Reduced graphene oxide(RGO)reinforced Mg biocomposites for use as orthopedic applications:Mechanical properties,cytocompatibility and antibacterial activity

Somyeh Azri,Ali Shmsipur,*,Hmid Rez Bkhsheshi-Rd

a Department of Materials and Metallurgical Engineering,Amirkabir University of Technology,Tehran,Iran

b Advanced Materials Research Center,Department of Materials Engineering,Najafabad Branch,Islamic Azad University,Najafabad,Iran

Received 21 April 2021;received in revised form 15 September 2021;accepted 21 September 2021

Abstract Magnesium(Mg)has attracted wide interest in orthopedic applications as they exhibit great biodegradability and strong biocompatibility,while corrosion is the main concern for Mg that should be addressed prior to biomedical applications.In this work,ZM31(Mg-3Zn-1Mn)/xRGO(x=0,0.5,1 and 1.5 wt%)biocomposites were synthesized by semi-powder metallurgy method.The results showed that the RGO acting as an effective reinforcing fille to prevent deformation and showed better compressive strength(282.3±9 MPa)and revealed enhancement in failure Strain(7.8±2.1%)at 1 wt% RGO concentration compared to Mg alloy(244.5±9 MPa and 7.1±1.5%respectively).Moreover,fracture analysis indicated a more ductile fracture of the nanocomposites after the incorporation of RGO.Crack bridging,crack deflectio and crack branching are dominant mechanisms for reinforcement of Mg-based containing RGO.Mg composites containing 0.5 wt% RGO showed a low corrosion rate(2.75 mm/year),while more incorporation of RGO resulted in an increased corrosion rate(4.38 mm/year).In addition,the degradation rate of ZM31 alloy(2.57 mg·cm-2·d-1)obviously decreased with the addition of 0.5 wt%RGO(1.84 mg·cm-2·d-1)in the SBF.Besides,continuous apatite layers were created on the composites in the SBF solution.Also,the cell culture examinations showed good cell viability and adhesion on composites with 0.5 and 1 wt% RGO,which was demonstrated by the SEM and MTT assay The alkaline phosphatase(ALP)activity of the ZM3-0.5RGO composite was considerably higher than that of ZM31 matrix alloy in 24 h and 48 h,implying higher osteoblastic differentiation.The antibacterial behavior toward both bacteria(E.coli and S.aureus)exhibited that escalating RGO concentration in Mg-matrix composites leads to further inhibition of bacteria growth.These finding suggested that ZM31-0.5RGO biocomposite could be a more promising candidate for orthopedic implants.? 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

Keywords:Mg alloys;Reduced graphene oxide;Mechanical properties;Corrosion resistance;Antibacterial activity;Biocompatibility.

1.Introduction

The need for superior metallic biomaterials for the synthesis of implants is progressively escalating,and it is expected to rise in order to meet the requirements for people experiencing bone fractures,as frequently need the biomaterial implants to be recovered[1,2].The biomaterials that are virtually inert are known as the firs generation of biomaterials,whereas the surface bioactive materials are classifie as the second generation of biomaterials.Finally,the degradable biomaterials are categorized as the third generation of biomaterials with a temporary structure[3].

Biodegradable implant materials might be slowly dissolved,consumed or excreted in the human body and permit tissue to connect with the implant and ultimately substitute it so that there would be no requirement for an extra operation to eliminate implants after the surgical regions were healed[4].The biodegradable polymers,including poly-glycolic acid and poly-lactic acid,are widely applied as implant materials since their low mechanical strength significantl decreases their load-bearing and tissue-bearing capabilities[5,6].In this perspective,compared to the polymer-based materials,metallic materials such as the Mg-based,Fe-based and Zn-based alloys possess greater efficien y as biodegradable materials for load-bearing applications due to the fact that they have higher strength and ductility than the polymers[7,8].As for biodegradable orthopedic implants,an ideal biodegradable material is required to have the strength higher than 200 MPa,the elongation greater than 10% and a degradation rate less than 0.5 mm/year in simulated body fluid at 37°C,to achieve an efficien lifetime of 90-180 days[9].Also,low weight and osseointegration are expected properties in implant materials.The implant material must be strong enough to withstand a variety of biomechanical forces[1].

In comparison with these metallic materials,Mg and its alloys have a more interesting combination of favorable properties because of their low density(1.78 g/cm3),high specifi strength,significan mechanical characteristics including,Young’s modulus(40-45 GPa)and compressive yield strength(65-100 MPa),which match the natural bone(density 1.8 g/cm3;elastic modulus 3-20 GPa,and compressive yield strength(130-180 MPa)better than other metallic implants[10,11].As a result,the fracture toughness of magnesium(15-40 MPa·m1/2)is greater than that of ceramic biomaterials such as Al2O3(4.0 MPa·m1/2)and ZrO2(6-15 MPa·m1/2)[12].In addition,its elastic modulus(41-45 GPa)is close to that of the bone(3-20 GPa)that of iron(～211.4 GPa)or zinc(～90 GPa),avoiding the stress-shielding effect[1].It is well known that stress transfer between an implant device and a bone is not homogeneous when the implant device and the bone have different Young’s modulus;this is known as stress shielding.In such cases,bone atrophy occurs,causing the implant to loosen and the bone to refracturing[13].In this regard,a mismatch in elastic modulus can cause the implant to bear a greater portion of the load and induce stress shielding of the bone[1].The other significan benefi of magnesium is its greater cell interaction,besides its suitable mechanical characteristics[14].Nevertheless,pure magnesium will corrode very easily in the SBF solution containing Cl-ions with a pH of around 7.4 and hence lose its mechanical integrity so that it poses the question of Mg’s suitability as fracture fixatio devices[15-17].Several methods have been introduced to resolve corrosion issues and low mechanical characteristics of pure Mg.These include the composite formation with appropriate bioactive reinforcements,alloying,or surface modifications In this context,Zn,Zr,Sr,Ca,Nd,Ce,Y,Gd,Mn,and Al are commonly used as alloying elements to improve corrosion resistance and mechanical properties[1,16,18].However,alloying elements may not be biocompatible and may pose serious health risks during degradation.According to the literature,Al in the body causes neurotoxicity and Alzheimer’s disease,and rare earth(RE)elements can cause shortness of breath,chest pain,pneumoconiosis,etc.[19,20].In a recent study,after the degradation of the Mg-10Gd pin,serious Gd accumulation was identifie in the kidneys and spleen of rabbits[21].Because of the toxicity of several alloying elements,researchers developed bioactive particle reinforced Mg-based metal matrix composites(MMCs)as biodegradable metallic materials for orthopedic implants.The preparation of metal matrix nanocomposites(MMNCs)is gaining great attention[14,22].In fact,the nanomaterials with special characteristics showed great promise as metal reinforcing materials[23].

