简介概要

In vitro biodegradability of Mg-2Gd-xZn alloys with different Zn contents and solution treatments

来源期刊：Rare Metals2019年第7期

论文作者：Meng Zhang Wei-Lin Deng Xiao-Ning Yang Yong-Kang Wang Xiang-Yu Zhang Rui-Qiang Hang Kun-Kun Deng Xiao-Bo Huang

文章页码：620 - 628

摘要：This study aims to investigate the addition of Zn on the corrosion property and cytocompatibility of Mg-2Gd-xZn（x= 0,3,4 and 5;wt%）alloys,which were prepared by gravity permanent mold casting and solution treatment,respectively.The results show that the intermetallic phases of these ternary alloys are mainly composed of Mg₁₂GdZn and Mg₃GdZn₃.The content of secondary phases as well as the grain size is greatly dependent on the Zn addition.Compared to the binary Mg-2 Gd alloy,the corrosion resistance of the most ternary alloys is significantly improved.Furthermore,the in vitro cell culture study demonstrates the potential cytocompatibility of the developed ternary alloys.It indicates that a series of Mg-2Gd-xZn（x= 0,3,4 and 5;wt%）with medically acceptable corrosion rate are developed and show great potential use as a new type of biodegradable implants.

稀有金属(英文版) 2019,38(07),620-628

In vitro biodegradability of Mg-2Gd-xZn alloys with different Zn contents and solution treatments

Meng Zhang Wei-Lin Deng Xiao-Ning Yang Yong-Kang Wang Xiang-Yu Zhang Rui-Qiang Hang Kun-Kun Deng Xiao-Bo Huang

College of Materials Science and Engineering, Taiyuan University of Technology

Shanxi Key Laboratory of Advanced Magnesium-Based Materials, Taiyuan University of Technology

作者简介：*Xiao-Bo Huang,e-mail: huangtyut@163.com;

收稿日期：16 November 2018

基金：financially supported by the National Natural Science Foundation of China (Nos. 31300808 and 31400815);the Scientific and Technological Innovation Programs of Higher Education Institutions in Shanxi (No. 201417);the Natural Science Foundation of Shanxi Province (No. 2013021011-1);

In vitro biodegradability of Mg-2Gd-xZn alloys with different Zn contents and solution treatments

Meng Zhang Wei-Lin Deng Xiao-Ning Yang Yong-Kang Wang Xiang-Yu Zhang Rui-Qiang Hang Kun-Kun Deng Xiao-Bo Huang

College of Materials Science and Engineering, Taiyuan University of Technology

Shanxi Key Laboratory of Advanced Magnesium-Based Materials, Taiyuan University of Technology

Abstract：

This study aims to investigate the addition of Zn on the corrosion property and cytocompatibility of Mg-2Gd-xZn(x= 0,3,4 and 5;wt%)alloys,which were prepared by gravity permanent mold casting and solution treatment,respectively.The results show that the intermetallic phases of these ternary alloys are mainly composed of Mg₁₂GdZn and Mg₃GdZn₃.The content of secondary phases as well as the grain size is greatly dependent on the Zn addition.Compared to the binary Mg-2 Gd alloy,the corrosion resistance of the most ternary alloys is significantly improved.Furthermore,the in vitro cell culture study demonstrates the potential cytocompatibility of the developed ternary alloys.It indicates that a series of Mg-2Gd-xZn(x= 0,3,4 and 5;wt%)with medically acceptable corrosion rate are developed and show great potential use as a new type of biodegradable implants.

Keyword：

Mg-2Gd-xZn; Corrosion resistance; Biodegradability; Cytocompatibility;

Received： 16 November 2018

1 Introduction

Magnesium (Mg) alloys show great potential as bone implants,compared to the traditional metallic implant materials,such as stainless steels,titanium alloys and cobalt chromium alloys.This is due to their excellent combination of Young,s modulus and low density that is similar to natural bones [ 1, 2] .More importantly,their superior biodegradability ensures their absorption after the healing of wound tissues,avoiding a secondary surgical procedure to remove the implant [ 3, 4, 5] .However,Mg and its alloys are likely to be corroded in the human body fluids,due to their high negative standard electrode potential [ 6] .The degradation products,including soluble magnesium hydroxide (Mg(OH)2),magnesium chloride(MgCl₂) and hydrogen gas,cause severe ion imbalance and pH instability in organism [ 7, 8, 9] .

