Benjamín Mllán-Ramos ,Danela Morqueho-Marín ,Phaedra Slva-Bermudez ,Davd Ramírez-Ortega ,Osmary Depablos-Rvera ,Juleta Garía-López ,Marana Fernández-Lzárraga,f,José Vtora-Hernández,Detmar Letzg,Argela Almaguer-Flores,Sandra E.Rodl

a Instituto de Investigaciones en Materiales,Universidad Nacional Autónoma de México,CDMX 04510,Mexico

b Posgrado en Ciencia e Ingeniería de Materiales,Universidad Nacional Autónoma de México,CDMX 04510,Mexico

cUnidad de Ingeniería de Tejidos,Terapia Celular y Medicina Regenerativa,Instituto Nacional de Rehabilitación Luis Guillermo Ibarra,CDMX 14389,Mexico

d Posgrado en Ciencias Médicas,Odontológicas y de la Salud,Universidad Nacional Autónoma de México,CDMX 04510,Mexico

e Instituto de Ciencias Aplicadas y Tecnologia,Universidad Nacional Autónoma de México,CDMX 04510,Mexico

fPosgrado de Doctorado en Ciencias en Biomedicina y Biotecnología Molecular,Escuela Nacional de Ciencias Biológicas,Instituto Politécnico Nacional,CDMX 11340,Mexico

g Institute of Material and Process Design,Helmholtz-Zentrum Geesthacht,Max-Planck-Strasse 1,Geesthacht 21502,Germany

h Magnesium Innovation Centre MagIC,Helmholtz-Zentrum Geesthacht,Max-Planck-Strasse 1,Geesthacht 21502,Germany

i Laboratorio de Biointerfases,Facultad de Odontología,División de Estudios de Posgrado e Investigación,Universidad Nacional Autónoma de México,CDMX 04510,Mexico

Abstract Biodegradable magnesium alloys are promising candidates for temporary fracture fixation devices in orthopedics;nevertheless,its fast degradation rate at the initial stage after implantation remains as one of the main challenges to be resolved.ZrO2-based coatings to reduce the degradation rate of the Mg-implants are an attractive solution since they show high biocompatibility and stability.In this work,the degradation,cytotoxicity,and antibacterial performance of ZrO2 thin films deposited by magnetron sputtering on a Mg-Zn-Ca alloy was evaluated.Short-term degradation of ZrO2-coated and uncoated samples was assessed considering electrochemical techniques and H2 evolution(gas chromatography).Additionally,long term degradation was assessed by mass-loss measurements.The results showed that a 380 nm ZrO2 coating reduces the degradation rate and H2 evolution of the alloy during the initial 3 days after immersion but allows the degradation of the bare alloy for the long-term.The ZrO2 coating does not compromise the biocompatibility of the alloy and permits better cell adhesion and proliferation of mesenchymal stem cells directly on its surface,in comparison to the bare alloy.Finally,the ZrO2 coating prevents the adhesion and biofilm formation of S.aureus.

Keywords: Zirconium dioxide;Magnetron sputtering;Short-term degradation;H2 evolution.

1.Introduction

Magnesium and its alloys are promising candidates to produce biodegradable implants for bone repair applications since their elastic modulus and specific density are similar to those of human bones [1,2],providing less stress shielding and appropriate mechanical support than other metallic alloys.Another advantage of the Mg alloys is that they degrade within the human body reducing the need for a second surgery to remove the metallic support after the bone has been restored.However,despite many efforts,the Mg alloys degrade and lose their mechanical integrity before the bone fractures have healed [3,4].The healing time is between 4 and 12 weeks for small to medium size bones and 12–24 weeks for larger weight-bearing bones.During the last years,an extensive search for Mg alloys with improved biocompatibility,high strength,ductility and lower degradation rates has led to the production of Mg-Zn-Ca alloys [5–15].Both alloy elements contribute positively to bone remodeling and present no risk to humans.It has been shown that addition of Zn and Ca improves the ductility of Mg alloy sheets [10,12,13]by avoiding the formation of the basal plane texture.To preserve the degradation rate under acceptable limits,the alloying elements must be in solid solution [6,7,16].The biodegradation rate of Mg-Zn-Ca alloys range between 8.2 and 2.10 mm/year,which depends not only on the components,but also on the processing method,grain sizes and the test solution used [6].A recent work [17]has reported biodegradation rates below 0.5 mm/year,which is already an ideal rate to permit bone regeneration [3].Hou et al.[17],showed that the mirror polished plates of Mg-Zn(0.7 wt%)-Ca(0.6 wt%)alloy(ZX11)produced by twin roll casting presented a degradation rate<0.20 mm/year and retained the mechanical integrity up to 35 days under immersion,ideal for the fracture heal of small bones.The alloy was not cytotoxic to human primary osteoblasts,and the overall responsein vitrowas superior to other MgZnCa alloys which have shown positive results in clinical tests [18].Meanwhile,Martynenko et al.[19]prepared Mg-1.03Zn-0.66Ca alloys by rotary swaging(RS)to refine the grain structure and achieved degradation rates of 0.29 and 0.44 mm/year for the quenched and RS sample,respectively.

The biocompatibility of the biodegradable implants is largely controlled by the release of degradation products,thus,lower degradation rates led to improved biocompatibility,as well as the preservation of the mechanical integrity.For Mg and its alloys,the production of H2during the degradation is also an important factor determining the performance of the Mg alloys for orthopedic implants [20].The H2evolution is due to the degradation of Mg in aqueous solutions(Mg+H2O→Mg(OH)2+H2↑).Abdel-Gawal and Shoeib [6]evaluated H2release in Mg-Zn-Ca alloys of varying composition,finding degradation rates between 2.17 and 5.47 mm/year after 10 days of immersion,values that were similar to those obtained by the mass-loss test.Atrens et al.[4]indicated that the steady-state corrosion rates estimated from the mass-loss experiments are usually in agreement with the amount of evolved H2measured by the volumetric technique.Kim et al.[21]reported the biological effect of the H2formation during the Mg degradation for bothin vitroandin vivotests finding that the gas production rate is higher at the initial stage of implantation(first day).The released gas was composed mainly of H2with minor concentrations of CO and CO2and although not systemic toxicity was detected by the Mg ions,the gas induced superficial skin necrosis and long-term osteolytic lesions.Therefore,for biomedical applications,not only reduced long-term degradation rates should be achieved,but also the initial abrupt corrosion of the implant-that leads to high H2evolution rates must be reduced.

One strategy to overcome this situation is the use of polymeric or ceramic-based coatings [22,23].Regarding coatings,Yin et al.[22]described that the challenges(corrosionresistant,self-degradable,biocompatible and osseointegrable)required for the coatings have not been achieved yet.Among the different options of ceramic coatings,TiO2is very attractive,since it corresponds to the oxide layer present in the state-of-the-art Ti implants.However,as it happens with many other cathodic coatings,we found that coupling Mg alloys with thin TiO2coatings was significantly detrimental for the corrosion[24].The low standard electrode potential of Mg led to the formation of a galvanic pair with the Ti/TiO2film that enhanced the localized corrosion of the substrate,increasing the H2evolution and the cytotoxic response [24].An alternative oxide ceramic coating with reported biocompatibility and osseointegration capability is ZrO2[25–27].Several studies have reported that the ZrO2coatings deposited on Mg-based alloys by different techniques offer corrosion protection and show excellent biocompatibility and potential to induce osteoblast differentiation [28–36].Most of these ZrO2coatings have thickness above 1.5μm and they clearly provide electrochemical protection efficiencies(EPE)around 80–100%(Review in Supplementary Information).Though,very thick coatings are not attractive for the biomedical applications since it is desirable to maintain the long-term degradation of the magnesium alloy and avoid the overstated release of other metallic ions into the body fluids.Interestingly,recent reports working with atomic layer deposition(ALD)show significant efficiency of thin ZrO2coatings [35,37,38].In our experience [39],ZrO2films deposited by Magnetron sputtering(MS)could also be attractive since the films are biocompatible and induce the differentiation of stem cells into the osteoblast phenotype [40],process that will promote the bone fracture healing.Very few works [34,36]have evaluated the performance of ZrO2films on Mg alloys deposited by MS.