A wide variety of distinctive characteristics,including large aspect ratio,exceptionally high Young’s modulus and strength,and great thermal characteristics are displayed by carbonaceous nanomaterials[24,25].Graphene nano-platelet(GNP),which consists of a single-atom-thick layer of sp2-bonded carbon atoms in a tightly packed two-dimensional honeycomb lattice,possesses an intrinsic strength of approximately 130 GPa,Young’s modulus of up to 1 TPa,a low density of 1 g/cm3,and a wide specifi surface area of 2630 m2/g[26,27],with low cost and inherent biocompatibility[28]which could be used as an impressive nanofille designated for MMNCs[23].The reduction of GO into RGO decreases water disperse-ability due to less oxygenated functional groups on the edges and planes[29],which stabilizes GO in the body and minimizes its potential cytotoxicity.Graphene nano-platelets are mostly biodegraded through enzymatic oxidation by peroxidase,which results in natural oxidative degradation in biological processes[9].Typically,biosafety and stability are more potent for RGO compared to GO in vivo[30].Several examinations have shown that RGO is biocompatible,which makes it ideally suited pertaining to tissue engineering,cell culture and other biomedical applications[31,32].Recent studies also recommended that a single atomic layer of graphene is impermeable to gas molecules and creates an atomic hurdle in a way that it cannot be passed through it by helium gas[33,34].At low concentrations of up to 50μg·mL-1,graphene particles were found to be nontoxic and safe for biomedical applications[35,36].Also,the volume fraction of graphene in metallic-based biomaterials should be less than 10% because a high concentration of graphene results in significantl increased stiffness of the composite,which may be incompatible with natural bone biomechanically[9].

In this context,Munir et al.[33]prepared and characterized Mg-based composites encapsulated with various amounts of GNPs from 0.1 to 0.3 wt% fabricated with powder metallurgy(PM)and their results displayed that Mg-xGNPs withx＜0.3 wt% may enhance the anti-corrosion behavior and biological response.Shuai et al.[4]fabricated and characterized Mg-GO composites by selective laser melting with 3D honeycomb nanostructure.Their result demonstrated that this structure could serve as a good barrier to inhibit the corrosion attack and GO accelerates the apatite formation.However,there is no study regarding the encapsulation of RGO into Mg-3Zn-1Mn(ZM31)magnesium matrix composite to enhance cytocompatibility and antibacterial activity.Zn is a biocompatible element with about the solubility of 6.2 wt%in Mg.It has a solid solution-strengthening as well as an aging strengthening effect.Also,Zn minimizes the corrosive effects of iron and nickel impurities that may be present in Mg alloys.The solubility limit of Mn in Mg is about 2.2 wt%.It provides grain refinemen as well as enhancement in tensile strength and corrosion resistance of Mg[1].However,further addition of Zn and Mn beyond the solubility limit results in the creation of galvanic cells and a reduction in corrosion resistance.Nearly all physiological functions are severely harmed in the presence of zinc deficien y.Mn is nontoxic and plays an important role in the activation of various enzyme systems[17].According to[8],Mg-Zn-Mn based-alloys are one of the magnesium based-alloys that have a low in vitro corrosion rate.The main objective of the present work was to fabricate the ZM31 composite reinforced with RGO via the semi powder metallurgy(SPM)method.The antibacterial properties,bioactivity and cytocompatibility,including viability,cell adhesion,and proliferation,were assessed in more detail.Besides,the mechanical properties and anti-corrosion behavior of the synthesized nanocomposites were evaluated.In addition,the reinforcing mechanisms in the ZM31/RGO biocomposites were demonstrated.

2.Experimental procedures

2.1.Materials

Mg(99.8% purity,50μm mean particle size),Zn powder(98.8% purity,7.5μm mean particle size),and Mn(99.5%purity,45μm average particle size)powders were purchased from Sigma-Aldrich,USA.In addition,the nanofille,graphene oxide nano-platelets(GO)(with an average length of 1-10μm)were obtained from US-Nano Co.The reduced graphene oxide(RGO)nano-platelets were developed by the chemical reduction method of GO by simply using the hydrazine hydrate reducing agents at 80 °C for 12 h,according to Ref[37].

2.2.Fabrication of composites

The Mg based-RGO composites were produced using the solution-based powder metallurgy process(as shown in Fig.1).The process was followed by mechanical alloying with an alloy of Mg-3Zn-1Mn.The powders were milled in the planetary ball mill(NARYA-MPM2×250 H,Iran)under the argon atmosphere at 300 rpm for 25 h.About 30 gr of powder was placed in a stainless steel milling vial and stainless steel balls were added with the ball-to-powder ratio of 20:1.The details of mechanical alloying factors are outlined in Table S1,as presented in the supporting information.

The magnesium alloy was applied as a matrix and mixed in ethanol for 1 h by means of magnetic stirring at 600 rpm.At the same time,RGO nano-platelets were ultrasonicated separately in ethanol for 3 h.A drop of a specifi solution of RGO nano-platelets was applied to the above powder slurry in ethanol.To achieve a uniform mixture,the mixing process proceeded for 1 h.To acquire the composite powder,the mechanically agitated mixture was filtere and vacuum dried in a vacuum oven(Vacumat 200,Vita Zahnfabrike,Germany)at 70°C for 24 h.To obtain green billets withφ10×10 mm dimensions,the composite powder Mg-3Zn-1Mn-xRGO(x=0,0.5,1,1.5 wt%)was compacted in a stainless steel mold at room temperature under 660 MPa pressure.After compacting,the green billets were sintered in an argon-controlled atmosphere in the box furnace(TF5/25-1500,AZAR furnace,Iran)at 600°C for 2 h.The chemical composition of the composite is outlined in Table S2.

2.3.Microstructural characterization

A fiel emission scanning electron microscope(FESEM,Tescan,Mira 3 Czech Republic)and optical microscopy(OM,Olympus BXiS Japan)were used to perform microstructural and fractographical characterizations.Prior to optical microscope assessment,1.5 g picral acetic(purity 99.2%),100 mL ethanol(purity 99.7%),6 mL acetic acid(purity 99.6%),and 10 mL distilled water were mixed together as etchant solution.Polished samples were etched for 20 s in the picral etchant.The FESEM instrument was equipped with an energy-dispersive X-ray spectroscopy detector(EDS,DXPeX10 P Digital X-Ray Processor).The X-ray diffraction(Siemens D5000)with Cu-Kαradiation(45 kV,40 mA)was applied to reveal the phase components.To investigate the morphology of RGO nano-platelets,milled matrix powder and composite powders,transmission electron microscopy(TEM;Phillips 208 m)was used.A Raman spectrometer(Takram P50C0R10 with a laser wavelength of 532 nm)was employed to determine the structural changes of RGO in the nanocomposites.To determine the information on the chemical composition of GO and RGO,X-ray photoelectron spectroscopy(XPS)data were obtained by X-ray photoelectron(XPS,Bes Tec,Germany)employing twin anode XR3E2 X-Ray source system.