Microalloying is one of the most effective methods to control the dlegradation of Mg-based biodegradable alloys [ 6] .Up to now,various Mg-based alloys with increased corrosion resistance have been developed [ 3] .These biodegradable Mg-based alloys include alloying with essential elements or low toxic elements and modifying industrial Mg alloys with rare earth elements (REEs) [ 10, 11] .The addition of REEs to Mg alloys has recently drawn researchers,attention in the field of biomaterials due to their strengthening efficacy [ 10, 12] .Generally,alloying with REEs is used to improve the high-temperature strength and creep resistance of the Mg-based alloy.Iin particular,several Mg-based biodegradable alloys containing gadolinium (Ged) have been investigated,showing a good combination of mechanical and corrosion resistance [ 13, 14, 15, 16] .Owing to the high solubility of Gd in Mg (up to23.49 wt%) [ 17] ,the mechanical properties of Mg-based alloys can be adjusted by varying Gd conntents [ 18, 19, 20, 21] .The binary Mg-Gd alloys show improved creep resistance than the commercially used Mg alloys [ 22, 23, 24] .The corrosion behavior of binary Mg-Gd alloys in 1 wt%NaCl solution at 2 wt%-15 wt%0 Gd has been investigated.The results showed that the degradation rate of the alloys increased as Gd increasedl up to 10 wt%and then decreased with further Gd (10 wt%-15 wt%) [ 3, 10, 25] .

Recently,some ternary alloys based on Mg-Gd-X systems have also been proposed,as the ternary intermetallic compounds might have potential influence on the properties of these alloys [ 26] .Among these systems,the MgGd-Zn is a new and promising one as its microstructure consists of different phases depending on the content ratio of Zn to Gd,which results in different performances [ 11, 26] .Zinc (Zn) is an abundant essential element in the human body vital for bone formation [ 5, 27, 28] .Moreover,Zn has a relatively high solubility in Mg (6.2 wt%) and the mechanical properties of the Mg-Zn alloys can be improved by increasing the content of zinc up to 4 wt% [ 6, 11] .Zhang et al. [ 6] found that a moderate addition of zinc could improve the corrosion potential and resistance of Mg alloys in simulated body fluid (SB^F).Srinivasan et al. [ 26] have evaluated the microstructure,mechanical and corrosion properties of Mg-Gd-Zn alloys in the as-cast condition.The results showed that the physicochemical properties of this alloying system are related not only to the doped elements but also to the treatment conditions.Since this alloying system shows excellent performance as a type of biodegradable alloy,a thorough knowledge of the preparation-structure-property correlation of these alloys is essential.

In this study,we attempt to develop an Mg-2Gd-xZn(x=0,3,4 and 5;wt%) alloy system by gravity permanent mold casting and subsequent solution treatment.The effects of heat treatment craft and addition of Zn with gradient content on the microstructure,degradation behavior and cytocompatibility of these alloys were investigated.This new type of biodegradable implant with both medically acceptable corrosion rate andl good cytocompatibility may have promising applications in clinic.

2 Experimental

2.1 Preparation of Mg-2Cd-xZn alloys

The Mg-2Gd-xZn (x=0,3,4 and 5;wt%) alloys were fabricated by gravity permanent mold casting.Firstly,the high-purity magnesium was put into the mild steel crucible with a 60-mm internal diameter when its temperature reached to 600℃,and then,a RJ-6 anti-oxidizing flux including 54 wt%-56 wt%KCI,27 wt%-29wt%CaCl₂,14 wt%-16 wt%BaCl₂ and 1.5 wt%-2.5 wt%NaCl was added.When the 1magnesium completely melted,the intermediate alloy Mg-Gd and pure were added.After that,the semi-melted alloys were heated to 760℃and the temperature was maintainedl for 15 min.When the alloy was completed melted,it was stirred for20 min at 300 r·min^-1.The temperature of the alloy was then reduced to 730℃.Boron nitride (BN) was used as a mold release agent.The as-cast (AC) samples were homogenized at 420℃for 24 h and then water quenched to form the as-solution-treated (AS) alloys.The as-cast Mg-2Gd-xZn alloys were denoted as AC1 (x=0),AC2(x=3),AC3 (x=4),AC4 (x=5),respectively.Similarly,the as-solution-treated samples were marked as AS1 (x=0),AS2 (x=3),AS3 (x=4),AS4 (x=5).The chemical compositions of the AC and the corresponding AS alloys analyzed by integrated coupled plasma optical emission spectroscopy (ICP-OES) are given in Table 1.