Therefore,the aim of our work is to reduce the initial fast degradation and H2release from the Mg-Zn-Ca(ZX11)alloy using a ZrO2coating deposited by magnetron sputtering,assuring that the biocompatibility is not compromised.For this,we have produced thin ZrO2coatings using reactive magnetron sputtering and evaluated the electrochemical corrosion,mass-loss,H2evolution,as well as their biocompatibility and antibacterial activity.

2.Materials and methods

2.1.Magnesium alloy

The ZX11 alloy strips were produced by twin roll casting(TRC)technique at the Helmholtz-Zentrum Geesthacht.More details on the alloy production and composition can be found elsewhere [15].The ZX11 5.3 mm thick strips were further processed by hot rolling at 450 °C.Four rolling passes where necessary to reach the final gauge thickness of 1.8 mm.Before the first pass and at intermediate passes,the sheets were heated for 15 min at 450 °C.After the final rolling pass,the samples were air-cooled.To recrystallize the microstructure,samples were subsequently annealed at 350 °C for 30 min in a convection air furnace.Details on the microstructure after thermomechanical treatment can be found in [24].

2.2.Films deposition and characterization

Samples of ZX11(Mg-0.7Zn-0.6Ca)alloy were mechanically and progressively fine-ground using a series of SiC papers from #360 up to #2000 grade,rinsed with acetone,cleaned with isopropyl alcohol in ultrasonic bath during 15 min and dried with flow air.

The ZrO2films were deposited on the alloy pieces with dimensions of 10×30×1.8 mm3(named as rectangular)and 10×10×1.8 mm3(named as square)by magnetron sputtering starting from a metallic Zr target with diameter of 10 cm.The deposition conditions were chosen based on our previous results [39,41,42].Deposits were done under a reactive Ar/O2atmosphere with ratio of 8:2 in a total volumetric flow of 10 standard cubic centimeter per minute.The base and work pressure were 4×10?4and 3 Pa,respectively.The radiofrequency(RF)power applied to the target was 200 W.Deposition times between 20 and 60 min were evaluated.The square samples were coated on all faces(both sides and laterals)and used for the immersion and biological tests.

The thickness and topography of the samples were measured using a Zygo NexviewTMoptical profilometer and the images were analyzed through MxTMversion 6.4.0.21 software.

Surface images of the samples were obtained by a field-emission gun scanning electron microscopy(FESEM)JEOL7600F.Surface composition was measured by X-ray photoelectron spectrometry(XPS)using a Physical Electronics VersaProbe II system with a scanning XPS microprobe.The measurements were done using the Al Kα(1486.6 eV)X-ray source(100 μm beam)at different zones and samples to assure the homogeneity and reproducibility of the deposits.

The crystallinity of the coatings was determined by X-ray diffraction in grazing incidence mode(GI-XRD).Additionally,the identification of the ZrO2phase was verified through micro-Raman spectroscopy.Details of these measurements are shown in the Supplementary Information.

2.3.Electrochemical characterization

The corrosion behavior of the ZX11 and ZrO2-coated alloy was carried out using a three electrodes cell,with a saturated Calomel electrode(SCE)as reference,a graphite rod as counter electrode and the ZX11 sample as the working electrode in 150 mL of Dulbecco’s Modified Eagle’s Medium/Ham F-12 50/50 Mix(DMEM/F-12;Corning?,USA)+10% v/v fetal bovine serum(FBS,Gibco?,USA)at 37 °C.A thin-film 3-electrodes cell in which only a defined area(1.13 cm2)of the sample’s face is exposed to the electrolyte was used.This geometry allows to perform two independent measurements on each rectangular coated and uncoated sample.

All the tests were carried out in a Gamry Reference 600 potentiostat in the following order,first the open circuit potential(OCP)was monitored during 3 h,then electrochemical impedance spectroscopy(EIS)was done in a frequency range of 100–100 kHz with a 10 mV amplitude perturbation.To ensure that the sample surface does not suffer major changes,the OCP was measured again,observing that it stabilized quickly in 10 min;subsequently,linear polarization resistance(LPR)was measured at a scan speed of 0.16 mV/s in±25 mV range versus OCP.After that,the OCP was registered again during 10 min,and finally,potentiodynamic polarization(PDP)curves were obtained.The cathodic and anodic branches were measured separately in different zones of the sample.The cathodic polarization was done from 0 to-1.0 V vs.OCP and the anodic from 0 to +1.0 V vs.OCP,respectively,at scan speed of 1 mV/s.

2.4.Hydrogen evolution test

Hydrogen evolution of the ZX11 and ZrO2-coated alloy was determined in a closed system consisting of a 127 mL glass reactor coupled to a gas chromatograph(Figs.1 and S1).Prior to any measurement N2was introduced in the system to displace any residual O2.The hydrogen evolution was measured every 20 min for 5 h using the gas chromatograph(Shimadzu GC-2014)device equipped with a Shimadzu Molecular Sieve Column CU7669-1 and a thermal conductivity detector.N2was used as the carrier gas.The coated and uncoated ZX11 square samples were adhered to a quartz tube inside the reactor using a carbon double-side tape that expose the frontal and lateral faces(area 1.72 cm2)but isolated the back face from the medium.The solution was 50 mL of DMEM/F-12 +10% v/v FBS that was maintained at 37°C and subject to continuous stirring at 500 rpm.The volume to area ratio(V/A)was 29 mL/cm2.

Fig.1.Scheme of the closed system designed for the evaluation of the H2 production during the degradation of the ZX11 and ZrO2-coated alloy using a gas chromatographer.

2.5.Immersion test

The square samples coated on both faces and laterals(total exposed area 2.72 cm2)were weighed(W0)to obtain their initial mass and then sterilized(UV light).Immersion times of 2,4,15 and 30 days were selected.For each time,four samples were allocated in a 3D-printed acrylonitrile butadiene styrene(ABS)sample holder(Fig.S2)specifically designed to allow homogenous exposure of the surface to the culture medium.Sample holders were individually submerged in 220 mL of DMEM/F-12 supplemented with 10% v/v of FBS and 1% v/v antibiotic/antimycotic and maintained at 37 °C.The volume of the solution was estimated according to the ASTM G3172(2004)standard to avoid the alkalization of the medium.The medium was changed every seven days.At the end of the immersion time,the samples were taken out,water rinsed,dried at 50 °C in air and weighed(Ww).One sample of each group was reserved for surface analysis evaluation,meanwhile,the other three were rinsed using a chromium oxide solution to remove the degradation products.The chromium solution was freshly prepared by dissolving 180 g/L chromium(VI)oxide in distilled water.Then,the samples were immersed in the chromium oxide solution during 20 min at room temperature,before rinsing with water and 100% ethanol.Finally,samples were left to dry overnight and weighed(Wt).The degradation rate(PW)in mm/year at each immersion time was calculated according to the following equation:

where?mcorresponds to the mass loss(W0-Wt)in gram,Ais the surface area of the sample in cm2,ρMgis the alloy density(1.748 g/cm3)andtis the immersion time in hours.