2.4.Mechanical properties

In conjunction with ASTM standard E9-09,the cylindrical sintered nanocomposites with a diameter of 6 mm and a height of 9 mm were pressed by the SANTAM(STM-50)universal testing device at a crosshead speed of 0.5 mm/min to determine the compressive strength at room temperature,and every specimen was measured three times.The indentation tests were conducted using a Vickers hardness tester(LECO M-400)on the sintered samples at a peak load of 300 gf and a duration of 15 s.For the average value,each specimen was analyzed in fi e various locations.

2.5.Corrosion behavior

The immersion and electrochemical tests were employed to measure the corrosion properties of nanocomposites.The corrosion tests were carried out in the simulated body flui(SBF).Kokubo’s solution comprising 142.0 mmol/L Na+,5.0 mmol/L K+,2.5 mmol/L Ca2+,1.5 mmol/L Mg2+,4.2 mmol/L HCO3-,147.8 mmol/L Cl-,1.0 mmol/L HPO42-and 0.5 mmol/L SO42-was used as the SBF solution.Before the corrosion measurement started,the surface of prepared specimens was ground with 600-2000 grade SiC abrasive paper and then sonicated in an acetone bath for 3 min.Finally,it was dried in a stream of hot air.A potentiometric polarization test with a Metrohm Autolab PGSTAT30 at a voltage range of-250 to+250 mVSCEand an open circuit potential at a rate of 0.5 mV/s in the SBF was performed to evaluate the corrosion rate.In this process,a graphite electrode was used as the counter electrode and a saturated calomel electrode(SCE)as the reference electrode and the samples were examined with a certain surface of 0.785 cm2exposed to the electrolyte as the working electrode.Electrochemical impedance spectrometry(EIS)was employed using an AC impedance analyzer(Solortron,1260)after 30 min immersion of the composites in SBF to obtain stability in the potential.This test was performed at an open circuit potential in the range of 105to 10-2Hz,in accordance with the ASTM G106 standard,using a sinusoidal signal with a potential amplitude of 10 mV.

Fig.1.Schematic of the preparation process of ZM31-RGO nanocomposite by semi-powder metallurgy(SPM)method.

The mass loss tests were conducted in conjunction with ASTM standard G31-72[38]for the composites.The cleaned composites with a diameter of 10 mm and a thickness of 5 mm were weighted with a precision of 0.0001 g and the ZM31/xRGO composites were soaked in SBF from 12 to 168 h at 37 °C in a water bath.The composites were rinsed with distilled water after each immersion time and dried at room temperature.Prior to weight loss measurements,the surface corrosion products of composites were cleaned using chromic acid solution(300 g/L Cr2O3+10 g/L AgNO3)next were weighed[39].Each test was replicated three times to attain reproducible results.During processing,the pH values of the solution were measured using a pH meter(PHS-3C,Shanghai Lei Ci Device Works,China).Furthermore,the volume of H2released as a result of the magnesium dissolution was evaluated according to Ref[40].First,the composites containing RGO were immersed in SBF and subsequently funneled and a burette,which was fille with SBF,were put upside-down directly above them to trap the H2gas.

2.6.In-vitro biocompatibility

A direct cell adhesive assay was carried out for the samples.Initially,in a Dulbecco’s modifie Eagle medium(DMEM)containing 10% Fetal Bovine Serum(FBS),1%penicillin/streptomycin and 1.5% geneticin human osteogenic sarcoma MG-63 line cells were cultivated under the standard cell-culture conditions.Before the cell experiment,all specimens were sterilized under ultraviolet radiation for 2 h.In 12-well plates,1 mL MG-63 cells with a density of 1×104cells/mL were distributed to the composites and cultured for 24 h.The composites were then taken out from the plate upon immersion and washed 3 times with PBS.The cells attached to the composite surface were fi ed with 2.5% glutaraldehyde and dehydrated in 70% ethanol for 2 h,and subsequently,the cell morphology was evaluated according to Ref[4].

An indirect 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium-bromide(MTT,Sigma,Saint Louis,USA)assay based on the extraction technique was used to evaluate the in vitro nanocomposite cytotoxicity containing RGO.The extracts were prepared in accordance with ISO 10993-12.Every specimen was firs immersed in a serumfree DMEM cell culture medium for 72 h in a humidifie atmosphere with 5% CO2at 37 °C for preparation of extracts of different samples.Following incubation,a 0.4μm membrane filte was used to filte the extracted according to standard.The ratio of the surface area of alloy samples to the extraction medium was 3 cm2/mL.The extract was then withdrawn,centrifuged and serially diluted(50%)for the cytotoxicity test or,alternatively,refrigerated at 4 °C before the cytotoxicity test.The MG-63 cells were seeded in 24 well culture plates in DMEM with 10% FBS at 1×104cells/mL of medium in each well and the plates were incubated to allow attachment inside the incubator at 37 °C in the humidifie atmosphere with 5% CO2.The DMEM with 10% FBS was used as the control group.Following the incubation of the cells for 24 and 48 h,the extracts were replaced by media,50μL of MTT solution were added to each well,followed by 4 h of incubation at 37 °C with a humidifie atmosphere of 5% CO2.In order to dissolve formazan crystals,100μL of dimethyl sulfoxide(DMSO)was added to the well.Then the supernatant was transferred to a 96-well plate.Finally,the absorbance was read at 545 nm using an ELISA Reader(Stat Fax-2100,Miami,USA).

Nuclear staining with DAPI(4ˊ,6-diamidino-2-phenylindole,blue fluorescenc in live cells)was performed to study the MG-63 cell line proliferation nanocomposites containing RGO under fluorescenc microscopy(BX60,Olympus,Japan).Besides an alkaline phosphatase(ALP),which is an important component for bone formation,a staining process was conducted to evaluate the osteogenic differentiation capability of MG-63 cells in 24 h and 48 h post-culture extracts.1×104cells/mL were seeded and positioned separately in a 24-well plate.The cultured cells were washed with PBS 3 times,the cells on the specimens were lysed in 1% Triton X-100 for 1 h,and 400μL of p-nitrophenyl phosphate(p-NPP,pH 9.8)was added into cell lysis and incubated at 37 °C for 15 min as per the manufacturer’s instructions(Jiancheng,Nanjing,China).To determine the cell viability,the absorbance(optical density,OD)of each sample was read at 520 nm in the automatic microplate reader(Thermo Scientifi Multiskan GO,USA)and ALP activities were calculated by extrapolation from a standard curve.The relative ALP activity was normalized to the protein concentration of each sample and described as IU per gram of protein.Accuracy of results was ascertained by repetition of each test 3 times.

