Short Communication

Self-protection against corrosion of aged magnesium alloy in simulatedphysiological environment

Guosong Wu 1, Ying Zhao 1, Xuming Zhang, Jamesh Mohammed Ibrahim, Paul K. Chu ⇑Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong

a r t i c l e i n f o

Article history:Received 8 August 2012Accepted 17 November 2012Available online 29 November 2012

Keywords:A. MagnesiumB. PolarizationB. EISC. Interfaces

a b s t r a c t

A self-protection behavior of Mg – 7.5 Al – 0.8 Zn – 0.2 Mn alloy in cell culture media is reported. Usingaging treatment, the b-phases (Mg17Al12) are precipitated from the supersaturated a-phases and distrib-uted along grain boundaries or grains. The aged alloy suffers from severe corrosion in the early immersionstage but after a critical immersion time, its corrosion resistance increases rapidly in the later stage. Themechanism is discussed based on the role transition of b-phase and the precipitation of phosphates bylocalized basification. Moreover, the aged alloy also exhibits a better biocompatibility.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Magnesium-based materials are considered for osteosyntheticapplications because their Young’s modulus (E = 41–45 GPa) issimilar to that of bones (E = 3–20 GPa) and their natural biodegrad-ability obviates the need for a second surgery. Magnesium is alsoessential to human metabolism and present in large amounts inthe human body. Generally for a normal adult, the daily intake ofMg is about 300–400 mg and excess magnesium is excreted inthe urine. However, because of the poor corrosion resistance, mag-nesium implants cannot withstand long-term attack in the physio-logical environment [1–3]. Generally, the surface morphology,microstructure, and composition determine the efficacy of artificialimplants and also alter protein adsorption which mediates theadhesion of desirable and undesirable cells [4]. After implantationinto the human body, the corrosion dynamics is quite complex andMg-based implants should be designed to control hydrogen evolu-tion, localized basification and degradation especially in the earlystage. Hence, it is crucial to study and fathom the dynamic inter-face between the implant surface and body tissues/fluids and its ef-fect on cell growth.

Mg–Al–Zn–Mn alloys such as AZ80 and AZ91 are model materi-als having simple phases such as a-phase (Mg matrix), b-phase(Mg17Al12), and AlxMny [5,6]. Witte et al. [7] found that as-castAZ91D alloys had fast in vivo degradation rates. Zhou et al. [8] con-ducted aging treatment to increase and homogenize the b-phases

and managed to improve the corrosion resistance in simulatedbody fluids. In common aqueous solutions, the improvement canbe attributed to the transformation of the b phase from the cathodeof the galvanic cell to the protective barrier [5]. However, in thepresence of large quantities of ions and proteins in the physiolog-ical environment, the corrosion process involving the participationof non-matrix phases is more complicated and cell response to thedynamic surface is also not well understood. In this study, thein vitro degradation of aged Mg alloy was studied in a completedcell culture media. An unusual self-protection behavior was ob-served and discussed and the biological responses were alsoinvestigated.

2. Experimental details

Mg – 7.5 wt% Al – 0.8 wt% Zn – 0.2 wt% Mn alloy extruded plateswere cut into 10 � 10 � 5 mm3 pieces. They were put in an ovenand heated from 23 to 440 �C at a rate of 10 �C/min. After keepingat 440 �C for 24 h in air, they were quenched in 13 �C water. Theywere then put back into the oven and heated from 23 to 200 �C.After heating at 200 �C for 48 h, they were taken out and quenchedin 13 �C water again. Afterwards, the samples were mechanicallyground using up to 1200 grit SiC paper (micron grading: 5 lm).X-ray diffraction (XRD) with Cu Ka radiation and scanning electronmicroscopy (SEM) were conducted to characterize the phase com-position and microstructure.

A complete cell culture medium (a mixture of Dulbecco’s mod-ified eagle medium and 10 vol.% fetal calf serum) was used as a testsolution in this study. The Dulbecco’s modified eagle medium(DMEM) powder was bought from Life Technologies Corporation

0010-938X/$ - see front matter � 2012 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.corsci.2012.11.029

⇑ Corresponding author.E-mail address: paul.chu@cityu.edu.hk (P.K. Chu).