The topography of all samples,after removal of degradation products,were analyzed by optical profilometry.

2.6.In vitro evaluation

2.6.1.Viability of cells exposed to the alloy extracts

The square pieces were sterilized(UV light),independently immersed in 1.5 mL of preparedculture medium(DMEM/F-12 supplemented with 10% v/v FBS and 1% v/v antibiotic-antimycotic),and incubated under cellculture conditions(37 °C,5% CO2)for 72 h.According to the ISO 10993:12 standard,the relation of specimen weight to extraction medium was set at 0.2 g/mL.Then,the extracts were collected,sterile filtered(0.22 μm pore size filter)and diluted with fresh supplemented DMEM/F-12 to three different dilutions(5X,10X and 15X),according to the proposed procedure to test the cytotoxicity of biodegradable metals such as Mg and its alloys by Fischer et al.[43].The Mg concentration in the extracts was evaluated as described in the Supplementary Information.

The effect of the extracts on cells viability was independently evaluated by the MTT assay,the Crystal Violet technique,and the LIVE/DEAD(calceinAM/ethidium homodimer)assay,as described in detail in the supplementary file.Human mesenchymal stem cells in pass 5 to 6(Ad-MSC;Mesenchymal Stem Cells derived from human adipose tissue;ATCC? PCS-500-011TM)were used.Their characterization is shown in Fig.S3.

2.6.2.Viability of cells cultured on the surface of the samples

The UV light sterilized ZX11 and ZrO2-coated alloy square pieces were placed in a 24-well tissue culture plate and incubated withculture mediumfor 4 h atculture conditionsto stabilize the samples before cell seeding.Subsequently,Ad-MSC were seeded on the surfaces at a density of 2×104cells/cm2.Cell-seeded samples were placed back in the incubator and incubated in fresh culture medium and conditions for 72 h.Then,cell viability was evaluated by the LIVE/DEADTMViability/Cytotoxicity Kit for mammalian cells(Invitrogen?,USA)according to the kit manufacturer instructions.Finally,samples were rinsed twice with Phosphate Buffer Saline 1X(PBS;Gibco?,USA)and visualized by Fluorescence Microscopy(Axio Imager Z2,Carl Zeiss).Images were processed using the AxioVision software? and cell viability(green/red,live/dead cells)was qualitatively evaluated from micrographs.

2.6.3.Culture medium pH monitoring

In order to evaluate the pH evolution of the medium under cell culture conditions,the ZX11 and ZrO2-coated alloy square samples were sterilized and immersed in 1.5 mL of culture medium and kept at culture conditions for 72 h;according to the ISO 10993 standard with a relation of specimen weight to medium of 0.2 g/mL.Then,at 1,3,5,24,48 and 72 h of incubation,the pH of the medium was monitored with a 340 Beckman pH Meter coupled to a micro pH electrode(Hanna Instruments).

2.6.4.Bacterial test

The effect on the planktonic bacterial growth of two aerobic reference strains associated with opportunistic and implantable device infections:Escherichia coli(ATCC 33780)andStaphylococcus aureus(ATCC 25923)was tested using the culture media containing the lixiviated products from squares of ZX11 and ZrO2-coated alloy samples.In brief,a bacterial suspension with 105cells/mL of eitherE.coliorS.aureus,was added to the samples and incubated at 37°C in an orbital shaker at 120 rpm under aerobic conditions for 1 and 7 days.After incubation,the effect of the lixiviated products of the coated and uncoated alloys was estimated by counting of the number of colony-forming units(CFU’s)present in the incubation broth media taken from the inoculated samples.All experiments were performed in triplicate.

Fig.2.Micrographs obtained by SEM of(a)an uncoated substrate of ZX11 alloy and(b)a ZrO2 film on an alloy substrate.

Additionally,the square samples(ZX11 and ZrO2-coated alloy)incubated with the bacteria were prepared to observe the bacterial adhesion and biofilm morphology using the scanning electron microscopy(SEM).Further details can be found in the supplementary file.

2.7.Statistical analysis

All the data are presented as the mean±standard error.The statistical significance was determined by the T-test and significant differences were determined using Bonferroni’s modification of Student’s t-test.

3.Results

3.1.ZrO2 Film characterization

The first evaluation was the selection of an adequate thickness for the ZrO2film.As mentioned before,the purpose is to provide corrosion resistance to the Mg alloy using a minimum thickness.The selection was done using electrochemical impedance spectroscopy measurements for films deposited at 20,40 and 60 min in 0.89 wt.% NaCl solution.The results shown in Fig.S4 indicated that the films deposited at 60 min with a thickness of 386±27 nm duplicated the corrosion resistance of the alloy,value that we considered as a good improvement.Thus,60 min deposition time was selected to produce the ZrO2films for further evaluation.

The surface morphology of the ZX11 and ZrO2-coated alloy pieces is shown in Fig.2(a,b),where it can be observed that the lines left on the substrate during the grinding are coated by a granular-like ZrO2film.This granular morphology is typical of magnetron sputtered films deposited on similar deposition conditions(no bias and intermediate pressures)[44,45].

Fig.3 shows an example of the(a)low and(b,c)highresolution XPS spectra of a ZrO2film,measured without Ar+ion cleaning.The low-resolution spectrum reveals photoelectron peaks from Zr,O,and a minor C contribution,without signals from the Mg substrate(see Fig.3a),even when different collection angles were used(Fig.S5).Similar spectra were obtained in different regions and samples,confirming the uniformity of the coatings,which covered the peaks and valleys of the Mg substrate as can be observed in the highmagnification SEM images in Fig S5.

The high-resolution spectrum of the Zr 3d is shown in Fig.3b,a good fit of the spin-orbit doublets was achieved considering two oxidation states for the Zr atoms.The major contribution belongs to the Zr4+state since the binding energy of the Zr 3d5/2orbital is placed at 182.30 eV with the corresponding Zr 3d3/2orbital at 2.38 eV above [46].Small spin-orbit signals from a suboxide or Zr-OH were found at 180.24 and 182.62 eV [47,48].The chemical shift between the Zr doublets is about 2.0 eV,and according to [48,49],it corresponds to the Zr2+oxidation state.The presence of the Zr-OH is ruled out because the Zr 3d chemical shift should be only 1 eV [47].The analysis of the O 1s orbital showed a major peak centered at 530.2 eV corresponding to the ZrO2(Fig.3c)and a small signal(12%)at 532.2 eV,which is associated to the O bonded to the adsorbed carbon.Similar bonding configuration was observed for different zones of the same sample or even among different samples deposited under the same conditions.The average atomic O/Zr ratio,estimated after correcting for O bonded to C,was 2.1±0.1,which suggests a slight excess of oxygen.This excess oxygen is more likely not bonded and allocated at grain boundaries or within the micro-porosity of the films.

The structural identification of the ZrO2films was performed using XRD and Raman spectroscopy.The XRD pattern indicates that the film has a nanocrystalline nature(crystallite size is about 10 nm)with the ZrO2monoclinic phase,as it is shown in Fig.4a.The Raman spectrum shown in Fig.4b confirms the monoclinic phase according to Barberis et al.[50].No substrate heating was used during film deposition but the prolonged deposition time induced substrate heating and crystallization of the film.

Fig.3.(a)XPS low-resolution spectrum of a ZrO2 film on ZX11 alloy.High-resolution spectra of(b)Zr 3d and(c)O 1s orbitals.

