential and reduce the degradation rate of Mg in simulated body fluid(SBF),thus the degradation rate of the single phase Mg–6Zn alloy was slower than that of high-purity Mg in SBF [11].Furthermore,Zn can also significantly improve the mechanical properties of Mg[15].In short,Mg-Zn binary alloys or Mg-Zn-based alloys are considered to be suitable for biomedical applications [5,13,15–18].

Many studies have reported the influence of Zn contents on the mechanical properties and corrosion resistance of binary Mg-Zn alloy [19–21].Apart from corrosion resistance and mechanical properties,biocompatibility is another vital performance index of the implanted materials [4,22,23].However,the effect of Zn contents on the protein adsorption behavior of binary Mg-Zn alloy is still unclear.The protein adsorption is the initiating event when biomaterials enter the human body [24].Then,cell adhesion,activation,migration and proliferation will be regulated by the protein adsorption layer formed on the biomaterial surface [25].Therefore,the protein adsorption directly determines the biocompatibility of biomaterial.Vascular stents are required to have excellent blood compatibility,that is,the protein adsorption on the stents surface should be inhibited to avoid platelet adhesion and thrombosis [26].

Among plasma proteins,fibrinogen(Fg)has been recognized as a key mediator of inflammatory responses as well as platelet activation,blood coagulation and leukocyte binding [23,25].In addition,the Fg adsorption trend on the material surface was consistent with that of platelets.Such as,compared with pure Mg,the Mg-Ce and Mg-Ca alloys that promote Fg adsorption also promote platelet adsorption,and Mg-Mn alloy that restrains Fg adsorption also inhibits platelet adsorption [4,8,27].It is probably because that Fg consists of three protein chains,namelyα,β,andγ,and has platelet binding sites at the C terminal region of eachγchain[23].Therefore,the biomaterials with ultralow Fg adsorption should be more blood compatible for cardiovascular stents[25].

When the Zn content exceeds its solid solubility limit in Mg,the second phase containing Zn will be precipitated on the sample surface [11,15,19,20].In order to obtain different Zn content on the sample surface,according to the solid solubility of Zn in Mg and the references [11,15,19,20],binary Mg-xZn(x=1,2,3,5 and 10 wt.%)alloys were used for the Fg adsorption experiments in this work.Mg-xZn(x=1,2 and 3 wt.%)were single-phase solid solutions,and the second phases were precipitated on the Mg-xZn(x=5 and 10 wt.%)alloy surface.Pure Mg was adopted as a control material.In order to reveal the proteins adsorption mechanisms on the Mg-Zn alloy,computer simulation was also used in this paper to study the Fg adsorption behavior on the surface of Mg-xZn(x=0.7,1.4 at.%)(0001).The recombinant 30kDa C-terminal fragment of the human Fgγchain(FgγC30)was used as a model protein.By combining the molecular dynamics(MD)and density functional theory(DFT)simulations with the fluorescent Fg adsorption experiments,the microscopic adsorption mechanism of protein on Mg-Zn alloy was illustrated.It would provide scientific guidance for alloy design of novel biodegradable Mg alloys in the future.

2.Computational and experimental methods

2.1.Computational methods

2.1.1.DFT simulation details

The simulation model containing six layers atoms(144 atoms),with the bottom two layers fixed and the top four layers fully relaxed,was built by substituting Mg atoms with Zn atoms in the Mg(0001)surface.The supercell was separated by the vacuum region ofin thezdirection.The simulations based on projector augmented wave(PAW)method were performed with the Vienna Ab initio Simulation Package(VASP)[28–30].The cutoff energy of the plane-wave basis set was 550 eV in all simulations.Exchange-correlation of electrons was treated in the generalized gradient approximation(GGA)with the Perdew–Burke–Ernzerh(PBE)[31].The interatomic forces were set to be less 0.02for the convergence threshold of optimized configuration.The Brillouin zone was employed with a 3×3×1 k-point grid.The optB86b-vdW functional was used in the calculations [32].