1 Co-first authors.

Corrosion Science 68 (2013) 279–285

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and its composition is shown in Table 1 [9]. The medium was pre-pared with the powder, distilled water, and 3.7 g of NaHCO3 per li-ter. The electrochemical experiments were performed on a ZahnerZennium electrochemical workstation using the conventionalthree-electrode technique. The potential was referenced to a satu-rated calomel electrode (SCE) and the counter electrode was a plat-inum sheet. The samples with a surface area of 10 � 10 mm2 wereexposed to the media at 37 �C. The open-circuit potentials (OCP)were monitored in real time for 24 h and polarization tests wereconducted following the OCP measurement at a scanning rate of1 mV/s. Electrochemical impedance spectroscopy (EIS) was con-ducted at different immersion time. The data were recorded from100 kHz to 100 mHz with a 5 mV sinusoidal perturbing signal atthe open-circuit potential. Scanning electron microscopy (SEM)was used to examine the surface morphology and X-ray photoelec-tron spectroscopy (XPS) was employed to determine the surfacecomposition after immersion for 24 h.

Protein adsorption and the cell morphology and proliferationwere monitored to evaluate the biocompatibility of the aged alloy.In the protein adsorbance assay, the samples were first sterilizedin 75% (v/v) ethanol for 40 min and rinsed three times with sterilephosphate buffered saline (PBS). A 1 ml complete cell culturemedium was pipetted onto each sample placed on 24-well plates.After incubation at 37 �C for a certain time, they were transferredto a new 24-well plate and washed with 1 ml PBS twice. 150 ll of1% sodium dodecyl sulfate (SDS) solution was then added to thewells and shaken for 1 h to detach the proteins from the samples.

The protein concentration in the collected SDS solutions wasdetermined using a MicroBCA protein assay kit (Pierce). MouseMC3T3-E1 pre-osteoblasts were seeded on each sample in 24-wellplates at a density of 5 � 104 cells per well for cell morphologyobservation and a density of 3 � 104 cells per well for cell prolif-eration assay. After culturing for 24 h, the samples were rinsed,fixed, stained, and examined by fluorescence microscopy. A cellcount kit-8 (CCK-8 Beyotime) was employed to determinequantitatively the viable pre-osteoblasts. Those cells were cul-tured with renewing the medium every day. After culturing for1, 3, and 7 days, the samples with the seeded osteoblasts wererinsed twice with sterile PBS and transferred to fresh 24-wellplates. The culture medium with 10% CCK-8 was added tothese samples and after incubation for 4 h, the solution wasaspirated and the absorbance was measured at 450 nm using aspectrophotometer.

3. Results and discussion

The Mg17Al12 phase is the only precipitate generated duringaging [10]. XRD reveals that the b-phase increases after aging(Fig. 1a). Before the heat treatment, a small number of fine b-phases are distributed along the grain boundaries and some AlxMny

particles are randomly embedded in the matrix (Fig. 1b). The b-phases dissolve in a–Mg forming a supersaturated solid solutionvia quenching during the solution process. Subsequently, the b-phases precipitate into two forms during aging. The first is discon-tinuous precipitation by that coarse lamellar b-phases grow fromthe grain boundaries into the grains and the process ceases inthe early precipitation process. The second is continuous precipita-tion by which fine lath-like b-phases form in the remaining regionsof the matrix [11,12]. The details of these features are shown inFig. 1c and d and the inset in Fig. 1d describes the morphology ofcontinuous precipitation in the grains.

The corrosion behavior of the aged alloy in the cell culture med-ia is significantly altered. The OCP evolution (Fig. 2) reveals a sim-ilar trend in both the as-received and aged samples, but the agedalloy has a more stable OCP value in the later stage. Polarizationafter 24 h of immersion (Fig. 3) discloses that the corrosion resis-tance is improved as manifested by the lower corrosion currentdensity and higher DE value. The corrosion current density is de-rived from cathodic Tafel region extrapolation as shown in the in-sert of Fig. 3 and DE represents the difference between thecorrosion potential and transition potential in the anodic region.Figs. 4–6 show the EIS data. The Nyquist plots are presented inFig. 4 and Bode plots are displayed in Figs. 5 and 6. It should benoted that the OCP is changing during the measurement periodbased on the result shown in Fig. 2. In each EIS measurement,the OCP was fixed and the testing time lasted only for about5 min. Thus, although the obtained data are distorted to some ex-tent, this approximate in situ measurement is still feasible to revealthe EIS evolution trend in our investigation. A general equivalentcircuit model, Rs(RfCPEf)(RtCPEdl) [13], is proposed to fit the EISdata, where Rs is the solution resistance between the referenceelectrode and working electrode, CPEf and CPEdl are constant phaseelements representing the capacitance of the surface film and dou-ble layer, respectively, and Rf and Rt denote the surface film resis-tance and charge transfer resistance, respectively. We furtherpropose Rp, the sum of Rf and Rt, to evaluate the corrosion resis-tance. Based on the fitted data in Fig. 7, the evolution process hastwo regimes. In regime A, Rp of the aged sample is much smallerthan that of the as-received one whereas in regime B, Rp increasesrapidly and becomes much higher.