Fig.4.(a)Grazing incidence XRD pattern and(b)Raman spectrum of the ZrO2 film on ZX11 alloy.The spectrum is broad but Lorentzian deconvolution allows the identification of the phase.

3.2.Electrochemical evaluation

The open circuit potentials(OCP)of the ZX11 and ZrO2-coated alloy are showed in Fig.5a.The data presented is the average of six measurements performed on six different samples following the procedure described in Section 2.3;that is,3 h of OCP followed by EIS,10 min of OCP followed by LPR and 10 min of OCP before the final PDP test.As soon as the alloy enters in contact with the electrolyte the corrosion process starts.During the first hour of immersion,the OCP of the ZX11 alloy changed rapidly towards more negative potentials(decreasing approximately 80 mV).After reaching -1.859 Vvs.SCE,the trend is reversed and the OCP moves towards less negative values.On the other hand,the ZrO2-coated alloy initiated at-1.854 Vvs.SCE and shifts into less negative potentials during the following three hours.The variation of the OCP value after the EIS measurements was 17 and 4 mV for the uncoated and coated alloy,respectively.Meanwhile,a minor variation was observed between the LPR and PDP measurements.This indicates that both surfaces attain certain stability after the 3 h of immersion,and they were not substantially affected by neither the EIS nor the LPR acquisitions.

Nyquist plots of the ZX11 and ZrO2-coated alloy present three-time constants,Fig.5b.The first capacitive semicircle at high frequencies is commonly associated to the resistance and capacitance of the accumulated corrosion products.The middle frequency semicircle is referred to the charge transfer resistance and capacitance of the double layer [33,35].The inductive loop at low frequencies is associated to the adsorption-desorption of the corrosion products onto the surface and related to pitting corrosion of the ZX11 alloy [51].However,as it is usually referred,a larger semicircle indicates a larger impedance and better corrosion resistance of the system.Thus,from the qualitative comparison obtained from the average of six independent measurements,it can be resolved that the ZrO2thin film offers corrosion protection to the alloy.This agrees with the 2-fold decreased of the modulus of the impedance at low frequency(10 mHz)observed in the Bode representation(Fig.5c)for the coated alloy,data reported in Table 2 as Bode-R.Although a significant difference was not observed for the phase shift,Fig.5d.

The EIS spectra were simulated using the equivalent circuit(EC)shown in Fig.5b,where Rsis the solution resistance,Rfis the resistance of the corrosion products layer,CPEfis the constant phase element related to the capacitance of the passive layer of the bare alloy or the ZrO2thin film for the coated alloy.The charge transfer resistance and double layer capacitance are represented by Rtand CPEt,respectively.Additionally,to fit the response at low frequencies an inductance,L,and its respective resistance,RLwere included.The fitted parameters are shown in Table 1.

Fig.5.(a)Average of six independent measurements of the OCPs of the ZX11 and ZrO2-coated alloy immersed in DMEM/F-12 +10% v/v FBS as a function of the time.The OCPs were measured before the EIS,LPR and the PDP tests for each sample.Average values of the EIS results for ZX11 and ZrO2-coated alloy:(b)Nyquist plot presenting the measured data as symbols and the result of the fitting using the equivalent circuit as lines,(c)Impedance modulus and(d)Phase angle plots.

Table 1 Fitted parameters of the EC for coated and uncoated alloy samples.a1 is the exponent of the CPEf and a2 is the exponent of the CPEdl.

After EIS measurement,the OCP was monitored again during 10 min and then,the linear polarization resistance(LPR)was assessed.The slope of the Log ivs.E corresponds to the polarization resistance of the system(Rp),Fig S6.The results showed a higher Rpfor the coated sample in comparison with the uncoated sample,see Table 2.

In order to further evaluate the corrosion performance,potentiodynamic polarization(PDP)measurements were carried out and the corresponding PDP curves are showed in Fig.6(a,b).Since Mg is a very reactive substrate,anodic and cathodic branches were obtained separately,and each figure represents the average of three measurements.Cathodic branches are qualitatively very similar,but Tafel extrapolation used to get the corrosion potential(Ecorr)and corrosion current density(icorr)indicated small differences,Table 2.TheEcorrof the uncoated and ZrO2-coated samples is the same but theicorrof the ZrO2-coated samples diminished approximately 30% in comparison with the uncoated alloy.The anodic branches could not be fitted since both show a passivation region that cannot be fitted assuming a Tafel behavior.However,it shows that for voltages between -1.4 and -1.7 Vvs.SCE,the current is slightly lower for the coated sample.

Fig.6 also contains the topographical analysis of the samples after being submitted to the succession of electrochemicalmeasurements:OCP-EIS-LPR-PDP.It shows separate images for the cathodic and anodic branches.The images were obtained using the optical profilometry and for each condition,three figures are shown:the whole exposed area(inset),a higher magnification image of the affected zones,and a profile obtained along the whole area.Observing the four figures,it can be seen that under anodic conditions,the main features correspond to accumulation of material on the surface.Meanwhile,for the cathodic conditions,holes are the main features.The accumulated materials correspond to the well-known corrosion products during Mg degradation(Mg(OH)2,MgCl2,MgCO3).The effect of the ZrO2coating is observed in a reduction in both lateral dimensions and maximum height of the corrosion products.The difference is more significant for the cathodic exposed areas.There are less holes for the coated sample and the deep is reduced from -11.3 to -8.4 μm.Nevertheless,the coated sample presented holes of larger lateral dimensions,which might be related to localized corrosion areas associated to zones where the film was detached,as shown later.The variation of the root-mean-square roughness(Sq)of the samples for the whole area(10.2×10.2 mm2),before and after the electrochemical tests is presented in Table 3.The data shows that despite the initial Sqvalues are lower for the uncoated alloy,there is a larger increment for the uncoated sample during both the anodic and cathodic PDP curves in comparison with the ZrO2coated samples.The increase in the average roughness is representative of the damage suffered by the surface during the tests.

Table 2 Resistances obtained from the Bode diagram and LPR fit and fitted parameters of the cathodic PDP curve of ZrO2 coated and bare samples.

Table 3 Surface roughness before and after PDP and H2 evolution assessment.

The electrochemical parameters obtained from EIS,LPR and PDP presented in Table 2 indicate that the ZrO2thin film deposited on the ZX11 alloy offers corrosion protection,and its protective properties are more notorious from the LPR and EIS tests.The electrochemical protection efficiency(EPE)estimated from the Bode-R and LPR-Rpresults shows that the thin ZrO2coating provides about 40% protection.It is important to mention that we used a large V/A ratio(88.5 mL/cm2)to limit the alkalization of the medium and mimic the physiological conditions.The pH variation at the end of the electrochemical tests was 0.42±0.08 for the ZX11 and 0.46±0.05 for the ZrO2-coated alloy,i.e.the pH remained around natural physiological values,resulting in a continuous corrosion of the samples.

Additionally,the roughness of the coated samples after PDP evaluation is lower in comparison with the uncoated samples since the ZrO2coating diminishes the corrosion rate and consequently the precipitation of corrosion products on the surface.

3.3.Hydrogen evolution

Hydrogen evolution(HE)during Mg degradation is one of the major issues limiting the application of Mg alloys into the biomedical field.It is normally measured using the volumetric method,but such method is not reliable for high-corrosion resistance alloys as the ZX11,due to the low amount of H2produced.Therefore,in this work,we selected a different approach to obtain the instantaneous degradation rate of samples immersed in a solution of DMEM/F-12+10% v/v FBS at 37°C through the H2evolution measured by gas chromatography.To quantify the H2generated during the immersion time,a calibration curve was previously performed.The calibration curve relates the integrated area of the chromatographic hydrogen peak to different known H2pressures(using pure H2gas),then,the moles of H2are estimated using the ideal gas expression.

