Supplementary Information

Biodegradability Engineering of Bio-absorbable Mg alloys: Tailoring the

electrochemical property and microstructure of constituent phases

Pil-Ryung Cha, Hyung-Seop Han, Gui-Fu Yang, Yu-Chan Kim, Ki-Ha Hong, Seung-Cheol

Lee, Jae-Young Jung, Jae-Pyeong Ahn, Young-Yul Kim, Sung-Youn Cho, Seok-Jo Yang and

Hyun-Kwang Seok.

1. Microstructures of as-cast and extruded Mg-Ca-Zn alloys

Figure S1(a)-(e) shows the microstructures of as cast Mg-5wt%Ca-Xwt%Zn (X= 0, 0.5, 1,

1.5, 3) alloys. As-cast Mg-5wt%Ca is composed of the primary α-Mg and eutectic Mg/Mg2Ca

phases. High contrast represents the Mg2Ca phase in the eutectic phase which constructs a

well-connected network in the whole specimen. The microstructure was changed significantly

by the addition of Zn as shown from Fig. S1 (b)-(e). The network lamella structure almost

disappeared and the Mg2Ca phase was condensed only by the addition of 1wt.% Zn.

Additionally, a new eutectic structure (α-Mg + Mg6Ca2Zn3) appeared and its volume and size

was increased with the increase of Zn content. Figure S1(f) and (g) shows the microstucture

of extruded Mg-5wt%Ca-Xwt%Zn (X= 1, 3) alloys. The secondary phase with fine lamellar

contrasts was observed in the extruded samples and refinement of the microstructure and

dispersion of the lamellar phase were observed after extrusion. Each phasees were confirmed

by XRD (Fig. S2).

Role of Mg6Ca2Zn3 in the corrosion

Here we present why we concluded that Mg2Ca(Zn) phase was responsible for the reduced

corrosion rate insead of the ternary intermetallic compound Mg6Ca2Zn3. As shown in Fig. 1

of the manuscript, corrosion mainly occurs along Mg2Ca phase in the binary Mg-Ca alloy and

corrosion rate decreases significantly with the addition of Zn content. Most important fact in

this observation is that the corrosion of Mg2Ca is reduced sharply with the addition of Zn

content. As shown in EDX results on Fig. 2 of the manuscript, corrosion rate shows inverse

proportion to the concentration of Zn in Mg2Ca phase of the Mg-Ca-Zn alloy. If the ternary

intermetallic compound is mainly responsible for the reduction of corrosion rate, higher

corrosion rate of the 4wt% Zn alloy compared to the 3wt% Zn one cannot be explained (Fig.

2e of the manuscript) since the phase fraction of ternary intermetallic compound increased as

the content of Zn increased (Fig. S3). In particular, the ternary intermetallic compounds were

rarely found, and mostly Mg2Ca(Zn) phases were observed when the corroded fronts of Mg-

Ca-Zn alloys were closely observed using SEM. Based on the above results, we concluded

that the low corrosion rate of our Mg-Ca-Zn alloy resulted mainly from the corrosion

potential change of the Mg2Ca(Zn) phase.

Figure S1: Microstructures of alloys: (a-e) as-cast Mg-5wt%Ca-xwt%Zn (x= 0, 0.5, 1, 1.5

and 3); and (f-g) extruded Mg-5wt%Ca-xwt%Zn (x= 1 and 3) (longitudinal (bottom) and

radial (top) direction, respectively).

20 25 30 35 40 45 50 55 60 65 70 75 80a)

2theta / degree

Inte

nsity

b)

f)

e)

d)

c)

Mg6Ca2Zn3

Mg2CaMg

Figure S2: XRD patterns obtained from samples of Figure S2.

1 2 3 40

1

2

3

4

5

6

7

8

9

10

Zn Contents (wt%)

Phas

e fra

ctio

n (%

)

Mg2Ca Ca2Mg6Zn3

Figure S3: Variation of phase fractions of Mg2Ca and Mg6Ca2Zn3 intermetallic compounds

with the Zn content in Mg-5wt%Ca-xwt%Zn alloys at 293 K (Pandat thermodynamics

software).

