Enhanced Osseointegration of Zn-Mg Composites by Tuning theRelease of Zn Ions with Sacrificial Mg-Rich Anode DesignHongtao Yang,† Xinhua Qu,‡ Wenjiao Lin,§ Dafu Chen,⊥ Donghui Zhu,∥ Kerong Dai,*,‡

and Yufeng Zheng*,†,#

†Department of Materials Science and Engineering, College of Engineering, Peking University, Beijing 100871, China‡Department of Orthopedics, Shanghai Key Laboratory of Orthopedic Implant, Shanghai Ninth People’s Hospital, ShanghaiJiaotong University School of Medicine, Shanghai 200011, China§R&D Center, Lifetech Scientific (Shenzhen) Co. Ltd., Shenzhen 518057, China⊥Laboratory of Bone Tissue Engineering, Beijing Research Institute of Traumatology and Orthopaedics, Beijing JiShuiTan Hospital,Beijing 100035, China∥Department of Biomedical Engineering, College of Engineering, University of North Texas, Denton, Texas 76207, United States#International Research Organization for Advanced Science and Technology, Kumamoto University, 2-39-1 Kurokami, Chuo-Ku,Kumamoto 860-8555, Japan

*S Supporting Information

ABSTRACT: Relatively slow degradation rate and delayedosseointegration induced by excessive release of Zn2+ ions aretwo main disadvantages of the use of pure Zn ion bioabsorbableorthopedic implants. In light of this, we designed a cathodicprotection strategy by incorporating Mg, acting as a sacrificialanode, into Zn to form Zn-Mg composites. The performance ofnovel Zn-Mg composites with regard to degradation behaviorand biocompatibility was evaluated systematically under in vitroand in vivo conditions. Macro-galvanic coupling that formedbetween the Mg-rich phase (anode) and the Zn matrix phase(cathode) accelerated the degradation of Zn-Mg composites ascompared to that of pure Zn. Composition analysis revealedZnO as the dominant product of Zn-Mg composites, followed by calcification matrix formation during the bone healing process.Cytotoxicity assay showed prominently improved cell viability after addition of Mg. Histological analysis manifested delayedosseointegration for the pure Zn group. In contrast, direct contact between new bone and Zn-5Mg composite in multiplelocations and increased bone bonding areas were found over time. The synergic biological effect of co-releasing Zn2+ and Mg2+

ions by preferential corrosion of sacrificial Mg-rich phase contributed to the ameliorated bone integration. Thus, introducingsacrificial Mg-rich anode is an effective design strategy to increase the degradation rate of pure Zn while simultaneouslyimproving its bone integration ability.

KEYWORDS: Zn-Mg composites, sacrificial Mg-rich anode, enhanced osseointegration, degradation mechanism, orthopedic implants

■ INTRODUCTION

Zinc, which is an essential trace element for human health,plays critical roles as an enzymatic cofactors, a structuralcomponent of proteins, and a transcriptional regulator in awide range of cellular and biochemical processes. The genesassociated with zinc finger proteins account for 3% of thehuman genome, implying the importance of zinc across avariety of functions.1 More importantly, zinc is involvedsignificantly in bone formation and bone resorption. Zincshowed a stimulatory effect on osteogenesis and mineralizationthrough activation of aminoacyl-tRNA synthetase in osteo-blastic cells and stimulation of cellular protein synthesis.2

Meanwhile, zinc acted as a potent NF-κB activation antagonistin osteoclast precursors and suppressed osteoclast differ-

entiation.3 Furthermore, nutritional zinc can prevent boneloss induced by bone disorders.4

The practice of fabricating zinc and its alloys for use as boneimplants had not been reported until recently.5,6 Three mainfindings have been presented: First, pure Zn was able to inducenew bone formation in rat femur condyle, whereas delayedosseointegration was found 8 weeks post-operation, which wasproblematic in bone implants. Excessive release of Zn2+ ionsduring degradation was believed to play a dominant role in thedelayed osseointegration.6 Second, the degradation rate of pure

Received: September 19, 2018Accepted: December 20, 2018Published: December 20, 2018

Article

pubs.acs.org/journal/absebaCite This: ACS Biomater. Sci. Eng. 2019, 5, 453−467

© 2018 American Chemical Society 453 DOI: 10.1021/acsbiomaterials.8b01137ACS Biomater. Sci. Eng. 2019, 5, 453−467

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Zn is slow; it may take more than a decade for a 5 mmdiameter pure Zn screw to degrade completely under boneenvironments.6 Third, bulk Zn-1Mg alloy exhibited apromotion effect in osteogenesis after 1 week’s implantationin mouse femora.5

Introducing Mg into Zn can be an effective methodology toshape the fate of Zn during degradation with regard to theabove three aspects. First of all, galvanic cells, composed of Mgas the anode and Zn as the cathode, were found to form duringdegradation, induced by the significant potential differencebetween the two (Zn, −0.762 V vs SHE; Mg, −2.372 V vsSHE).7 As a result, accelerated degradation is expected afterthe incorporation of Mg. More importantly, the preferentialdissolution of sacrificial Mg anode can lead to prior release ofMg2+ ions, which circumvents the toxicity caused by excessiverelease of Zn2+ ions. Second, Mg plays a beneficial role in thebone healing process. The extraction of Mg promoted cellviability, bone alkaline phosphate (ALP) activity, and mRNAexpression of osteogenic differentiation-related genes, includ-ing ALP, osteopontin (OPN), and RUNX2 of human bonemarrow mesenchymal stem cells (hBMSCs).8 Mg alsoimproved the adhesion of osteoblastic cells on the biomaterials’surface by modifying the affinity of the related integrin to theirrespective ligands.9 Animal tests demonstrated that Mg-basedimplants enhanced new bone formation, including enhancedperiosteal and endosteal bone formation and good osseo-integration between degradation product and new bone.10

Apart from this, Mg was reported as a strengthening elementfor alloying with pure Zn, forming the hard intermetallic phaseMg2Zn11 and refining the grain size.11 Alloying is a commonlyused method to add Mg into Zn. Nevertheless, Zn-Mg alloysdisplayed little difference from pure Zn in terms of degradationbehavior.11,12 The formation of a tiny and highly dispersedintermetallic phase (Mg2Zn11) that rich in Zn weakened theeffects of galvanic corrosion. Therefore, spark plasma sintering(SPS) was chosen as an alternative way to incorporate Mg intoZn in order to create effective galvanic cells in the presentstudy, with the degradation behavior, osteogenesis, and boneintegration ability of the Zn-Mg composites being systemati-cally evaluated.