Where,nis the number of moles of H2,Pis the pressure of the system at each H2injection entering in the closed system,Vtotalis the total volume of the closed system reactor andVsolutionis the volume of the reaction solution(50 mL of DMEM/F-12 +10% v/v FBS),Ris the ideal gas constant(0.082 L atm/ mol K)andTis the reaction temperature(310.15 K or 37 °C).

Fig.6.PDP plots(a)anodic and(b)cathodic branches.Topography of the samples after the anodic and cathodic PDP evaluation of the ZX11(c,d)and ZrO2-coated alloy(e,f).Figures present the whole exposed area captured in an image of 10.2 × 10.2 mm2,an amplified area of 1070 × 1070 μm2,and the profile obtained at the center of the whole exposed areas(black line shown).

The results are expressed as the amount of H2in units of μmol/cm2as a function of the immersion time.Considering that the H2evolution occurs under open circuit potential and corresponds to the cathodic reaction(2H2O+2e?→H2+2OH?)that induces the dissolution of Mg(Mg →Mg+++2e?),the charge neutrality demands that the rate of cathodic reaction(HE rate)equals the rate of anodic reaction and so,the dissolution of one magnesium atom generates one molecule of hydrogen gas.In other words,the evolution of one mol of hydrogen gas corresponds to the dissolution of one mol of magnesium [52–54].In this way,the mass loss of Mg can be calculated from the results of the HE in a H2:Mg molar proportion of 1:1,as shown in Fig.7 for both evaluated samples.The protective effect of the ZrO2led to a significant reduction in the amount of evolved H2observed immediately after the initial measurement at 20 min.This response is preserved along the 300 min of the test,where the ZrO2-coated alloy accumulated half the amount of evolved H2compared with the uncoated alloy.Fig.7 shows that the H2release is not linear along the evaluation period,reflecting the initial corrosion of the Mg and the subsequent passivation of the surface by the formation of the Mg(OH)2layer.However,for simplicity,the plot can be divided into 2 or 3 stages,where varying degradation rates can be estimated from the slopes allowing comparison with previous works [55,56].In the case of the ZX11 alloy,the HE plot can be divided into three stages.In the first 60 min(Stage I)high initial H2production was observed with a rate of 0.296 μmol/cm2min equivalent to a mass loss rate of 7.2 μg/cm2min,followed by a gradual decrease after 180 min(Stage II,H2rate of 0.095 μmol/cm2min),and finally,a stabilization is observed for the remaining period(300 min Stage III)with a HE rate of 0.01 μmol/cm2min equivalent to mass loss rate of 0.38 μg/cm2min.On the other hand,for the ZrO2coated sample,only two stages were observed.During the first 180 min(Stage I),slow HE was observed,with a rate of 0.070 μmol/cm2min(1.7 μg/cm2min),value that is 4 times lower than for the uncoated sample.Finally,in the stage II,the HE rate was 0.037 μmol/cm2min(0.9 μg/cm2min),2.5 lower than the rate of the uncoated ZX11 alloy in the stage II.These results indicate that the deposition of the ZrO2layer hampers the access of the solution to the surface of Mg and cathodic sites.Therefore,the rate of H2production decreases.

Fig.7.(a)Hydrogen evolution as a function of immersion time for the ZX11 and ZrO2-coated alloy and corresponding mass loss of Mg on the right y-axis that was calculated from the results of the hydrogen evolution assuming that one mole of hydrogen gas corresponds to the dissolution of one mole of magnesium.[53]Topography of the samples before(b,d)and after(c,e)the H2 evolution test.

Following Shi et al.[53],the average corrosion rate estimated from the H2evolution experiments,PAH,over the whole immersion time was also obtained and expressed in different units for comparison(Table 4).For the rate in mm/year,the following equation was used:

whereVH(mL/ cm2)was obtained as

whereρis the hydrogen gas density at 37 °C and 1 atm(0.0000781 g/mL),(nH2)is the number of moles of H2in the total immersion time(5 h),MMH2is the molecular mass of H2gas(2.015 g/mol)and S is the surface area(1.72 cm2).

It is important to point out that the method used in this work to quantify the H2generated is different from the common funnel and eudiometer test,since it only quantifies the H2,not the total volume of evolved gases [57].Moreover,because the large volume-to-area used(V/A=29 mL/cm2)and stirring conditions,there was not a significant increase in the pH and accumulation of corrosion products,resulting in a continuous dissolution of the Mg.The lower H2evolutionof the ZrO2-coated alloy shows the potential effectivity of the coating on the inhibition of the magnesium degradation during the first hours of implantation(see Table 4).

Table 4 Average and Instantaneous corrosion rates estimated from the HE measured by gas chromatography in a closed system consisting of a 127 mL glass reactor with 50 mL with a solution of DMEM/F-12+10% v/v FBS at 37 °C during 5 h of immersion.

Fig.7 shows the morphological changes observed for both samples after the 300 min degradation in the DMEM/F12+10% v/v FBS culture medium,where the H2evolution was monitored.Before the test,both samples showed the linear pattern left by the grinding process(Fig.7(b,d)).However,this pattern is lost for the uncoated ZX11 alloy(Fig.7c)indicating the aggressive degradation suffered.Meanwhile for the coated alloy,the grinding pattern remains,presenting only some areas of localized corrosion(Fig.7d).Table 3 reports the variation in the surface average roughness after the H2evolution test.The Sqvalue increased from 0.2 to 3.5 μm for the uncoated alloy,meanwhile,for the ZrO2-coated alloy the increment was only from 0.3 to 1.6 μm,indicating a smaller damage to the surface.

Table 5 Comparison of the different degradation rates(in mm/year)of the ZX11 and ZrO2-coated alloys estimated by the PDP(Pi),HE(PAH)and the immersion(PW)tests.

3.4.Degradation in physiological conditions

The degradation rate(Pwin mm/year)and mean degradation depth(μm)was evaluated from the mass loss test as a function of time for the samples immersed in supplemented cell culture medium at 37 °C(Fig.8a).

The ZrO2-coated alloy exhibited slightly lower Pwin comparison to the uncoated alloy for the three immersion times,however,this difference is not statistically significant.Nevertheless,the degradation rate changed over time with the same trend for both samples,showing a faster degradation rate within the first four days of immersion(1.32 and 1.25 mm/year for the ZX11 and ZrO2-coated alloys,respectively)that significantly decreased over the immersion time.The degradation rate became quite stable after 14 days of immersion,and by day 30 the degradation rate was 0.28 and 0.30 mm/year for the ZrO2-coated and uncoated ZX11 alloys,respectively.Fig 8b shows the mean degradation depth as a function of the immersion time,the slope in this plot gives the long-time degradation rate inμm/day [58].The ZX11 alloy exhibited a linear trend with a slope(0.37 μm/day)similar to that reported by Hou et al.[17]for the same alloy,although the test conditions were completely different,since we used a larger immersion volume/area(V/A)ratio(20 mL/cm2)in which the surfaces are not passivated by the corrosion products.However,for the ZrO2-coated ZX11 alloy,a nonlinear behavior is observed,presenting a very low rate up to the 15 days,with a sudden increment at longer time.This enhanced degradation observed at immersion times longer than 15 days and in comparison to the values obtained by the HE and electrochemical tests might be related to the establishment of a galvanic coupling between the Mg and the ZrO2as reported for thin ALD samples [38],but in the ALD films,the loss of protection was observed at shorter immersion times(50 h).

