lbmd - study on bio-corrosion and cytotoxicity of a sr-based bulk...

Study on bio-corrosion and cytotoxicity of a Sr-based bulk metallicglass as potential biodegradable metal

H. F. Li,1* K. Zhao,2* Y. B. Wang,1 Y. F. Zheng,1 W.H. Wang2

1State Key Laboratory for Turbulence and Complex System and Department of Advanced Materials and Nanotechnology,

College of Engineering, Peking University, Beijing 100871, China2Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China

Received 26 April 2011; revised 7 August 2011; accepted 5 September 2011

Published online 24 November 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.31958

Abstract: In this study, a novel Sr-based bulk metallic glass

(BMG) with nominal composition of Sr40Mg20Zn15Yb20Cu5

(at.%) was fabricated with high-purity raw materials by induc-

tion-melting method. Its mechanical properties, corrosion

behavior, and cellular compatibility were studied in vitro as a

feasibility evaluation of potential biodegradable metal. The

compression strength of Sr40Mg20Zn15Yb20Cu5 alloy was

(408.2 6 20.0) MPa, which was much higher than human bone

(164–240 MPa). Its Young’s modulus (20.6 6 0.2) GPa was

quite close to human bone (5–23 GPa). MTT assay and

PNPP method were employed to measure the proliferation abil-

ity and ALP activity of the experimental Sr-based BMG. The

results 0–1 grade cytotoxicity and high ALP activity revealed that

it was harmless to osteoblast-like MG63 cells. Additionally, the

cells cultured on the surface of Sr40Mg20Zn15Yb20Cu5 alloy plate

exhibited polygonal and dorsal ruffle configuration, and were

well attached to the substrate by cellular extension, indicating

their healthy status. The combination of increased mechanical

strength, greater corrosion resistance, and excellent biocompati-

bility makes the Sr40Mg20Zn15Yb20Cu5 BMG very attractive mate-

rial for biodegradable orthopedic implant applications. VC 2011

Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 100B:

368–377, 2012.

Key Words: Sr-based bulk metallic glasses, mechanical prop-

erty, corrosion behavior, cytotoxicity

How to cite this article: Li HF, Zhao K, Wang YB, Zheng YF, WangW.H.. 2012. Study on bio-corrosion and cytotoxicity of a sr-basedbulk metallic glass as potential biodegradable metal. J Biomed Mater Res Part B 2012:100B:368–377.

INTRODUCTION

Metallic materials are more suitable to support the repairor replacement of bone tissue that has become diseased ordamaged, compared with ceramics or polymeric materials,due to their combination of high mechanical strength andfracture toughness.1

Stainless steels, titanium and cobalt-chromium-basedalloys are commonly used metallic biomaterials at present.However, these current metallic biomaterials have manylimitations: First, the elastic moduli of current metallicbiomaterials are not well matched with that of naturalbone tissue, which might result in stress shielding effectsthat can lead to reduced stimulation of new bonegrowth and remodeling thus decreasing implant stability.2

Second, the possible release of toxic metallic ions and/orparticles through corrosion or wear processes would lead toinflammatory cascades which reduce biocompatibility andcause tissue loss.3–5 Third, current metallic biomaterials are

most essentially bioinert in vivo, remaining as permanentfixtures, which in the case of screws, pins, and platesused to secure serious fractures, have to be removedby a second surgical procedure after the tissue has fullyhealed.6 Repeat surgeries increase costs to the healthcare system, bring great pain and further morbidity tothe patient.

Biodegradable implant materials in the human body canbe gradually dissolved, absorbed, consumed or excreted, sothere is no need for the secondary surgery to removeimplants after the surgery regions have been healed.7

Therefore, the study of innovative biodegradable implantmaterials have attracted considerable attention as potentialimplant materials at the forefront in the area of biomaterialsin recent years. Nevertheless, current biodegradableimplants made of polymers1 or conventional magnesiumand magnesium alloys8 have an unsatisfactory mechanicalstrength, and therefore limited their applications.

*Both authors contributed equally to this work.