2.7.In-vitro antibacterial activity

The antibacterial performance of composites against Staphylococcus aureus(S.aureus)and Escherichia coli(E.coli)was evaluated by the agar disk diffusion method.To accomplish this,the sterile swab dipped into the microbial suspension was flushe(pressing swabs to the side of the pipe)and the cultivation areas were in the shape of lawns.The composites were placed in an agar medium in an incubator at 37 °C for 1 day.From the inhibition zone around them,it can be seen whether the samples have antibacterial properties or not.In addition,according to Ref[7],as a reference group,the bacteria suspension was inoculated on the agar plate at a concentration of 1×104colony forming units(CFU)/mL to compare the antibacterial characteristics of the composites.

2.8.Statistical data analysis

All data obtained are reported as mean value±standard deviation(SD)(forn=3).T-tests were performed between various groups to assess the p values assumed to be significan when*p＜0.05 and**p＜0.01 were used.

Fig.2.FESEM images of(a)Mg powder;(b)Zn powder;(c)Mn powder;(d,e)Wrinkled surface morphology of as received RGO nano-platelets with various magnifications and(f)TEM image of RGO.

3.Results and discussion

3.1.Microstructure and composition

The morphologies of the pure Mg,Mn,Zn and RGO powders are shown in Fig.2.As it can be seen from Fig.2a-c.The Mg and Mn powders displayed an irregular morphology while Zn powders possess a typical spherical morphology.The pure RGO nano-platelets powders likewise revealed a fla e-like morphology with a mean particle size of 1-10μm and a thickness of around 5 nm(see Fig.2d-f).It is obvious that the multilayer RGO platelets are wrinkled,which twisted and overlapping the RGO stacks.

The SEM and TEM images of ball-milled ZM31 alloy are also shown in Fig.3a,b.Here,the morphology of milled powder is spherical with an agglomerate particle size of around 5-10μm.Interestingly,Fig.3c depicted the dispersion of 0.5 RGO nano-platelets within the Mg-based matrix,which implies that the approach used is efficien for the uniform dispersing of RGO inside the Mg alloy matrix.It can be due to the fact that the residual oxygen groups on RGO nanoplatelets are assisted to disperse it uniformly in ethanol by the presence of ultrasonic waves before their addition into ZM31 alloy powders.Additionally,many studies reported homogenous dispersion of graphene in the Mg matrix in ethanol using a similar method[41-44].Moreover,as it is evident from Fig.3d,the RGO nano-platelets are well decorated with distributed Mg particles.The strong interaction between Mg alloy and RGO occurred since oxygen-mediated bonding(as illustrated in Fig.3g).In this respect,Mg reacts with the oxygen in functional groups,resulting in the formation of interfacial MgO nanoparticles on the interface between magnesium and RGO.The oxygen used in oxygen-mediated bonding originated from the residual oxygen-containing functional groups(-COOH,C=O,and-OH)of RGO.It was reported[45]that the oxygen functional groups are leading to a strong interfacial bonding in composite by promoting the electron exchange between carbon atoms and metal matrix or directly interacting with metal.Thus,RGO reinforcement closely combines with Mg atoms.Further SEM and TEM images of composite powders are shown in Fig.S1.It is worth noting that further increasing the content of RGO up to 1.5 wt% leads to agglomerations of RGO,as shown in Fig.S1-e,f,which seriously deteriorates the mechanical properties of the composites.

Fig.3.FESEM and TEM images of(a,b)ZM31 alloy and(c,d)ZM31-0.5RGO nanocomposite powders,schematic illustrations:(e)GO;(f)RGO;(g)the schematic showing the oxygen-mediated bonding between Mg and RGO.

XPS can provide a more precise assessment of the reduction achieved during heating treatment.Fig.4a-c shows the XPS survey spectra and high resolution C 1s XPS spectra of GO and RGO,respectively.The carbon and oxygen atomic percentage data are also labeled in the inset of Fig.4b,c.Oxygen-containing functional groups were discovered to be attached to graphene platelets,and the XPS spectra of asreceived GO(Fig.4b)can be deconvoluted into four peaks:the carbon atoms corresponding to sp2carbon(C=C/C-C,284.8 eV),epoxy/hydroxyls(C-O/C-O-C,286.6 eV),carbonyl(C=O,287.4 eV),and carboxylates(O-C=O,288.6 eV)[46,47].As XPS analysis proved(as shown in Fig.4c),the intensity of the peak corresponding to oxygen functionalities significantl decreases after the reduction of GO.But these oxygen groups still present in RGO.It was believed that by the chemical reduction using reductants,producing electrically conducting platelets,a portion of oxygencontaining functional groups connected to the carbon plane decomposed[37].The XRD analysis finding of the RGO nano-plates,ZM31 alloy and ZM31-xRGO composites were shown in Fig.4a.The XRD patterns revealed thatα-Mg is the primary phase of the ZM31 alloy.There are three peaks at 2-theta of 32°,34°,and 37°,corresponding to the(1 0 0),(0 0 2)and(1 0 1)planes of HCP Mg crystal,respectively(Standard cards No.190,239).Owing to the low amount of RGO,the XRD patterns is not able to indicate RGO peaks,merely via escalating RGO in 1.5 wt% peaks(002)and(101)were found at 24° and 42.9° respectively.There are no signs of the in situ creation of carbide phases in the composite with RGO,implying the chemical stability of RGO at a high sintering temperature.

For the ZM31-xRGO composites,the Raman spectra are revealed to assess the structural changes of RGO nanoplatelets throughout SPM,as shown in Fig.4b,at 1355 cm-1and 1592 cm-1,the RGO powder presented two main peaks,corresponding to the vibration of carbon atoms with dangling bonds at in-plane terminations(D-band)and the sp2-bonded carbon atoms in the 2D hexagonal lattice(G-band)of RGO,respectively[48].It can be found that after SPM,the two bands persisted without shifting in the spectra of ZM31-xRGO composites,confirmin the presence of RGO in the composites.It is widely recognized that the comparative intensity ratio of D-band to G-band(ID/IG)might offer specifi data about the internal quality of graphene[49].Hence,the ID/IGvalues of ZM31-xRGO composites were measured and the results indicated that with increasing RGO amount,this ratio was increased.The escalation intensity ratio of ID/IGproposed that layer stacking and re-agglomeration of the RGO in the Mg matrices triggered the accumulation of non-sp2defects at high temperatures in the sintering process[33].

Yang et al.[50]have revealed the intensity ratio of ID/IGis amplifie from 0.993 for GO to 1.107 for RGO,which revealed that the mean size of sp2carbon domain was diminished related to RGO.The amount of RGO plays a crucial role in the Mg matrix distribution.On top of that,the RGO illustrates that two wide peaks positioned at the high-frequency region of about 2706 cm-1(2D or G′)might be in connection with the second order of zone boundary phonons,and the band at 2926 cm-1is linked to the vibration of D+G band[51].As a consequence of the existence of specifi functional groups,RGO displays better properties compared to graphene due to the outstanding interfacial adhesion between RGO and Mg matrix.