2. Mechanical and corrosion properties of the Mg-Ca-Zn alloys with different Zn

contents

Element alloying technology is one of the effective measurements to improve the corrosion

resistance as well as mechanical properties of magnesium. However, some alloying elements

can cause a high risk of side effects during in vivo service so the alloying elements must be

biocompatible. Among the possible elements, calcium was proposed by many researchers to

alloy with magnesium to achieve greater mechanical properties. The addition of at least 5wt. %

Ca is needed to obtain the suitable properties of Mg alloys for use in field of orthopedics.

Unfortunately, the biodegradation rate of Mg-Ca binary alloy was accelerated seriously

compared to that of pure Mg when the addition of Ca content was more than 1wt. % [S1]. In

order to improve the bio-corrosion resistance of Mg-Ca alloy without sacrificing the benefits

of calcium element, zinc element was considered as a third alloying element because zinc has

the potential to improve the corrosion resistance after aluminum. Zhang et al. developed Mg-

Ca-Zn-Mn alloys and implied that the corrosion resistance had close relationship with the

atomic ratio of Zn/Ca [S1]. Furthermore, Zn is highly essential microelement and component

of many proteins, nucleic acid synthetase of human body.

Biodegradation behavior of Mg-5wt.%Ca-xwt.%Zn alloys was investigated by immersion

test in Hank’s solution. The corrosion amount of Mg-5wt.%Ca-xwt.%Zn (x=0, 1, 1.5, 3, 5)

alloys was evaluated by measuring the amount of hydrogen evolution during the immersion

test.

Figure S4 shows that the amount of hydrogen evolution for each as-cast alloy as a function of

immersion time. For Mg-5%Ca binary alloy, the amount of hydrogen evolution was 3.47

ml/cm2 during 3.37 hours immersion. However, after the addition of Zn content, for all the

Mg-5wt.%Ca-xwt.%Zn (x=1, 1.5, 3, 5) ternary alloys, the amount of hydrogen evolution was

less than 0.02ml/cm2 during the same immersion time. The addition of Zn affected the

biodegradation behavior not only for short immersion time but also for long time immersion.

The hydrogen evolution of Mg-5wt.%Ca-xwt.%Zn (x=1, 1.5, 3, 5) ternary alloys during 300

hours immersion was still less than that of Mg-5wt.%Ca binary alloy during 3.37 hours

immersion. So it was obvious that the biodegradation behavior of Mg-5wt.%Ca alloy can be

controlled effectively by the addition of the Zn content.

0 50 100 150 200 250 300 350 4000.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

2.50 Mg5Ca Mg5Ca1Zn Mg5Ca1.5Zn Mg5Ca3Zn Mg5Ca5Zn

Hydr

ogen

am

ount

(ml/c

m2 )

Immersion Time [hr]

Figure S4: Hydrogen evolution amount of as-cast Mg-5%Ca-x%Zn alloy.

In order to show the Zn effects on the biodegradation behavior from quantitative calculation,

the immersion time of each sample for one fixed hydrogen evolution amount was compared

with each other. According to Figure S4, to reach the corroded state corresponding to

0.4ml/cm2 hydrogen gas, the immersion time for each alloy was required as follows: 1.02

hours for Mg-5wt.%Ca, 198.92 hours for Mg-5wt.%Ca-1wt.%Zn, 297.12 hours for Mg-

5wt.%Ca-1.5wt.%Zn, 286.64 hours for Mg-5wt.%Ca-3wt.%Zn, and 172.91 hours for Mg-

5wt.%Ca-5wt.%Zn. It showed that the biodegradation rate was decreased significantly when

the addition of Zn content was increased from 1 wt.% to 1.5 wt. % and it was almost

maintained when the addition of Zn content was increased from 1.5wt.% to 3wt.%. However,

the biodegradation rate was increased slightly when the addition of Zn content was more than

3wt.% in Mg-Ca alloy.

Figure S4: Hydrogen evolution rate of extruded Mg-5wt.%Ca-xwt.%Zn alloy.

Mg5Ca Mg5Ca0.5Zn Mg5Ca1Zn Mg5Ca1.5Zn Mg5Ca3Zn0

50

100

150

200

250

300

350

400

450

155 157175 174

194

365 370386

400 406

17.7 16.5 16.5 18.5 18.5

Stre

ngth

(MPa

)

Y.S U.C.S El

Figure S5: Mechanical properties of Mg alloys with different Zn contents

Figure S5 shows the variation of yield stress (YS), ultimate compressive stress (UCS) and

elongation (El) in several different Mg alloys. YS and UCS increase with increasing Zn

contents while elongation property stays similar.