Correspondence to: Y. F. Zheng; e-mail: [email protected] and W.H. Wang; e-mail: [email protected]

Contract grant sponsor: Research Fund for the Doctoral Program of Higher Education; contract grant number: 20100001110011

Contract grant sponsor: National High Technology Research and Development Program of China (863 Program); contract grant numbers:

2011AA030101, 2011AA030103

Contract grant sponsor: National Basic Research Program of China (973 Program); contract grant number: 2012CB619102

Contract grant sponsor: National Natural Science Foundation of China; contract grant numbers: 30870623, 51041004

Contract grant sponsor: Program for New Century Excellent Talents in University; contract grant number: NCET-07-0033

368 VC 2011 WILEY PERIODICALS, INC.

Bulk metallic glasses (BMGs) are a neoteric class ofalloys with a fully amorphous microstructure comparedwith conventional crystalline alloys, and show a promisingpotential for biomedical applications owing to the combina-tion of superior strength, high elastic strain limit, relativelylow Young’s modulus, excellent corrosion resistance, andwear resistance. They have attracted much attention as acandidate for biomaterial in recent years. Mg-based andCa-based BMGs are the two typical biodegradable bulkmetallic glasses. The present authors8 and Zberg et al.9

studied the mechanical properties, corrosion behavior, invitro and in vivo biocompatibility of Mg-Zn-Ca bulk metallicglassed and their studies demonstrated that amorphousMg-Zn-Ca alloys showed better compression strength andcorrosion resistance than pure Mg (99.95%), besides, theyhad excellent cell and tissue compatibility. Furthermore,Mg-Zn-Ca BMGs with Zn content higher than 28% (at.%)would not generate hydrogen both in vitro and in vivo.9 Thepresent authors10 evaluated the Ca65Mg15Zn20 BMG’s feasi-bility for potential skeletal applications by both in vitro testson ion release and cytotoxicity and in vivo implantation, andfound that Ca65Mg15Zn20 BMG had no detectable cytotoxiceffect over a wide range of concentrations (0–50%) and ani-mal tests proved that there was no obvious inflammationreaction at the implantation site and CaMgZn BMG implantsdid not result in animal death. However, one recognizabledrawback of Ca65Mg15Zn20 BMG is the relatively rapid deg-radation rate, which might result in a loss of mechanicalstrength of the BMG.

The Sr-based BMGs are a rather new class of metallicglasses which were firstly reported in 2009 by the presentauthors.11 Sr-based BMG alloys are of interest as potentialbiodegradable materials due to following reasons: (1) Physi-cal properties; such as low density, low Young’s moduluswhich is comparable to the modulus of human bones11; (2)Biological effect/function of elements Sr, Mg, and Zn; (i)Strontium (Sr) can inhibit bone resorption and stimulatebone formation in both rodents,12 and osteoporoticpatients.13,14 Sr-based drug treatments for osteoporosis,such as Protelos can reduce the risk of fracture in patientsafter one year of treatment. In vitro studies have shownthat Protelos inhibits osteoclast activity15 and stimulatesosteoblast proliferation.16 Sr has an affinity for bone and isincorporated into it by surface exchange and ionic substitu-tion. Strontium salts were found to stimulate bone forma-tion and inhibit bone resorption both in vitro and in vivo.17

Strontium-containing hydroxyapatite (Sr-HA) was synthe-sized as a filling material which can be used locally to mixwith bone cement.18,19 Sr-HA bioactive bone cement pro-motes osteoblast attachment and mineralization in vitro,20

as well as bone growth and osseointegration in vivo.21,22

Local delivery of strontium with HA in cement has beenshown to induce new bone formation and effective in reduc-ing fracture risk in osteoporosis.20 The development of thedrug strontium ranelate, which has recently been shownto reduce the incidence of fractures in osteoporoticpatients,23,24 is the main reason for the growing interest instrontium-enriched calcium phosphate bioceramics and

bone cements.25,26 Strontium is present in the mineralphase of bone, especially in the regions of high metabolicturnover.27 In vitro, it increases the number of osteoblastsand reduces the number and activity of osteoclasts16,28; invivo, it inhibits bone resorption and improves bone forma-tion.29–31 Strontium can replace calcium in the HA structurefor the whole range of compositions, inducing a linear varia-tion in the lattice constants.32,33 (ii) Magnesium is essentialto human metabolism and is the fourth most abundant cat-ion in the human body, with estimated 25 g magnesiumstored in human body and approximately half of the totalcontent stored in bone tissue. Magnesium is a cofactor formany enzymes and stabilized the structures of DNA andRNA.6,34 (iii) Zinc (Zn) is the second most prevalent traceelement in the human body and is reported to stimulatefracture healing of bone,35 reduces postmenopausal boneloss,36 improves bone mineralization,37,38 and improvesskeletal strength.39 Moreover, Zn is an effective agent inkilling bacterial strains commonly associated with infectionin orthopedic surgeries.40

As a degradable metal with excellent mechanical proper-ties such as low Young’s modulus comparable to themodulus of human bones, higher compression strength thancortical bone, a natural ionic presence (Sr, Mg, Zn, etc.) withsignificant functional roles in vitro and in vivo biological sys-tems, Sr-based BMG has great potential to serve as biocom-patible, osteoconductive, degradable implants for orthopedicapplications.

