**3. Results and discussion**

#### **3.1 Phase compositions and microstructures evolution of the as-cast Mg-Zn-Ca alloys 3.1.1 The effects of Zn content on phase compositions and microstructures of the as-cast alloys**

In this study, in order to investigated the effects of Zn and Ca on the phase compositions and microstructures evolution of the as-cast Mg-Zn-Ca alloys, respectively, the initial content of Ca design as 0 wt. % and then changed the content of Zn to study the effects of Zn on phase compositions and microstructures. The chemical compositions of the Mg-xZn alloy obtained by ICP-AES were listed in Table 1. The impurity contents of the Mg-x Zn alloy were very low for better degradation properties and biocompatibility. X-ray diffraction (XRD) analyses were used to investigate the existing intermetallic phases in the Mg-x Zn Ca alloys (Fig. 1). As shown in Fig. 1, there was only α-Mg diffraction peaks phase in the Mg-1.0Zn alloy. Diffraction peaks from the Mg2Zn phase was not detected. With the Zn concentration increasing, MgZn phase's patterns were began to detect in Mg-5.0 Zn and Mg-6.0 Zn alloy.


Table 1. Chemical compositions of the as-cast Mg-Zn alloy

The microstructures of the as-cast Mg-x Zn alloys were shown in Fig.2. Fig. 2(a) was taken from Mg-1.0 Zn alloy, in which the microstructure consists of the α-Mg . The maximum solubility of Zn in the magnesium was about 2 wt. % at room temperature in the equilibrium state, when no more than 2 wt. % Zn was added, the Zn was solid solution in Mg matrix. When the contents of Zn was more than 4 wt. % , the microstructure obviously changed, there were more second phases precipitated and the morphogenesis of second phases were small particle. As shown in Fig.2 (f), with the increasing of Zn content, lamellar eutectic appears in the as-cast microstructure. The eutectic structures were very coarse and mostly distributed in the grain boundary and less in the areas of inter-dendrite,

Research on Mg-Zn-Ca Alloy as Degradable Biomaterial 189

Fig. 2. Optical microstructure of as-cast Mg-Zn alloys (a) Mg-1.0Zn; (b) Mg-2.0Zn; (c) Mg-

**3.1.2 The effects of Ca content on phase compositions and microstructures of the** 

In present study, the mechanical properties show that when Zn content is 4wt. %, the MgxZn has good mechanical properties. Thus, the initial content of Zn designs as 4.0wt. % and then changed the content of Ca to study the effects on phase compositions and microstructures .The chemical compositions of the Mg-4.0 Zn-x Ca alloy obtained by ICP-AES were listed in Table 3. X-ray diffraction (XRD) analyses were used to investigate the existing phases in the Mg-4.0Zn-xCa alloys, and the results were shown in Fig.3. The results showed that α-Mg and MgZn phases were detected in the Mg-4.0 Zn alloy, and it also indicated that the diffraction peaks from the MgZn phase were very weak and that of the Mg were strong. There was no obvious change in diffraction peak when 0.2 wt. % Ca and 0.5wt. % Ca was added into the Mg-4.0 Zn alloy. With the Ca concentration increased to 1.5 wt. %, Ca2Mg6Zn3 phases began to be detected in Mg-4.0 Zn-xCa. When the Ca concentration increased to 2.0 wt. %, Mg2Ca, and Ca2Mg5Zn13 phases began to be detected in

Chemical composition (wt.%)

Al Zn Mn Si Fe Ca Mg

Mg-4.0Zn 0.023 3.926 0.058 0.031 0.004 0.007 Balance Mg-4.0Zn-0.2Ca 0.033 1.852 0.030 0.039 0.007 0.180 Balance Mg-4.0Zn-0.5Ca 0.029 2.732 0.022 0.036 0.007 0.452 Balance Mg-4.0Zn-1.0Ca 0.019 3.925 0.021 0.032 0.008 0.915 Balance Mg-4.0Zn-1.5Ca 0.027 5.223 0.031 0.034 0.009 1.635 Balance Mg-4.0Zn-2.0Ca 0.024 5.977 0.019 0.033 0.012 2.158 Balance

Table 2. Chemical composition of the as-cast Mg-4.0Zn-xCa alloys

3.0Zn; (d) Mg-4.0Zn; (e) Mg-5.0Zn; (f) Mg-6.0Zn

**as-cast alloys** 

the alloy.