The different degradation process of the ZX11 and ZrO2-coated alloy samples(Fig.8c)is also evident by observing the variation of the surface morphology(optical micrographs in Fig.S7).At 4 days of incubation in cell culture medium,the ZX11 alloy exhibited a series of shallow cavities or“holes”that were uniformly distributed all over the surface of the alloy.The linear pattern observed before the test(Fig.7(b,d))was completely lost.For the ZrO2-coated alloy,a localized degradation phenomenon occurring only in certain areas of the samples’ surface was observed.Most of the coating remained adhered to the surface of the alloy up to 30 days,as could be visually observed and confirmed by XPS analysis of the samples before the CrO3cleaning procedure(not shown).The depth of the grooves in the ZrO2-coated alloy at 30 days of immersion was larger than that of the cavities in the uncoated ZX11 alloy;however,this did not cause a larger overall degradation rate(mm/year).For the coated samples,the localized corrosion occurs in areas where the ZrO2coating delaminates,and the under-film Mg corrodes faster either due to the microenvironment in the cavity(pitting corrosion process)or the formation of the Mg-ZrO2galvanic pair as mentioned before.On the other hand,on the uncoated alloys,the degradation process was more homogeneous over the whole surface of the samples,thus the maximum depth of the cavities seemed not to increase over degradation time and the mass loss was homogeneously distributed all over the surface.

Two secondary but relevant effects are also observed during the degradation of Mg alloys in physiological conditions:the dissolution of Mg with the subsequent increase in Mg concentration in solution and the variation in the solution pH.

Fig.9a shows the concentration of Mg in solution after 72 h of immersion in the cell culture medium.This period was chosen in accordance with the protocol used for the biological evaluation.Since the DMEM/F-12 medium also contains Mg,its concentration was also determined for comparison.Fig.9a shows a reduction of about 30% in the Mg concentration in the immersion medium due to the protective effect of the ZrO2coating.

Fig.9.(a)Mg concentration released to the DMEM culture medium after 72 h of incubation at 37 °C.?, p < 0.05 vs.DMEM-F12;#, p < 0.05 between indicated samples.(b)pH variation of medium for samples immersed in the culture medium using a low solution volume to sample surface ratio as suggested by the ISO 10993.

The pH variation of the ZX11 and ZrO2-coated alloy over 72 h are shown in Fig.9b.Similarly,to the H2evolution,three stages can be identified.Stage 1 corresponds to a drastic increase in pH within the first three hours of immersion,which agrees with the observed increment in H2release by chromatography.For the ZX11 alloy,pH increased from 7.4 to 8.5 compared to the coated sample,which in the same period reached 8.2(Stage 1).In Stage 2,from 3 to 48 h,the pH of the ZX11 alloy increased from 8.5 to ～9.1(0.6 increase in pH).Meanwhile,for the coated sample,the increment was smaller(8.2–8.6).Finally,for the third stage(48–72 h),the pH remained relatively constant,for both the ZX11 and the coated samples.No longer immersion times were verified,because these tests were performed following the ISO 10993 standard,in which low V/A ratios are used,therefore under the static conditions,the alkalization of the solution proceeds.The alkalization decreases the solubility of the corrosion products,which are precipitated on the surface and inhibits further degradation.The lower pH value of the ZrO2-coated alloy in comparison with the uncoated sample demonstrated the barrier properties of the ZrO2coating at short immersion times.

3.5.Biocompatibility evaluation

3.5.1.Calcein evaluation

Fig.10 shows the viability of Ad-MSC cells cultured with different dilutions of the alloy’s extracts,as directly assessed by the calcein AM/ethidium homodimer assay.

After 3 days of cell culture,qualitative evaluation of the micrographs showed a larger number of viable cells(in green)in the ZrO2/ZX11 sample exhibiting an elongated fusiform morphology,similar for all extracts and dilutions studied,and similar to the possitive Ctrl(Fig.10a).Quantitative analysis of the number of viable cells per micrograph at 3 days of culture evidenced a larger number of viable cells when culture with the extracts of the ZrO2-coated alloy,at all extracts dilutions,in comparison with the Ctrl and the extracts of ZX11;Fig.10b.Statistically signficant differences between the ZX11 alloy and the ZrO2-coated alloy were only observed for the 15X extracts dilutions at day 3.However,this enhanced cell viability percentage is not confirmed by the quantitative MTT and violet crystal assays shown later.

At day 6 of culture,an increase in the number of viable cells was observed for all extracts and dilutions studied(Fig.10c)in comparison with their corresponding samples at 3 days of culture.Nevertheless,cells cultured with the extracts of ZX11 and those of the Ctrl seemed to have a more rhombohedral-like shape,in comparison to cells cultured with the extracts of ZrO2-coated alloy,which displayed a more elongated-fusiform morphology.At 6 days of culture,the quantification of the number of viable cells per micrograph also showed a larger average number of viable cells when culture with the extracts of the ZrO2coated alloy or the ZX11 alloy,in comparison with the Ctrl.However,differences at this culture period were not significant(Fig.10d)but for the 5X extracts dilutions of the ZrO2coated alloy and the ZX11 alloy,in comparison with the Ctrl.

3.5.2.MTT and violet crystal assays

The viability of Ad-MSC cells cultured with different dilutions of the alloys extracts was also indirectly evaluated at 3 days of culture by the metabolic activity of the cells(MTT assay)and the number of cells adhered(Violet Crystal assay)to the culture plate;Fig.11.

The MTT assay(Fig.11a)showed that cell viability percentage was not affected by any of the extracts(from both ZX11 and ZrO2-coated alloy),at the different dilutions studied,exhibiting a 100% viability in all cases,with respect to the Ctrl that corresponded to standard culture conditions for cell growth.There were not statistically significant differences in cell viability for any of the extracts in comparison with the Ctrl.In the same sense,the Violet Crystal technique showed no significant differences in cell viability percentage under the different culture conditions studied(Fig.11b),confirming that the extracts did not affect the viability of the cells at the different dilutions studied,in comparison with the Ctrl.

Despite the small differences in the average viability values obtained from the semi quantitative analysis of the calcein assay(Fig.10),the quantitative MTT and violet crystal assays(Fig.11)did not show statistically significant differences(p <0.05)between the ZrO2coated and uncoated ZX11 alloy .The reason for this is that quantification of the number of viable cells per field from the calcein assay is a localized technique that might led to larger quantitative variations mainly due to the specific fields per culture well that are chosen to be quantified,while MTT and crystal violet are global quantitative techniques that evaluate the total number of cells per culture well plate.Thus,the conclusion from these tests is that the ZX11 alloy and the ZrO2coated ZX11 alloy did not induce any cytotoxic effect to the mesenchymal stem cells up to 6 days and at the three dilutions tested.

3.5.3.Adhered cells

The capability of the ZX11 and ZrO2-coated alloy samples to sustain cell culture was assessed by the calcein/ethidium homodimer assay in Ad-MSC cells cultured on the samples at 3 days of culture.Before this study,the hydrophilichydrophobic character of the samples was evaluated(Fig.S8),indicating that there was not a significant difference in the water contact angle between both samples(109.52°±4.87° for the ZX11 and 110.43°±9.85° for the ZrO2).

