Evaluation on the corrosion resistance,antibacterial property and osteogenic activity of biodegradable Mg-Ca and Mg-Ca-Zn-Ag alloys

Hewei Chen,Bo Yun,Rui Zho,Xio Yng,Zhnwen Xio,Antonic Auror,Bit An Iuli,Xingdong Zhu,*,Antonic Vsile Iulin,c,*,Xingdong Zhng

a National Engineering Research Center for Biomaterials,Sichuan University,Chengdu 610064,China

b Faculty of Materials Science and Engineering,University Politehnica of Bucharest,Bucharest 060042,Romania

c Academy of Romanian Scientists,Bucharest 050094,Romania

Abstract The rapid degradation of magnesium(Mg)-based implants in physiological environment limits its clinical applications,and alloying treatment is an effective way to regulate the degradation rate of Mg-based materials.In the present study,three Mg alloys,including Mg-0.8Ca(denoted as ZQ),Mg-0.8Ca-5Zn-1.5Ag(denoted as ZQ71)and Mg-0.8Ca-5Zn-2.5Ag(denoted as ZQ63),were fabricated by alloying with calcium(Ca),zinc(Zn)and silver(Ag).The results obtained from electrochemical corrosion tests and in vitro degradation evaluation demonstrated that the three Mg alloys exhibited distinct corrosion resistance,and ZQ71 exhibited the lowest degradation rate in vitro among them.After addition of Zn and Ag,the antibacterial potential of Mg alloys was also enhanced.The in vitro cell experiments showed that all the three Mg alloys had good biocompatibility.After implantation in a rat femoral defect,ZQ71 showed significantl higher osteogenic activity and bone substitution rate than ZQ63 and ZQ,due to its higher corrosion resistance as well as the stimulatory effects of the released metallic ions.In addition,the average daily degradation rate of each Mg alloy in vivo was significantl higher than that in vitro,as could be due to the implantation site located in the highly vascularized trabecular region.Importantly,the correlations between the in vitro and in vivo degradation parameters of the Mg alloys were systematically analyzed to fin out the potential predictors of the in vivo degradation performance of the materials.The current work not only evaluated the clinical potential of the three biodegradable Mg alloys as bone grafts but also provided a feasible approach for predicting the in vivo degradation behavior of biodegradable materials.? 2021 Chongqing University.Publishing services provided by Elsevier B.V.on behalf of KeAi Communications Co.Ltd.This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/)Peer review under responsibility of Chongqing University

Keyword:Mg alloys;Degradability;Antibacterial property;Osteogenic ability;Bone defect repair.

1.Introduction

In the last few decades,nondegradable metals,including titanium,titanium alloys and stainless steels,are usually preferred to orthopedic implants owing to their outstanding mechanical properties.However,in clinical practice,most of these currently applied metals exhibit a significantl higher mechanical strength than bone,leading to severe stressshielding problems[1].Additionally,the release of toxic ions from metal implants through corrosion or wear processes often causes inflammator osteolysis[2].Biodegradable metals with favorable elastic modulus and good biocompatibility have become a better choice for orthopedic implants.Among current biodegradable metals,magnesium(Mg)and its alloys have been extensively investigated and successfully used as various types of internal fixatio devices in orthopedic surgeries[3,4].However,rapid degradation of Mg in the physiological environment gives rise to a series of deleterious effects,such as excessive hydrogen evolution,local basicity and loss of mechanical integrity,which impede its clinical applications[5].Hence,it is vital to control the degradation rate of Mg to satisfy the clinical requirements.

A substantial amount of work has been done to regulate the degradation rate of Mg,mainly including surface coating,surface treatment and metallurgical alloying strategies[6-8].Surface coatings on Mg are usually achieved via chemical conversion and deposition.Zou et al.constructed a lamellar zinc-loaded montmorillonite(Zn-MMT)coating on the surface of Mg alloy by a hydrothermal method,and their results showed that the Zn-MMT coating exhibited better corrosion resistance and higher antibacterial activity than bare Mg alloy counterparts[9].Yu et al.developed a combined treatment with microarc oxidation and hydrothermal treatment to fabricate a fluoride-dope hydroxyapatite coating on the surface of AZ31 magnesium.Their results showed that the composite coating exhibited a higher corrosion resistance than the uncoated substrate and significantl improved the cell responses of MC3T3-E1[10].Surface treatments via either chemical or physical methods have also been developed to improve the degradation property of Mg implants.In a recent study,Wang et al.found that 2D nanofla es fabricated in situ on Mg by a combined method with an alkali treatment and a hydrothermal treatment could improve the carrion resistance,antibacterial activity and biocompatibility of Mg[11].Though controlled surface engineering,including surface coating and modifica tion,can improve corrosion resistance to a certain degree at the early stage after implantation,corrosion and subsequent loss of mechanical integrity of modifie Mg implants can inevitably occur following the degradation of the surface coating and modifie surface.Addition of appropriate alloying elements,such as zinc(Zn),calcium(Ca),manganese(Mn),and strontium(Sr)into a Mg matrix has been suggested as another effective method to retard the corrosion process and improve the mechanical strength[12].Among these alloying elements,Ca and Zn have been extensively investigated due to their vital role within the human body and numerous essential functions for bone regeneration[13].Li et al.investigated the effect of calcium content on the microstructure,mechanical properties and biocompatibility.Their results showed that Mg-1Ca had good biocompatibility and a low corrosion rate[14].Chen et al.investigated the in vitro and in vivo biocompatibility of a Mg-Zn alloy and found that it exhibited an increased cell attachment and mineralization ability and an improved gene expression of collagen 1α1 and osteocalcin[15].Bakhsheshi-Rad et al.further introduced Zn into Mg-Ca alloys and found that the addition of Zn resulted in improved corrosion resistance and hardness due to the formation of the Ca2Mg6Zn3phase in Ma-1Ca-Zn alloys[16].

In addition to controlling the degradation rate,exploiting Mg implants with inherent antibacterial properties has attracted enormous interest from both industry and academia[17,18].Bacterial infection often occurs after clinical device implantation,resulting in an early failure of implanted devices and a series of complications[19].Previous studies have demonstrated that pure Mg has an intrinsic bacterial killing ability due to natural degradation,leading to an elevation in Mg ion concentration,local pH and hydrogen production[17].However,as previously mentioned,the uncontrollable degradation rate of Mg severely hampers its clinical applications.There have been efforts to simultaneously improve the corrosion resistance and antibacterial properties of Mg alloys by employing antibacterial elements,such as copper(Cu)and silver(Ag)[20,21].Recently,Qin et al.also reported that the addition of proper amounts of Zn,Zr and neodymium(Nd)could effectively enhance the corrosion resistance,biocompatibility and antimicrobial properties of Mg[22].Therefore,introducing an appropriate amount of Ca,Zn and Ag into a Mg matrix may not only improve the corrosion resistance of Mg but also enhance its osteogenic activity and antimicrobial ability.However,there are few studies on Mg-Ca-Zn-Ag quaternary alloys,and their degradation,antimicrobial and biological performance in vitro and in vivo have not yet been reported.