■ MATERIALS AND METHODSMaterials. Pure Zn powders (99.9%, 45−109 μm, Alfa) and pure

Mg powders (99%, 38−125 μm, Tangshan Weihao) were used asstarting materials for the present study. Zn-Mg powders (1, 2, and 5wt% Mg, remainder Zn) and pure Zn powders were sintered into 30× 7 mm2 solids via SPS (SPP-1050 system, Sumitomo Coal MiningCo. Ltd., Japan). The mixed powders were sintered at 380 °C under50 MPa pressure for 6 min in a vacuum. Cylinders (2 × 4 mm2 and 2× 5 mm2) for the mechanical test and animal test and disk samples(10 × 1 mm2) for the rest of the tests were cut from the as-sinteredspecimens, respectively. All specimens were polished metallo-graphically to 2000 grit, followed by washing in acetone and ethanolwith an ultrasonic cleaner.Microstructure Characterization. Specimens were ground with

SiC papers and polished with 0.1 μm diamond paste. Themetallographic structure was observed via an optical microscope(Olympus BX51 M) after the samples were etched with a 4% HNO3solution in ethanol. The density of experimental samples wasmeasured by a balance (XS105, Mettler Toledo). X-ray diffraction(XRD) was analyzed on samples by using a Rigaku DMAX 2400instrument with Cu Kα radiation at a scan rate of 6°/min over ascanning angle from 20° to 80°. The chemical composition of thesamples was analyzed by using a scanning electron microscope

(Hitachi S-4800, Japan) equipped with an energy-dispersivespectrometer (Bruker Quantax, Germany).

Mechanical Tests. Compressive tests were performed on auniversal test machine (Instron 5969) at a rate of 2 × 10−4/s.Microhardness was analyzed to determine the Vickers hardness of thesamples. The loading force was 0.1 kN, with a 15 s holding time.

Electrochemical Tests. An electrochemical workstation (Met-rohm Ltd., Switzerland) was used for all the electrochemical tests. Athree-electrode system was used, comprising the working electrode(specimens), reference electrode (saturated calomel electrode), andcounter electrode (platinum electrode). The tests were carried out inHanks’s solution6 at room temperature. The diameter of the exposedsurface of the working electrode was 6 mm. The open-circuit potential(OCP) value was recorded during immersion of the system inHanks’s solution for 5400 s. Electrochemical impedance spectroscopy(EIS) was measured in a frequency range from 105 to 10−2 Hz with a10 mV perturbation. For potentiodynamic polarization (PDP), thescanning rate was set at 1 mV/s, and five samples were tested for eachgroup. After the tests, the corrosion products were removed in a 200g/L CrO3 solution, and scanning electron microscopy (SEM) withenergy-dispersive spectrometry (EDS) was used to examine thecorrosion morphology.

Scanning Kelvin Probe. A Scanning Kelvin Probe (SKP, M370)was used to measure the voltage potential of samples in air; 25 μmwas chosen as the step size at the x/y-axis with a 100 ± 2 μm workingdistance. The scanning area was 500 μm × 500 μm. Threemeasurements were taken for reproducibility. The tests were carriedout at 20 °C with 40% relative humidity.

Immersion Tests. In vitro degradation was studied by immersionof samples in Hanks’s solution (at 37 °C, pH 7.4) for 50 daysaccording to the ASTM-G31-72 method.13 The exposure ratio foreach sample was 20 mL/cm2. A pH meter (Mettler FiveEasy pHFE20K) was used to follow the change in pH value at selected timepoints. Samples were cleaned with distilled water after immersion, andthe corrosion morphologies were observed by SEM and opticalmicroscopy. The corrosion products were further examined by XRD.Epoxy was used to embed the samples, and the blocks were cuttransversely to expose the sample in cross section. The cross sectionswere examined by SEM with EDS. The weight loss was evaluated byusing an electronic balance after the corrosion products wereremoved. Five duplicate sample were measured for each group.Inductively coupled plasma optical emission spectroscopy (ICP-OES,iCAP6300, Thermo Scientific, USA) was used to examine the ionconcentrations in Hanks’s solutions.

Cytotoxicity Tests. Cell viability was evaluated according to theISO 10993-5 (2009) method. The cytotoxicity of experimentalsamples was evaluated by using MC3T3-E1 cells. The cells wereincubated in minimum essential medium (MEM), supplemented with100 U/mL penicillin, 100 μg/mL streptomycin, and 10% fetal bovineserum (FBS), at 37 °C in a 95% humidified atmosphere of 5% CO2.The cytotoxicity test was performed for both sample extracts and ionsolutions. Extracts were prepared by incubating the samples in culturemedium with 10% FBS at 1.25 mL/cm2 for 24 h. Zn2+ and Mg2+ ionculture media were prepared by dissolving ZnCl2 and MgCl2 (Sigma-Aldrich, US) in MEM. Afterward, the prepared medium was filteredusing a 0.22 μm membrane filter and diluted with MEM to differentconcentrations. The medium was supplemented with 10% FBS, 100U/mL penicillin, and 100 μg/mL streptomycin and stored at 4 °Cbefore use. Culture medium and MEM with 10% dimethyl sulfoxide(DMSO) were used as negative control and positive control,respectively. A total of 3 ×103 cells were seeded in the 96-wellplate and left for 24 h to allow attachment. Next, 100 μL extracts wasadded to replace the original medium. Subsequently, 100 μL of a 10%Cell Counting Kit-8 (CCK-8) solution (Dojindo, Kumamoto, Japan)was added to each well and incubated for an additional 1 h afterincubation for 1, 2, and 4 days. The optical density was then measuredby using a microplate reader (Bio-Rad 680) at 450 nm. ICP-OES wasused to detect the released ions in the extracts.