2.1.2.MD simulation details

MD simulations of FgγC30(PDBID:1FID)[33]adsorbing on the Mg(0001)surface with different Zn content(0.7 and 1.4 at.%)was performed.The structure of FgγC30 was acquired from the RCSB(Research Collaboratory for Structural Bioinformatics)protein data bank [34].The Nterminus,lysine and arginine residues were treated as the protonated state,while C-terminus,glutamic acid and aspartic acid residues were treated as the deprotonated state.The net charge of FgγC30 was?5e.MD simulations were performed using Large-scale Atomic/Molecular Massively Parallel Simulator(LAMMPS package)[35].In total,the supercell of Mg-Zn alloys(4320 atoms)with dimensions of,were kept fixed during the MD simulations.The initial orientation of FgγC30 on surfaces was same to our previous work for Mg-Zn alloy [22],which were determined by parallel-tempering method.To ensure the spontaneous adsorption behavior,the initial distance between FgγC30 and the surfaces was set to be.Water molecules were added into the final simulation box forming a water layer for a thickness of(almost 16,046 molecules).The counter ion was added into the water box to get a neutral system.The Chemistry at Harvard Molecular Mechanics(CHARMM22)force field parameters was used for water molecules,counter ions and protein [36].Water molecules were depicted using the transferable intermolecular potential 3 point(TIP3P)model.The interaction potentials involving Mg and Zn atoms were obtained from the literature [37,38].The parameters of Lennard-Jones(L-J)potential for nonbonding atoms cross interactions,such as the counter ions,water molecules,protein and substrates,followed the Lorentz-Berthelot mixing rule [39],which had been widely used in different materials for calculating the intermolecular potential [40,41].The MD simulations were performed in the non-volcanic tremor(NVT)ensemble using thethermostat [42].The periodic boundary condition was implemented in three directions and time step was set to be 1 fs.The particle-particle-particle-mesh(PPPM)method was used to compute the long-range electrostatic interactions [43].Finally,simulation for each molecular assembly was performed at 310K in 16ns.The adsorption trajectories of FgγC30 were visualized by the Visual Molecular Dynamics(VMD)program [44].

2.2.Experimental methods

Alloy ingots with a nominal composition of Mg-xZn(x=1,2,3,5 and 10 wt.%)were produced using high-purity Mg and Zn,which were melted in an electrical resistance furnace under the protection of a mixed gas atmosphere of CO2+0.2% SF6.The melt was cast into a preheated steel mold(200°C)with dimension of 100mm×100mm×30mm.The samples with the dimension of 10mm×10mm×3mm,which cut from the as-cast ingot(pure Mg and Mg-Zn alloys),were used for the fluorescent Fg adsorption experiments.

Fig.1.Adsorption of fluorescent Fg on samples surfaces after immersion 20min,40min and 60min.(a1,a2,a3)pure Mg,(b1,b2,b3)Mg–1Zn,(c1,c2,c3)Mg–2Zn,(d1,d2,d3)Mg-3Zn,(e1,e2,e3)Mg-5Zn and(f1,f2,f3)Mg-10Zn alloy.The bright spots in the images are Fg.

All samples were ground with SiC paper and polished,followed by ultrasonically cleaning for 5min in ethanol.The 3D surface morphology of the samples is shown in Fig.S1(Supporting Information).It can be seen that the surface morphology and roughness of the polished samples are almost identical,which eliminate the effect of surface roughness on protein adsorption.

To characterize the surface energy of the surfaces,contact angles were measured by sessile-drop method.3 μL of deionized water were dropped on the sample surface and the liquid drop was photographed.5 different regions per sample were measured and the mean value was collected as the contact angle of each sample.The contact angles of all the samples are close to each other(Fig.S2 and Table S1),indicating the surface energy of the five alloys are similar [45,46].

The Mg–xZn alloys samples were exposed to Hank’s solution for 24 h at room temperature.The corroded samples were rinsed by distilled water and dried in air.Then the corrosion morphologies were observed using scanning electron microscope(SEM),which is shown in Fig.S3(Supporting Information).The corrosion resistance of Mg-xZn(x=1,2 and 3)is higher than that of Mg-xZn(x=5 and 10).