In our previous study,we prepared three Mg alloys,including Mg-0.8Ca(ZQ),Mg-0.8Ca-5Zn-1.5Ag(ZQ71),and Mg-0.8Ca-5Zn-2.5Ag(ZQ63),by alloying with Ca,Zn and Ag and investigated their hydrogen evolution rates in vitro.Our results showed that both the ZQ71 and ZQ63 alloys exhibited lower hydrogen release rates than the ZQ alloy[23].In this study,we further evaluated the degradation behavior,antibacterial performance and cytocompatibility of Mg alloys in vitro.In addition,the in vivo degradation and biological performance of the Mg alloys were also investigated by implantation in a rat bone defect model.Moreover,considering the great differences between the results from the in vitro versus in vivo studies,we utilized a multivariate analysis to assess the correlation of the degradation performance in vitro and vivo.

2.Materials and methods

2.1.Sample preparation and characterization

Three ZQ alloys were prepared by a melt-casting method according to the procedure described in our previous study[23-25].In this study,the plate samples with dimensions of 3×3×2 mm3were prepared for materials characterization and in vitro study.The cylinder samples with dimensions ofΦ2.5×4 mm2were prepared for in vivo animal study.

The surface morphologies and elemental composition of the samples were examined by a field-emissio scanning electron microscopy(FE-SEM,S4800,Hitachi,Japan)equipped with an energy dispersive spectrometry(EDS).Phase composition of the samples was determined by X-ray diffractometer(XRD,DX-1000,China).For the above tests,three parallel samples were used.

2.2.Electrochemical corrosion tests

Electrochemical corrosion tests including open circuit potential(OCP),electrochemical impedance spectroscopy(EIS)and potentiodynamic polarization(PDP)were carried out at room temperature in phosphate buffered saline solution on a PARSTAT 2773 electrochemical workstation(Princeton Applied Research,USA).The measurements were performed on a three electrode electrochemical cell with a saturated calomel electrode(SCE)as the reference electrode,a hollow graphite cylinder as the counter electrode,and the Mg alloy sample as the working electrode.The EIS tests were carried out at the open circuit potential by applying a 10 mV ac signal in the frequency range rate of 10 kHz to 10 mHz.The PDP curves were obtained at a scan rate of 1.67 mV s-1over a potential range of-2.2 V to-0.6 V.All potentials data were recorded in the SCE,and all tests were done in triplicate to obtain the average data.

2.3.In vitro biocompatibility and degradation evaluation

2.3.1.Cell culture

In order to closely mimic the in vivo application situation,the exposure culture method was used to investigate the biocompatibility and degradation of ZQ,ZQ63 and ZQ71 in vitro.Using this method,each sterilized sample was placed in a Transwell(Cat #3413;Corning,USA)unit and directly into the established MC3T3-E1(Cell Bank of Chinese Academy of Sciences,Shanghai,China)culture in vitro.Similar method has been reported by a previous study,in which the method was used to evaluate the cytocompatibility and degradation of Mg-4Zn-1Sr alloy and Mg pins in vitro[26].Cell medium comprised of a-minimum essential medium(ɑ-MEM,Gibco,USA),10%(v/v)standard fetal bovine serum(FBS,Gibco,USA)and 1%(w/v)penicillin/streptomycin(Gibco,USA)was used.Cell were seeded in 24-well plates at a density of 1×104cells per/well and then cultured for 1 day.Afterward,the medium was refreshed and the Transwell inserts with Mg alloy sample at the center were placed into each MC3T3-E1-containing well and cultured for 1,2 and 3 days.The blank culture plate was used as the control.To closely mimic in vivo conditions where the circulation system regularly takes away soluble degradation products from the local site of implantation,the medium was collected for analyses and replaced with fresh media every day.The plates were maintained at 37 °C in a humidifie incubator under at 5% CO2throughout the study.All samples were sterilized by gamma radiation before the experiment.

2.3.2.Cell morphology and viability

The morphology of MC3T3-E1 cells co-cultured with each sample was observed by a confocal laser scanning microscope(CLSM,TCS-SP5,Leica Microsystems,Germany).On day 1,2 and 3,the cells were stained with fluorescei diacetate(FDA,Sigma,USA)for live cells(green)and propidiumiodide(PI,Sigma,USA)for dead cells(red)after being washed twice with phosphate buffered solution(PBS).For further observing morphology and quantifying cell area,cells were stained with Phalloidin-TRITC(Sigma)and 4′,6-Diamidino-2-phenylindole dihydrochloride(DAPI,Sigma)to visualize cytoskeleton and the cell nucleus.The mean area of single cell at 1 day was quantitatively evaluated by using Image Pro-Plus 6.0 software(Media Cybernetic,USA),in which six randomized non-overlapping field in each sample were selected.

The viability of MC3T3-E1 cells co-cultured with each sample was evaluated by counting kit-8 assay(CCK-8,Dojindo,Japan).At each time point,the samples were taken out and 1 mL of 10% CCK-8 solution per/well was added into the plates.After incubation for 2 h at 37 °C,200μL of supernatant in each well was transferred into a 96-well plate.The optical density(O.D.)was measured at a wavelength of 450 nm by a multifunctional,full-wavelength microplate reader(Varioskan Flash,Thermo Scientific USA).For the above assays,all experiments were performed in triplicate.

2.3.3.Degradation of Mg alloys

The in vitro degradation of various Mg alloys at each time point was evaluated through measurements of sample mass,pH value,and ionic concentrations of the collected media.At each time point,the mass of the dried samples was measured and used to determine a percent fina mass relative to the initial mass.The pH of the medium was measured immediately after collection using a calibrated pH meter(FiveEasy Plus,METTLER TOLEDO,China).Inductively coupled plasma-atomic emission spectroscopy(ICP-AES,IRIS Adv,Thermo,USA)was used to quantify the amount of Mg2+,Ca2+and Zn2+ions in the collected medium and the amount of Ag+ions in PBS.Surface topographies of the samples at each time point were observed by SEM.

2.4.Antibacterial activity in vitro

Escherichia coli(E.coli)andStaphylococcus(S.aureus)

were chosen to evaluate the antibacterial properties of various Mg alloys in vitro.E.coliandS.aureusstrains in frozen stock were cultured in Luria-Bertani(LB)broth at 37oC and 150 rpm overnight.After measuring OD value,E.coliandS.aureuswere diluted to 2.9×105CFU mL-1and 3.6×104CFU mL-1with the LB medium,respectively.And then,1 mL of the LB medium was placed in the well containing the Mg alloy samples.The blank plate co-cultured with the bacteria was used as the control sample.The absorbency of the culture medium was measured every 12 h by a spectrophotometer(UNIC-7200,China)at 410 nm for

E.coliand 650 nm forS.aureus.