For cytoskeletal observation, cells were incubated on a glass slideand cultured with selected ion solutions for 12 h. The cells’

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morphology was evaluated by staining with FITC-phalloidin (Sigma,USA) and DAPI (Sigma, USA). The slides were then observed undera confocal laser scanning microscope (CLSM, Leica TCS, Germany).Hemocompatibility. The hemocompatibility of the studied

materials was examined on the basis of hemolysis rate, plateletcount, and coagulation time. Diluted healthy human blood wasprepared with saline at a volume ratio of 4:5. Samples were incubatedwith 10 mL of physiological saline at 37 °C for 30 min, followed byincubation for another 60 min with addition of 0.2 mL of dilutedblood. Distilled water was used as positive control and saline solutionas negative control. The tubes were then centrifuged for 5 min at3000 rpm. The absorbance of supernatant in each well of the 96-wellplates was analyzed at 545 nm by using a microplate reader (Bio-Rad680).Platelet-rich plasma was used for the adhesion test, obtained by

centrifuging healthy human blood at 1000 rpm for 10 min. A total of0.2 mL of plasma was added onto the surface of each sample andincubated at 37 °C for 60 min. Phosphate-buffered saline (PBS) wasused to rinse the samples before the fixation by 2.5% glutaraldehydesolution for 2 h at room temperature. Afterward, samples weredehydrated in a graded series of alcohol solutions for 10 min each,followed by drying in a desiccator. SEM was performed to view themorphology of the platelets.Platelet-poor plasma was used to study coagulation time, obtained

by centrifuging the healthy human blood at 3000 rpm for 15 min.Samples were then incubated in 500 μL of platelet-poor plasma at 37°C for 30 min. Activated partial thromboplastin time (APTT),prothrombin time (PT), and thrombin time (TT) were measured byadding reagents. ICP-OES was used to detect the ion concentrationsin the plasma.Antibacterial Test. The protocol for the antibacterial test was

previously reported6 using Staphylococcus aureus (ATCC 25922).Luria-Bertani (LB) broth was used to culture S. aureus at 37 °C for 16h. For the test, 1 mL of bacteria suspension was added into each

sample at a density of 107 colony-forming units (CFU)·mL−1 andincubated for 24 at 37 °C. The bacteria in the suspension and on thesample surface were then measured by the spread plate method. Theantibacterial rates were calculated as follows:(1) antibacterial rate forplanktonic bacteria (Rp, %) = (B − A)/B × 100%, and (2)antibacterial rate for planktonic bacteria (Ra, %) = (D − C)/D ×100%. A and C represent the average number of bacteria in the culturemedium and on the sample surface of test groups (Zn-Mg compositeand pure Zn), respectively. B and D represent the average number ofbacteria in the culture medium and on the sample surface of thecontrol group (Ti6Al4V), respectively.

Animal Model and Surgery. Femoral condyles in a rat modelwere investigated according to our previously published protocol inthis study.6 Zn-5Mg composite was selected to perform in vivo testsbased on its superior in vitro performance, with pure Zn as control.Thirty-two Sprague−Dawley rats (male, 180−235 g) were providedby Ninth People’s Hospital, affiliated with Shanghai JiaotongUniversity. Briefly, rats were anesthetized and their legs were bothshaved and depilated. A hole (2 mm in diameter) was drilled throughthe distal side of the epiphysis gap in the bone. Normal saline wasused to wash the bone cavity and remove debris. Samples were theninserted into the predrilled hole in a blind manner. Each rat wasimplanted with one sample and sacrificed at 4 or 8 weeks aftersurgery. Radiography was used to assist the surgical procedure andobserve the healing process post-operation and before sacrifice. TheAnimal Ethical Committee at the Ninth People’s Hospital, affiliatedwith Shanghai Jiaotong University, approved all the implantationprotocols in this study.

Micro-Computed Tomography (Micro-CT). After the rats weresacrificed, the femoral condyles with implants were retrieved and fixedin 10% formalin. The implants were scanned by micro-CT (Skyscan1172, Bruker, Belgium) at a 20 μm resolution. Three-dimensionalreconstruction was conducted by using NRecon software (Skyscan,

Figure 1. Microstructure of Zn-5Mg composites. (a) Backscattered electron images of Zn-5Mg composites, magnifying the areas marked with redand blue rectangles. Elemental mappings (below) correspond to the image on the top right with the highest magnification (zinc, red; magnesium,light blue; oxygen, yellow). (b) Quantitative analysis of P1, P2, and P3 in the image with the highest magnification in (a). (c) X-ray diffractionpatterns of the studied materials.

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Belgium). The quantitative analysis was performed by using CTAnsoftware.Histological and Cross-Sectional Analysis. The retrieved

bones were fixed in 10% buffered formalin and dehydrated ingradient ethanol, followed by incubation in xylene for 3 days. Methylmethacrylate was used to embed the samples. The samples were thensectioned into 200 μm slices and polished to 50−70 μm for vanGieson staining. For cross-sectional analysis, the sections were furtherpolished to 7000 grit and observed by SEM with EDS. Soft tissuesections were prepared by decalcifying the samples and embeddingthem in paraffin. Hematoxylin and eosin (H&E), Masson’s trichrome,and tartrate-resistant acid phosphatase (TRAP) staining wasperformed following the manufacturer’s protocol.Statistical Analysis. All the numerical data in this study are

presented as mean ± standard deviation. Statistical analysis was doneby using the one-way ANOVA test. Statistical significance was definedas P values less than 0.05.

■ RESULTSMicrostructure. Figure S1 in the Supporting Information

shows the optical micrographs of experimental Zn-Mgcomposites and pure Zn, both consisting of crystalline phaseswith a large particle size range. Representative backscatteredelectron (BSE) images and elemental mapping of Zn-Mgcomposites are presented in Figure 1a. Mg-rich phasesexhibited a dark contrast feature distributed homogeneouslyin the light feature representing the Zn matrix phase. Themagnified image indicated that the Mg-rich phases arecomposed of an inner core and an outer shell layer. Elementalmapping demonstrated that the intensity of the Mg signaldecreased from the inner core to the outer shell. Oxygenenrichment was detected at phase boundaries. Selected areaanalysis showed that the ratios of Mg:Zn were 1:2 and 2:11 inthe inner core and outer shell positions, respectively (Figure1b). XRD patterns identified the formation of Mg2Zn11 andMgZn2 intermetallic phases in Zn-Mg composites (Figure 1c).It can be proposed that Mg-rich phases were composed of aninner core of MgZn2 and an outer layer of Mg2Zn11. Therelative densities of all experimental samples were higher than99.5%, indicating a high densification rate of materialsfabricated by the SPS process (Table 1).

Mechanical Properties. The mechanical properties ofexperimental samples are presented in Figure S2 in theSupporting Information. Zn-Mg composites showed signifi-cantly higher compressive yield strength (CYS) than pure Zn(Figure S2a, p < 0.05). The CYS increased from 54 ± 17.18MPa for pure Zn to 186.63 ± 26.54 MPa for the Zn-5Mgcomposite. Addition of Mg increased the microhardness ofpure Zn significantly (Figure S2b, p < 0.05). A superb plasticityof pure Zn was seen in the compressive stress−strain curves(Figure S2c). With addition of Mg contents up to 2 wt%, theexcellent compressive plasticity was maintained, whereas the

Zn-5Mg composite displayed a limited plasticity. The limitedplasticity is presumed to be induced by the increased volumefraction of hard and brittle intermetallic phases. The CYS ofZn-Mg composites is comparable to that of natural bone(130−180 MPa). Therefore, the composites are sufficient forsome applications without load-bearing requirements. Defor-mation processes can be applied to further improve themechanical performance of Zn-Mg composites.