2.2 Characterization of Mg-2Gd-xZn alloys

The specimens were ground,polished and etched in alcohol solution containing 4 vol%nitric acid.Then,the micro structure and the composition of the secondary phases of Mg-2Gd-xZn (x=0,3,4 and 5;wt%) alloys were observed by optical microscope (OM,4XC) and scanning electron microscope (SEM,MIRA3-XMU) attached with an energy-dispersive spectroscope (EDS),respectively.The average grain size was measured by line intercept with the Nano Measurer (Fudan University,China).The content of the secondary phase was ensured by multi-images analysis with image J 2×.In addition,X-ray diffractometer (XRD,DX-2700,Haoyuan) was used to analyze the phase compositions of the investigated samples,with Cu Ka radiation at voltage and current of 40 kV and30 mA,respectively.The diffraction patterns were analyzed with Jade-6.5 software with the aid of JCPDF-2004 database.

下载原图

Table 1 Chemical composition of Mg-2Gd-xZn alloys (wt%)

2.3 Immersion test

The immersion test was carried out in the simulated body fluid (SBF) at (37.0±0.5)℃according to ASTM (2004)NACE TM0169/G31-12a standard (the ratio of surface area to solution volume was 1 cm²:30 ml) to evaluate the corrosion resistance of the investigated alloys.After immersion for 9 days,the specimens were dipped in the solution of 200.g L^-1 CrO₃ and 10 g-L^-1 AgNO₃ at 60℃for 10 min to remove the corrosion products.The corrosion rate (CR) was calculated according to the formula as follows:

where K is the constant (8.76×10⁴),t is the impregnation time in hours,A is the area in cm²,W is the mass loss in g and D is the density in g·cm^-3.In addition,the threedimensional surface morphologies after the immersion test were observed by a confocal laser scanning microscope(CLSM,C2 Plus,Nikon).

2.4 Electrochemical tests

The potentiodynamic polarization was usually carried out to evaluate the corrosion behavior of alloys.All the specimens were polished with silicon carbide (SiC) abrasive paper up to3000 grit and then washed with acetone and ultrapure water.CHI760E electrochemical workstation with a conventional three-electrode cell was used for the electrochemical measurements at (37.0±0.5)℃.Before test,a stable opencircuit potential (OCP) was achieved by dipping the sample in the electrochemical corrode solution (SBF) for 40 min.The scan range of the polarization was from-0.5 to 1.0 V at a scan rate of 1.0 mV·s^-1 relative to the open-circuit potential.The CorrView version 3.0 was used to educe the corrosion potentials (E_corr) and the corresponding corrosion current density (i_corr).Finally,the instantaneous corrosion rate (P_i,mm·year^-1) can be directly calculated according to the formula as follows [ 29] :

2.5 Cytocompatibility assessments

The live/dead cell staining assay was used to investigate the cellular viability by testing the integrity of cytomembranes.The mouse MC3T3-E1 preosteoblasts were cultured on the samples at a concentration of 1×10⁵cells·well^-1.The plates were incubated at 37℃in an atmosphere of 5%CO2 and 95%air in the CO₂ incubator.After incubation for 24 h,each specimen in the aperture was stained with 300μl calcein AM/EthD-1 according to the protocol (Live/Dead,Life Technologies).Then,the cellular state was evaluated by CLSM.

3 Results and discussion

3.1 Characterization of alloys

Figure 1 shows XRD patterns of the Mg-2Gd-xZn (x=0,3,4 and 5;wt%) alloys.The investigated binary alloys are mainly composed of a-Mg solid solution in both groups(AC and AS).In view of the limited available data for MgGd-Zn phases,the diffraction patterns of Mg-Y-Zn phases were used to identify the existence of Mg-Gd-Zn phases in this study [ 26] .With the addition of zinc,peaks corresponding to Mg₁₂GdZn and Mg3GdZn₃ are mainly observed in the AC group,which is consistent with the previous report [ 17] .Except for Gd phases,the same types of secondary phases are detected in the AS group.Compared to the as-cast alloys,the detectable peaks in AS alloys shifted to a lower degree,which is deduced to be due to the dissolution of Gd and Zn in Mg matrix [ 25] .