After immersion for 24 h, large scar-like patterns correspondingto the b-phase rich areas emerge from the surface of the aged sam-

Table 1Composition of the Dulbecco’s modified eagle medium (DMEM) powder.

Components Concentration (mg/L)

Amino acidsGlycine 30

L-Arginine hydrochloride 84

L-Cystine 2HCl 63

L-Glutamine 584

L-Histidine hydrochloride-H2O 42

L-Isoleucine 105

L-Leucine 105

L-Lysine hydrochloride 146

L-Methionine 30

L-Phenylalanine 66

L-Serine 42

L-Threonine 95

L-Tryptophan 16

L-Tyrosine disodium salt dihydrate 104

L-Valine 94

VitaminsCholine chloride 4

D-Calcium pantothenate 4

Folic acid 4Niacinamide 4Pyridoxine hydrochloride 4Riboflavin 0.4Thiamine hydrochloride 4i-Inositol 7.2

Inorganic saltsCalcium chloride (CaCl2) (anhydrous) 200Ferric nitrate (Fe(NO3)3�9H2O) 0.1Magnesium sulfate (MgSO4) (anhydrous) 97.67Potassium chloride (KCl) 400Sodium chloride (NaCl) 6400Sodium phosphate monobasic (NaH2PO4–H2O) 125

Other components

D-Glucose (Dextrose) 4500

Phenol red 15

280 G. Wu et al. / Corrosion Science 68 (2013) 279–285

ple (Fig. 8). Besides, localized corrosion associated with the areaswith AlxMny is found on both the aged and as-received alloys.The XPS survey spectra in Fig. 9 show that the aged alloy is similarto the as-received alloy. C, O and N are observed from the surfaceand Ca, P, Mg and Al are detected at a depth of 100 nm. At depthslarger than 500 nm, the Mg and Al peaks increase but the Ca, P, andO peaks diminish, indicating that the composition of a Ca–P layer isnot subjected to proteins in the media. The high-resolution spectrain Fig. 10 further disclose that Ca and P are related to phosphates[14]. Both metallic and oxidized state sub-peaks are observed fromthe Mg 1s and Al 2p spectra. At 100 nm, the Mg 1s peak is com-posed of the Mg0 peak and two Mg2+ peaks (one from oxide/hydroxide [15] and the other from phosphate [16]). At a depth of500 nm, the phosphate sub-peak disappears. In general, a surfacefilm consisting of MgO/Mg(OH)2 is easily formed on magnesium al-loy in common aqueous solutions. MgO is not stable in an aqueous

solution based on thermodynamics and has a trend to becomemagnesium hydroxide. If Cl� exists in the environment, OH� willbe substituted by Cl� to form soluble chloride [17,18]. Therefore,this surface film has a poor protective effect in the corrosion pro-cess. Recently, it has been found that phosphates can be precipi-tated on Mg-based materials during the immersion in simulatedbody fluids, which can gradually modify the corrosion resistancewith the immersion time [19,20]. Generally, the blocking of defectsis considered as one of the important self-healing processes [21].The precipitation of phosphates can provide the possibility to re-pair the corrosion products on Mg-based materials. Thus it canbe naturally envisaged as a self-protection process against theaggressive species. In this study, aging treatment significantlymodified the distribution of b-phases in Mg–Al–Zn–Mn alloy andconsequently triggered an interesting evolution of corrosion resis-tance in cell culture media.

Fig. 2. Open circuit potential versus time of the as-received and aging-treatedsamples. Fig. 3. Polarization curves of the as-received and aging-treated samples.

Fig. 1. (a) XRD patterns and (b–d) microstructures of the as-received and aged samples. Panel (d) shows the magnified selected-area in panel (c) and the insert in panel (d)shows the magnified image of a randomly selected area in the grains.

G. Wu et al. / Corrosion Science 68 (2013) 279–285 281

Fig. 4. Nyquist plots of the as-received and as-aged samples.

Fig. 5. Bode plots of the as-received sample.