Electrochemical Tests. The results of electrochemicaltests are depicted in Figure 2. In addition, the calculatedelectrochemical parameters obtained by Tafel extrapolation arelisted in Table 2. The addition of Mg resulted in an apparentdecrease in corrosion resistance of pure Zn. A decrease incorrosion potential combined with an increase in corrosioncurrent density with Mg contents was observed in potentio-dynamic polarization curves. A passivation behavior wasobserved in Zn-Mg composites based on the current plateausin anodic polarization. Two time constants could bedetermined from the feature of the Nyquist plots. Twocapacitive loops were identified on all experimental samplesexcept for the Zn-5Mg composite. The capacitive loop at highfrequencies could be related to the uniform corrosion layer,while the capacitive loop at low frequencies could be inducedby charge transfer and electrochemical double-layer effects.The tail-shaped curve developed in the low-frequency range forthe Zn-5Mg composite could be assigned to mass-transport-controlled events. In contrast to pure Zn, the reduceddiameters of semicircles in Zn−Mg composites suggested adecreased corrosion resistance. Figure 2c presents therepresentative corrosion morphology with elemental mappingsafter removal of corrosion products. Corrosion pits dispersedon the pure Zn surface showed that grain boundaries had beenattacked seriously. In contrast, typical galvanic corrosion wasobserved for Zn-Mg composites, indicating that the Mg-richphases were corroded severely but the Zn matrix was leftalmost intact. The distribution of the surface potential wasinvestigated using the SKP technique (Figure 2d,e). Theaverage potential value of pure Zn was −624 mV, and themaximum potential difference (ΔV) on the surface was 81.8mV. In contrast, the average potential value of the Zn-5Mgcomposite shifted negatively to −992 mV, with a maximumΔV up to 513.8 mV. The potential map revealed a greatincrease in localized potential gradient after Mg was added intoZn, which resulted in the formation of galvanic corrosion inZn-Mg composites.

Immersion Tests. The macroscopic corrosion morphologyof experimental specimens after immersion in solution ispresented in Figure 3a. Localized corrosion attack was foundmainly at the edge of the pure Zn round disc sample. The Zn-Mg composites typically featured increasing amounts ofcorrosion pits and white corrosion product uniformlydistributed on the sample surface. Figure 3b shows themicrocorrosion morphology in the uniform corrosion region ofsamples. An intact and homogeneous corrosion layer formedon all the samples. Addition of Mg induced large amounts ofmicrocracks in the corrosion layer compared with pure Zn.The corrosion rates based on weight loss are shown in Figure3c. The Zn-5Mg composite exhibited an increased degradationrate as compared to that of pure Zn. Figure 3d shows theevolution of pH value with time. For all the samples, the pHvalue increased rapidly in the first week of immersion, followedby a relatively steady state. Zn-Mg composites exhibited higherpH values compared to that of pure Zn. Concentrations of

Table 1. Relative Density of Studied Materials afterSintering

material sintering conditionsdensity(g cm−3)

relative density(%)a

pure zinc 40 MPa, 380 °C, 6 min 7.078 99.13 (0.301)Zn-1Mg 6.919 99.91 (0.061)Zn-2Mg 6.965 100 (0.003)Zn-5Mg 6.648 100 (0.119)aStandard deviations are presented in the parentheses.

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Zn2+ ions declined significantly with increasing Mg contents,while Mg2+ ions followed the opposite trend (Figure 3e,f).

The chemical composition of corrosion products is analyzedand displayed in Figure 4. Elemental mapping of the crosssections demonstrated distinct features between Zn-Mgcomposites and pure Zn. Pure Zn formed a compact anduniform corrosion layer with approximately 5 μm thickness.The corrosion layer−metal interface was relatively intact. Thecorrosion products were composed of zinc, oxide, andphosphate. In contrast, increasing amounts of corrosionproducts were found covering the surface of the Zn-5Mgcomposite. The corrosion layer−metal interface was zig-zaggedwith some micropores present. Importantly, corrosion attackhappened preferentially in the Mg-rich region. Elementmapping revealed a double-layer structure including an outer

Figure 2. Corrosion behaviors of studied materials based on electrochemical tests. (a) Potentiodynamic polarization curves. (b) Nyquist plots. (c)Corrosion morphologies of samples after cleaning. SKP maps of (d) pure Zn and (e) Zn-5Mg composite exposed to air.

Table 2. Corrosion Properties of Studied Samples Based onElectrochemical Testa

materials Icorr (μA·cm−2) Ecorr (V)corrosion rate(mm·year−1)

pure zinc 5.730 (2.777) −0.978 (0.026) 0.085 (0.041)Zn-1Mg 7.550 (0.900) −1.066 (0.104) 0.114 (0.012)Zn-2Mg 13.891 (3.927)b −1.101 (0.050)b 0.209 (0.059)b

Zn-5Mg 27.638 (4.833)b −1.312 (0.012)b 0.427 (0.077)b

aStandard deviations are presented in the parentheses. bp < 0.05. Ecorr,corrosion potential; Icorr, corrosion current density.

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layer rich in Zn, O, and P and an inner layer consisting of Znand O. Mg was hardly found in the corrosion products. Theelemental composition of corrosion products was furtherinvestigated quantitatively (Figure 4b). Zn and O were theprimary elements in corrosion products, followed by P and Ca.In contrast to pure Zn, an increase in O, Ca, and Mg with asimultaneous decrease in Zn was detected in corrosionproducts of Zn-Mg composites. XRD identified ZnO andCaCO3 as the major corrosion products for all experimentalsamples (Figure 4c).Cytotoxicity Tests. To quantitatively measure the survival

of MC3T3-E1 cells after culturing in the extracts ofexperimental samples, we examined the cell viability over 4days (Figure 5a). Zn-Mg composite extracts demonstrated acytocompatibility superior to that of pure Zn. Pure Zn showeda significant toxicity to MC3T3-E1 cells on the first day of

culture, and the cell viability decreased over time. In contrast,Zn-Mg composites improved the viability of MC3T3-E1 cellsto higher than 85%, further increasing over time, indicating agood cytocompatibility. The ion concentrations of Zn-Mgcomposite extracts revealed a decrease in Zn2+ ion concen-tration but an increase in Mg2+ ion concentration as comparedto the pure Zn group.To further investigate the effects of Zn2+ and Mg2+ ions on

cell viability and morphology, MC3T3-E1 cells were treatedwith a series of solutions with different Zn2+ and Mg2+ ionconcentrations (Figure 5b−d). Zn2+ ion at a concentration of23.9 μg/mL exhibited pronounced toxicity on MC3T3-E1cells, while 1-fold diluted Zn2+ ion at a concentration of 12.1μg/mL promoted cell proliferation significantly. Zn2+ ionconcentrations ranging from 12.1 to 1.76 μg/mL showed nocytotoxicity on cells. In contrast to the Zn group, the samples