The SEM images and the EDS mapping of Mg,Zn,Mn and C of ZM31 Mg alloy matrix and ZM31/xRGO nanocomposites are shown in Fig.5.In fact,the Fig.5a-d show that the RGO is embedded in the ZM31 matrix structures.The presence of RGO within the composite was verifie via EDS analysis,where carbon Kαpeak was detected at 0.277 eV.When the pre-dispersion was finished each RGO can be furnished on the surface of Mg particles as observed in the figures It is in fact,a desirable dispersion of RGO in this stage.The explanation behind observations is attributed to the existence of oxygen atoms,which implies that the maintaining of specifi functional groups after the reduction process enables the RGO to form a strong bonding between Mg-matrix and RGO.To further verify whether RGO plays a role in the reduction of the grains of ZM31 alloy,we studied the optical images of the composites,as shown in Fig.S2(see supporting information).As is obvious from this figure with the increasing amount of the RGO content from 0 to 1.5 wt%,the mean grain size of the composites has diminished.

Fig.4.XPS survey spectra of(a)GO and RGO;High resolution C 1 s XPS spectra of(b)GO;and(c)RGO.The inset in(b)and(c)show the carbon and oxygen atomic percentage data of GO and RGO,respectively;(c)XRD patterns of RGO and ZM31-xRGO(x=0,0.5,1 and 1.5 wt%)nanocomposites;and(d)Raman spectra of RGO and ZM31-RGO nanocomposites.

3.2.Mechanical properties

The results of microhardness and compressive strength studies are shown in Fig.6a-c and Table S3(see supporting information).The Vickers hardness of ZM31-xRGO composites escalated from 49±2 to 71±3 HV with an increasing amount of RGO from 0 to 1.5 wt%,mainly due to the fact that the grain refinemen and high strength of RGO may resist the indentation-induced plastic deformation.Young’s modulus,σUCS,and failure strain values of magnesium alloy were enhanced with an increase in weight fractions of RGO.The Young’s modulus,ultimate compressive strength,and failure strain for the ZM31 alloy were measured as 30.2 GPa,244.5 MPa,and 5.6%,respectively.TheσUCSwas 259.4,282.3,and 231.1 MPa for the ZM31-RGO composites containing 0.5,1,and 1.5 wt%RGO,respectively.Also,the compression failure strain of the Mg-RGO composites containing 0.5,1,and 1.5 wt%RGO was 6.3,6.5 and 5.4%,respectively.Thus the compressive strength and failure strain were gradually enhanced and reached a maximum value of 282.3 MPa and 6.5 % at about 1 wt% of RGO,showing an improvement compared to the ZM31 alloy(244.5 MPa and 5.6%).Failure strain values of composites increase with increasing RGO contents up to 1 wt%,which is due to the graphitic behavior of RGO at higher loading fractions.Although the RGO occurrence increased the compressive mechanical properties,theσUCSand failure strain were reduced with increasing RGO in nanocomposites containing more than 1.5 wt% RGO owing to the formation of agglomeration of RGO nano-platelets.In fact,it is reported that the grain refinemen and load transfer via the Mg/RGO interfaces,from the Mg matrix to the stiffer RGO nano-platelets additives are an effective strategy to improve the mechanical properties of Mg alloys[49,52,53].Furthermore,both Mg and graphene have a coefficien of thermal expansion of 26×10-6K-1and 1×10-6K-1,respectively.As a result of strain generated by the mismatch of thermal expansion,dislocations are created at their interfaces.The induced dislocation densities will result in increased strength of composites by preventing the movement of dislocations between the matrix and graphene interfaces[44,54].On the other hand,the extreme addition of RGO of 1.5 wt% was challenging to distribute uniformly and have a negative effect on the mechanical properties.The results show that the compressive strength of ZM31-0.5RGO(259.4 MPa)is higher compared to Mg-0.3GNP(169±18 MPa)[33],Mg-0.2GNP(183±4 MPa)[33],Mg-3Zn-5HA(116±5 MPa)[55],Mg-0.5Zr/0.1GNPs(219±3 MPa)[56],Mg-1Al-Cu/0.18GNP(225 MPa)[57]and cortical bone(164-240 MPa)[58],which meet the requirement for biomedical applications.

Fig.5.SEM images,EDX and map of(a)ZM31 alloy;(b)ZM31-0.5RGO;(c)ZM31-1RGO;and(d)ZM31-1.5RGO.

Fig.6.(a)Microhardness values;(b)Compressive stress-strain curves of the matrix and nanocomposites;and(c)Changes of ultimate compressive strength(UCS)and failure Strain of ZM31 alloy,ZM31-0.5RGO,ZM31-1RGO and ZM31-1.5RGO samples(*p＜0.05).

The SEM morphologies of fracture surfaces of ZM31 and nanocomposites are shown in Fig.7.All samples show fracture at about 45° in relation to the loading axis of the compression.It can be seen that ZM31(Fig.7a,e)displayed several cleavage characteristics that have the sign of a standard pattern of brittle fracture.Twinning shear bands control magnesium-based composites deformation behavior.The shear bands are attributed to work hardening behavior and heterogeneous deformation as work-hardening rates are higher for samples failed by shear bands deformation[59].These cleavage cracks intersected with twist grain boundaries resulted in the creation of“rivers”,with the direction in which the cleavage cracks spread locally[60].However,the addition of RGO led to mixed failure modes in the Mg matrix that showed evidence of ductile material.The surface fracture of the ZM31-RGO nanocomposites with encapsulation of 0.5,1,and 1.5 wt% RGO is shown in Fig.7(b,f,c,g and d,h),respectively.The fracture surfaces of ZM31-RGO composites were rather shallow dimples and almost fine morphological fractures compared to the ZM31 alloy matrix.The existence of a great amount of reinforcement particles(RGO)in the composite matrix can lead to agglomeration in the composite that has an unfavorable impact on the mechanical characteristics.However,with further addition of RGO(1.5 wt%),agglomerates developed because of the solid van der Waals attractions between carbon atoms,which negatively affected their strengthening ability.

Fig.7.SEM images of fracture surfaces of ZM31 and ZM31-xRGO composites after compression testing:(a,e)ZM31;(b,f)ZM31-0.5RGO;(c,g)ZM31-1RGO;and(d,h)ZM31-1.5RGO nanocomposites.

Fig.8.SEM images of fracture surfaces cracks:(a)Crack bridging;(b)Crack deflection and(c,d)Crack branching mechanisms of ZM31-1RGO composites;(e)EDS mapping of the fracture surface of ZM31-1RGO nanocomposite;and(f)Reinforcing mechanisms schematic(A colourful version of this figur can be viewed online).