Materials

Fig. 1. XRD patterns of as-cast Mg-Zn alloys (a)Mg-1.0Zn; (b)Mg-2.0Zn; (c)Mg-3.0Zn; (d)Mg-4.0Zn; (e)Mg-5.0Zn; (f)Mg-6.0Zn

Fig. 1. XRD patterns of as-cast Mg-Zn alloys (a)Mg-1.0Zn; (b)Mg-2.0Zn; (c)Mg-3.0Zn; (d)Mg-

4.0Zn; (e)Mg-5.0Zn; (f)Mg-6.0Zn

Fig. 2. Optical microstructure of as-cast Mg-Zn alloys (a) Mg-1.0Zn; (b) Mg-2.0Zn; (c) Mg-3.0Zn; (d) Mg-4.0Zn; (e) Mg-5.0Zn; (f) Mg-6.0Zn

#### **3.1.2 The effects of Ca content on phase compositions and microstructures of the as-cast alloys**

In present study, the mechanical properties show that when Zn content is 4wt. %, the MgxZn has good mechanical properties. Thus, the initial content of Zn designs as 4.0wt. % and then changed the content of Ca to study the effects on phase compositions and microstructures .The chemical compositions of the Mg-4.0 Zn-x Ca alloy obtained by ICP-AES were listed in Table 3. X-ray diffraction (XRD) analyses were used to investigate the existing phases in the Mg-4.0Zn-xCa alloys, and the results were shown in Fig.3. The results showed that α-Mg and MgZn phases were detected in the Mg-4.0 Zn alloy, and it also indicated that the diffraction peaks from the MgZn phase were very weak and that of the Mg were strong. There was no obvious change in diffraction peak when 0.2 wt. % Ca and 0.5wt. % Ca was added into the Mg-4.0 Zn alloy. With the Ca concentration increased to 1.5 wt. %, Ca2Mg6Zn3 phases began to be detected in Mg-4.0 Zn-xCa. When the Ca concentration increased to 2.0 wt. %, Mg2Ca, and Ca2Mg5Zn13 phases began to be detected in the alloy.


Table 2. Chemical composition of the as-cast Mg-4.0Zn-xCa alloys

Research on Mg-Zn-Ca Alloy as Degradable Biomaterial 191

Fig. 4. Microstructure of as-cast Mg-4.0Zn-xCa alloys (a) Mg-4.0Zn; (b) Mg-4.0Zn-0.2Ca; (c)

The tensile tests were carried out at room temperature on the as-cast Mg-x Zn alloys. The tensile strength and elongation of present alloy were shown in Table 3. The typical stresstrain curves of Mg-x Zn alloys were depicted in Fig.5. As shown in Table 3 and Fig.5, after 1.0 wt.% Zn was added to the pure Mg, the mechanical properties of as-cast Mg-1.0Zn like

Mg-4.0Zn-0.5Ca; (d) Mg-4.0Zn-1.0Ca; (e) Mg-4.0Zn-1.5Ca; (f) Mg-4.0Zn-2.0Ca

**3.2 Mechanical property evolution of the as-cast Mg- Zn-Ca alloys 3.2.1 The effects of Zn content on mechanical property of as-cast alloys** 

Fig. 3. XRD patterns of as-cast Mg-4.0Zn-xCa alloys (a) Mg-4.0Zn; (b) Mg-4.0Zn-0.2Ca; (c) Mg-4.0Zn-0.5Ca; (d) Mg-4.0Zn-1.0Ca; (e) Mg-4.0Zn-1.5Ca; (f) Mg-4.0Zn-2.0Ca