Figure 3. In vitro corrosion behavior of samples after immersion. (a) Optical images of experimental samples after immersion in Hanks’s solution.(b) Surface morphology of uniform corrosion regions of samples. (c) Corrosion rates and (d) pH values. (e) Zn2+ and (f) Mg2+ ion concentrationsin the medium; *p < 0.05.

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with a concentration gradient of Mg2+ ions added up to 27.9μg/mL was unable to reverse the toxicity induced by the highconcentration of Zn2+ ions. Moreover, addition of Mg2+ ionsshowed no effect on cell proliferation at the Zn2+ ionconcentration of 11.5 μg/mL. Cytoskeleton reorganizationand cell morphology were examined with typical Zn2+ andMg2+ ion concentrations. Compared to control, cells treatedwith a Zn2+ ion concentration of 23.9 μg/mL showed anunhealthy round and dead morphology, while cells treated witha Zn2+ ion concentration of 12.1 μg/mL tended to displaypolygonal or spindle shape and were well spread andproliferated. Addition of Mg2+ ions had little effect on thecell morphology compared to the Zn and 1/2 Zn group.Hemocompatibility. Figure S3a in the Supporting

Information displays the morphologies of adhered plateletson Zn-Mg composites and pure Zn. Platelets on the Zn-Mgcomposites showed a more activated state than those on pureZn. The platelets adhered on pure Zn were round andinactivated. In contrast, small amounts of pseudopodia couldbe seen stretched from platelets in Zn-Mg composites,indicating the beginning of an early dendritic state. A slightlyincreased number of platelets in Zn-Mg composites wasobserved as compared to the number in pure Zn. Moreimportantly, the platelets were distributed homogeneously onthe sample surface. A low risk of severe hemolysis was expectedfor the studied materials on the basis of the hemolysis rates,which were much lower than the safe value of 5% (FigureS3b).Coagulation times were measured to investigate the effects

of Zn-Mg composites and pure Zn on the speed of coagulation(Figure S3c). In contrast to control, a significant prolongationin APTT and PT and a reduction in TT were found in the Zn-Mg composite and pure Zn groups (p < 0.05). Adding Mg hadlittle impact on altering the speed of clotting by means ofAPTT, PT, and TT. The concentration of Zn2+ ions released inplatelet-poor plasma is shown in Figure S3d. In comparison tocontrol, incubated experimental samples demonstrated asignificant increase in Zn2+ ion concentrations. Meanwhile, adecline in Zn2+ ion concentration with Mg contents was found.

Antibacterial Property. The morphology of adhered S.aureus on experimental specimens is displayed in Figure S4a.Large amounts of bacteria cells on the Ti6Al4V surface wereintact and typically grape-shaped, indicating biofilm formation.For Zn-based composites, much fewer bacteria cells withdamaged cell surface were visible, suggesting an inhibition onbacteria adhesion and biofilm formation. Figure S4b,c showsthe antibacterial rates against planktonic and adherent bacteria,respectively. The Rp values of composite groups wereapproximately 95%, with no statistical significance. The Ravalues of composite groups ranged from 80% to 90%, in whichpure Zn exhibited a higher Ra value than that of Zn-Mgcomposites. All these results indicated that Zn-basedcomposites can inhibit the bacteria colonization effectively.The pH value and released Zn2+ ions in culture medium weremeasured, as shown in Figure S4d. In contrast to Ti6Al4V,incubation of Zn-based composites in culture mediumincreased the Zn2+ ion concentrations significantly (p <0.05). Meanwhile, the pH values of the culture mediumshowed no statistical significance in all groups.

Degradation Analysis of the Implants. To investigatethe osteogenesis, osseointegration, and material degradation,the Zn-5Mg composite was selected for in vivo tests with pureZn as control. The X-ray radiographs at selected time pointsare presented in Figure 6a. The contours of the implants areclearly visible, indicating their excellent radiopacity. Nodislocation was observed with all the implants. No formationof gas cavities was visible around the pure Zn and Zn-Mgcomposite rod samples. Figure 6b depicts cross sections offemurs with the implants at weeks 4 and 8. At week 4, newbone formation was observed for both groups. In addition, thebone mass around the implants increased over time. The Zn-5Mg composite showed a better bone integration ability thanthat of pure Zn. Tight and direct bone bonding to the implantscould be seen for the Zn-5Mg composite at week 4. Incontrast, pure Zn was surrounded by an intervening layer.Moreover, the contact areas of new bone and Zn-5Mgcomposite increased at week 8, indicating an improvedosseointegration over time. The 3D reconstruction images of

Figure 4. Corrosion products’ compositional analysis. (a) Elemental mappings of the corrosion interface of selected materials. (b) Quantitativeanalysis and (c) X-ray diffraction patterns of the corrosion products.

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implants revealed a uniform and slow degradation progress atthe macroscopic scale (Figure 6c). Both implants maintainedmechanical integrity after 8 weeks’ implantation. According tothe calculated implant volume, the Zn-5Mg composite groupdegraded slightly faster than the pure Zn group withoutsignificance (Figure 6d). The volumes of the pure Zn and theZn-5Mg composite implants remaining dropped to 98.30 ±1.02% and 97.61 ± 1.11%, respectively, after 8 weeks.Degradation Products Analysis. To understand the

compositional changes of corrosion products during thematerial degradation, a cross section of the bone−implantinterface was analyzed by BSE images and EDS analysis, asshown in Figure S5 and Figure 7. In general, the degradation ofboth implants proceeded in a localized mode at the microscalelevel, and the implants maintained their mechanical integrity atweek 8 post-operation. However, the elemental distribution atthe bone−implant interface varied greatly between the samplesfrom the pure Zn and Zn-Mg composite groups. For the pureZn group, the corrosion attack began at the grain boundariesand penetrated farther inside. In contrast to the compact