Among the series of Sr-based BMGs reported by ourprevious study,11 Sr40Mg20Zn15Yb20Cu5 has the best corro-sion resistance and it didn’t have any remarkable degrada-tion and weight change after immersed in deionized waterfor more than one week. On the contrary, other reportedSr-based BMGs11 such as Sr60Mg18Zn22, Sr60Li5Mg15Zn20had too rapid degradation rate and fully degraded within 1min in deionized water.

The aim of this study is to evaluate Sr40Mg20Zn15-Yb20Cu5 BMG’s potential as biomaterials for orthopedicapplications by the comprehensive in vitro studies on themicrostructure, mechanical property, corrosion rate, andcytotoxicity.

MATERIALS AND METHODS

Preparation of materialsThe Sr-based BMGs with nominal compositions (at.%) ofSr40Mg20Zn15Yb20Cu5 were prepared by induction-meltingmethod. The base elements Sr (99.0%), Zn (99.9%), Mg(99.9%), Yb (99.5%), and Cu (99.9%) were melted in aquartz tube under high vacuum (better than 3.0 � 10�3Pa).After that, the melt was subsequently cast into a liquidnitrogen cooled copper mould with a cavity of 50 mm �5 mm � 2 mm. The as-cast Sr-based samples were furthercut into 5 mm � 5 mm � 2 mm plates by a low speed pre-cision diamond saw (SYJ-150, MTI, China) for the corrosionand biocompatibility tests with the surface being polishedup to 2000# grit. All the samples were then ultrasonicallycleaned in acetone, absolute ethanol and deionized waterfor 15 min, respectively. For the cytotoxicity tests, the

ORIGINAL RESEARCH REPORT

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | FEB 2012 VOL 100B, ISSUE 2 369

samples were sterilized by ultraviolet-radiation for at least2 h for one side and then turn over the samples for another2 h’s ultraviolet-radiation sterilization.

Microstructural characterization and compositionanalysisX-ray diffractometer (XRD, DMAX 2400, Rigaku, Japan) usingCu Ka radiation and transmission electron microscopy(TEM, Tecnai F20, FEI, Holland) were employed for theidentification of the amorphous structure of as-castSr40Mg20Zn15Yb20Cu5 BMG samples, and the actual chemicalcomposition was analyzed by energy-disperse spectrometer(EDS, QUANTAX 400, Bruker, Germany). Differential scan-ning calorimetry (DSC, DSC822e, Mettler Toledo, Switzer-land) was performed under a purified argon atmospherewith a constant heating rate of 20 K/min to identify thethermodynamic parameters of Sr40Mg20Zn15Yb20Cu5 BMG.

Mechanical testsUniaxial compression testing was conducted with a mechan-ical testing system (8562, Instron, America) at a constantnominal strain rate of 1 � 10�4�s�1 at room temperature.The test samples with 2 mm in diameter and 4 mm inlength were prepared according to ASTM standards.

The travel time of ultrasonic waves propagating throughthe sample was measured using an ultrasonic system(MATEC 6600, America) with a measuring sensitive of0.5 ns and a carrying frequency of 10 MHz. The elastic con-stants (including the Young’s modulus E, the shear modulusG, the bulk modulus K) were derived from the acoustic dataand the density q. The density q was measured by Archi-medes’ principle in absolute alcohol.

Immersion testThe immersion test was carried out in Hank’s solution41

(NaCl 8.0g, CaCl2 0.14g, KCl 0.4g, NaHCO3 0.35g, glucose1.0g, MgCl2�6H2O 0.1g, Na2HPO4�2H2O 0.06g, KH2PO4 0.06g,MgSO4�7H2O 0.06g dissolved in 1 L deionized water)according to ASTM-G31-72.42 After different immersiontimes, the samples were taken out from Hank’s solution, thecorrosion products on the sample surfaces were removed,gently rinsed with deionized water, and then dried at roomtemperature. This procedure must be handled carefully inorder to minimize the measurement error. The weight wasmeasured on an electronic balance (AL204, Mettler Toledo,Switzerland) with a measuring sensitivity of 0.1 mg. The pHvalue of the solution was also recorded during the immer-sion tests (PHS-3C pH meter, Lei-ci, China). Three sampleshad been used for weight loss and pH value measurements.Changes on the surface morphologies and microstructure ofthe samples before and after immersion were characterizedby scanning electron microscopy (SEM, S-4800, Hitachi,Japan), equipped with energy-disperse spectrometer (EDS,QUANTAX 400, Bruker, Germany) attachment. The amountof hydrogen generated by Sr-based BMGs was measured inaccordance to Ref. 43. The inductively coupled plasmaatomic emission spectrometry (ICP-AES, PROFILE SPEC, Lee-man, America) was employed to measure the concentrations