The microstructures of the as-cast Mg-4.0 Zn-x Ca alloys were shown in Fig.4. Fig. 4(a) was taken from the Mg-4.0 Zn alloy, which consisted of the dendrite α-Mg matrix and some polygonal shaped second phases which distributed in the areas of inter-dendrite and grain boundary. The second phases were very coarse in the Mg-4.0 Zn. Fig. 4(b) was taken from the Mg-4.0 Zn-0.2 Ca alloy, which indicated that the microstructure had an evidently change compared with Mg-4.0 Zn alloy, and the second phase changed its shape and distributed in the areas of inter grain. With the increase of Ca concentration, however, lamellar eutectic appeared in the as-cast microstructure, eutectic structure was mostly distributed in the grain boundary and little in the areas of inter-dendrite, as shown in Fig. 3 (e) and (f) which were taken from Mg-4.0 Zn-1.5 Ca and Mg-4.0 Zn-2.0 Ca alloys, respectively. It's easy to fond out that the morphogenesis of second phases have an obviously change by an increase in Ca content. At first the second phase was polygonal particles in Mg-4.0 Zn alloy, and then when less than 0.5 wt. % Ca was added in Mg-4.0 Zn alloy, the second phase changed its morphology, and it was small round particle. Finally when more than 0.5wt. % Ca was added, the second phase was lamellar structure.

In the initial stages of solidification, Zn and Ca were complete melts in the magnesium. Subsequently, as the solidification develops, the solute atoms are rejected by the growing α-Mg and enriched in the residual liquid, which began to form clusters precipitation in the grain boundary and inter dendrite arm space. When the Ca concentration was increasing to 1.5 wt. %, it was apt to forming lamellar eutectic.

Fig. 3. XRD patterns of as-cast Mg-4.0Zn-xCa alloys (a) Mg-4.0Zn; (b) Mg-4.0Zn-0.2Ca; (c)

The microstructures of the as-cast Mg-4.0 Zn-x Ca alloys were shown in Fig.4. Fig. 4(a) was taken from the Mg-4.0 Zn alloy, which consisted of the dendrite α-Mg matrix and some polygonal shaped second phases which distributed in the areas of inter-dendrite and grain boundary. The second phases were very coarse in the Mg-4.0 Zn. Fig. 4(b) was taken from the Mg-4.0 Zn-0.2 Ca alloy, which indicated that the microstructure had an evidently change compared with Mg-4.0 Zn alloy, and the second phase changed its shape and distributed in the areas of inter grain. With the increase of Ca concentration, however, lamellar eutectic appeared in the as-cast microstructure, eutectic structure was mostly distributed in the grain boundary and little in the areas of inter-dendrite, as shown in Fig. 3 (e) and (f) which were taken from Mg-4.0 Zn-1.5 Ca and Mg-4.0 Zn-2.0 Ca alloys, respectively. It's easy to fond out that the morphogenesis of second phases have an obviously change by an increase in Ca content. At first the second phase was polygonal particles in Mg-4.0 Zn alloy, and then when less than 0.5 wt. % Ca was added in Mg-4.0 Zn alloy, the second phase changed its morphology, and it was small round particle. Finally when more than 0.5wt. % Ca was

In the initial stages of solidification, Zn and Ca were complete melts in the magnesium. Subsequently, as the solidification develops, the solute atoms are rejected by the growing α-Mg and enriched in the residual liquid, which began to form clusters precipitation in the grain boundary and inter dendrite arm space. When the Ca concentration was increasing to

Mg-4.0Zn-0.5Ca; (d) Mg-4.0Zn-1.0Ca; (e) Mg-4.0Zn-1.5Ca; (f) Mg-4.0Zn-2.0Ca

added, the second phase was lamellar structure.

1.5 wt. %, it was apt to forming lamellar eutectic.