corrosion layer formed in vitro, degradation products generatedin vivo were distributed in a dispersive way. Elemental mappingindicated Zn and O as the major elements of corrosionproducts. Direct contact between newly formed bone andimplant was hardly found in the pure Zn group. As for the Zn-5Mg composite group, corrosion attack happened preferen-tially at the phase boundaries between the Mg-rich phase andthe Zn matrix phase (Figure 7a). Large amounts of degradationproducts diffused away from the implant surface to thesurrounding tissue. According to the elemental mapping, fourdistinct regions were identified at the bone−implant interface,based on chemical composition. New bone was rich in Ca andP in addition to fibrous tissue, which mainly contained C andO. The degradation products mainly consisted of Zn and O,while Zn and Mg were the major elements in the compositematrix. Moreover, the lack of Mg in the degradation productssuggested that the Mg-containing products dissolved duringdegradation. In contrast to the pure Zn group, tight and directbone bonding to degradation products was observed in the Zn-5Mg composite group. To further investigate the composi-

Figure 5. Cytotoxicity analysis. Cell viability after culturing with (a) sample extracts, (b) different Zn2+ ion concentrations, (c) Zn2+ ions (thehighest concentration) with different Mg2+ ion concentrations, and (d) Zn2+ ions (the 1-fold diluted concentration) with different Mg2+ ionconcentrations for 1, 2, and 4 days and corresponding Zn2+ and Mg2+ ion concentrations in culture media; *p < 0.05, compared with pure Zngroup. (e) Cell morphologies after culturing with different Zn2+ and Mg2+ ion concentrations for 12 h. Control group was treated with cell culturemedium.

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Figure 6. Micro-CT analysis. (a) Radiographs and (b) in vivo 2D images of implants. (c) In vivo 3D images and (d) remaining volume of implants.

Figure 7. Cross-sectional analysis of Zn-5Mg composite implant after 8 weeks’ implantation. (a) Backscattered electron images of a cross section ofthe Zn-5Mg composite implant with magnification of the area marked with the red rectangle. Elemental mappings correspond to the magnifiedarea. (b) Magnification of the blue rectangle in the bottom part of (a) and corresponding line analysis. (c) Quantitative composition analysis ofregions 1−8 in magnification of (a). NB, new bone; DP, degradation products; FT, fibrous tissue.

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tional change in the bonding area between bone anddegradation products, an EDS line scan was performed (Figure7b). The peaks of Ca, P, and O were detected and remainedconstant in region 6. However, the relative peak intensities ofCa and P decreased, while the Zn peak increased sharply fromregion 6 to region 4, and no Ca and P peaks were observed inregion 4. O peaks gradually increased from region 6 to region4. In contrast, peak intensities of Zn decreased, while those ofCa and P increased drastically from region 4 to region 5.Additionally, Zn, O, Ca, and P peaks were all detected inregion 5. According to the EDS analysis (Figure 7c), region 4was composed of Zn and O, which should be the typicaldegradation product ZnO. Region 6 was the new bone tissuewith a typical Ca/P atom ratio of 1.61 (hydroxyapatite, Ca/P =1.6). In contrast to region 4, the calcification matrix appearsdeposited in region 5 as a result of increased Ca and Pcontents.Histological Analysis. Figure 8 manifests tissue response

adjacent to the implants after 4 and 8 weeks using van Giesonstaining. At week 4, a layer of woven bone tissue with plenty ofosteocytes formed surrounding both implants. Increased bonemass with a maturation process could be observed extendingoutward from the implants over time. In addition, the thickness

of trabeculae in the vicinity increased over time. As forosseointegration, the Zn-5Mg composite showed an over-whelmingly better performance than pure Zn throughout theimplantation period. Direct bone bonding to pure Zn wasfound only at limited sites, while a layer of fibrous connectivetissue was observed surrounding the implant. In contrast, newbone was in direct contact with the Zn-5Mg composite inmultiple locations at week 4. Moreover, an increasing amountof directly contacted areas suggested improved boneintegration over time. H&E, Masson’s trichrome, and TRAPstaining analyses are shown in Figure 9. For the pure Zn group,a fibrous connective tissue with increasing thickness over timewas seen. Meanwhile, an inflammatory response, evident byinfiltration of lymphocytes and macrophage, was still visible atweek 8. In contrast, H&E demonstrated a mild inflammatoryresponse adjacent to the implant, and this inflammationsubsided from week 4 to week 8 in the Zn-5Mg compositegroup. Masson’s trichrome staining showed the formation ofcollagen and neoangiogenesis in the tissue surrounding of bothimplants. TRAP staining revealed limited osteoclast activity inboth groups.

Figure 8. Van Gieson staining of (a) pure Zn and (b) Zn-5Mg composite implants at selected time points. The red rectangles are highlighted in themagnifications.

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■ DISCUSSIONDegradation Mechanism with Bone Healing Process.

The degradation of implants is closely related to thesurrounding physiological environment. The bone healingprocess is complex and highly regulated, with consecutive andclosely linked stages.14,15 According to the histological analysis(Figures 8 and 9), three stages were typically seen during thedegradation of Zn-based implants. The degradation mecha-nism that occurs during the bone healing process is presentedin Figure 10a.Stage Ι: Hematoma, Inflammatory Cells Infiltration,

Hypoxia, and Low pH (5.2−6.9).16−18 According to thePourbaix diagram for physiological condition,19 the degrada-tion of Zn at this stage should be characterized by hydrogenevolution reaction (eq 1), which is balanced by dissolution ofZn (eq 2). In contrast to pure Zn, a galvanic cell was formedbetween the Zn matrix and Mg-rich phases due to the potentialdifference in the Zn-5Mg composite. Consequently, Mg-richphases were corroded preferentially (eqs 3 and 4), anddegradation of the composite implant was accelerated bygalvanic corrosion. Thermodynamically, Zn2+ and Mg2+ ionswere predicted to be the dominant products at this stage.20

2H O 2e H 2OH2 2+ → +− −(1)

Zn Zn 2e2→ ++ − (2)

2Mg 2Mg 2e→ ++ −(3)

2Mg 2H O 2Mg 2OH H22

2+ → + ++ + −(4)

Stage II: Soft Callus, Fibrous Tissue Encapsulation, LowOxygen Tension, and Neutral pH (∼7.4).17,18 The increases inpH and oxygen concentration could promote the oxygenreduction reaction (eq 5) during degradation of Zn. Thedissolved oxygen showed an impact on corrosion of Znthrough the oxygen reduction reaction in the pH range from 4to 12.20 As degradation progressed, the pH increased byformation of OH−, which promoted the generation ofZn(OH)2, followed by a gradually change to ZnO (eq 6).Mg(OH)2 (eq 7) should be another product in addition toZnO during the degradation of the Zn-5Mg composite.However, the generated ZnO and Mg(OH)2 were unstableunder neutral physiological conditions. The high concentrationof Cl− (150 mmol L−1) promoted the dissolution of theseproducts and converted them to soluble chlorides, whichprevented the formation of an effective protection layer.20−23 Itshould be noted that ZnO was detected as the predominantdegradation product in both implants under in vivo conditions

Figure 9. H&E, Masson’s trichrome, and TRAP staining of (a) pure Zn and (b) Zn-5Mg composite implants at selected time points. The yellowcircles indicate neoangiogenesis.