of alloying element ions which had dissolved from the alloyplates. An average of three measurements was taken forhydrogen generation.

Electrochemical testsA three-electrode cell was used for electrochemical meas-urements. The auxiliary electrode was a platinum and thereference electrode was a saturated calomel electrode (SCE).The electrochemical tests were conducted with an electro-chemical workstation (CHI604D, Chenhua, China) at thetemperature of 37�C in Hank’s solution. The open circuitpotentials (OCP) were monitored for 7200 s. The potentio-dynamic polarization tests were carried out at a scanningrate of 1 mV/s and the initial potential was about 300 mVbelow the corrosion potential (Ecorr) after OCP measure-ments. After polarization testing, samples were gentlyrinsed with deionized water and dried in air. Changes in thesurface morphologies were characterized by scanning elec-tron microscopy (SEM, S-4800, Hitachi, Japan).

Cytotoxicity testHuman osteoblast-like cell (MG63) was adopted to evaluatethe cytotoxicity of Sr-based BMGs. MG63 cells were culturedin minimum essential medium (MEM), 10% fetal bovineserum (FBS), 100 U mL�1 penicillin and 100 mg ml�1 strep-tomycin at 37�C in a humidified atmosphere of 5% CO2. Thecytotoxicity tests were carried out by indirect cytotoxicityevaluation and direct cell adhesion experiment. For indirectcytotoxicity evaluation, the extracts were prepared by incu-bating Sr40Mg20Zn15Yb20Cu5 BMG samples in serum freemedium in a humidified atmosphere with 5% CO2 at 37�Cfor 72 h to create a ‘‘BMG extract’’. The supernatant fluidwas withdrawn and centrifuged to prepare the extractionmedium. The extraction medium was serially diluted to 75,50, 25, 10, and 5% concentration and refrigerated at 4�Cbefore the cytotoxicity test. The control groups involved theuse of MEM medium as negative controls and 10% dime-thylsulfoxide (DMSO) MEM medium as positive controls.5 � 103 cells/100 lL of medium were seeded in each welland incubated in 96-well cell culture plates for 24 h toallow cell attachment. The medium was then replaced with100 lL of extracts. After incubating the cells in a humidifiedatmosphere with 5% CO2 at 37�C for 1, 3, and 5 days,respectively, the 96-well cell culture plates were observedunder inverted phase contrast microscope. After that, 10 lL3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-mide (MTT, Sigma-Aldrich, America) was added to eachwell. The samples were incubated with MTT for 4 h at37�C, then 100 lL formazan solubilization solution(10%SDS in 0.01M HCl) was added in each well overnightin the incubator in a humidified atmosphere. The opticaldensity (OD) measurements of the samples were conductedby microplate reader (Bio-RAD680, America) at 570 nmwith a reference wavelength of 630 nm. The cell relativegrowth rate (RGR) was calculated according to the followingformula:

RGR ¼ ODtest=ODnegative � 100% (1)

370 LI ET AL. STUDY ON BIO-CORROSION AND CYTOTOXICITY OF SR-BASED BMG

The concentration of alloying element ions in the extrac-tion medium was measured by inductively coupled plasmaatomic emission spectrometry (ICP-AES, PROFILE SPEC,Leeman, America).

For direct cell adhesion experiment, 2 � 104 cells wereseeded onto the Sr40Mg20Zn15Yb20Cu5 BMG surfacesdirectly. After 1 day’s culture in a humidified atmospherewith 5% CO2 at 37�C in 24-well plates, the parallel sampleswere rinsed three times with phosphate buffer solution(PBS, pH ¼ 7.4) and fixed in 2.5% glutaraldehyde solutionfor 2 h at room temperature, followed by dehydration in agradient ethanol/distilled water mixture (50, 60, 70, 80, 90,and 100%) for 10 min each and dried in hexamethyldisila-zane (HMDS) solution. The surface of cell adhered experi-mental samples was observed by scanning electron microscopy(SEM, S-4800, Hitachi, Japan).