Surface morphology and structure of the bacterial cultured on the Mg alloy samples were observed by SEM.Prior to observing,the samples were fi ed in 2.5% glutaraldehyde overnight at 4 °C and rinsed with PBS two times,and then dehydrated in ascending concentrations of alcohols from 30%to 100%.Finally,Gold was sputtered on the sample surface for SEM observation.All experiments were performed in triplicate.

2.5.In vivo animal study

2.5.1.Surgical procedure

All procedures in the experiments were approved by the Institutional Animal Care and Use Committee of Sichuan University.Fifteen female Sprague-Dawley rats aged 8 weeks(200±20 g)were obtained from the Animal Center of Sichuan University(Chengdu,China).A femoral condyle defect model was established in osteoporotic rats based on our previous work.In brief,the rats were anaesthetized by intraperitoneal injection of pentobarbital(Nembutal 2 mg/100 g).A sagittal incision was made on the skins of the distal femoral and then the femoral condyle was exposed by blunt dissection.A cylindrical hole(diameter:2.5 mm and depth:4 mm)was created perpendicular to the distal femur of rats by using a dental drill with low rotational drill speed.Finally,the femoral condyle defects were generated and randomly fille with sample.All the rats as-operated were evenly distributed into three groups including ZQ,ZQ63 and ZQ71.After surgery,two fluorochromes i.e.tetracycline(30 mg/kg at 14 and 13 days before death,Sigma)and calcein(6 mg/kg at 4 and 3 days before death,Sigma)were administered to assess the osetogenic activity at 4 weeks.At 4 weeks postoperatively,the rats were euthanized by an intraperitoneal overdose injection of pentobarbital.The harvested bone specimens were fi ed in a 4% paraformaldehyde solution for 7 days before further analysis.

2.5.2.Micro-computed tomography(μ-CT)evaluation

A micro-computed tomography imaging system(μ-CT,SCANCO VivaCT80,Switzerland)was used to monitor the degradation of Mg alloys and bone formation within the defect region of the rats in situ during the implantation period.The cross-sections of the specimens(n=5)were imaged by using a scanning procedure with a spot size of 7μm and a maximum voltage of 70 kV.After being reconstructed with a high-resolution protocol,the resultant grayscale images had an isotropic voxel size of 25μm(pixel matrix:2018×2048).The obtained grayscale images were further reconstructed and analyzed using Scanco software.During the reconstruction,a global threshold was used to segment the newly formed bone from each implant.After thresholding and segmentation,the degradation rates of implants were calculated by normalizing the degraded material volume(ΔMV)to the initial material volume(MV),and the new bone substitution rate were calculated by normalizing the bone volume(BV)to theΔMV.In addition,in order to predict the lifetimes of various Mg alloys in vivo,a linear model with y-intercept set to unity was used to fi the ratio ofΔMV/MV as a function of time in weeks.

2.5.3.Histological staining

The fi ed bone specimens(n=5)were dehydrated in ascending concentrations of alcohol from 75% to 100% and then embedded in polymethylmethacrylate(PMMA).Three transverse sections for each embedded specimen were cut into?100μm-thick sections using a microtome(SAT-001,AoLiJing,China),followed by grinding and polishing to a fi nal thickness of?25μm.Then,the sections were stained with hematoxylin and eosin(H&E)for histological observation by a light microscope(Bx60,Olympus,USA)equipped with a digital CCD camera.For quantitative analysis of newly formed bone,the acquired microscope images were histologically evaluated using Image Pro-Plus software(Media Cybernetic,USA).During the process,the“segmentation”tool was used to quantitatively measure theΔMA/MA,which was calculated as the percentage area of degraded materials,and the BA/ΔMA,which was define as the percentage area of bone substitution.

The nonstained sections were observed by CLSM to determine the mineral apposition rate of new bone formation by monitoring the length between the two labels(yellow:tetracycline label,green:calcein label)over time,as described in some previous reports.For quantificatio of the mineral apposition rate,three randomized nonoverlapping areas in the ingrown bone area of each specimen were analyzed using IPP 6.0 software.

2.6.Analyzing the degradation rates of various Mg alloys in vitro and in vivo

Based on the measured Mg2+ion concentrations in the

MC3T3E1/ɑ-MEM culture system and the volume change from 3D reconstructions ofμ-CT images,the average daily degradation rates in vitro and in vivo were calculated according to the following equations(Eqs.(1),2 and 3,respectively):

avg.dailydeg.rate

In Eq.(1),[Mg2+]was the measured concentration of Mg2+ion at each time point(i=1,2 and 3).[Mg2+]ɑ-MEM,totalwas the total measured concentration of Mg2+in blankɑ-MEM over the 3 day period.Vmwas the volume value of the culture medium,and V0was the initial volume value of Mg alloy sample.In Eq.(2),V0was the initial volume value of Mg alloy implant,V28was the volume value of Mg alloy implant at day 28 postoperatively.ρwas the weight density of various Mg alloy implants.

In addition,a multivariate analysis was used to assess the correlation of the degradation performance in vitro and vivo.Pairwise correlations between in vitro degradation and in vivo degradation results were investigated using Pearson correlation analysis for each pair of results,and the resulting pairwise correlations were visualized as a confusion matrix.

2.7.Statistical analysis

All data are expressed as the mean±standard deviation(SD)of three independent experiments,unless otherwise stated in the caption.Statistical analysis was carried out using SPSS 16.0 software,and one-way ANOVA with Tukey’s post hoc test was performed to determine the significan differences between groups.Significan correlations were identifie forp-values＜0.05.Pairwise correlations between in vitro degradation and in vivo degradation parameters were investigated using Pearson correlation analysis for each pair of parameters,and the resulting pairwise correlation were visualized as a confusion matrix.The difference was considered statistically significan at ap-values＜0.05.

Fig.1.Characterization of the Mg alloys.SEM images and surface elemental analysis,and corresponding X-ray diffraction patterns for ZQ,ZQ71 and ZQ63 alloys.

3.Results

3.1.Surface characterization of the Mg alloys

The surface topography and elemental composition of the Mg alloys were investigated by SEM coupled with EDS,and the results are shown in Fig.1.As shown in Fig.1a,the binary ZQ alloy consisted of aɑ-Mg(gray area)matrix and Mg2Ca intermetallic phase,and the intermetallic phase with a lamellar structure was continuously distributed at the grain boundaries.The addition of Ag resulted in the precipitation of Ca2Mg6Zn3and Mg3Ag within the ZQ71 grains.Compared to that for ZQ71,ZQ63 showed a similar morphology,but its grain boundaries were more pronounced due to the higher percentage of Ag.A homogeneous element distribution on the surface of various alloys was observed.The presence of these phases was further confirme by XRD analyses(Fig.1b).The characteristic peaks of Mg and Mg2Ca were present for all the samples,and typical characteristic peaks of Ca2Mg6Zn3and Mg3Ag were present for both ZQ71 and ZQ63.Moreover,the amount of Mg3Ag phase increased with increasing Ag content up to 2.5 wt.%in the Mg alloys,but the amount ofɑ-Mg phase decreased.