In order to decrease the content of secondary phases to the utmost extent,the as-cast alloys were solution treated at420℃for 24 h.Figure 2 shows the distribution of secondary phases in the Mg-2Gd-xZn (x=0,3,4 and 5;wt%)alloys.The secondary-phase particles are mainly continuously located at the grain boundaries resembling a network structure,and some small-sized spherical particles distribute along the inner regions of grains.In both groups,the content of secondary phases increases with the increase in Zn content (Fig.3).Compared to the AC alloys,the continuous precipitation of second phase at the grain boundary becomes intermittent after the solution treatment.Meanwhile,the secondary-phase particles in the AS alloys are obviously less than those in the AC alloys,which indicates that some secondary-phase particles may be dissolved into the Mg matrix after the solution treatment [ 14] .

Fig.1 XRD patterns of Mg-2Gd-xZn (x=0,3,4 and 5;wt%) alloys

Fig.2 SEM images of a-d AC alloys and e-h AS alloys:a,e Mg-2Gd;b,f Mg-2Gd-3Zn;c,g Mg-2Gd-4Zn;d,h Mg-2Gd-5Zn

Fig.3 Secondary-phase area fraction of investigated alloys

Figure 4 shows SEM image and EDS spectra of as-cast Mg-2Gd alloy.EDS analysis performed on the dark region(marked as Spots 1 and 2 in SEM image) confirms the presence of small amounts of Gd in the Mg matrix.SEM image and EDS spectra of as-cast Mg-2Gd-5Zn alloys are presented in Fig.5.The bright secondary phases marked as Spots 1 and 2 are observed in the microstructure,which have the stoichiometry composition equivalent to Mg₁₂GdZn and Mg₃GdZn₃,respectively.

The microstructure of Mg-2Gd-xZn (x=0,3,4 and 5;wt%) alloys is observed by OM (Fig.6),and the grain size of Mg-2Gd-xZn alloys (x=0,3,4 and 5;wt%) was measured by the Nano Measurer (Fig.7).With the increase in Zn content in the ternary alloys,the grain size is significantly decreased.It indicates that the growth of grain is hindered by a large amount of secondary phases during casting process [ 3, 30] .Compared to the AC specimens,the grain size in the AS alloys is slightly enlarged.

3.2 Corrosion properties

3.2.1 Immersion tests

Figure 8 shows the result of the weight loss of the Mg-2Gd-xZn (x=0,3,4 and 5;wt%) alloys in the immersion test,and the corrosion rate is calculated according to Eq.(1).With the increase in Zn content in the ternary alloys,the corrosion rate decreases in both groups.However,the corrosion rate suddenly increases when the Zn content in the alloy is 5 wt%.Compared to the AC alloys,the AS alloys show a slightly higher corrosion resistance.

Figure 9 shows the three-dimensional morphologies of these alloys after the immersion test.In the as-cast alloys,it is found that the increase of zinc content in Mg-2Gd-xZn(x=0,3,4 and 5;wt%) leads to the corrosion on the substrate more uniform.Compared with the as-cast alloys,the corresponding AS alloys exhibit slightly heterogeneous corrosion morphology after the immersion test,because the volume of secondary phase decreases and the net structure phases distributing along the grain boundary become discontinuous.Meanwhile,the development of the intercrystalline corrosion and pitting corrosion on the AS1,AS2 and AS4 alloys makes the corrosion pit link together.Moreover,it is found that the Mg-2Gd-4Zn alloy shows a smoother corroded surface than other alloys in both groups,which may be related to the comprehensive effect of the secondary-phase content and grain size.

Fig.4 a SEM image of as-cast Mg-2Gd alloy and corresponding EDS spectra of b Spot 1 and c Spot 2

Fig.5 a SEM image of as-cast Mg-2Gd-5Zn alloy and corresponding EDS spectra of b Spot 1 and c Spot 2

3.2.2 Polarization behavior

Figures 10 and 11 and Table 2 present the electrochemical test results of the alloys in SBF at (37.0±0.5)℃.As compared with the AC samples,the AS samples show lower cathodic currents and higher negative value of E_corr.Both the highest E_corr and the lowest cathodic current density are achieved at the AS3 sample,which suggests that the cathodic reaction resistance is improved by the solution treatment.The cathodic polarization curve is assumed to represent the corrosion behavior such that a lower polarization current indicates a lower corrosion rate [ 31] .Earlier reports [ 30, 32, 33] suggested that the reduction in the volume of secondary phases,which will act as microelectrodes during electrochemical corrosion processes,has been confirmed to contribute to the decreased corrosion rate.Meanwhile,the grain-refined Mg alloys exhibit better corrosion resistance due to increased grain boundary density,which can contribute to the formation and adhesion of the passive film along the grain boundary [ 34] .