282 G. Wu et al. / Corrosion Science 68 (2013) 279–285

It is suggested in the previous studies that b-phases play acrucial role in the corrosion behavior of Mg–Al alloys in NaCl solu-tions. If the volume fraction of b-phases is small, they will serve asa galvanic cathode to accelerate the corrosion of the a-phases. Butwhen a continuous b-phase network is formed in the corrosionprocess, the b-phases will act as a barrier to resist the corrosion[22–24]. Recently, Abidin et al. [25] investigated the corrosionbehaviors of pure Mg, AZ91, ZE41 and Mg2Zn0.2Mn alloys inHank’s solution and NaCl solution. Due to the formation of a moreprotective surface film in Hank’s solution, the corrosion in Hank’ssolution is slightly influenced by microstructure in contrast to thatin NaCl solution. In this study, we consider the synergistic effect ofmulti-factors and propose a model (Fig. 11) to explain the unusual

Fig. 6. Bode plots of the as-aged sample.

Fig. 7. Polarization resistance versus time obtained from electrochemical imped-ance spectroscopy.

Fig. 8. Surface morphology of the as-received (a) and aged (b) samples afterimmersion for 24 h. The inset in (b) shows the magnified b-phase rich area.

G. Wu et al. / Corrosion Science 68 (2013) 279–285 283

self-protection phenomenon. Owing to redistribution of theb-phases, galvanic cells are quickly formed in the (a + b) areas witha-Mg as anodes and b-phase as cathodes in the initial stage. Theanodic reaction is: Mg ? Mg2+ + 2e� and the cathodic reaction is:2H2O + 2e�? 2OH� + H2". The total reaction is: Mg + 2H2O ?Mg(OH)2 + H2" [26,27]. Dissolution of Mg increases the OH� con-centration near the surface. Therefore, new chemicalreactions occur as follows: 2OH� + H2PO�4 ? PO3�

4 + 2H2O, OH� +HPO2�

4 ? PO3�4 + H2O. When the solubility limit is exceeded, Ca3

(PO4)2 (or Ca10(PO4)6(OH)2) and Mg3(PO4)2 precipitates from thesolution [28,29]. After these secondary corrosion products combinewith the primary corrosion product, Mg(OH)2, to cover the reactedsurface, the electrochemical activity is reduced [30]. In the b-phaserich area, the b-phase constitute a barrier network that can furtherresist corrosion. Song and Xu [31] considered that AlxMny phasewas also an effective cathode to the a–Mg matrix having a detri-mental effect on the corrosion performance. In the as-receivedsample, the fine b-phases can be covered more easily by the corro-sion products but coarse AlxMny phases become the dominatingfactor influencing corrosion. But if they exist in the b phase-richareas, the detrimental effect is weakened by the b-phase barriernetwork. Outside these areas, corrosion is retarded when theymeet the next b-phase network in the process. Therefore, com-pared to the untreated alloy, the aged alloy undergoes a moreeffective self-repair and even self-strengthens in the simulatedphysiological environment.

The biological behavior of the aged alloy is also altered. Asshown in Fig. 12a, more proteins from bovine calf serum adsorbon the aged alloy at time points of 5 min, 30 min, and 24 h. The cellmorphology observed after culturing for 24 h reveals that moreosteoblasts attach onto the surface besides better spreading(Fig. 12b). The cell viability assay shows that osteoblast prolifera-tion is improved on the aged alloy after culturing for 1, 3, and7 days (Fig. 12c). However, it should be noted that the effects ofother factors such as cell growth and pre-cleaning with PBS are stillunknown and need further investigation.

4. Conclusion

An interesting self-protection behavior is observed from agedMg–Al–Zn–Mn alloy in cell culture media. Compared to theuntreated sample, the corrosion resistance is lower in the initialstage but increases rapidly after a critical immersion time andmaintains a larger value in the later period. The synergistic effectrendered by the b-phase and shielding of corrosion product isbelieved to induce this unusual behavior which also improvesthe biocompatibility.

Fig. 9. XPS survey spectra of the as-received and aged samples after immersion for24 h.

Fig. 10. High resolution XPS spectra of the aged sample after immersion for 24 himmersion.

284 G. Wu et al. / Corrosion Science 68 (2013) 279–285

Acknowledgments

This work was financially supported by Hong Kong ResearchGrants Council (RGC) and General Research Funds (GRF) Nos. CityU112510 and 112212 and City University of Hong Kong Applied Re-search Grant (ARG) No. 9667066.

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Fig. 11. Schematic diagram of the self-protection mechanism.

Fig. 12. (a) Adsorbed proteins on the samples. (b) Fluorescent images of osteoblasts cultured on samples for 24 h, and (c) Cell viability assay of cells incubated on the samples.

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