3.2.Electrochemical characterization

3.2.1.OCP and PDP analysis of the Mg alloys

The corrosion resistance of the Mg alloys was firs evaluated by exposing the samples to a PBS solution and measuring the OCP values.As shown in Fig.2a,the OCP of the three Mg alloys reached a relatively stable state after soaking in PBS solution for 900 s.With the addition of Zn and Ag,a significantl increased OCP was observed for both the ZQ71 and ZQ63 alloys,but the OCP of ZQ71 was higher than that of ZQ63,suggesting a higher electrochemical stability and lower corrosion tendency.Fig.2b shows representative potentiodynamic polarization curves for ZQ,ZQ71 and ZQ63 after immersion in PBS solution at 37 °C.All Mg alloys presented similar polarization curves in terms of their shape.For a quantitative comparison,the polarization parameters,including the corrosion potential(Ecorr)and corrosion current density(icorr),were calculated by using a Tafel extrapolation of the cathodic polarization curve,as summarized in Table 1.After the addition of different contents of Ag,the Ecorrof ZQ increased from-1.54±0.02 V vs.SCE to-1.47±0.02(ZQ71)and-1.49±0.01 V vs.SCE(ZQ63),respectively.Moreover,theicorrof ZQ,ZQ71,and ZQ63 were 1.29×10-6,9.76×10-7,and 5.29×10-7A cm-2,respectively,indicating that theicorrvalue for ZQ71 and ZQ63 decreased approximately one order of magnitude compared to that of the ZQ alloy.

3.2.2.EIS analysis of the Mg alloys

EIS measurements were performed at the open-circuit potential to trace the corrosion behavior of the Mg alloys immersed in PBS solution at 37 °C,and the results are shown in Fig.2c-e.The Nyquist plot(Fig.2c)shows that all the samples were characterized by a capacitive loop in the highfrequency range and an inductive loop in the low-frequency range.In general,a capacitive loop can be attributed to a charge transfer process and formation of a corrosion product layer,whereas an inductive loop can be related to the dissolution of Mg.The diameter of the high-frequency semicircle gives the charge transfer resistance Rtat the electrode/electrolyte interface,which is used to evaluate the corrosion property of the alloys.All Mg alloys showed similar EIS spectra,except for the difference in the diameter of the capacitive loop.This indicated that the corrosion mechanism of the three Mg alloys was similar,while the corrosion resistance was clearly enhanced due to the larger diameter of the capacitive loop for both ZQ71 and ZQ63 than that for ZQ.Moreover,improved corrosion protection properties were also confirme by the larger low-frequency impedance modulus(|Z|).As shown in Fig.2d,the|Z|value of ZQ was 86.46±1.45Ωcm2,which is far lower than that of ZQ71(130.75±3.95Ωcm2)and ZQ63(113.59±2.68Ωcm2).Fig.2e shows Bode phase plots for various Mg alloys.All Mg alloys presented a single phase peak in the low-frequency range,while the phase angle of ZQ was lower than those of ZQ71 and ZQ63.The above results demonstrate that the addition of Ag significantl enhanced the corrosion resistance of the Mg alloy.

Fig.2.Electrochemical testing results of ZQ,ZQ71 and ZQ63 alloys.(a)The open circuit potential,(b)Potentiodynamic polarization curves,(c)Nyquist plots,(d)Bode plots of|Z|vs.frequency,and(e)Bode plots of phase angle vs.frequency of the Mg alloys in PBS solution at 37 °C.(f)The electrochemical equivalent circuit model of the Mg alloys.

Table 1Corrosion parameters of potentiodynamic polarization for different samples in PBS solution at 37 °C.

Table 2EIS parameters of different samples.

To further elucidate the corrosion characteristics of the three Mg alloys,an electrochemical equivalent circuit model was proposed to simulate the electrochemical reactions occurring on the sample(Fig.2f).In the equivalent circuit,Rsdenotes the solution resistance;Rfand Qfare the resistance and capacitance of the oxide film respectively;Lfrepresents the inductance formed by the adsorption of products on the sample surface;and Rctis the charge transfer resistance.In addition,the conventional capacitance element in the circuit is replaced by a constant phase element(CPE)denoted by Q,reflectin surface dispersion of a nonideal capacitance.The impedance of the CPE is define by:

whereω(=2πf)is the angular frequency;nis an empirical exponent that changes in the range of 0 to 1,wherenis 0 for a pure resistor andnis 1 for a pure capacitor;and Q is a frequency-independent real constant representing the total capacitance of the CPE and having units of F sn.The EIS data were fitte with the commercial software ZsimpWin3.1 to the equivalent circuit shown in Fig.2f,and the fitte values are listed in Table 2.As shown in Table 2,the ZQ71 and ZQ63 alloys had higher Rfvalues and lower CPE values than the ZQ alloys,indicating that the oxide film on the surface of the ZQ71 and ZQ63 alloys protected the substrate from corrosion and defects more effectively.In addition,the Rctvalues of the ZQ71 and ZQ63 alloys were obviously higher than that of the ZQ alloy.The above results show that alloying could significantl improve the corrosion resistance of magnesium alloys,which is consistent with other chemical measurements.

3.3.In vitro biocompatibility and degradation evaluation

3.3.1.Cell morphology and viability

Representative fluorescenc microscopy images of MC3T3-E1 cells cocultured with different Mg alloys for 3 days are shown in Fig.3a-c.Based on the CLSM observation(Fig.3a)of the cell cytoskeleton and nucleus stained by rhodamine-phalloidin and DAPI,the morphology of MC3T3-E1 cells cocultured with all Mg alloys was not significantl different.After spreading,the cells had a round or elliptical nucleus and a well-organized cytoskeleton.The quantitative analysis for the cell spreading area after culturing for 1 day(Fig.3b)also showed that all three Mg alloys exhibited similar cell spreading areas that were comparable to that of the control group.Fig.3c shows the CLSM images of MC3T3-E1 cells grown in coculture with different Mg alloys.For each group,the cells grew well and presented a normal cell morphology,indicating good in vitro biocompatibility of Mg alloys.At days 2 and 3,more live cells were present on ZQ71 and ZQ63 than on ZQ.Fig.3d shows the CCK-8 assay for cell viability in each group.The cells exhibited good proliferation with a prolonged incubation time.At day 1,there was no significan difference in the cell proliferation rate between the four groups.At days 2 and 3,a higher cell proliferation rate was found on both ZQ71 and ZQ63 than on ZQ.Especially at day 3,ZQ71 showed the highest cell proliferation rate among the four groups.