Alkaline phosphatase activitiesTo assay alkaline phosphatase (ALP) activity, cells werecultured in 5, 10, 25, 50, 75, and 100% concentrationgradient Sr40Mg20Zn15Yb20Cu5 BMG extractions and onSr40Mg20Zn15Yb20Cu5 disks directly for 3, 7, and 11 days atan initial seeding density of 4 � 104 cells/800 lL ofmedium per well (24-well cell culture plates). ALP activitywas evaluated as the amount of p-nitrophenol (pNP) pro-duced on hydrolysis of p-nitro-phenyl phosphate (pNPP) inthe presence of ALP as catalyst. Briefly, 100 lL of 1-steppNPP (Sigma) was added to each well containing a 100 lL

aliquot of supernatant. After 30 min incubation at 37�C thereaction was terminated by adding 50 lL of 2M NaOH. Theabsorbance was measured at 405 nm (Bio-RAD680,America). ALP activity results are presented as percentagesof negative group.

RESULTS

Microstructure and mechanical propertiesFigure 1(a) shows the XRD patterns of Sr40Mg20Zn15Yb20Cu5BMG. The typical amorphous features of Sr40Mg20Zn15-Yb20Cu5 BMG samples can be confirmed by the absence ofdetectable crystalline diffraction peaks, together with thebroad scattering signals around 30� and 52� , respectively.From the EDS analysis [the inset of Figure 1 (a)], itcan be seen that the actual composition of the Sr40Mg20-Zn15Yb20Cu5 BMG is very close to its nominal composition.

Figure1(b) shows the high-resolution TEM (HRTEM)micrograph and the corresponding selected area diffraction(SAED) pattern of Sr40Mg20Zn15Yb20Cu5. The high-resolutionTEM image shows an isotropic disordered pattern, which istypical of an amorphous structure, and no ordered structureand distinguishable crystallites are observed even on thenanoscale. Furthermore, the selected area electron diffrac-tion (SAED) pattern shows a broad halo without any diffrac-tion spots, which also indicates the formation of asingle amorphous phase. The microstructural analyses byTEM prove that the as-cast Sr40Mg20Zn15Yb20Cu5 alloy iscompletely amorphous.

FIGURE 1. (a) XRD pattern and EDS spectrum (inset); (b) high-resolution TEM (HRTEM) micrograph and the corresponding selected area diffrac-

tion (SAED) pattern (inset); (c) DSC measurement; (d) stress-strain curve of Sr40Mg20Zn15Yb20Cu5 BMG. [Color figure can be viewed in the online

issue, which is available at wileyonlinelibrary.com.]



FIGURE 2. (a) Weight loss; (b) pH value; (c) Volume of hydrogen generation of Sr40Mg20Zn15Yb20Cu5 BMG as a function of the immersion time

in Hank’s solution. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

FIGURE 3. (a)Surface morphology; (b) EDS spectrum of the rectangular area in (a); (c) EDX maps of the whole surface of Sr40Mg20Zn15Yb20Cu5 BMG

after being immersed in Hank’s solution at 37�C for 72 h. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]


Figure 1(c) shows the corresponding DSC curves ofSr40Mg20Zn15Yb20Cu5 BMG. The as-cast Sr40Mg20Zn15-Yb20Cu5 BMG sample has distinct glass transition and sharpcrystallization peaks in its DSC trace, confirming the glassystructure. The glass transition temperature (Tg) and the firstcrystallization temperature (Tx1) of Sr40Mg20Zn15Yb20Cu5BMG are indicated by arrows.

Figure 1(d) presents the typical compression stress–straincurves of Sr40Mg20Zn15Yb20Cu5 BMG. The fracture strength rf

for Sr40Mg20Zn15Yb20Cu5 BMG is (408.2 6 20.0) MPa.

Corrosion behaviorFigure 2(a) illustrates the weight loss of Sr40Mg20Zn15-Yb20Cu5BMG as a function of immersion time. From the data showed inFigure 2(a), we can calculate the average weight loss rate ofSr40Mg20Zn15Yb20Cu5 BMG is (0.1795 6 0.0146) mg cm�2 h�1.

Figure 2(b) shows the variation of the pH value ofimmersion solution as a function of immersion time. It canbe seen that the pH rises rapidly at the first 10 h, and thenchanges slower. After 100 h immersion in Hank’s solution,the pH value of Sr40Mg20Zn15Yb20Cu5 BMG is (9.23 6 0.26).