Fig. 4. Microstructure of as-cast Mg-4.0Zn-xCa alloys (a) Mg-4.0Zn; (b) Mg-4.0Zn-0.2Ca; (c) Mg-4.0Zn-0.5Ca; (d) Mg-4.0Zn-1.0Ca; (e) Mg-4.0Zn-1.5Ca; (f) Mg-4.0Zn-2.0Ca

#### **3.2 Mechanical property evolution of the as-cast Mg- Zn-Ca alloys 3.2.1 The effects of Zn content on mechanical property of as-cast alloys**

The tensile tests were carried out at room temperature on the as-cast Mg-x Zn alloys. The tensile strength and elongation of present alloy were shown in Table 3. The typical stresstrain curves of Mg-x Zn alloys were depicted in Fig.5. As shown in Table 3 and Fig.5, after 1.0 wt.% Zn was added to the pure Mg, the mechanical properties of as-cast Mg-1.0Zn like

Research on Mg-Zn-Ca Alloy as Degradable Biomaterial 193

Mg–4.0Zn 58±1.0 216.8±15 15.8±5.5 Mg–4.0Zn–0.2Ca 58.1±1.0 225±5 17.5±1.0 Mg–4.0Zn–0.5Ca 70±3.0 180±5 12.3±1.5 Mg–4.0Zn–1.0Ca 83±2.0 175±10 8.7±1.0 Mg–4.0Zn–1.5Ca 83±3.0 167±5 7.1±2.5 Mg–4.0Zn–2.0Ca 90±4.0 143±5 2.1±0.5

Table 4. Mechanical properties of Mg-4.0wt.%Zn-xCa alloys at room temperature

microporosity.

deteriorate.

**3.3 In-vitro degradation tests** 

The mechanical properties of magnesium were affected by each alloying constituent. Zinc was an effective alloying ingredient in magnesium. Because zinc had a relatively high solid solubility in magnesium at high temperature, a good mechanical properties were achieved by solid solution strengthen. Binary Mg-Zn alloys like Mg-Al alloys, also respond to age hardening, and contrary to Mg-Al alloys, coherent GP zone and semeicoherent intermediate precipitate were formed to have an enhanced effect. However, in the Mg-Zn alloys, the maximum solubility of zinc in the magnesium drops to 1.6 wt. % (i.e. 0.6 at. %) at room temperature in the equilibrium state [23]. When the zinc content was more than 4.0 wt. %, in the solidification process, the melt zinc atoms would be rejected by the growing α-Mg and enriched in the residual liquid, these rich areas were often prone to formation of

In Mg-Zn alloys, the progressive addition of Ca had been found to substantially increase the temperature difference between liquid and solid phase lines, which was conducive to the grain refinement in the solidification process. At the same time, the introduction of Ca to Mg-Zn alloys result in precipitation of desolventizing phase, Ca2Mg6Zn3 and Ca2Mg5Zn13, which could enhance the strength and toughness of alloy [24][25][26]. The current work showed that an addition of small amount of Ca to Mg-4.0 Zn alloys had a marked increase in the tensile strength, but Ca content was excess of 0.5 wt. % make the tensile strength prone to decrease. The precipitates in the Mg-4.0 Zn-0.2 Ca and Mg-4.0 Zn-0.5 Ca alloys were Ca2Mg6Zn3 and Ca2Mg5Zn13 phases, which were small particles in the alloys. Thus, the tensile property of Mg-4.0 Zn-0.2 Ca and Mg-4.0 Zn-0.5 Ca alloys were improved. However, the maximum solubility of Ca in the magnesium was only 0.2 wt. % at room temperature and 1.2 wt. % at high temperature in the equilibrium state, when more than 1.0 wt. % Ca was added, the precipitates in the grain boundary began to continuously precipitated and the morphogenesis of the precipitates were changed to lamellar structure, made the tensile properties decline. When the Ca concentration was up to 2.0 wt. %, in the grain boundary tends to form eutectic structure which caused the tensile property

**3.3.1 The effects of Zn content on in-vitro degradation of the as-cast alloys** 

The representative potentiodynamic polarization curves of Mg-xZn alloys in Hank's solution were shown in Fig.8, with pure Mg as contrast. As shown in Fig.8, the corrosion potential of the Mg-x Zn alloys was higher than that of pure Mg. The corrosion potential of