3.3.2.In vitro degradation of the Mg alloys

Fig.4 summarizes the results for the in vitro degradation of the Mg alloys after 3 days of culturing with MC3T3-E1 inɑ-MEM.The SEM images shown in Fig.4a and Fig.S1 were obtained from the corroded surfaces of the ZQ,ZQ71 and ZQ63 samples.For the ZQ substrate,after culturing in ɑ-MEM for 1 day,the surface experienced severe corrosion and exhibited a rough and corroded morphology,in which numerous cracks and degradation products were observed.With a longer culturing time inɑ-MEM,the degradation products started to form clusters,and localized corrosion pits began emerging on the surface of ZQ.However,for the ZQ71 substrate,the surface was smooth and dense,and no visible corrosion was observed after culturing inɑ-MEM for 2 days.Few cracks and degradation products occurred on the surface of ZQ71 at day 3.In addition,the ZQ63 substrate also exhibited smaller cracks and corrosion pits than ZQ during the whole culture period.

Fig.3.In vitro cytocompatibility of the Mg alloys in exposure culture with MC3T3-E1 cells during a 3-day period.(a)CLSM images of the cell cytoskeleton(red)and nucleus(blue)stained by rhodamine-phalloidin and DAPI,and(b)corresponding quantitative analysis for the cell spreading area after culturing for 1 day.(c)CLSM images of the cells stained by FDA and PI.(d)Cell viability of MC3T3-E1 cultured with different Mg alloys at day 1,2 and 3.(For interpretation of the references to color in this figur legend,the reader is referred to the web version of this article.)

Fig.4b shows the mass and pH change of the Mg alloys after culturing with MC3T3-E1 inɑ-MEM for 3 days.With a prolonged culturing time,the mass retention rates of all Mg alloys,which represents the corrosion rate directly,decreased gradually.During the whole culturing period,ZQ exhibited the lowest mass retention rates among the three Mg alloys,indicating rapid degradation of the substrate.At day 3,no significan difference in mass retention rates was found between ZQ71 and ZQ63.The pH value of the culture medium containing ZQ alloy increased with time and reached up to approximately 8.45 after culturing for 3 days,which is significantl higher than that of the control group.However,a downward overall trend of the pH values was found in the culture medium containing the ZQ71 and ZQ63 alloys,and the ZQ71 group showed a significantl lower pH value than the ZQ63 group at day 3.

Fig.4.Characterization of the Mg alloys after in vitro degradation in exposure culture with MC3T3-E1 for a 3-day period.(a)SEM images of different Mg alloys at day 1,2 and 3.(b)Percent fina mass relative to initial mass of the Mg alloys and pH of MC3T3-E1 culture media incubated with the Mg alloys.(c)Mg2+,(d)Ca2+and(e)Zn2+ion concentration in MC3T3-E1 culture media incubated with the Mg alloys.

The released ion concentrations,including Mg2+,Ca2+,Zn2+and Ag+,were measured by ICP,and the results are shown in Fig.4c-e.Compared to that for the control group,the Mg2+ion concentration of the culture medium containing the three Mg alloys increased with prolonged culturing time.On days 1 and 2,the ZQ71 group exhibited significantl lower Mg2+ion concentrations than the ZQ and ZQ63 groups.A similar trend was observed for the Zn2+ion concentration.However,there were no significan differences in the Ca2+ion concentration among the three material groups during the whole culturing period.In addition,the Ag+ion concentration of the culture medium was not detected due to the low concentration and interference of medium.Thus,we detected the Ag+ion concentration in PBS using the same procedure.As shown in Fig.S2,the Ag+ion concentration of the PBS containing ZQ71 and ZQ63 alloys increased with prolonged culturing time,and the ZQ63 group showed significantl higher Ag+ion concentrations than ZQ71 group.

3.4.In vitro antibacterial activity

The antibacterial properties of the Mg alloys were evaluated quantitatively,and the results are shown in Fig.5a and b.At 12 h,the antibacterial rates of ZQ,ZQ71 and ZQ63 againstS.aureuswere 57.46±3.71,53.97±4.39,and 58.05±2.32%,respectively.When the culturing time was increased to 48 h,the antibacterial rates of ZQ,ZQ71 and ZQ63 againstS.aureusincreased to 94.47±1.59,95.02±0.6,and 95.18±1.6%,respectively.There were no significan differences in the antibacterial rate among the three Mg alloys,suggesting a similar antibacterial activity againstS.aureus.However,forE.coli,ZQ showed significantly higher antibacterial activity than ZQ71 and ZQ63 at 12 h.With prolonged culturing time,the antibacterial rates of ZQ71 and ZQ63 againstE.coliincreased gradually and reached 87.85±0.51 and 86.62±3.77% after 48 h of culturing,respectively,which were significantl higher than that for ZQ(81.54±2.03%).The bacterial morphology and membrane integrity of theS.aureusandE.colicultured on various Mg alloys were observed by SEM,and the results are shown in Fig.5c.In general,theS.aureuscells had a spherical shape with a smooth surface,and theE.colicells were rod-shaped with round ends.As shown in Fig.5c,the bacterial cells for bothS.aureusandE.coliattached loosely to the surface of the ZQ sample,their surface was coarse and distorted,and the cell membrane was damaged with intracellular material leaking out(white arrows).The bacterial cells on ZQ71 and ZQ63 exhibited a similar morphology,suggesting a similar antibacterial effect.

Fig.5.In vitro antibacterial activity of the alloys.(a),(b)The antibacterial rates of different alloys against S.aureus and E.coli.(c)SEM images of bacteria in the Mg alloys after incubation at 37 °C for 12 h.

3.5.In vivo degradation and osteogenic activity of the Mg alloys

3.5.1.Animal weight change and serum analysis

To investigate the in vivo degradation and osteogenic ability of different Mg alloys,cylindrically shaped samples were implanted into metaphyseal defects in rat femurs.We firs monitored the changes in body weight and serum ion concentrations,including Mg2+,Ca2+and Zn2+,at 4 weeks postoperatively(Fig.S3).The body weight of the rats increased steadily,and no significan difference was found among all groups.The Mg2+,Ca2+and Zn2+serum ion concentrations in all alloy groups were similar to those in normal rat serum,indicating that all Mg alloys did not induce systemic changes in Mg2+,Ca2+and Zn2+ionic concentrations in the rat serum.No statistically significan differences were detected for any of the ions measured for any of the groups.