The hydrogen generation of Sr40Mg20Zn15Yb20Cu5 BMG as afunction of immersion time is shown in Figure 2(c) and theresults demonstrates that the average hydrogen generation ratefrom Sr40Mg20Zn15Yb20Cu5 BMG is 0.04175 mL cm�2 h�1.

Figure 3 shows the SEM micrographs and EDS analysisof sample surface after immersion test in Hank’s solutionfor 72 h. At low magnification [Figure 3(a)], the corroded

Sr40Mg20Zn15Yb20Cu5 BMG has compact surface morphology,with a layer of corrosion product coating on it. At high magni-fication, it can be seen that the morphology of corrosion prod-uct on top of Sr40Mg20Zn15Yb20Cu5 BMG immersed in Hank’ssolution is sphere [see the inset of Figure 3(a)]. EDS analysisresults [Figure 3(b,c)] show the presence of oxygen, calcium,phosphorus, carbon, zinc, magnesium, and strontium on thesurface of 72 h immersed Sr40Mg20Zn15Yb20Cu5 BMG samplein Hank’s solution. From the EDS analysis, it can be concludedthat the surface products of Sr40Mg20Zn15Yb20Cu5 BMG inHank’s solution are mainly hydroxyapatite (HA) and phos-phates and carbonates of Ca, Mg, and Sr.

The open circuit potential (Eocp) of Sr40Mg20Zn15Yb20Cu5BMG in Hank’s solution is recorded for 7200 s. Its evolutionwith time was shown in Figure 4(a). During the initial5000 s of immersion time, the Eocp increases with time,then keeps at a relatively stable value. The electrochemicalpolarization curve of Sr40Mg20Zn15Yb20Cu5 BMG in Hank’ssolution is shown in Figure 4(b). As analyzed from the Tafelregions of the polarization curve [Figure 4(b)], the corrosioncurrent density (icorr) of Sr40Mg20Zn15Yb20Cu5 BMG is about22.786 lA cm�2. Figure 4(c) shows the SEM micrographs ofSr40Mg20Zn15Yb20Cu5 BMG sample surface after polarizationexperiments. The surface film of Sr40Mg20Zn15Yb20Cu5 BMGis relatively flat and compact with less micro-cracks, sug-gesting that the Sr40Mg20Zn15Yb20Cu5 BMG is protectivefrom the penetration of the electrolyte and its surface filmmight reduce the corrosion rate effectively.

FIGURE 4. (a) OCP curve, (b) potentiodynamic polarization curve and (c) SEM morphology of Sr40Mg20Zn15Yb20Cu5 BMG after polarizations in

Hank’s solution at 37�C. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]



FIGURE 5. (a) The element releasing into Hank’s solution and (b) MEM solution of Sr40Mg20Zn15Yb20Cu5 BMG. [Color figure can be viewed in

the online issue, which is available at wileyonlinelibrary.com.]

FIGURE 6. (a) Cytotoxicity of MG63 cells cultured in the control and 5%, 10%, 25%, 50% and 100% concentration Sr40Mg20Zn15Yb20Cu5 BMG

extraction mediums; (b) Optical morphologies of MG63 cells that were cultured in the negative control and 100% concentration

Sr40Mg20Zn15Yb20Cu5 BMG extraction mediums for 1, 3, and 5 days; (c) The morphology of MG63 cells cultured on Sr40Mg20Zn15Yb20Cu5

BMG directly for 1d. (d) Normalized ALP activities of MG63 cell lysates in the control and 5%, 10%, 25%, 50% and 100%

concentration Sr40Mg20Zn15Yb20Cu5 BMG extraction mediums after 7 days. (e) Normalized ALP activities of MG63 cell lysates cultured directly

on the Sr40Mg20Zn15Yb20Cu5 BMG disks after 3, 7, and 11 days. [Color figure can be viewed in the online issue, which is available at

wileyonlinelibrary.com.]


Ion releaseFigure 5(a,b) demonstrates the ion concentration ofSr40Mg20Zn15Yb20Cu5 BMG in Hank’s (a) and MEM (b) solu-tions after the immersion period of 3 days. It can be seen inFigure 5 that, after 3 days immersion, the ions released intothe solutions are mainly Sr and Mg ions, on the contrary,Zn, Yb, and Cu ion concentrations are relatively low.