The fluorescent Fg adsorption on the sample surface during immersion was directly performed under cell culture conditions.The immersion time was 20min,40min and 60min,respectively.In order to ensure experimental repeatability,three samples were used.Surface morphologies and chemical compositions of Mg and Mg-Zn alloys were characterized via confocal laser scanning microscopy and SEM equipped with energy-dispersive X-ray spectroscopy(EDS).The adsorbed Fg was labeled via inverted phase contrast microscope.While the compositional analyses of Mg-Zn alloys were performed using X-ray photoelectron spectroscopy(XPS).

Fig.2.Average number density of Fg adsorption on six regions of Mg alloys surface.

3.Results and discussion

3.1.Understanding the protein adsorption behavior by experiments

The fluorescent Fg adsorption on pure Mg and Mg-xZn(x=1,2,3,5 and 10 wt.%)alloys surfaces during immersion was depicted in Fig.1.The bright spots in the figures were fluorescent Fg.Fig.2 showed the number density of Fg.The adhesion protein number density is the ratio of adherent protein number(bright spots)to the total area of image.The results revealed that Fg successfully adsorbed on pure Mg and Mg-xZn surfaces.The Fg adsorption amount on different alloys was significantly different.Compared with pure Mg,more Fg were adsorbed on Mg-xZn(x=1,2,3 and 5)surface,whereas few Fg was observed on Mg-10Zn surface.In addition,the Fg adsorption amount on Mg-xZn(x=1,2 and 3)surface increased significantly with the immersion time.After 60min of immersion,Fg exhibited significantly bigger spots and more uniform adsorption,which reflected the stronger protein adsorption on these surfaces.However,the Fg adsorption amount on Mg-5Zn alloys was less than that on Mg-3Zn alloy,and no difference was observed for protein adsorption on Mg-10Zn alloy surface with the immersion time.Therefore,it can be concluded that the protein adsorption amount tends to increase at first and then decrease with the Zn content increase.

Fig.3.The SEM morphology and EDS images of the samples after immersion 60 min:(a1,a2,a3)Mg-1Zn,(b1,b2,b3)Mg-2Zn,(c1,c2,c3)Mg-3Zn,(d1,d2,d3)Mg-5Zn and(e1,e2,e3)Mg-10Zn.

The SEM morphology and element type of Mg-Zn alloys after 60min immersion were characterized,as shown in Fig.3.As the EDS point scan data of region Ⅰhad high content of protein elements(C,N and O),the white bumps on the surface were adsorbed proteins.The region Ⅱwas the matrix phase(αMg)of the sample.There was no Zn element in the EDS point scan data of region Ⅰand region Ⅱof Mg-1Zn,Mg-2Zn and Mg-3Zn alloy,indicating that Zn atoms were not distributed in the surface layer,while theαMg matrix of Mg-5Zn and Mg-10Zn contained relatively high concentration of Zn.Other researchers had also found that no Zn was detected in theαMg matrix surface of Mg-4wt.%Zn alloy[12],while a small amount of Zn was detected on the Mg-6wt.%Zn alloy [18].To further investigate the distribution of Zn element on the surface,XPS analysis was performed,and the results were shown in Fig.4.The whole range of the binding energy survey and Zn 2P spectra(Fig.4a and c)indicating the Zn element was not detectable for Mg-1Zn and Mg-3Zn,while Zn present in Mg-10Zn,which was consistent with the results of EDS.This suggests that whether Zn precipitates on the surface depends on its content.In this paper,MD and DFT simulation was used to reveal the mechanism of the protein adsorption and the Zn distribution.