3.5.2.μ-CT evaluation

To directly compare sequential implant degradation and bone remodeling,we rigorously tracked each sample after implantation at the respective time points by in situμ-CT.Fig.6a shows the cross-sectional view of three-dimensionally reconstructedμ-CT images of metaphyseal bone in the defective region(red dotted line).Theμ-CT images confirme gradual degradation of all Mg alloys in vivo and new bone formation in response to the degradation of the implant.Additional residual materials(white)and bone formation(gray)were observed in the defects of the ZQ71 and ZQ63 groups from week 1 onwards.All three implants showed homogeneous degradation from week 1 to week 2,and then the implants lost structural integrity and degraded into smaller segments.At week 4,only a small part of the spaces created by the degradation of ZQ alloy was substituted by the newly formed bone,but the spaces in the ZQ71 group were almost completely substituted.In addition,theμ-CT images also confirme the presence of peri-implant gas pockets(dark region)for all groups.For the ZQ group,the gas pockets gradually increased and spread into the cortical bone region with prolonged implantation time.However,for the ZQ71 and ZQ63 groups,the gas pockets seemed to be smaller than those in the ZQ group.

The volume reduction rate of the implants(ΔMV/MV),the percent volume of the degraded materials at each time point(ΔMV)relative to their paired initial volume(MV),are plotted in Fig.6b.The volume reduction rate of ZQ was much faster than that of ZQ71 and ZQ63 during the whole implantation period.At week 4,theΔMV/MV of the ZQ,ZQ71 and ZQ63 groups were 97.8±0.7,78.8±5.6 and 90.4±1.9%,respectively.TheΔMV/MV in ZQ was significantl higher(p＜0.01)than that in ZQ71 and ZQ63,and ZQ71 exhibited higher(p＜0.05)ΔMV/MV than ZQ63.In addition,the new bone substitution rates and the percent volume of the newly formed bone at each time point(BV)relative to the volume of the degraded materials(ΔMV)are plotted in Fig.6c.A significan decrease in BV/ΔMV for the ZQ and ZQ63 groups occurred at 3 and 4 weeks postoperatively,while that of the ZQ71 group was almost unchanged from weeks 1 to 4,suggesting that the growth rate of new bone in the ZQ71 group could closely match the degradation rate of the alloy material.At week 4,the BV/ΔMV of the ZQ71 group was 2.6-and 1.6-folds significantl higher than that of the ZQ and ZQ63 groups,respectively.

3.5.3.Histological evaluation

To verify the aboveμ-CT results,we further analyzed histological staining data for the implants retrieved at 4 weeks postoperatively(Fig.7a).The typical light microscopic image of the H&E stained sections from the ZQ group showed that the ZQ alloy was almost completely degraded,and plenty of hydrogen gas-induced cavities were observed in the defect region and the trabecular region where bone marrow tissues were absent(Fig.7a).For the ZQ71 and ZQ63 groups,no obvious gas pocket was observed in the defect region and peri-implant region.Importantly,a considerable amount of continuous mature bone with few remaining materials was observed in the ZQ71 and ZQ63 groups but not in the ZQ group.In vivo sequential fluorescen labeling with tetracycline and calcein showed an increased fluorescen response in the ZQ71 and ZQ63 groups,indicating an increased dynamic mineral apposition rate(Fig.7b).Further quantitative analysis for material degradation and new bone formation was performed on the H&E stained sections,and the results are shown in Fig.7c and d.At 4 weeks postoperatively,theΔMA/MA values for ZQ,ZQ71 and ZQ63 were 93.8±4.4,72.6±15.7 and 72.0±13.3%,respectively.The ZQ71 and ZQ63 groups showed a much lowerΔMA/MA values than the ZQ group,but there was no significan difference in theΔMA/MA values between ZQ71 and ZQ63.In addition,the BA/ΔMA values for ZQ,ZQ71 and ZQ63 at 4 weeks postoperatively were 8.9±4.5,27.2±9.1 and 21.2±7.3%,respectively.The ZQ71 and ZQ63 groups showed far higher BA/ΔMA values than the ZQ group,and the ZQ71 group exhibited the highest BA/ΔMA.

3.6.Degradation rates of the Mg alloys in vitro and in viv o

Fig.8a shows a scatter plot of the implant volume as a function of time during the implantation period.According to the linear-fitte curves,all Mg alloys showed a decreasing trend in implant volume over time,and ZQ71 exhibited a slower degradation rate than the other groups.Extrapolation of these curves indicated that ZQ,ZQ71 and ZQ63 implants would completely degrade at 29,32 and 39 days postoperatively,respectively.Fig.8b shows the average daily degradation rates of the Mg alloys in vitro and in vivo that was normalized by the corresponding initial volume.The average daily degradation rates of ZQ from both in vitro and in vivo measurements were significantl higher than those of ZQ71 and ZQ63 under the corresponding conditions.In addition,the in vitro average daily degradation rates of ZQ,ZQ71 and ZQ63 in the MC3T3-E1/ɑ-MEM system were 23.0±6.2,17.1±6.1 and 17.0±4.0 mg cm-3d-1,respectively,which were significantl lower(p＜0.001)than their respective in vivo average daily degradation rates(62.4±2.8 mg cm-3d-1for ZQ,46.6±3.1 mg cm-3d-1for ZQ71 and 54.1±2.8 mg cm-3d-1for ZQ63).

Fig.6.Micro-CT evaluation for the in vivo degradation and bone formation.(a)Reconstructed micro-CT images of metaphyseal bone in the defective region(red dotted line).Upper right corner of each sample:the new bone formation within the defective region(gray).Bottom right corner of each sample:the residual alloy materials(white).(b)The volume reduction rate of the implants:the percent volume of the degraded materials at each time point(ΔMV)relative to their paired initial volume(MV).(c)The new bone substitution rates:the percent volume of the newly formed bone at each time point(BV)relative to the volume of the degraded materials(ΔMV).(For interpretation of the references to color in this figur legend,the reader is referred to the web version of this article.)

After all data were documented,Pearson correlation anal -ysis was performed to compare the in vitro degradation and in vivo degradation parameters(Fig.8c).Three regions in the confusion matrix(heatmap)were of interest,in which deep blue and red represent strong correlation coefficients close to 1 or-1,and the cross mark indicates no significan correlation.1○Three electrochemical parameters including Ecorr,icorr,and|Z|were significantl correlated with the pH value of the culture medium and mass retention rates(Mretention)of Mg alloys,indicating that the in vitro degradation behavior of Mg alloys was closely related to their corrosion resistance.2○Among the in vitro degradation indexes,only the pH value was positively associated with the volume reduction rate of implants(ΔMV/MV)and their in vivo average daily degradation rates.3○The overall in vivo degradation indexes were strongly correlated with the electrochemical parameters and were not significantl correlated with the in vitro degradation parameters.This suggested that electrochemical tests may be a more effective tool in predicting the in vivo degradation behavior of implants than in vitro degradation models.

Fig.7.Histological analysis for the bone regeneration.(a)H&E staining of histological sections from different alloys at 4 weeks postoperatively(B:new bone;Black arrow:material).(b)In vivo sequential fluorescen labeling of new bone formation(Yellow:tetracycline label,Green:calcein label).(c)The area reduction rate of the implants:the percent area of the degraded materials at each time point(ΔMA)relative to their paired initial area(MA).(d)The new bone substitution rates:the percent area of the newly formed bone at each time point(BA)relative to the area of the degraded materials(ΔMA).(For interpretation of the references to color in this figur legend,the reader is referred to the web version of this article.)