3. Mechanical and corrosion properties of the extruded Mg-Ca-Zn alloys with different

Ca contents

The related studies on biodegradable Mg alloys have been mostly focused on relatively low

contents of Ca (less than < 2 – 3wt%). This originates from the consensus developed in the

paper [S2] stating that the increase of Ca content to more than 3wt% resulted in the increase

of mechanical strength accompanied by steep decline in elongation and sharp increase of

corrosion rate. This is due to the brittleness of Mg2Ca phase. Under this consensus, various

Mg-Ca-Zn alloy systems with less than 2-3wt% Ca have been published. Currently, due to

this high brittleness, there are only few reported studies showing the tensile test results (yield

strength and elongation) of Mg-Ca/Mg-Ca-Zn alloys with high Ca content (>2wt%Ca). The

brittleness also prevented microstructural modification of alloys with high Ca contents by

using mechanical methods such as extrusion and ECAP. However, enough ductility was

achieved for extrusion of Mg-Ca/Mg-Ca-Zn cast alloys in this study. The microstructural

modification by extrusion provided high yield strength for ductile Mg-Ca-Zn alloys as shown

in Fig. S6. YS and ultimate tensile strength (UTS) increased with increasing Ca contents

whereas the elongation decreased. Mg-5wt%Ca-1wt%Zn alloy deeply investigated in our

manuscript shows high YS (~ 200MPa) and good ductility (~10%).

Mg0.8Ca1Zn Mg1Ca1Zn Mg3Ca1Zn Mg5Ca1Zn Mg7Ca1Zn0

50

100

150

200

250

300

136.07 129.75

178.38192.17

217.32

240.27 231.18249.34 256.15

272.98

30.12 28.85 15.63 8.994.25

Stre

ngth

(MPa

)

Y.S U.T.S El

Figure S6: Mechanical properties of extruded Mg-Ca-Zn alloys with different Ca contents

The corrosion rate of the various alloys with different Ca contents were investigated via

hydrogen evolution as shown in Fig. S7. The result showed that the corrosion rate of Mg-

5wt%Ca-1wt%Zn alloy was very close to that of extremely pure Mg. The reduced corrosion

rate of Mg-5wt%Ca-1wt%Zn is mainly due to the Mg2Ca(Zn) phase as mentioned in our

manuscript.

0 100 200 300 400 5000.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

Hydr

ogen

am

ount

(ml/c

m2 )

Time (hr)

E-pure Mg E-Mg-0.8Ca-1Zn E-Mg-1Ca-1Zn E-Mg-3Ca-1Zn E-Mg-5Ca-1Zn E-Mg-7Ca-1Zn

Figure S7: Hydrogen evolution amount over time of the Mg-xwt.%Ca-1wt.%Zn alloys (x=0.8,

1, 2, 3, 5, 7).

In summary, the optimized electro-chemical tailoring was performed to make the corrosion

rate of our Mg alloys close to that of extremely pure Mg as much as possible by modifying

corrosion potential of two constituent phases. Furthermore, clinically acceptable corrosion

property (no observation of bubble formation in vivo) was achieved through the optimized

microstructural modification. As shown in Fig. S8, the application of both electro-chemical

tailoring and microstructural modification produced low corrosion rate as extruded pure Mg

with far superior mechanical properties.

Figure S8: Tensile yield strength versus bio-corrosion rate map of various Mg alloys

4. Weight Loss Measurements

The weight loss measurement is a commonly practiced method to measure the corrosion rate

of magnesium, and the result can be compared with measurements done via hydrogen

evolution to validate the general trend. The weight loss measurements for this study were

performed using the same method from previously reported studies [S3, S4]. All the samples

were washed with distilled water after immersion in Hanks’ solution. The immersion at room

temperature in chromic acid cleaning solution containing 200gL-1 CrO3 + 10gL-1 AgNO3

followed to remove the corrosion products without removing any metallic Mg alloys.