3.2.Understanding the protein adsorption behavior by MD simulations

In order to obtain accurate results of MD simulation,it is necessary to determine the stable structure of Zn in Mg alloy.The atom structure model is shown in Fig.S4(Supporting Information).DFT calculation shows that the system energy of Mg-0.7Zn(?171.190 eV/144 atoms)with Zn in the surface layer is larger than that(?171.238 eV/144 atoms)when Zn in the inner layer,which indicates that the system is more stable when Zn atoms in the inner of theαMg.This reveals the thermodynamically mechanism that Zn is not observed on the surface of Mg-1Zn,Mg-2Zn and Mg-3Zn alloys.However,the kinetics of Zn self-diffusion from the surface to the interior of the alloy remains to be further studied.

The Zn elements will precipitate on the Mg-Zn alloy surface with the increase of Zn content [12,18].When the Zn content is high,there will be various models depend on the Zn atomic spacing.In order to study the influence of increased Zn content on Fg adsorption,four models of Mg-1.4Zn(at.%)are constructed(Fig.S4 in Supporting Information).In order to screen out the most stable structure,the energies of various models were calculated by DFT(Fig.S4 and Table S2 in Supporting Information).It can be seen that when the Zn atomic spacing is,the corresponding energy is the lowest and the structure is the most stable.The MD simulation results of the other three models of Mg-1.4Zn(at.%)are shown in Fig.S6 and Fig.S7(Supporting Information).It should be noted that the simulation results of these four models are close to each other(seeing Figs.S6,S7 and Fig.5).

Fig.4.XPS analysis of the Mg-Zn alloy:(a)entire range of the binding energy survey,(b)Mg 2S spectra and(c)Zn 2P spectra.

Fig.5.The overall configurations,top view of Fgγ C30 with anchoring residues and the pair correlation functions between Mg/Zn with N/O in the protein after 16ns MD simulations for modal Ⅰ(a1,a2,a3),modal Ⅱ(b1,b2,b3),modal Ⅲ(c1,c2,c3)and modal Ⅳ(d1,d2,d3),respectively.Only residues within distance 3.5 ?A from the surface are represented.The gray atoms are Zn.The figures in a2-d2 represent number of residues anchored to the surface.

According to the above results,four types of Zn content models were constructed in this paper(Fig.S5 in Supporting Information):Mg-0.7Zn(modal Ⅰ)and Mg-1.4Zn(modal Ⅱ)with Zn atoms in the inner layer(low Zn content model),Mg-0.7Zn(modal Ⅲ)and Mg-1.4Zn(modal Ⅳ)with Zn atoms in the surface layer(high Zn content model).After 16ns running,the equilibrated structure of FgγC30 was obtained(Fig.5).The distribution and content of Zn will significantly affect the FgγC30 adsorption.It can be seen from Fig.5 that when Zn atoms were in the inner layer(modal Ⅰand modal Ⅱ),the anchored residues gradually increase and their distribution were more concentrated with Zn content increase.When Zn atoms were in the surface layer(modal Ⅲand modal Ⅳ),the anchored residues gradually decrease and their distribution were dispersive with Zn content increase.Furthermore,the number of anchored residues to the surface with Zn in the inner layer was more than that of Zn in the surface layer.Therefore,the protein adsorption is stronger when the Zn atoms are distributed in the alloy inner.

Fig.6.(a)-(d)The parameters of Fgγ C30 during MD simulation for RMSD,moving distance of the mass center(?dcom)of Fgγ C30,interaction energy and atomic distribution of Fgγ C30 along the z direction,respectively.

Here,the influence of Zn content on protein adsorption is further analyzed at the atomic scale.The O/N elements are negatively charged and have larger charges than the other atoms in the protein.Therefore,O/N elements play a leading role in the proteins adsorption process.Fig.5 a3-d3 show the pair correlation functions g(r)for Mg/Zn and O/N atoms in FgγC30.Several sharp peaks are observed in the RDF of Mg-O and Mg-N when the distance is less than.However,no peaks are presented in the RDF of Zn-O and Zn-N.These implying that the O/N elements prefer to be adsorbed on Mg atoms rather than on Zn atoms in the Mg-Zn alloys.