4.Discussion

Since biodegradability was evaluated in the human body in 1930s,Mg-based biodegradable implants have been extensively investigated and successfully commercialized in 2010 as biodegradable screws for bone fixatio and fragmentation due to their excellent biodegradability and biocompatibility and appropriate mechanical properties[27].However,the rapid degradation rate of Mg-based materials impedes their further clinical applications.Alloying appears to be an effective approach to control the biodegradation rate of Mg-based materials in biomedical applications.In the present study,a Mg-Ca-Zn-Ag quaternary alloy was prepared by powder metallurgy.The biodegradation behavior,antibacterial performance and osteogenic properties of the Mg alloys in vitro and in vivo and the correlation to the biodegradation behavior were systematically investigated.

Fig.8.Evaluation for the degradation rates of the Mg alloys in vitro and in vivo,and their correlation.(a)A scatter plot of the implant volume as a function of time during the implantation period.(b)The average daily degradation rates of the Mg alloys in vitro and in vivo normalized by the corresponding initial volume.(c)Confusion matrix of in vitro degradation and in vivo degradation parameters according to their pairwise correlations.Deep blue and red represent strong correlation coefficients close to 1 or-1,and the cross mark indicates no significan correlation.(For interpretation of the references to color in this figur legend,the reader is referred to the web version of this article.)

4.1.Biodegradation behavior in vitro and in vivo

It is well known that the microstructure and chemical composition play a crucial role in regulating the degradation behavior of Mg alloys.During solidificatio of the Mg-0.8Ca alloy,the Mg2Ca eutectic phase with a lamellar structure formed in the grain boundaries.The addition of Zn and Ag into Mg-0.8Ca not only led to the emergence of Ca2Mg6Zn3and Mg3Ag intermetallic phases but also resulted in a higher degree of microstructural homogeneity and grain finishin[23],as confirme by the SEM and XRD results(Fig.1).These differences in microstructure and composition further resulted in different corrosion rates of the three Mg alloys.Although the results from electrochemical corrosion tests of various Mg alloys showed a similar trend,we could consider that they had similar characteristics in the initial corrosion response during the electrochemical testing.Further quantitative results showed that all three Mg alloys exhibited higher Ecorrand lowericorrvalues than pure Mg[28],and ZQ71 had the highest Ecorrand lowesticorrvalue among the three Mg alloys(Fig.2).According to ASTM standard G102-89,the corrosion rate(CR,mm/year)of the samples is directly proportional to theicorrvalue[26,29].Therefore,the corrosion rates of the pure Mg and three Mg alloys were observed to be in the following order:pure Mg＜ZQ＜ZQ63＜ZQ71,indicating that alloying significantl improved the corrosion resistance of Mg.This findin is in agreement with a previous study by Mao et al.,who demonstrated that the addition of Ca and Zn could improve the corrosion resistance of Mg alloys by enhancing the density of the oxide fil on the outer layer[30].In general,the corrosion occurring in Mg alloys can be attributed to the introduction of impurities and alloying elements that make the active Mg matrix and other phases form galvanic corrosion[31].In the galvanic couple of the Mg-Ca alloy,the Mg2Ca phase was the anode,and theɑ-Mg phase was the cathode.After introducing Zn,the Ca2Mg6Zn3phase formed and acted as the cathode,and the Mg2Ca phase acted as the anode.In this case,the Ca2Mg6Zn3phase was corroded slower than the Mg2Ca andɑ-Mg phases due to lower reactivity[16,32].Upon the addition of Ag,a Mg3Ag intermetallic phase formed,which caused the formation of a microgalvanic cell between theɑ-Mg and the precipitates of Mg3Ag,resulting in a slight increase in the corrosion rate[12].

In addition to intrinsic material factors,including microstructure and chemical composition,the culture system and method used in vitro could affect the degradation behavior of Mg-based materials.To better reflec the biodegradation responsivity under physiological conditions,we used an MC3T3-E1/ɑ-MEM culture system to investigate the biodegradation behavior of the Mg alloys[26].The results from the in vitro degradation showed a mass gain at day 1 and then a mass loss at day 2 and day 3 for both ZQ and ZQ63,but ZQ71 showed a mass loss at day 3.The sample mass gain suggested the deposition of insoluble degradation products on the surface of the Mg alloys,which was also confirme by the SEM observation(Fig.4a).During the in vitro culture period,ZQ71 showed a smaller mass loss and lower pH and[Mg2+]in the culture medium than ZQ63 and ZQ(Fig.4b and c),indicating a significantl lower in vitro degradation rate.The decreased in vitro degradation rate of ZQ71 could be directly attributed to its enhanced corrosion resistance,as demonstrated by the electrochemical corrosion tests.

The implantation results further confirme the positive effect of the alloying process on enhancing the corrosion resistance of the Mg-based materials.Hydrogen gas released during the fast degradation of Mg-based materials induces cavities in the peri-implant bone tissues.Additional hydrogen gas pockets inside the adjacent trabecular bone and cortical bone were observed viaμ-CT for the ZQ group(Fig.6a).In addition,a large hollow bone marrow area was formed in the defect region of the ZQ group due to the rapid degradation of implant materials.In contrast,a well-maintained adjacent bone structure was present in both the ZQ71 and ZQ63 groups,indicating that the addition of Zn and Ag regulated the degradation rate of the ZQ alloy(Fig.6d and Fig.7c).Histological analyses at 4 weeks postoperatively further confirme this result.Importantly,compared to that for ZQ63,ZQ71 exhibited a lower implant volume reduction rate,suggesting the dominant role of Zn instead of Ag in improving the corrosion resistance.

According to the results summarized from the in vitro and in vivo studies,the in vivo average daily degradation rates of all three Mg alloys were significantl higher than their respective degradation rates in vitro,consistent with previous studies[33,34].Compared to the cortical bone region,the trabecular region with rich vasculature and body fluid promotes the rapid degradation of Mg-based materials.In a recent study,Cipriano et al.found that the in vivo average daily degradation rates of Mg-based intramedullary were significantl higher than their in vitro degradation rates due to the site of implantation in the highly vascularized intramedullary region[26].In contrast,a systematic literature survey by Martinez Sanchez et al.indicated that the in vivo corrosion rate of many Mg alloys could be 1 to 4 times lower than the results from in vitro immersion tests[35].Many factors,including the selection of the corrosion solution,implantation site,experimental methods and time periods,could influenc the degradation rates of Mg-based materials.