Samples were then washed with distilled water and dried with high-pressure air to be dried in

the desiccator for 1-2 days. The weight was measured, and the weight loss (mg cm-2d-1) was

evaluated as:

∆W =𝑊𝑏 −𝑊𝑎

𝐴𝐴

where Wa was the weight after the immersion test and Wb was the weight before the

immersion test. A was the surface area of sample (cm2) and t was the time of immersion (d).

The average corrosion rate, PW (mm y-1) was calculated by converting the weight loss using

[S3]:

P𝑊 = 2.1 ∆𝑊

While there is a linear relationship between these two measurements, the corrosion rate

evaluated via examination of the hydrogen evolution rate would be less than the measurement

done by the weight loss evaluation as summarized in the elaborate corrosion work done by

N.I. Z Abidin et al [S3]. Several reported evaluation of high purity (HP) Mg (99.99%)

corrosion via weight loss and hydrogen evolution is available [S4-S7] and they are

summarized in the table below.

Table S1: Corrosion rate of HP Mg (99.99%) obtained from weight loss and hydrogen

evolution measurments.

(mm/y)

Fe Content (ppm) PW(3d) PW(30d) PH(30d)

HP-Mg (99.99) by G. Song Et al (2007) 45 0.02

HP-Mg (99.99) by S. Zhang Et al (2009) 70 0.9 0.2

HP-Mg (99.99) by S. Zhang Et al (2010) 70 0.4 0.1

HP Mg used for these studies included 45~70 ppm of Fe whereas HP Mg used for our study

contained only ~14 ppm of Fe. The corrosion rate of HP Mg with lower Fe contents (15 ppm)

was much lower than that of the above HP Mg (45~70 ppm of Fe). Fe impurity of the Mg

greatly affects the corrosion rate and this phenomenon is evident from the result of our study.

Table S2: Corrosion rates of extruded HP Mg (99.98%) and extruded Mg-5wt%Ca-1wt%Zn

alloy obtained from weight loss and hydrogen evolution measurements. Bold characters

represent the values obtained from weight loss meansurement.

(mm/y)

PH(4d) PH(8d) PH(14d) PW(4d) PW(8d) PH(4d)

Hanks' Hanks' Hanks' Hanks' Hanks' 3%NaCl

HP-Mg(14ppm) 0.09 0.05 0.03 0.26 0.16 3.21 Mg-5wt%Ca-1wt%Zn

(27ppm) 0.08 0.05 0.01 0.13 0.08 0.44

As shown in Table S2, newly developed ternary Mg alloy showed significantly lower

corrosion rate when compared to HP Mg used for this study. Weight loss evaluation showed

higher corrosion rate when compared to the evaluation via hydrogen evolution but the trend

of the corrosion rate was the same in both measurements.

5. Work functions of pure Mg and Mg2Ca

The microstructure of Mg-Ca alloys is composed of pro-eutectic Mg and Mg/Mg2Ca eutectic

phases. The rapid corrosion of Mg-Ca alloys in Hank’s solution is accompanied by the local

selective corrosion of Mg2Ca phase in the eutectic microstructure (Fig. 1 b in the manuscript).

The local corrosion of Mg2Ca phase is typical behavior of Galvanic corrosion where we can

conjecture that Mg and Mg2Ca have different corrosion potentials in Hank’s solution. We

conducted the first principle calculations to obtain the information for the corrosion potentials

of Mg and Mg2Ca and to investigate the effect of Zn.

Which of material properties can give us the information for the corrosion potential of each

phase? There is a clue in the prevailing equipment used in the measurement of the corrosion

potential. Scanning Kelvin Probe (SKP), one of the corrosion potential measurement

equipments, measures the work function of a material to obtain its corrosion potential. It has

been reported for several materials that measured work function bears a proportionate

relationship to the corrosion potential [S8]. The proportional relationship between work

function and corrosion potential may be explained as follows. The Fermi level of electron in a

material means its chemical potential [S9]. Based on the Fermi levels of materials, it can be

approximately determined the electron transfer from one material to the other one in two

materials junction because electron moves from higher chemical potential area to lower one.

As relative heights of the Fermi levels of materials between materials are correlated to their

work functions, the work function can be conjectured to be related to the corrosion potential.