The SEM images shown in Fig.8 characterized the fracture surfaces of the Mg-1RGO composites and the cracks to identify the possible mechanisms for improving the mechanical characteristics.In fact,due to the difference in elastic modulus,the load is transferred from the matrix to RGO as soon as a matrix crack is started and propagated.The successful mechanical interlocking and load transfer to the nanofiller was made possible by the wrinkled surface of RGO nano-platelets.The crack bridging toughening mechanism is demonstrated in Fig.8a where the two crack surfaces are bridged via RGO,which slows down the Mg matrix rupture.Further resistance to interfacial friction between RGO and Mg-matrix could properly postpone the crack propagation.

Figure 8b reveals the crack deflectio function.A crack propagates initially in the Mg-matrix and,subsequently,when it reaches graphene along with the RGO-Mg interface,it will be accompanied by the deflectio to the Mg matrix.Intended for further propagation of the crack,further energy is needed as the crack plane is no longer perpendicular to the stress axis.In addition,a tortuous route for crack growth is created through the deflectio approach,which enables considerably more dissipation of energy.Figure 8c,d revealed the mechanism of crack branching in the composites.When a crack spreads and reached the RGO,it was arrested as crack propagation and came across to RGO and could cause a longer path to release the stress,which may further improve the fracture toughness.

The possible reinforcing mechanisms are shown in Fig.8f.Here,the RGO might be examined at the de-bonding location,i.e.the interface place,as confirme via the EDS results presented in Fig.8e.As seen,the fl xural crack propagation path is observed in the surface of ZM31-1RGO composite,which is attributed to the high intrinsic strength of RGO to hinder the penetration of crack to form straight crack.

3.3.In vitro corrosion measurements

Fig.9a,b show the potentiodynamic polarization curves and the Nyquist plots at room temperature for various composites in SBF.According to Table S4 presented in the supporting information,the corrosion current densities for ZM31 alloy,ZM31-0.5RGO,ZM31-1RGO and ZM31-1.5RGO were evaluated as 129.3,96.41,134.2,and 153.7μA·cm-2,respectively.From the results,it can be seen that the ZM31-1.5RGO composite has poorcorrosion resistance.Meanwhile,the ZM31 alloy experienced a higher corrosion current density(icorr)than the Mg-0.5RGO composite,whereas the icorris elevated via more encapsulation of RGO into the Mg matrix.Likewise,the low RGO amounts ZM31-RGO composites experienced lower corrosion rates(CR)compared to the ZM31 alloy in the following order:ZM31-1.5RGO(4.38 mm/year)＞ZM31-1RGO(3.83 mm/year)＞ZM31 alloy(3.69 mm/year)＞ZM31-0.5RGO(2.75 mm/year).In comparison to the ZM31 alloy,the ZM31-1.5RGO composite presented a greater corrosion rate,which could be related to the galvanic corrosion created between RGO platelets and Mg matrix.In the composite containing 0.5 wt% RGO,the RGO nano-platelets dispersed uniformly in Mg matrix and completely protect the surface of composites,thereby inhibiting the corrosive solution onto the Mg matrix and consequently,decreases the corrosion rate of the composite.The higher concentration of RGO nano-platelets(i.e.,1.5 wt%)in the Mg matrix composites serve as cathodic sites owing to their agglomeration in Mg throughout the distribution procedure,causing micro-galvanic reactions during their corrosion examination that negatively affect their corrosion resistance.Results indicate the reasonable corrosion rate of ZM31-0.5RGO composite introduced in this study is 2.75 mm/year which is low compared to other Mg-based biomaterials such as ZK30-0.9GO(11.1 mm/year)[61],Mg-2Zn(7.47 mm/year)[62],Mg-0.3GNPs(11.00 mm/year)[33]and Mg-0.5Zr/0.1GNPs(11 mm/year)[56]in the SBF which introduces it as a proper candidate for implants.

These results confir properly with the EIS that the capacitive loop for ZM31-0.5RGO is greater compared to the other composites(Fig.9b).Typically,a greater capacitive loop means a lower corrosion rate in the Nyquist plots[63].To further evaluate the EIS spectra of composite encapsulated with different amounts of RGO nano-platelets,an equivalent electrical circuit model is performed and fittin lines are presented in Fig.9b.In this model,the CPEdland Rctare the double-layer capacitance and the charge transfer resistance,respectively,while Rsrepresents the electrolyte resistance and Rlshows a high-medium frequency capacitive loop and L has named as the inductive function.Typically,because of the high resistance of charge transfer,the sample can resist well against corrosion.The resulting kinetic parameters are listed in Table S5(as presented in the supporting information).The charge transfers resistance(Rct)of ZM31 of 95.58Ω·cm2escalated to 198.26Ω·cm2in ZM31-0.5RGO.Indeed,the compositing of ZM31 with RGO motivated the formation of a protective layer of Mg(OH)2on the surface in SBF,which formed to enhance the protection for the ZM31 matrix.Additionally,grain refinemen can diminish the mismatch stress between the surface layer and Mg substrate to hinder pitting formation[61].Graphene is likewise regarded as a suitable thin-layer material to prevent corrosion,as the surfaces of the sp2carbon atoms create a natural diffusion barrier between protected metal and reactants.Hence,the inhibit infiltratio of SBF solution enters the Mg surface and subsequently,reduces the corrosion rate[34]and consequently increases the corrosion resistance of the Mg alloy.

The immersion experiments were carried out regarding the H2release and the mass loss measurements to investigate the long-term degradation behavior of composites shown in Fig.9c-e.The pH value was found to increase quickly during the initial 24 h of incubation,while the pH value slightly increased in the subsequent period(see Fig.9c).During the immersion,the preliminary pH value increased rapidly,which can be attributed to the release of excess OH-.As a result of reaction equilibrium between all the ions existing in the corrosion solution,the pH of the solution stabilizes after 24 h.The pH curves showed a lower pH value of 9.62±0.3 for the ZM31-0.5RGO compared to those of 9.88±0.4 for ZM31-1RGO and 10.14±0.6 for ZM31-1.5RGO nanocomposites and 9.78±0.7 for ZM31 alloy after 168 h.The existence of carboxyl groups and hydroxyl functionalities in the RGO could react to form acids with atmospheric moisture.These functional groups may have contributed to a lower pH

Fig.9.(a)Potentiodynamic polarization curves;(b)Nyquist plots of ZM31-xRGO(x=0,0.5,1 and 1.5 wt%)composites with equivalent circuit used to model the results of composites;(c)pH values;(d)Hydrogen evolution volume of ZM31-xRGO composites by immersion tests;and(e)Weight loss rate of ZM31-xRGO nanocomposites after immersion in the SBF solution for 168 h.

when RGO is exposed to SBF[31].Theα-Mg matrix reacts with the SBF solution along with the release of hydrogen,as the corresponding reaction is illustrated in Eq.(1)[3,6]:

As shown in Fig.9d,numerous bubbles generated from the surface of the ZM31 alloy were detected at the preliminary stage of incubation for the hydrogen evolution examination,whereas small bubbles were detected on that of ZM31-0.5RGO.In addition,it can be shown that the total hydrogen of ZM31-0.5RGO composite release was 24.7 ml·cm-2after 168 h,which was lower compared to that of the ZM31(35.34 ml·cm-2),implying a lower corrosion rate.Furthermore,the degradation rates of ZM31 alloy and nanocomposite after 168 h immersion determined from the mass loss and the results are shown in Fig.9e.As seen,the degradation rate of ZM31 Mg alloy(2.57 mg·cm-2·d-1)obviously decreased with the addition of 0.5 wt% RGO(1.84 mg·cm-2·d-1),representing a comparatively higher degradation resistance.This was in good agreement with the polarization test results.