Given the diversity of culture systems,model construction and evaluation methods,in vitro simulated experiments are very challenging in predicting the in vivo degradation performance of Mg-based materials.Studies performed under different experimental conditions often exhibit diverse or even conflictin results.Correlation analysis has been widely used to identify some predictor parameters related to postsurgery survival times and predict the in vivo bone forming capacity of CaP-based bioceramics[36,37].Our Pearson correlation analysis revealed that electrochemical tests can be a useful tool in predicting the degradation behavior of Mg-based materials.Compared to those from in vitro simulated experiments,the parameters generated from the electrochemical tests were more effective in predicting the degradation behavior of the implants.

4.2.Antibacterial activity in vitro

The results of the in vitro evaluation of the antibacterial activity of various Mg alloys showed that the antibacterial effect of all Mg alloys gradually increased with extended culture time(Fig.5),indicating that their antibacterial activity was closely related to the pH value and[Mg2+][11].The synergetic effects of Mg ions and alkalinity may impair the expression of some adaptive genes,which are a key component of bacterial defense against environmental stresses,thus leading to bacteria inactivation[17].After 12 h of culturing,ZQ exhibited a higher antibacterial activity againstE.colithan ZQ71 and ZQ63,suggesting that the increased pH and Mg ions played more important roles than the released Zn and Ag ions in restraining bacterial growth in the early stage of culturing.With prolonged culturing time,ZQ71 and ZQ63 displayed similar but higher antibacterial potential than ZQ,although they produced lower pH values and[Mg2+],as confirme by a faster increase in the antibacterial rate.The Zn and Ag ions released with Mg alloy degradation might contribute to the enhanced antibacterial ability.It has been reported that released Zn ions could reduce bacterial adhesion and inhibit bacterial growth by generating reactive oxygen species[38].In addition,released Ag ions were able to exchange with imidazole,amino and carboxyl groups of the cell envelope proteins,and penetrate bacterial cell membranes to induce abnormal changes in the molecule structure of intracellular DNA,resulting in the loss of replication abilities[39,40].However,no statistically significan differences in the antibacterial activity between ZQ71 and ZQ63 were detected after 48 h of culturing,which might be attributed to the relatively low Ag ion concentration and short culture time.It is worth noting that the results from the antibacterial tests showed thatS.aureusinstead ofE.coliwas more sensitive to the ZQ71 and ZQ63 alloys.Similar to our results,Zou et al.also reported that Mg alloy with Zn-containing coating exhibited a significantl stronger antibacterial effect againstS.aureusthanE.coli[9].Higher susceptibility ofS.aureusthanE.coliis potentially relevant to the differences in the cell wall structure and the cell physiology.Gram-positive(S.aureus)cell walls,constituted with teichoic acids and lipoteichoic acids,are more vulnerable to Zn and Ag ions than Gram-negative(E.coli)cell walls[41].Moreover,Gram-negative bacteria are surrounded by an outer membrane that contains lipopolysaccharide molecules,which acts as an efficien barrier against released ions penetration[42].

4.3.Biocompatibility and osteogenic properties in vitro and in vivo

Good biocompatibility is an essential requirement for the clinical application of biomaterials.In general,the cytotoxicity of Mg-based materials is due to their high degradation rate.Rapid degradation of the Mg matrix causes hydrogen evaluation,high ion concentration and local alkalization,which adversely affect cell adhesion and growth[43].In the present study,all Mg alloys showed a similar cell morphology,spreading and proliferation compared to those of the control group,indicating good in vitro biocompatibility.The improved cytocompatibility of the Mg alloys is believed to be related to the enhanced corrosion resistance and the released functional ions.The enhanced corrosion resistance significantl slows the degradation of Mg alloys,which further creates a relatively stable interface for cell adhesion and growth[44].Furthermore,the released Ca and Zn ions also contributed to the enhanced cytocompatibility.As essential elements for the human body,Ca and Zn play important roles in many biological functions and have been demonstrated to promote cytocompatibility[45,46].

Our implantation results further confirme the good in vivo biocompatibility of the Mg alloys.Consistent with previous studies,all three Mg alloys did not induce obvious inflammator responses in vivo and caused any change beyond biological variations in Mg2+,Ca2+and Zn2+ionic concentrations in the blood during the implantation period.However,the Mg alloys presented different osteogenic properties in vivo.After 1 week of implantation,both ZQ71 and ZQ63 showed better integration with the surrounding bone tissues than ZQ,owing to the inhibition of the degradation rate in the initial stages of implantation,as seen in theμ-CT images(Fig.6a).During the whole implantation period,ZQ71 exhibited higher and more stable bone substitution rates than those for the ZQ63 and ZQ groups,suggesting a matched degradation rate with new bone formation over the long term.There was a relatively great difference in the quantitative results of new bone substitution rates between micro-CT and histological analysis due to the different evaluation method[47-49],but the overall changing trend was similar.The results further confirme that the net gain in the newly formed bone in the ZQ71 group could be ascribed to an enhanced corrosion resistance and osteogenic activity,in accordance with our in vitro results.In general,the overall bone remodeling in response to the degradation of Mg-based implants can be described as a result of two parallel processes.On the one hand,the rapid degradation of material releases hydrogen gas into the surrounding bone tissue,resulting in the formation of a gas cavity.After the addition of Zn and Ag alloying elements,the enhanced corrosion resistance significantl decreased the formation of gas cavities[50].On the other hand,the cavities created by the degradation of materials and released hydrogen gas gradually fil with newly regenerated bone,cartilage and fibrou tissue.The released metallic ions,including Mg2+,Ca2+and Zn2+,could stimulate extensive bone remodeling,leading to a net gain in bone volume and high-quality new bone[26,51].

5.Conclusion

In the present study,we evaluated the degradability,antibacterial property,biocompatibility and osteogenic activity of three Mg alloys(ZQ,ZQ71 and ZQ63).The addition of Zn and Ag could enhanced the corrosion resistance of ZQ alloy significantl,while a higher Ag content led to a slight decrease in it.As a result,ZQ71 showed a significantl lower degradation rate both in vitro and in vivo than ZQ and ZQ63 alloys.Possibly due to the implantation site located in the highly vascularized trabecular region,the average daily degradation rate of each Mg alloy in vivo was significantl higher than that in vitro.The further correlation analysis demonstrated that the electrochemical test could predict the degradation behavior of the Mg-based materials effectively.More importantly,ZQ71 showed a significantl higher osteogenic activity and bone substitution rate than ZQ and ZQ63,due to its lower degradation rate as well as the stimulatory effects of the released metallic ions.Therefore,the ZQ71 alloy might be a good candidate for biodegradable bone graft application.

Declaration of Competing Interest

The authors declare that they have no competing interests.

Acknowledgments

This work was financiall supported by Inter-Governmental S&T Cooperation Project Between China and Romania(2018LMNY003),Sichuan Science and Technology Innovation Team of China(2019JDTD0008)and the Fundamental Research Funds for the Central Universities(2021SCU12071).

Supplementary materials

Supplementary material associated with this article can be found,in the online version,at doi:10.1016/j.jma.2021.05.013.