Cytotoxicity testFigure 6(a) showed the MG63 cell viability cultured in 100,50, 25, 10, and 5% Sr40Mg20Zn15Yb20Cu5 BMG extractionmedium for 1, 3, and 5 days. It can be seen that cells culturedin low concentration extracts (below 25%) show a significanthigher absorbance than that of the control group, indicatingthat Sr40Mg20Zn15Yb20Cu5 BMG extraction at low concentra-tion can promote the cell proliferation. For the higher concen-tration extracts (above 50%), a reduced cell viability can begotten compared with negative group. However, the RGR val-ues are still above 75%, which means that the cell cytotoxicityis Grade 1 (no toxicity) according to ISO 10993-5: 1999.

Figure 6(b,c) showed the morphologies of MG63 cells cul-tured in the Sr40Mg20Zn15Yb20Cu5 BMG’s extraction media andon top of Sr40Mg20Zn15Yb20Cu5 BMG surface, respectively.Most of the cells were flattened, had polygonal configurationand dorsal ruffles, and were well attached to the substrate bycellular extension, indicating their healthy status.

Figure 6(d) showed normalized ALP activities of MG63 celllysates in the control and 5, 10, 25, 50, and 100% concentrationgradient Sr40Mg20Zn15Yb20Cu5 BMG extraction mediums after7 days. The results demonstrate significantly increased valuesof ALP activity for relatively low concentration extracts and the

highest values of ALP activity is also observed in the 10%extract, demonstrating a similar trend to that of MTT assay.

Figure 6(e) demonstrated the normalized ALP activities ofMG63 cell lysates cultured directly on the Sr40Mg20Zn15-Yb20Cu5 BMG disks after 3, 7, and 11 days. On the basis ofALP measurements, the Sr- based BMG substrates demonstratesignificant ALP activity and they are no less osteogenic in com-parison to the negative control group.

DISCUSSION

Microstructure and mechanical propertiesVarious mechanical properties of Sr40Mg20Zn15Yb20Cu5 BMGare listed in Table I and compared with those of human corti-cal bone, conventional crystalline Mg and Mg alloys and newlydeveloped Ca-based BMG. Since there is no dislocation-basedplastic-deformation mechanism in amorphous alloys, bulk me-tallic glasses reveal greatly improved strength and elasticity.9

From Table I it can be seen that the fracture strength ofSr40Mg20Zn15Yb20Cu5 BMG is much higher than that of corticalbone and conventional crystalline pure Mg and Mg-Ca alloys,besides, it is much higher than that of Ca-based BMG. Fromthe mechanical point of view, Sr40Mg20Zn15Yb20Cu5 BMG ismore favorable for orthopedic application due to its higherfracture strength and more similar Young’s modulus to humancortical bone compared with conventional crystalline pure Mgand Mg-Ca alloys and Ca- based BMG.

Corrosion behaviorTable II summarized the corrosion behavior of Sr-based BMGs,Ca-based BMG, and conventional crystalline Mg and Mg alloys.It can be seen in Table II that the corrosion rate of

TABLE I. Comparison of Mechanical Properties of Sr-Based BMGs, Ca-Based BMG, and Conventional Crystalline Mg and Mg

Alloys

MaterialsCompressive Strength

rf (MPa)Young’s modulus

E(GPa)Shear modulus

G(GPa)Bulk modulus

K(GPa)

Sr40Mg20Zn15Yb20Cu5 (BMG) 408.2 6 20.0 20.6 6 0.2 7.88 6 0.07 17.6 6 0.1cortical bone44 164-240 5-23Ca65Mg15Zn20 (BMG)45,46 300 26.4 10.1 22.6pure Mg47 (conventional crystalline) 198.1 64.5 44.5 6 0.8Mg–0.6Ca47 (conventional crystalline) 273.2 6 6.1 46.5 6 0.6Mg–1.2Ca47(conventional crystalline) 254.1 6 7.9 49.6 6 0.9Mg–1.6Ca47 (conventional crystalline) 252.5 6 3.3 54.7 6 2.4Mg–2.0Ca47 (conventional crystalline) 232.9 6 3.7 58.8 6 1.2

TABLE II. Comparison of Corrosion Behavior Among Sr-Based BMGs, Ca-Based BMG, and Conventional Crystalline

Mg and Mg Alloysa

Materials H2 generation rate (ml�cm�2�h�1) icorr (lA/cm2)

Sr40Mg20Zn15Yb20Cu5 0.04175 22.786Ca60Mg15Zn25 7.509 (in deionized water)48 12.667b 10