The bioactivity of the ZM31 alloy and their composites with RGO was assessed in vitro,in terms of apatite-forming ability in SBF.The SEM analysis revealed a number of small mineral clusters deposited on the surface of samples after 72 h of immersion(see Fig.10a).There were clearly deep corrosion pits and cracks on the surface of ZM31 alloy,revealing that the alloy presents localized corrosion.On the other hand,the corrosion product of other composites is relatively homogeneous.The high-magnificatio image from Fig.10a exhibits that the clusters were spheroidal and flat-li e crystallites.In these crystallites,EDS analysis was then carried out to evaluate the elemental composition.Apart from calcium,magnesium,oxygen,and phosphorus peaks were also observed(Fig.10b,c),which implies the existence of spheroidal and flat-li e crystallites bone-like apatite.Moreover,cracks on the corrosion surface were possibly caused by the contraction of the hydrated layer during the drying process[64].

At higher magnification one can distinguish a remarkable increase in both size and thickness of the apatite crystallites produced on the ZM31-0.5RGO compared to other nanocomposites(see Fig.10b).The EDS spectrum in Fig.10c also indicates an escalation in the atomic percentage of the phosphorus,suggesting the positive growth on the composites of the apatite film The graphene oxide rich in oxygen-containing groups can facilitate the apatite deposition on the Mg matrix by creating potential sites for nucleation and growth of the hydroxyapatite(HA)[4],which can result in a dense apatite fil and prevent the subsequent severe attack of SBF.Fig.10d revealed the XRD pattern of the Mg-0.5RGO after 14 days of immersion in SBF.The results clearly depicted that the corrosion products were Mg(OH)2and HA.Due to the presence of inorganic ions such as Cl-,H2PO4-and Ca2+in SBF,the corrosion products included HA and Mg(OH)2[5].

Fig.10.(a)SEM photomicrographs of ZM31-xRGO(x=0,0.5,1 and 1.5 wt% RGO)nanocomposites and EDS analysis of(b)ZM31-0.5RGO;(C)ZM31-1RGO after 72 h of immersion in the SBF;and(d)XRD pattern of ZM31-0.5RGO immersed in the SBF for 14 days.

3.4.Cell response

Biomaterials were used to facili tate the development of new tissue by providing active surface sites for direct cellular attachment,migration and proliferation[65].In this perspective,synthetic composites are expected to enhance the attachment and proliferation of the osteoblasts to ensure the effective performance of implants in orthopedics.It was reported[66]that the hydrophilicity of composites is reported to possess positive effects on their protein adsorption and cell attachment.As shown in Fig.11a,the water contact angles(WCA)on composites with different amounts of RGO were calculated to evaluate their hydrophobic characteristics.The WCA of ZM31 is 98±3°.When the RGO concentration is rising,the WCA of ZM31-RGO diminishes from 0.5 wt%(93±1°)to 1.5 wt%(87±1°),leading to increased hydrophilicity.The agglomeration of hydrophobic RGO in Mg matrices subjected more sites of the hydrophilic Mg matrices to the corrosive solution,increasing corrosion attack[67].When a higher concentration of GNPs(＞1 wt%)is added to Mg alloy,large agglomerations form due to the stacking of GNP layers,resulting in hydrophilic surfaces,which eventually result in galvanic and crevice corrosions in saltwater[67,68].

The DAPI labeled cells and their SEM images in Fig.11 clearly demonstrated the adhesion capabilities of the MG-63 cell lines on ZM31 alloy and composites.As can be seen,the cells might attach well to the ZM31 and ZM31-0.5RGO composites after 24 h of culture and the cell nuclei were thus stained with DAPI,as shown in Fig.11b,f and 11c,g respectively.These results imply the outstanding cell proliferation on the surface of composite encapsulated with low content of RGO.No major negative influenc on cell attachment and proliferation was seen in Fig.11c,g and Fig.11d,h,through 0.5 and 1 wt% RGO encapsulation into the matrix.Nevertheless,as the RGO amount increased to 1.5%,the number of cells attached to the ZM31-RGO composites diminishes significantl(Fig.11e,i).According to the corrosion examination result,the degradation rate of composites increases with increasing RGO contents.A high degradation rate dramatically increased pH and hydrogen release,leading to some toxicity and inhibition of cells to attach to the composite surface[69].Furthermore,the higher degradation rates increase considerably more corrosion products that could reduce the cell attachment of the specimens.

Fig.11f shows that t the cell number and state on the ZM31 alloy are lower and the cell shrinkage with spherical form is visible,while spindle cell morphologies were observed on ZM31 containing 0.5 and 1 wt% RGO composites,indicating a better cell propagation for composite with lower RGO contents(Fig.11g,h).The cells are flattene on surfaces of composites that have contacted neighboring cells with a broadly spaced cytoskeleton.Lim et al.[70]reported that the cells on graphene foams exhibit a spindle-shaped,elongated morphology with thin,aligned nuclei,which represent significan markers for osteogenic cell differentiation.The MTT assay was used for measuring the cell viability of cultured ZM31-xRGO nanocomposite extracts over 24 and 48 h,as shown in Fig.11j.The MTT assay presents that the cell viability increased with the extension culture time.Once diluting the extracts into 50%,a greater cells viability was found in ZM31-0.5RGO extract compared with ZM31 extract,particularly for longer terms.Degradation of the Mg alloy leads to enhancement of the pH value of the solution that serves as a primary barrier to cell proliferation.Electrostatic attractive forces,hydrogen bonding,the hydrophobic nature or the wettability of graphene family materials play a role in the interaction between proteins and these carbon nanostructures.The type of protein,surface charges on proteins and graphene,as well as the ionic strength,pH and temperature of the medium,all affect protein adsorption on graphene platelets[34,71].Theπ-πstacking caused by strong van der Waals forces has been identifie as a significan protein adsorption mechanism on the surfaces of graphene family nanomaterials.Strongπ-πstacking occurs primarily between the strong sp2bonding of carbon atoms in carbon nanomaterials and the benzene rings in amino acids(protein building blocks)[34,72].Also,Hatamie et al.[73]reported that the oxygencontaining functional groups including carboxylic,carbonyl and hydroxyl on the RGO layer might bind non-covalently through hydrogen surface connecting,electrostatic binding,andπ-πstacking with biologically active substances including extracellular growth factors.It then adsorbs extracellular to mediate cell attachment straight,facilitate the initial adhesion of stem cells and hence,the level of differentiation was increased[30,50].