Alloy Yield strength (MPa) UTS (MPa) Elongation (%)

pure Mg was still weak, in which the yield strength was 21MPa,UTS was 101MPa and the elongation was 6.9%. With the increasing of Zn contents, the yield strength , UTS and the elongation was increased. When the Zn content was up to 4.0wt.%, the mechanical properties reach to the peak value, the yield strength was 58.1MPa, UTS was 216.85MPa and the elongation was 15.8%. The mechanical properties of Mg-Zn binary alloy when ulteriorly increasing of Zn contents was declined. Its UTS was 182 MPa, and its elongation was only 7.2 % for Mg-6.0 Zn alloy. Direct estimation of stacking fault energy by thermodynamic calculations showed that Zn reduces stacking fault energy of the Mg-Zn alloys. Stacking fault energy is an important physical properties of the material, which directly affects the mechanical properties, dislocation cross slip, phase stability and the dynamic recrystallization of metal materials. It has been confirmed that the stacking fault energy in magnesium alloy plays an important role in mechanical properties and the dynamic recrystallization[22]


Table 3. Mechanical properties of Mg-xZn alloys at room temperature

#### **3.2.2 The effects of Ca content on mechanical property of the as-cast alloys**

The tensile strength and elongation of as-cast Mg-4.0 Zn-x Ca alloys were shown in table 4. The typical stress-train curves of as-cast Mg-4.0 Zn-x Ca alloys were depicted in Fig.6. The ultimate tensile strength (UTS) and elongation of as-cast Mg-4.0 Zn alloy were 180MPa and 9.5%, respectively. After 0.2 wt. % Ca was added, the UTS and elongation of as-cast Mg-4.0 Zn-0.2 Ca alloy were improved to 215 MPa and 17.5%, respectively. When 0.5 wt. % Ca was added, the Mg-4.0 Zn-0.5 Ca alloy has similar mechanical property as the Mg-4.0 Zn-0.2 Ca alloy. However, the mechanical properties of as-cast Mg-4.0 Zn-1.0 Ca alloy began to decline. When the Ca concentration was up to 2.0 wt. %, the alloy showed worse mechanical property, its UTS was 142 MPa and elongation was only 1.7 %.

Fig.7 showed the typical fracture surfaces of as-cast Mg-4.0 Zn-x Ca alloys. As it was showed that the fracture type was ductile fracture when Ca concentration was lower than 0.5 wt. %. Big dimples and tearing edges can be evidently observed on the fracture surface of the as-cast Mg-4.0 Zn-0.2 Ca (Fig.8 (a)).When the Ca concentration was 1.0 wt. %, the Mg-4.0 Zn-1.0 Ca alloy showed mixture fracture morphology. When the Ca concentration was up to 2.0 wt. %, the fracture type of the alloy was brittle fracture. The pearl-shaped fracture can be easily observed on the fracture surface of the as-cast Mg-4.0 Zn-2.0 Ca alloy (Fig. 5 (c)).

pure Mg was still weak, in which the yield strength was 21MPa,UTS was 101MPa and the elongation was 6.9%. With the increasing of Zn contents, the yield strength , UTS and the elongation was increased. When the Zn content was up to 4.0wt.%, the mechanical properties reach to the peak value, the yield strength was 58.1MPa, UTS was 216.85MPa and the elongation was 15.8%. The mechanical properties of Mg-Zn binary alloy when ulteriorly increasing of Zn contents was declined. Its UTS was 182 MPa, and its elongation was only 7.2 % for Mg-6.0 Zn alloy. Direct estimation of stacking fault energy by thermodynamic calculations showed that Zn reduces stacking fault energy of the Mg-Zn alloys. Stacking fault energy is an important physical properties of the material, which directly affects the mechanical properties, dislocation cross slip, phase stability and the dynamic recrystallization of metal materials. It has been confirmed that the stacking fault energy in magnesium alloy plays an important role in mechanical properties and the

Alloy Yield strength(MPa) UTS (MPa) Elongation (%)

Mg-1.0Zn 20±2 101.5±3 6.96±0.5 Mg-2.0Zn 27±2 145.9±5 12.23±1.5 Mg-3.0Zn 47±1.5 167.8±10 13.7±1.0 Mg-4.0Zn 58±1.0 216.8±15 15.8±5.5 Mg-5.0Zn 68±1.5 185±5 9.2±0.5 Mg-6.0Zn 69±1.5 182±5 7.2±0.5

Table 3. Mechanical properties of Mg-xZn alloys at room temperature

property, its UTS was 142 MPa and elongation was only 1.7 %.