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(Figure 7, S5). In contrast to the observation with the pure Znsample, only a trace amount of Mg was found in thedegradation products of Zn-Mg composites after theirimmersion (Figure 4), while the signal of Mg could hardlybe detected under a physiological environment. From athermodynamic point of view, the equilibrium constants(Ksp) for Mg(OH)2 and ZnO are 8.9 × 10−12 and 2.5 ×10−17, respectively.24,25 In light of this, ZnO is much morestable than Mg(OH)2 under physiological conditions. There-fore, it can be assumed that an amount of Mg2+ ions wasreleased into the surrounding tissue during degradation, whichwas confirmed by immersion tests (Figure 3). The fibroustissue that surrounds the implants can be treated as porousmedia, as it is composed of dispersed cells separated byconnective voids.26 Mass transfer across the fibrous tissueoccurs mainly via diffusion caused by concentrationgradients.27 No visible gas pocket was observed underradiography and micro-CT, which indicated that the evolutionrate of H2 was lower than the exchange rate of H2. The

concentration gradients between the implant surface, theadjacent tissue, and the atmosphere could be the main drivingforce behind the exchange. The evolving H2 diffuses into thesurrounding tissue. Diffusion through capillaries and transportvia the vascular system might constitute the essential exchangemechanism.28,29 For pure Zn, the significantly lower corrosionrates compared to Mg circumvented the rapid hydrogenrelease.11 The formation of intermetallic phases (MgZn2 andMg2Zn11) in Zn-Mg composites reduced the electricalpotential difference between Zn and Mg, therefore reducingthe galvanic corrosion driving force and preventing theformation of gas cavities as well.

O 2H O 4e 4OH2 2+ + →− −(5)

Zn 2OH Zn(OH) ZnO H O22 2+ → → ++ −

(6)

Mg 2OH Mg(OH)22+ →+ −

(7)

Stage III: Hard Callus (Woven Bone), Neoangiogenesis,Sufficient Oxygen Supply, and Increased Local pH.14,15,30

Figure 10. Schematic diagrams. (a) Degradation mechanism of Zn-based implants with bone healing process including hematoma, soft callus, andhard callus. (b) Galvanic corrosion between the Zn matrix and the sacrificial Mg-rich anode.

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Van Gieson staining revealed increasing amounts of newlyformed bone surrounding both implants at week 8. In contrastto pure Zn, the better osseointegration resulted in an increasedbone bonding with the implant over time in the Zn-5Mgcomposite (Figure 8). The mass-transfer process was predictedto be further impeded, especially in the Zn-5Mg compositegroup, which resulted in a reduction in corrosion rates overtime. Micro-CT revealed decreased corrosion rates withprolonged implantation time (Figure 6d). Similar resultswere reported for the long-term degradation of Mg-basedimplants as well.31,32 At this stage, the oxygen reductionreaction should become more predominant in the degradationof Zn. The degradation of implants and limited diffusioncondition contributed to a gradually rise in local pH value,which was beneficial to the stable existence of ZnO. Zn-Mgcomposites showed higher pH values compared with pure Zn(Figure 3d). Meanwhile, precipitation of calcium phosphatecompounds can be triggered by the increased pH values. A lowZn content region characterized by chemical compositionsimilar to that of bone was observed adjacent to the newlyformed bone (area 5 in Figure 7b) in the Zn-5Mg composite.According to the clinical study of Mg alloy implants, the lowZn content region could be crystalline calcium phosphates,which would ultimately get reabsorbed by the osteoclasts toinduce bone formation by the osteoblasts.24 The specificchemical composition of this region will be investigated in thefurther study.Sacrificial Mg-Rich Anode Design. Zinc is a widely used

sacrificial anode material in the marine environment for thecathodic protection of iron and steel.33,34 Inspired by this, wedesigned a cathodic protection system (Figure 10b) bychoosing Mg, a biodegradable and more “active” metal, as asacrificial anode to “protect” Zn during degradation, therebycircumventing the toxicity induced by excessive release of Zn2+

ions while accelerating the degradation. The microstructure ofZn-Mg composites was composed of the Zn matrix anduniformly distributed Mg-rich phases with an inner core ofMgZn2 and an outer shell layer of Mg2Zn11(Figure 1). Thesurface potentials of Zn-Mg composites decreased with Mgcontents. Moreover, a potential gradient was expected to formacross the Zn matrix and Mg-rich phases. The SKP revealed apronounced increase in the surface potential difference from81.8 to 513.8 mV after addition of Mg. As a consequence, theMg-rich phases, especially MgZn2, act as the anode, while theZn matrix, which has a more positive potential, acts as thecathode. Microgalvanic coupling formed between the Znmatrix and Mg-rich phases during degradation. The SEMimage with elemental mapping showed that the Mg-rich anodecorroded preferentially, leaving the Zn matrix unaltered(Figure 2c). Moreover, the discontinuous and isolateddistribution of the Mg-rich anode will not lead todisintegration of the composites during degradation. As aresult, the addition of Mg shifted the corrosion potentialnegatively and increased the corrosion current densitysignificantly on the macro-scale (Table 2). Uniformlydispersed corrosion pits covered with white products wereobserved in the corrosion morphology of Zn-Mg compositesafter immersion (Figure 3a). Moreover, the Zn2+ ionconcentrations decreased while Mg2+ ion concentrationsincreased significantly in all the media used in this study.Most importantly, accelerated degradation and enhancedosseointegration were observed in the Zn-Mg composite

group, which proved the effectiveness of the sacrificial Mg-rich anode design in Zn-based biomaterials.