Penna [47]found that the first phase of protein adsorption process was the biased diffusion of the peptide from the bulk phase toward the surface.Fig.6 shows the structural parameters changing of FgγC30 in the adsorption process.It can be seen from Fig.6a that the root-mean-square deviation(RMSD)value gradually tends to be stable after 8ns,indicating that the structure of FgγC30 no longer greatly change and the system is in equilibrium state.The larger the moving distance of the mass center(?dcom),the larger distance the protein moves towards the substrate.Fig.6b shows that FgγC30 moves rapidly towards the interface in the first 2ns due to the substrate attraction in modal Ⅰ-Ⅲ.The ?dcomreaches the maximum at 4ns,indicating that the stable adsorption state is achieved.However,?dcomis negative for the entire MD process in modal Ⅳ,indicating that the attraction of the substrate to the protein is weak.Fig.6c shows the interaction energy between FgγC30 and the surfaces.The interaction energy of the system with Zn atoms in the inner layers is less than that with Zn atoms in the surface layer,indicating that the Fg adsorption capacity is enhanced when the Zn exist in the inner layer of Mg-Zn alloy.Fig.6d shows the atomic distribution of FgγC30 along thezdirection.The more atoms distributed on the surface,the stronger the interaction between protein and substrate,and the more stable the adsorption structure.It can be seen from Fig.6d that when Zn atoms exist in the inner layer,the Fg adsorption capacity is stronger,and the adsorption capacity increase with increase of Zn content.However,the Fg adsorption capacity decrease significantly when the Zn atoms exist in the surface layer,and decrease significantly with the Zn content increase.These explain the sequence of Fg adsorption amount that observed in the experiment:Mg-3Zn>Mg-2Zn>Mg-1Zn>Pure Mg,and Mg-3Zn>Mg-5Zn>Mg-10Zn.

Fig.7.The RDF between Mg/Zn atoms and X(X=H,O in the water)atoms and the adsorption configurations of water molecules for modal Ⅰ(a1,a2),modal Ⅱ(b1,b2),modal Ⅲ(c1,c2,c3)and modal Ⅳ(d1,d2,d3),respectively.The density of water along the z direction was illustrated in(e).

3.3.Water molecule distribution on Mg-Zn alloys surfaces

In aqueous solutions,the concentration of water molecules on solid surface has significant impact on protein adsorptions[47,48].In the present work,the RDF,overall configurations and mass densities of water molecules on Mg-Zn alloys surfaces along thezdirection were calculated(Fig.7).The RDFs between O/H and Mg/Zn atoms are shown in Fig.7 a1-d1.The RDFs of Zn-O in the modals Ⅲand Ⅳexhibit the 1st peaks at,which is higher than that of Mg-O.RDFs of Mg-O 1st peaks at,representing the isolated water molecules that physically adsorbed at the Zn sites.The overall configurations of water molecules are shown in Fig.7 a2-d2.Compared with the water layers in modal ⅠandⅡ,a small amount of water molecules was observed between the 1st water layer and substrate surface in the modal Ⅲand Ⅳ.Fig.7 c3-d3 show the corresponding top view(only present a small number of water molecules between the 1st water layer and the substrate surface).It can be seen that a few water molecules are concentrated on Zn atoms.The mass densities of water molecules along thezdirection are shown in Fig.7e.There are two distinct water layers on the surface of Mg alloy,which is consistent with our previous simulation results [49].Interestingly,one and two water molecule distribution peaks appeared atr

The last phases of the protein adsorption process on material surface is the lockdown of the peptide on the surface via a slow,stepwise and largely sequential adsorption of its residues [47].Therefore,when the Zn atoms are precipitated on the surface,the strong Zn atoms-water molecules interaction prevents the protein from displacing the water layer above the interface,which makes it difficult to maximize the protein-interface interaction,and finally impedes the adsorption and rearrange of protein.

Fig.8.(a)–(d)The charge distributions of surface layer atoms for modal Ⅰ,modal Ⅱ,modal Ⅲand modal Ⅳ,respectively.The charge values represent the multiple of electron charge.The charge of each Mg atom at top layer is 0.054e in pure Mg system.