Fig.6 OM images of a-d AC alloys and e-h AS alloys:a,e Mg-2Gd;b,f Mg-2Gd-3Zn;c,g Mg-2Gd-4Zn;d,h Mg-2Gd-5Zn

Fig.7 Grain size of investigated alloys

Fig.8 Corrosion rate of AC and AS alloys in SBF at (37.0±0.5)℃after 9 days

It has been generally accepted that the bio-corrosion rate of Mg-based alloy is a function of its grain size and the content of secondary phase [ 30, 32] .In this study,the corrosion behavior of Mg-2Gd-xZn (x=0,3,4 and 5;wt%) depends dramatically on these two factors,which are influenced by the solution treatment and the content of Zn.Although the grain size is decreased by adding Zn in the ascast samples,the secondary phase area fraction is increased,which clearly overshadowed the beneficial effect of grain refinement on the corrosion performance,while the solution treatment without addition of Zn reduces the area fraction of secondary phase but enlarges the grain size.Taking both factors into consideration,the best corrosion performance can be obtained with a balanced grain size and secondary-phase area fraction.

3.3 Cytocompatibility

After culturing cells for 24 h on the specimen,the cytocompatibility of different Mg-2Gd-xZn (x=0,3,4 and 5;wt%) alloys is evaluated by a live/dead staining protocol.The live cells are stained in green,while the dead cells are in red.As shown in Fig.12,most dead cells and few live cells appear in the AC 1,AC4,AS 1 and AS4 samples,which indicates the serious cytotoxicity.In contrast,there are abundant live cells attaching and spreading on the surface of the other samples.Especially in the AS3 sample,few dead cells emerging on the surface indicate that the cells can accommodate the corrosion rate of this alloy.Combining the cytotoxicity results and the corrosion studies mentioned above,it is found that the cells growth condition is coincident with the corrosion rate of devised alloys.Furthermore,the present study demonstrates that the cytocompatibility of Mg-2Gd-xZn (x=0,3,4 and 5;wt%) alloys in vitro can be achieved by the solution treatment.However,animal experiments need to be performed in the future study in order to test the biocompatibility of these alloys in vivo.

Fig.9 CLSM three-dimensional images after immersion test in SBF at (37.0±0.5)℃for 24 h of a-d AC alloys and e-h AS alloys:a,e Mg-2Gd;b,f Mg-2Gd-3Zn;c,g Mg-2Gd-4Zn;d,h Mg-2Gd-5Zn

Fig.10 Potentiodynamic polarization curves of AC alloys immersed in SBF at (37.0±0.5)℃

Fig.11 Potentiodynamic polarization curves of AS alloys immersed in SBF at (37.0±0.5)℃

下载原图

Table 2 Electrochemical data evaluated by Tafel extrapolation,linear polarization for AC and AS alloys immersed in SBF at (37.0±0.5)℃

β_c,cathodic slope;β_a,anodic slope

Fig.12 Live/dead assay of cells viability after 24 h cultured on investigated samples of a-d AC alloys and e-h AS alloys:a,e Mg-2Gd;b,f Mg-2Gd-3Zn;c,g Mg-2Gd-4Zn;d,h Mg-2Gd-5Zn

4 Conclusion

The effects of Zn content and solution treatment on the degradability and cytocompatibility of Mg-2Gd-xZn (x=0,3,4 and 5;wt%) alloys were investigated in this study.The intermetallic phases in these ternary alloys mainly consisted of Mg₁₂GdZn and Mg₃GdZn₃ phases.The degradation rate of these alloys depended primarily on the grain size and the content of secondary phase,which were fundamentally determined by the solution treatment and the addition of Zn.By solution treatment to balance the secondary-phase content and grain size,the Mg-2Gd-4Zn alloy was demonstrated to exhibit an acceptable degradability.Furthermore,the in vitro cell culture studies demonstrate the potential cytocompatibility of the developed ternary alloys as a new type of biodegradable implants.