The Mg-1.5RGO composite presented a lower cell density and viability in comparison with other composites.As it was shown in previous sections,the composite shows the highest corrosion rate.Higher degradation rate of composites will lead to escalation of pH value of the culture medium and subsequent cell destruction.Moreover,the rapid hydrogen evolution and release of gas bubbles near the composite resulted in diminished cell adhesion and growth of MG-63 cells,which can interrupt tissue regeneration[74].Osteoblast differentiation is one of the most critical steps in entire cellular activity and bone formation capacity for better understanding of the influenc of RGO on the response of MG-63 cells.Thus,the effects of RGO on osteoblast differentiation were investigated using an ALP assay,which is the firs indicator of osteoblast differentiation.

Fig.11k presents ALP level of the MG-63 cells cultured on the composite extracts.On the Mg-0.5RGO composites,the ALP expression level depicted the highest potential to promote the osteogenic differentiation of MG-63 cells and the lowest activity of ALP from the ZM31 alloy was found.It can be noticed that the ALP function of all ZM31-RGO biomaterials is a time-dependent soaking fashion,which escalated the level of cell differentiation with the extension of soaking time in the ZM31-RGO composite extracts.The great activity of ALP of ZM31-RGO composites with the low content of RGO in this examination implied that they are capable of improving the cell differentiation process.According to the ISO 10993-5:2009,materials with a cytotoxicity of Grade 0(100%＜cell Viability)and Grade 1(75%＜cell viability＜99%)are non-toxicity[75].After 24 h cell viability of MG-63 cells incubated in ZM31-0.5RGO extract maintained at a value 95% while after 48 h,cell viability of MG-63 cells incubated in ZM31-0.5RGO extract increased to 97%,indicating that high cell viability of MG-63 cells in ZM31-0.5RGO extract.According to the results of the in vitro cytocompatibility study,it appeared that the degree of toxicity caused in MG-63 cells after subjection to these nanocomposites was low or absent.Also,the ZM31-0.5RGO composites showed better cell response,such as cell attachment,cell growth and differentiation.

3.5.Antibacterial activity

The implant-connected infections are considered as one of the most significan issues after their operation,contributing not only to implant failure but also to complications,morbidity and mortality[9,68].Thus,the antibacterial behavior of ZM31-xRGO(x=0,0.5,1 and 1.5 wt%)nanocomposites was examined against two types of bacteria,including

Staphylococcus aureus(S.aureus)andE.coli(E.coli)(Fig.12a).The the inhibition zone diameter for the composites ZM31,ZM31-0.5RGO,ZM31-1RGO and ZM31-1.5RGO was found to be 0.5 mm;2.3 mm;3 mm;and 4.2 mm againstE.coli,respectively.The size of composite inhibition zone of ZM31,ZM31-0.5RGO,ZM31-1RGO,and ZM31-1.5RGO was 0.3 mm,1.1 mm,2.3 mm and 3.1 mm versusS.aureusrespectively.

The encapsulation of RGO was observed to noticeably improve the antibacterial performance of ZM31,according to the findings Fig.12b demonstrates the comparison of the bacterial plate count method CFUs on composite samples and further demonstrating their variations in antibacterial performance.The reduction in the number of colonies indicates that ZM31-1.5RGO with high RGO amounts has the best preventing bacterial attachment and growth.According to both analyses,the antibacterial mechanism of ZM31-xRGO has attributed to both the physical destruction of the bacterial membranes through the sharp edges of RGO nano-platelets and the reactive oxygen species(ROS)formation.Safari et al.[57]and Saberi et al.[68]verifie the strong interaction between the sharp edges of graphene in the Mg-based matrix and the consequent destruction of the cell membrane of the bacteria.By destroying the cell structure or metabolic malfunction of an antibiotic agent,the antibacterial coating/fil frequently damages the bacteria.The inhibition of bacterial adhesion works mainly by preventing the formation of biofil[68,76].The significan quantities of the hydroxyl and Mg ions are generated into the medium throughout Mg-matrix degradation.Such great alkalinity itself acts as a serious danger to the growth of bacteria,while a high concentration of Mg ion will produce a large osmotic pressure in the bacteria[77].Mg corrosion is a redox reaction and the generation of ROS in bacteria and its function in the destruction process need to be determined[68,69].Overall,it’s concluded that in vitro antibacterial activity of Mg-RGO biocomposite is related to the amount of RGO.

4.Conclusion

1.ZM31-xRGO(x=0,0.5,1,and 1.5 wt%)composites with various content of RGO as the reinforcement phase was successfully developed by semi-powder metallurgy(SPM).

2.The hardness of ZM31-xRGO composites increased with increasing the RGO content and reached 71 HV higher than ZM31 alloy(49 HV).A similar trend was observed regarding the compressive strength(CS)and failure Strain,which escalated to282.3 MPa and 6.5% respectively for the ZM31-1RGO composite compared to the ZM31 alloy(244.5 MPa and 5.6%).Furthermore,crack bridging,crack deflection and crack branching were determined as crucial reinforcing mechanisms in the Mg composites containing RGO.

3.The ZM31-xRGO composites typically presented faster corrosion compared to the initial ZM31 alloy(3.69 mm/year),except for the ZM31-0.5RGO composite in SBF solution,which showed a slower corrosion rate of 2.75 mm/year,owing to the impressive anti-infiltratio characteristics and homogeneous distribution of RGO inside of the Mg-matrix,which result in a major reduction in current densities and subsequent corrosion rates.In Mg-1.5RGO,partial agglomeration could be found,which has a negative effect on corrosion resistance(4.38 mm/year).

4.The presence of apatite layers formed on the surface samples after 72 h was granted to support the bioactivity of ZM31-RGO composites.The ZM31-0.5RGO composite exhibited better cytocompatibility compared to the ZM31 alloy because of the lower degradation rate where MG-63 cells bind and well distributed on composite containing RGO.The antibacterial test results showed that the encapsulation of RGO into ZM31 alloy could obstruct bacterial growth by increasing the RGO concentrations from 0.5 to 1.5 wt%.

5.This study indicated the potential applications of ZM31-0.5RGO biocomposite in reducing corrosion and infections of the implants.It is expected to be an ideal and promising material for biomedical magnesium alloys.

Fig.12.Antibacterial activities of ZM31-xRGO(x=0,0.5,1 and 1.5 wt%)nanocomposites by(a)the disk diffusion test;and(b)plate count technique CFU results against both S.aureus and E.coli bacteria.

Supplementary materials

Supplementary material associated with this article can be found,in the online version,at doi:10.1016/j.jma.2021.09.016.