3.4.The effect of Zn on the charge distribution of alloy surface

The proteins-substrate interactions mainly include van der Waals and electrostatic interactions [22,26,50],the surface charge will significantly affect the proteins adsorption.The charge distribution of the first-layer atoms of Mg-Zn alloy was shown in Fig.8.When Zn atoms in the inner layer(Fig.8a),the surface layer atoms are all positively charged and the average charge(0.065e)is higher than that of pure Mg(0001)(0.054e).This is because the electronegativity of Zn is larger than Mg,and then has a stronger possession of electrons,which eventually makes the Zn atoms negatively charged and the neighboring Mg atoms positively charged.The protein diffusion towards the material interface is induced by a long-range coulomb force [48].Therefore,the positively charged surface will have a stronger adsorption effect on the negatively charged Fg,when Zn exist in the inner of Mg-Zn alloy.Furthermore,the average positive charge of surface atoms increases with the Zn content(Fig.8b),and the Fg adsorption amount will gradually increases accordingly.When Zn atom in the surface layer(Fig.8c),the average positive charge amount of surface atoms significantly reduced,it will reduce the proteins adsorption.The atoms on the surface are negatively charged on average with the increase of Zn content(Fig.8d),which will repel the negatively charged proteins,resulting in a significant reduction in protein adsorption.

Whether Mg-Zn alloy promotes or inhibits the Fg absorption depends on Zn content.The experimental results show that Zn is usually distributed in the alloy inner when Zn content is low,and Zn can be detected on the surface only when Zn content is high.The protein adsorption amount increase first and then decrease with the increase of Zn content.MD calculations show that the Zn distribution characteristics caused by different Zn content changed the atomic type,surface charge distribution and the water molecular layers number on the surface,which would affect the surface-proteins interaction strength and ultimately affect the proteins adsorption.The schematic diagram is shown in Fig.9.

Combining both the theoretical and experimental results,we can provide some guidance to the design of Mg-Zn biomaterials.To avoid the occurrence of blood clotting,Zn should be distributed on the alloy surface by regulating Zn content or heat treatment process.However,to accelerate blood clotting and osteoblast adsorption for bone implant materials,the Fg adsorption needs to be enhanced,and various methods are needed to make Zn distribute in the alloy inner.

4.Conclusions

In the present study,the effect of Zn content in Mg-Zn binary alloy on fibrinogen adsorption was studied by experiment and simulations.The calculated results were in good agreement with the experimental results,the all show that the Fg adsorption amount increased at first and then decreased with Zn content increase.The results can be concluded as follows:

Fig.9..Schematic illustration of the Fg adsorption on Mg-Zn alloys surfaces.

(1)Experimental results showed that whether Zn was distributed on the alloy surface depends on its content.The simulation results showed that the system energy was low and the structure was stable when Zn exists in inner layer of the alloy.

(2)When the Zn content was low,such as in Mg-1Zn,Mg-2Zn and Mg-3Zn alloys,Zn element was distributed in the inner layer,resulting in high positive charge of Mg atoms and a large number of anchoring residues on the surface.These promote the fibrinogen adsorption,and the promotion effect is more obvious with Zn content increase.

(3)When Zn content was high,such as in Mg-5Zn and Mg-10Zn alloy,some Zn elements can be distributed on the alloy surface,and the anchoring residues number on the surface was little,which inhibits the fibrinogen adsorption.The fibrinogen-surface interaction decrease with the Zn content increase,therefore,the inhibition effects of fibrinogen adsorption was enhanced with the Zn content increase.

(4)When Zn atoms on the surface of Mg-Zn alloy,a small number of water molecules with strong binding capacity appear in Zn sites,which will further hinder the diffusion and rearrangement of proteins on the surface.

Declaration of Competing Interest

The authors declare no conflict of interest.

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China(No.U1804251)and the National Key Research and Development Program of China(Nos.2017YFB0702500 and 2018YFC1106703).

Supplementary materials

Supplementary material associated with this article can be found,in the online version,at doi:10.1016/j.jma.2020.09.020.