WE4344 (conventional crystalline) 30.60as-cast pure Mg (99.95%)49(conventional crystalline) 0.032c0.108c (in SBF) 15.98 86.06 (in SBF)as-cast Mg-1Ca (conventional crystalline) 0.136 (in SBF)50 546.0944 (in SBF)as-extruded Mg-1.22Ca (conventional crystalline) 63.0051

a The date are all obtained in Hank’s solution except for special notes in parentheses.b The data are calculated from Figure 2(a) in.10

c The data are calculated from Figure 4. in.49



Sr40Mg20Zn15Yb20Cu5 BMG is much lower than that ofCa-based BMG and conventional crystalline Mg and Mg alloysin terms of H2 generation rate and icorr, thus, Sr40Mg20Zn15-Yb20Cu5 BMG has much better corrosion resistance than thatof Ca-based BMG and conventional crystalline Mg alloys.

The enhanced corrosion resistance is partly due to itsamorphous structure without any grain boundary, dislocation,and lattice defect.52 The alloying elements Yb and Cu also con-tribute to the enhanced corrosion resistance. In our previousstudy11on the corrosion properties of Sr- based BMGs with var-ious constituents, it was found that adding Yb and Cu can sig-nificantly improve the corrosion resistance of Sr-based BMGs.

Additionally, Chen KH and Fang HC et al.53–55 investi-gated the effect of addition of Yb on corrosion resistance ofAl alloys and their results demonstrated that Yb remarkablyenhanced resistance to stress corrosion cracking, exfoliationcorrosion and intergranular corrosion. Yamasaki M et al.56

studied the corrosion behavior of the melt spun Mg-Zn-Yballoy ribbons in 1% NaCl solution and found that Yb addi-tion significantly enhanced corrosion resistance of Mg alloys.Hu et al.57 and Ujiro et al.58 studied the effect of alloying Cuon the corrosion resistance of stainless steels and theirresults show that the alloying element Cu can remarkablyimprove the corrosion resistance in solutions with Cl�1.Zhang et al.59 studied the effect of alloying Cu on the corro-sion resistance of the Ni-P based amorphous alloys andfound that adding Cu can improve the corrosion propertiesof Ni-P and Ni-Sn-P alloys. Naka M et al.60 studied the effectof alloying Cu on the corrosion behavior of amorphous Fe-P-C alloys and their results demonstrated that Cu alloying ele-ment is effective in increasing the corrosion resistance ofamorphous alloys in various acidic and neutral solutions.

BiocompatibilityThe in vitro cytotoxicity of Sr40Mg20Zn15Yb20Cu5 BMG wasfound to be Grade 0-1 according to ISO 10993-5:1999, indicat-ing that the Sr-based alloy is safe as an implantable material.The results of ALP test showed that the present Sr-based BMGsamples exhibit excellent biocompatibility and the extracts atproper concentrations (lower than 50%) would promote cellproliferation. This promoting effect is probably attributed tothe Sr and Mg ions. The results of Qiu et al.61 suggested thatproper dose of strontium could stimulate the proliferation anddifferentiation of ROS17/2.8 cells in vitro. Zreiqat et al.62

reported that magnesium ions modified bioceramic substratacould enhance the adhesion of human bone-derived cell(HBDC), and the HBDCs grown on the Mg2þ-modified biocer-amic substrata were found to express significantly enhancedlevels of a5b1- and b1-integrin receptors, which mediated thecell adhesion to biomaterial surfaces.

CONCLUSIONS

In this study, Sr40Mg20Zn15Yb20Cu5 BMG samples are preparedand investigated in vitro as a biomedical degradable orthope-dic implant material. The following conclusions can be drawn.

(1) The Sr-based BMGs shows higher compression strengththan that of Ca60Mg15Zn25 BMG and conventional

crystalline pure Mg and Mg alloys and has similarYoung’s modulus to cortical bone.

(2) The corrosion resistance of Sr40Mg20Zn15Yb20Cu5 ismuch better than that of Ca60Mg15Zn25 BMG and con-ventional crystalline Mg and Mg alloys.

(3) The indirect and direct cytotoxicity tests shows thatSr-based BMG samples indicate satisfactory cell viabilityand ALP activity. Moreover, MG63 cells are found to bewell adhered on the surface of the Sr-based samples,indicting its good biocompatibility.

(4) The combination of increased mechanical strength,greater corrosion resistance, and excellent biocompati-bility makes the Sr40Mg20Zn15Yb20Cu5 BMG very attrac-tive material for biodegradable orthopedic implantapplications.

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