Representative micrographs of the viability assay on the surface of the coated and uncoated alloy samples are presented in Fig.12.The images show the attached cells on top of the large crystals produced due to the corrosion of the Mg alloy.The observation of the cells is difficult since the crystals presented a high luminescence.Nevertheless,the color(reddead/live-bright green)and agglomeration of the cells can be observed.It is possible to observe that the ZrO2-coated alloy presented a larger number of viable cells(showed in bright green)adhered to its surface,in comparison with the ZX11 bare alloy.In the same sense,cells adhered to the ZrO2-coated alloys formed groups(monolayers)and exhibited a more extended,fusiform morphology,while cells on ZX11 were isolated and exhibited a round morphology,suggesting a better cell adhesion on the ZrO2-coated alloy compared to ZX11.

Fig.10.In-vitro evaluation using calcein labeling for cell viability for three extracts dilutions and two cell culture times:(a)cells at two different amplifications after 3 days of culture in presence of the uncoated or coated samples extracts,(b)quantitative estimation of the number of viable cells per field at 3 days of cell culture,(c)cells at two different amplifications after 6 days of culture in presence of the uncoated or coated samples extracts,(d)quantitative estimation of the number of viable cells per field at 6 days of cell culture.?, p < 0.05 vs.Ctrl;#, p < 0.05 between indicated samples.

Fig.11.(a)MTT and(b)Violet crystal results for Ad-MSC cultured for 3 days in the presence of the extracts obtained from the ZX11 and ZrO2-coated samples after 72 h of immersion.Not significant difference was observed between the samples and control.

Fig.12.Viability of Ad-MS cells cultured on ZX11 and ZrO2-coated alloy after 72 h of culture.The bright green(calcein)circular features correspond to viable cells and the red(ethidium homodimer incorporated into the cells nuclei)features correspond to non-viable cells,white arrows are used to evidence both kinds of features.(a)and(b)correspond to 5X magnification.(c)and(d)correspond to 10X magnification.The green background fluorescence is due to the corrosion products.

3.6.Bacterial evaluation

The results from the interaction with two bacterial strains are presented in Fig.13 that includes the planktonic growth in the culture medium(Fig.13a,b)and the biofilm growth on the samples’ surface(Fig.13c,d).

Fig.13.Total number of the colony forming units(CFUs)obtained from the culture media containing the lixiviated products of the ZX11 and ZrO2-coated alloys after 1 and 7 days of incubation for(a) E.coli and(b) S.aureus.?p < 0.01,and ??p < 0.001,using T-test.Scanning electron micrographs of bacterial adhesion(1 day)and biofilm formation(7 days)on the ZX11 and ZrO2-coated alloy for(c) E.coli and(d) S.aureus.Scale bar represents 2 μm.

The number of CFUs quantified from the culture media containing the lixiviated products from the alloys,represent the effect of the degradation products in the planktonic bacterial growth.The total number of CFUs varied depending on the bacterial strain and the incubation time.However,for the ZX11 alloy,the number of CFUs was always reduced in comparison to the positive Ctrl at 1 and 7 days of incubation,regardless the strain tested,which is an indication of toxicity or bactericidal effect.The planktonic growth ofE.coli(Fig.13a)in the presence of the lixiviated products from the ZrO2-coated alloy(2.8 CFUs × 106/mL)was higher than for the ZX11(1.1 CFUs × 106/mL)(p <0.01),but similar to the Ctrl(5.2 CFUs × 106/mL).This effect was more pronounced after 7 days of incubation when a significant decrease in the planktonic growth ofE.coliwas detected for the uncoated alloy compared with the ZrO2-coated alloy(0.09vs.13.2 CFUs × 106/mL,respectively)(p<0.001).In the case ofS.aureus(Fig.13b)at 1 day of incubation,significant reduced number of CFUs were counted on the media obtained from the uncoated alloys compared with the coated ones(0.06vs.1.2 CFUs × 106/mL,respectively)(p<0.01).While,after 7 days of exposure to the lixiviated products of the alloys,the planktonic growth ofS.aureuswas significantly reduced with respect to the Ctrl(1.7×1010CFUs × 106/mL)for both uncoated and coated alloys(13.3vs.553 CFUs × 106/mL,respectively),p<0.001.

Since the reduced planktonic growth could be associated with a larger bacterial adhesion to the surface,we did also observe the alloy samples by SEM after the correspondingincubation periods.In the SEM images obtained at day 1,more adhesion ofS.aureusthanE.colicells can be observed on the uncoated ZX11 alloy,while no bacterial cells could be detected on the coated ZrO2alloy.However,after 7 days of incubation,the biofilm formation ofE.colion both the uncoated and coated alloys was evident,whileS.aureusbiofilms were only observed on the uncoated alloys and no visible colonization of this strain could be observed on the ZrO2-coated alloy.

The correlation between these two results indicated that planktonic bacteria was less affected for the ZrO2-coated alloy than the uncoated alloy.This agrees with the reduced corrosion products observed for the coated samples(less H2,lower pH variation and less Mg ionic concentration).Nevertheless,the bacteria attached to the surfaces was also reduced on the ZrO2coated samples.

4.Discussion

In this work,the effect of a 380 nm thick ZrO2coating deposited by magnetron sputtering on the degradation rate and biocompatibility of a Mg-Zn-Ca alloy was evaluated.Sheets of the Mg alloy(ZX11)were produced by TRC method and annealed at 350 °C to produce a fine microstructure without a preferential basal plane texture.Samples of the alloy were coated in one or both sides by a nanocrystalline ZrO2coating presenting the monoclinic phase.The degradation rate(DR)was evaluatedin-vitrousing different approaches:(i)electrochemical characterization,(ii)hydrogen gas evolution by gas chromatography and(iii)mass-loss in semi-static conditions.An estimation of the degradation rates in mm/year from the different techniques is presented in Table 5,although for the electrochemical test,the conversion of Icorrto Pi(corrosion rate estimated from the corrosion current density [59])for the coated sample is not completely accurate,since the film density is not taken in consideration.

ThePiandPAHrates indicates that the degradation rate is decreased by a factor of ～2 using the ZrO2coating.However,this trend is not observed for the long-time degradation obtained after the mass-loss tests.A possible explanation for the larger Pwvalues is that Cl?aggregates might have accumulated in the under-film pitting areas(Fig.7c)and as reported by Gonzalez et al.[60],these aggregates react with the chromic acid causing further degradation of the alloy during the cleaning time.The Cl?aggregates might be trapped during the immersion experiments since stirring is not applied.Moreover,Gonzalez et al.[60]also reported possible interactions between the chromic acid and Zr,that explains why the ZrO2film disappears after the chromic acid cleaning.The images in Fig.8 show that after long immersion time,the film suffers cracks.These are probably initiated in small pores,where the electrolyte passes towards the underlying Mg alloy,provoking an accelerated corrosion due to the differences in the standard electrode potentials between Mg and ZrO2,but also due to the localized character of the corrosion process.Such accelerated corrosion was also observed by Liu et al.[38]and Morquecho-Marin et al.[24].In the latter [24],the deleterious effect of the coating-substrate performance was immediately evidenced by a larger production of H2and enhanced cytotoxicity.In contrast,for the ZrO2-coated alloy in this work,the qualitative and quantitative biocompatibility tests,as well as the HE,the Mg ion release and pH variation,suggest that the ZrO2film provides certain degree of protection despite the similar PWvalues observed at longer degradation times.Therefore,we think that the absolute DRs estimated for the ZrO2-coated alloy during the mass-loss experiment might contain an unmeasurable contribution from the CrO3-Cl?interaction.