We performed the first principle calculations to obtain the work functions of pro-eutectic Mg

and Mg2Ca phases and to investigate the effect of Zn. Density functional theory (DFT)

calculations using the VASP program packages [S10] were used. The plane wave basis

expansions with an energy cutoff of 300 eV and the generalized gradient approximation

(GGA) with the PW91 exchange-correlation functional were used. The core-valence

interaction is described by the projector-augmented wave (PAW) method [S11]. We

constructed Mg2Ca slab structure to calculate work function as shown in Fig. 9, which are

constructed with 96 Mg atoms and 48 Ca atoms. Vacuum sizes are given larger than 20 Å to

minimize interaction between slabs. We first obtained the bulk Mg2Ca structure applying

Monkhorst-Pack sampling with a 8 × 8 × 4 k-point grid and then constructed a slab structure

with the resulting lattice constant and a 3 × 3 × 1 k-point grid. We fully optimized the atomic

configurations except 8 bottom atomic layers of which positions are fixed to mimic bulk

properties.

Figure S9 shows the atomic configuration of Mg2Ca and the top view of its (0001) surface

used in our first principle calculations. Mg2Ca has hexagonal structure and its (0001) surface

has minimum surface energy. On (0001) surface Ca atoms are located at higher positions

compared to Mg atoms. The work function of Mg2Ca in the most stable (0001) surface is 3.16

eV that is lower than that of pure Mg, say, 3.7 eV. This means that the Fermi level of Mg2Ca

is higher than that of pro-eutectic Mg and that the Galvanic circuit forms from Mg2Ca with

higher electrochemical potential to pro-eutectic Mg.

Figure S9: Atomic configurations of Mg2Ca used in the work function calculation. The upper-

right figure shows the top view of the calculation domain that corresponds to (0001) surface.

To calculate the effect of Zn on the work function of Mg2Ca, we replaced Ca atoms

protruding from (0001) surface with Zn atoms. The change of work function with the number

of replaced Zn atoms is listed in Table S3. Table S3 shows the variation of work function in

(0001) surface with the number of Zn atoms on the surface. In case of bare surface without

Zn, the WF is 3.16 eV and increases with Zn surface coverage such that it reaches 3.48 eV at

four surface Zn atoms. Therefore, the Galvanic potential between Mg and Mg2Ca decreases

with increasing Zn contents.

Table S3: Variation of the work function with Zn contents

Number of surface Zn atoms 0 1 2 3 4

Work function (eV) 3.16 3.26 3.34 3.42 3.48

6. Cytotoxicity test of excessive Mg, Ca and Zn ions

To investigate the effects of metal ions, cytotoxicity tests using HeLa and L929 cell line were

performed. The HeLa and L929 cells were maintained in the Dulbecco’s Modified Eagle’s

Medium (DMEM, Welgene, Korea) supplemented with 10% fetal bovine serum (FBS,

Welgene), 100 U/ml penicillin and 100ug/ml streptomycin (Welgene) at 37℃ in a humidified

atmosphere of 5% CO2, the media being changed every three days. For cytotoxicity test of

three metal ions, the Magnesium, calcium and zinc chloride (Sigma-Aldrich, St. Louis, MO)

was used. 5 x 103 of cells were seeded in 96 well plate and pre-cultured for 24 hours to allow

cell adhesion. Cell culture media was changed with fresh media containing up to 32mM of

magnesium, calcium ions and up to 1mM of zinc ions. Cells were incubated for 24 hours and

cell viability was measured by the CCK-8 kit (Dojindo, Japan). Figure S10 show the effect of

Mg, Ca and Zn ions on cell viability. Zinc ion showed the most toxic effect on both cell

models. Though it was slightly depend on the cell type, the cell viability started to be reduced

from about more than 60µM of zinc ion. In both magnesium and calcium ions, which had no

significant differences in cytotoxicity, a decline of the cell viability peered from about more

than 30mM.

Cytotoxicity of metal ions (L929)

Concentration (uM)C(-)

1

23.97.8

15.631.362.5125250500

10002000400080001600032000

Cell Viability (%)0 20 40 60 80

100

120

140M

g Ca Zn

Cytotoxicity of metal ions (HeLa)

Concentration (uM)

C(-)1

23.97.8

15.631.362.5125250500

10002000400080001600032000

Cell Viability (%)

0 20 40 60 80

100

120

140M

g Ca Zn

8.52 ppm

761.68ppm

Figure S10: The cytotoxicity for Mg, Ca and Zn ions in HeLa and L929 cell lines.

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