Enhanced Osseointegration for Zn-Mg Composites inComparison to Pure Zn. Osseointegration is essential forsuccessful implantation and internal fixation stability. Theextent and success of osseointegration depend on thebiocompatibility of the implant material.35 In this research,the implantation of pure Zn and Zn-5Mg composite materialscaused no severe inflammation, bone resorption, and necrosis(Figure 9). More importantly, new bone formation wasobserved surrounding both types of implants (Figure 8),which indicated the biosafety of Zn-based implants. However,delayed osseointegration, characterized by the lack of directcontact between bone and implant, was found in the pure Zngroup. In consideration of the negligible hydrogen release andmoderate pH increase of Zn during degradation,8,36−38 delayedosseointegration should be a great possibility, induced byexcessive release of Zn2+ ions. The in vitro cytotoxicity resultsverified this speculation (Figure 5). A relatively highconcentration of Zn2+ ions (23.9 μg/mL) jeopardized thecell viability and morphology of MC3T3-E1 cells significantly,while the cells exhibited increased proliferation rates andhealthy morphology when exposed to a lower Zn2+ ionconcentration (12.1 μg/mL). Therefore, the toxicity of Zn2+

ions is dose-dependent. An appropriate content of zinc iscritically important for the biological functions of cells.Previous studies have confirmed the cytotoxicity of Zn2+ ionat high concentrations. A Zn2+ ion concentration of 400 μM(26.2 μg/mL) or more resulted in cell death of mouseosteoblasts exposed to extraction medium.39 Proliferation ofhuman osteoblasts was significantly decreased on the surface of10% Zn-doped β-tricalcium phosphate (ZnTCP), while it wasincreased on 5% ZnTCP.40 In addition, human osteoblastsexposed to pure Zn exhibited a shrunken morphology andsignificant DNA damage after 7 days’ culture.41 Animal testsmanifested that ZnTCP with 0.316 wt% Zn induced newlyformed bone, covering the endosteal surface completely, whilea Zn content of 0.633 wt% evoked bone resorption.42 Althoughthe underlying mechanism for delayed osseointegration of pureZn remains obscure, we believe that controlling Zn2+ ionrelease during degradation is of critical importance to improvethe bone integration ability of Zn-based materials.Through incorporation of Mg into the Zn matrix, the

corrosion of Zn can be inhibited by preferential dissolution of asacrificial Mg-rich anode. Consequently, the excessive releaseof Zn2+ ions was replaced by co-release of Zn2+ and Mg2+ ions(Figures 3e,f and 5a). As a result, MC3T3-E1 cells exhibited asignificantly improved cell viability in Zn-Mg compositegroups. More importantly, direct bone bonding to the Zn-5Mg composite was observed at week 4, and improved boneintegration was found over time (Figure 8b). The superiorbone integration ability of the Zn-5Mg composite can bemainly attributed to the inhibited release of Zn2+ ions becauseof preferentially corrosion of Mg-rich phases during degrada-tion, although in some specific periods the Mg-rich phaseswere consumed and the corrosion of Zn was dominant. Thesurrounding new bone already formed a quite stable andmoderate corrosive environment. The relatively slow degrada-tion of implants at this stage would not have toxic effects onsurrounding tissues. Relatively low concentrations of Zn2+ ionspromoted mineralization of the extracellular matrix andosteogenic differentiation of hBMSCs. Meanwhile, enhancedexpression of bone-related genes, including ALP, collagen Ι,

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and osteopontin, was observed as well.43,44 Zn-implantedcoatings with reduced Zn ion release showed early-stage newbone formation and promoted osseointegration in the ratmodel.45 Besides, numerous studies have verified the beneficialrole of Mg2+ ions in osseointegration and osteogenesis. Mg2+

ions were able to improve bone cell adhesion and stability byincreasing the expression levels of α5β1 integrin.46,47 Mean-while, Mg2+ ions were found to upregulate the mRNAexpression levels of ALP and collagen Ι, which could promotethe osteogenic ability of BMSCs significantly.48 However, Mg2+

ions should play a subordinate role in the enhancedosseointegration of Zn-Mg composites as compared to Zn2+

ions due to the low content of Mg and its limited release. It isworth mentioning that the elevated pH value in Zn-Mgcomposite groups may contribute to the improved osseo-integration as well. Both the proliferation and ALP activity ofosteoblasts were significantly improved at high pH values (pH> 8).49 Previous studies have reported increased cell viability ofosteoblasts with less DNA damage and promotion effects innew bone formation on Zn-Mg alloys.5,41 Our results wereconsistent with the previous studies, which demonstrated anadvantage of Zn-Mg composites in osseointegration over pureZn during the bone healing process.

■ CONCLUSIONIn this study, Zn-Mg composites with Mg as sacrificial anodewere designed, and their feasibility as orthopedic implants wasevaluated with regard to degradation behaviors and bio-compatibility. The incorporation of Mg formed galvanic cellsbetween Mg-rich phases and the Zn matrix phase, therebyaccelerating the degradation rates of Zn-Mg composites. In thebone healing process, ZnO was produced predominantly,followed by calcification matrix formation during thedegradation of the Zn-Mg composites. The inhibition in therelease of Zn2+ ions resulting from preferential corrosion ofMg-rich phases contributed to the significantly improvedcytocompatibility. More importantly, our in vivo studymanifested good osteogenesis ability and superior osseo-integration of the Zn-5Mg composite as compared to pure Zn.In conclusion, Zn-Mg composites exhibit a great potential fordeployment in the application of bioabsorbable bone implants.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acsbiomater-ials.8b01137.

Figure S1, metallography of pure Zn and Zn-Mgcomposites; Figure S2, mechanical properties of Zn-Mg composites and pure Zn; Figure S3, hemocompat-ibility of Zn-Mg composites and pure Zn; Figure S4,antibacterial properties of Zn-Mg composites and pureZn; Figure S5, cross sectional analysis of pure Znimplant after 8 weeks’ implantation (PDF)

■ AUTHOR INFORMATIONCorresponding Authors*E-mail: yfzheng@pku.edu.cn.*E-mail: krdai@163.com.ORCIDDonghui Zhu: 0000-0002-3057-1889Yufeng Zheng: 0000-0002-7402-9979

FundingThis work was supported by the National Natural ScienceFoundation of China (Grant Nos. 51431002 and 51871004),NSFC/RGC Joint Research Scheme (Grant No. 5161101031),and National Key Research and Development Program ofChina (Grant No. 2016YFC1102402).

NotesThe authors declare no competing financial interest.

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ACS Biomaterials Science & Engineering Article

DOI: 10.1021/acsbiomaterials.8b01137ACS Biomater. Sci. Eng. 2019, 5, 453−467

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