It is also plausible that the ZrO2coatings are very protective only during the first days of immersion,as it was observed with the ZrO2coatings deposited by ALD.Liu et al.[38]showed that during the first day,the PAH-DR of AZ31 is reduced by a factor of 3 for the thinner ZrO2film(25 cycles)and a factor of 7 for the thicker film(100 cycles).However,as the immersion time continues,up to 4 days,the DR of the 25-cycles film is nearly double than for the uncoated alloy,effect explained by the penetration of the electrolyte and accelerated galvanic corrosion of the Mg.The DR drop of the thicker film remained,but it was reduced to a factor of 1.5X instead of the initial 7X.Similarly,Yang et al.[35]observed that the PW-DR of the thicker ZrO2film(400 cycles)was 6X factor lower than the uncoated alloy during the 5 days of immersion,but only 3X after 8 days.These two works showed that there is a reduction in the protection as the immersion time increases,which agrees with our results that include larger immersion times.Considering that the biocompatibility tests were performed from extracts obtained after 72 h of immersion and not cytotoxicity was detected(Figs.10 and 11),but the PW-DR of the ZrO2coated alloy at 4 days of immersion was similar to the uncoated alloy,the expected time for the protection is defined between 3 and 4 days.For longer immersion times,the penetration of the electrolyte into the substrate through the film porosity initiates the normal degradation of the alloy,explaining the similar Pwvalues.As mentioned in the introduction,this is the expected response from a protective coating for Mg alloys,that is,to reduce the initial reaction,but eventually allow the degradation of the alloy following its optimized degradation rate.More research needs to be performed to identity whether the 3 to 4 days of reduced corrosion and the percentage of reduction attained(40–50%)are enough for clinical applications,and to evaluate the effect of localized corrosion on the mechanical integrity.However,the biological results indicate that the ZrO2films deposited by magnetron sputtering are promising coatings to reduce the corrosion rate of Mg alloys during the initial stage(<4 days).Nevertheless,improvement at longer immersion time is still required.The advantage of the sputtering technique is that such improvement could be achieved changing some parameters during the deposition,not only increasing the thickness.Strategies to promote denser films include the use of substrate bias,adjusting the Ar/O2flow ratio to avoid the insertion of non-bonded oxygen as observed by the XPS data or promoting the formation of an amorphous film,instead of a nanocrystalline one.

The biocompatibility of the ZX11 and the ZrO2-coated alloy was confirmed using different methods,evaluation of the lixiviation products at 3 different dilutions(5,10 and 15X)obtained according to the ISO 10993 for 3 and 6 days of immersion,and direct cell attachment.Cell viability in presence of the lixiviation products,using the crystal violet and MTT assays,indicated 100% viability for the three dilutions studied without significant differences between uncoated and coated alloy.The calcein/ethidium homodimer assay was used to discriminate live from dead cells by simultaneously staining with green,fluorescent calcein-AM to indicate intracellular esterase activity and red-fluorescent ethidium homodimer-1 to indicate loss of plasma membrane integrity.The technique was used for the evaluation of the cell’s viability cultured in presence of the lixiviation products and for the direct observation of viable cells cultured directly on the surface of the samples.The results from the lixiviation products indicated that most of the cells were alive,in general,without significant differences between the uncoated and coated samples.For the cells cultured directly attached on the samples surface,observation of the cells resulted difficult due to the luminescence of the corrosion products.However,the larger number of viable cells and low number of dead cells on the coated samples was evident.From the biocompatibility tests,it can be concluded that the ZX11 alloy is not cytotoxic and the addition of a 380 nm ZrO2coating does not affect the response.However,the ZrO2coating allows a better adhesion and spreading of the cells on the surface of the samples,probably due to the reduced HE and alkalization of the surrounding media.Results that agree to the previous publications about ZrO2coatings on Mg alloys [33,35,61].Finally,the bacterial-surface interaction results are interesting;they showed that planktonic bacterial growth of both strains(E.coliandS.aureus)are affected by the degradation products on the culture media(H2,alkalization and Mg ions)of the ZX11 alloy,confirming previous reports where an antibacterial activity of Mg alloys have been reported against similar strains [62–64].The ZrO2-coated ZX11 degrade slowly,and this effect is reflected in the larger number of planktonic cells observed.However,when the biofilm,i.e.,the bacteria attached to the surface,was analyzed by SEM,the results indicated that the bacteria did not adhere to the ZrO2-coated alloy in the same amount as observed for the uncoated ZX11 alloy.This is agreement to our previous results [41],where similar nanocrystalline ZrO2coatings were deposited on pure titanium substrates and a lower bacterial adhesion was observed on the ZrO2coatings in comparison to TiO2coatings.The inhibition of biofilm formation is more important for implants than the planktonic activity,since once the biofilm is formed,standard antibiotic treatments are not effective and therefore the implant might fail by the well-known medical device-associated infections [65,66].

5.Conclusions

In this study,the barrier characteristics,biocompatibility and antibacterial properties of a 380 nm thick ZrO2coatings deposited by magnetron sputtering on twin rolled casted MgZnCa(ZX11)alloys were evaluated.

The effect of the coating on the short-term or instantaneous degradation rate was evaluated by H2evolution and electrochemical tests.Meanwhile,the longer degradation rate was monitored by measuring the mass-loss during immersion experiments in physiological solutions.The amount of H2produced(μmol/cm2)was decreased by a factor of two during the first 5 h of immersion,time in which H2production is usually larger because the Mg surfaces are not yet passivated by corrosion products.This result agreed with the ～2-fold reduction in the corrosion current density,the total impedance and polarization resistance,obtained from the electrochemical evaluation.Small defects(micro-pores or pinholes)in the coating allows the penetration of the electrolyte into the Mg substrate,initiating localized corrosion sites that eventually leads to the delamination of the ZrO2coating.In a period between 3 and 4 days,the protection of the ZrO2film is lost allowing the degradation of the substrate.Therefore,the longterm degradation rate of the ZrO2-coated alloy is very similar to that of the ZX11 alloy.

The amount of Mg released into the cell culture medium after 72 h of immersion was reduced by a 30% due to the presence of the ZrO2coating.In the same sense,the pH variation due to the degradation of the magnesium in the cell culture media was slightly reduced(factor between 1.3 and 2)during the first 48 h.

The biocompatibility was not compromised using the ZrO2coating and it allows the adhesion and proliferation of Ad-MSC cells directly on its surface.

Regarding the antibacterial effect of the ZrO2coatings,it seems to decrease the antibacterial effect of the Mg on the planktonic growth ofE.coliandS.aureusdue to the reduction in the degradation rate,pH variation and Mg ion release.However,the coating was especially effective in preventing the adhesion and biofilm formation ofS.aureus.

Acknowledgments

This work was supported by CONACYT-Frontera-1740,CONACyT-CB-288101,CONACyT-299703,and DGAPAPAPIIT-IN101419 projects.We thank technical support from O.Novelo,L.Huerta,C.Ramos,A.Tejeda and E.Hernández-Mecinas.Millán-Ramos B.acknowledges support from the CONACYT Ph.D.-scholarship(CVU 606030)and the program Doctorado en Ciencia e Ingeniería de Materials at the Universidad Nacional Autónoma de México.Morquecho-Marín D.acknowledges the support from the program Maestría y Doctorado en Ciencias Médicas,Odontológicas y de la Salud at the Universidad Nacional Autónoma de México and the Ph.D.scholarship from CONACyT(CVU 856985).Fernández-Lizárraga M.acknowledges the support from the program Posgrado de Doctorado en Ciencias en Biomedicina y Biotecnología Molecular,Escuela Nacional de Ciencias Biológicas at the Instituto Politécnico Nacional and the Ph.D.scholarship from CONACyT(CVU 739515).

Supplementary materials

Supplementary material associated with this article can be found,in the online version,at doi:10.1016/j.jma.2021.07.010.