**3.2.2 The effects of Ca content on mechanical property of the as-cast alloys** 

The tensile strength and elongation of as-cast Mg-4.0 Zn-x Ca alloys were shown in table 4. The typical stress-train curves of as-cast Mg-4.0 Zn-x Ca alloys were depicted in Fig.6. The ultimate tensile strength (UTS) and elongation of as-cast Mg-4.0 Zn alloy were 180MPa and 9.5%, respectively. After 0.2 wt. % Ca was added, the UTS and elongation of as-cast Mg-4.0 Zn-0.2 Ca alloy were improved to 215 MPa and 17.5%, respectively. When 0.5 wt. % Ca was added, the Mg-4.0 Zn-0.5 Ca alloy has similar mechanical property as the Mg-4.0 Zn-0.2 Ca alloy. However, the mechanical properties of as-cast Mg-4.0 Zn-1.0 Ca alloy began to decline. When the Ca concentration was up to 2.0 wt. %, the alloy showed worse mechanical

Fig.7 showed the typical fracture surfaces of as-cast Mg-4.0 Zn-x Ca alloys. As it was showed that the fracture type was ductile fracture when Ca concentration was lower than 0.5 wt. %. Big dimples and tearing edges can be evidently observed on the fracture surface of the as-cast Mg-4.0 Zn-0.2 Ca (Fig.8 (a)).When the Ca concentration was 1.0 wt. %, the Mg-4.0 Zn-1.0 Ca alloy showed mixture fracture morphology. When the Ca concentration was up to 2.0 wt. %, the fracture type of the alloy was brittle fracture. The pearl-shaped fracture can be easily observed on the fracture surface of the as-cast Mg-4.0 Zn-2.0 Ca alloy

dynamic recrystallization[22]

(Fig. 5 (c)).


Table 4. Mechanical properties of Mg-4.0wt.%Zn-xCa alloys at room temperature

The mechanical properties of magnesium were affected by each alloying constituent. Zinc was an effective alloying ingredient in magnesium. Because zinc had a relatively high solid solubility in magnesium at high temperature, a good mechanical properties were achieved by solid solution strengthen. Binary Mg-Zn alloys like Mg-Al alloys, also respond to age hardening, and contrary to Mg-Al alloys, coherent GP zone and semeicoherent intermediate precipitate were formed to have an enhanced effect. However, in the Mg-Zn alloys, the maximum solubility of zinc in the magnesium drops to 1.6 wt. % (i.e. 0.6 at. %) at room temperature in the equilibrium state [23]. When the zinc content was more than 4.0 wt. %, in the solidification process, the melt zinc atoms would be rejected by the growing α-Mg and enriched in the residual liquid, these rich areas were often prone to formation of microporosity.

In Mg-Zn alloys, the progressive addition of Ca had been found to substantially increase the temperature difference between liquid and solid phase lines, which was conducive to the grain refinement in the solidification process. At the same time, the introduction of Ca to Mg-Zn alloys result in precipitation of desolventizing phase, Ca2Mg6Zn3 and Ca2Mg5Zn13, which could enhance the strength and toughness of alloy [24][25][26]. The current work showed that an addition of small amount of Ca to Mg-4.0 Zn alloys had a marked increase in the tensile strength, but Ca content was excess of 0.5 wt. % make the tensile strength prone to decrease. The precipitates in the Mg-4.0 Zn-0.2 Ca and Mg-4.0 Zn-0.5 Ca alloys were Ca2Mg6Zn3 and Ca2Mg5Zn13 phases, which were small particles in the alloys. Thus, the tensile property of Mg-4.0 Zn-0.2 Ca and Mg-4.0 Zn-0.5 Ca alloys were improved. However, the maximum solubility of Ca in the magnesium was only 0.2 wt. % at room temperature and 1.2 wt. % at high temperature in the equilibrium state, when more than 1.0 wt. % Ca was added, the precipitates in the grain boundary began to continuously precipitated and the morphogenesis of the precipitates were changed to lamellar structure, made the tensile properties decline. When the Ca concentration was up to 2.0 wt. %, in the grain boundary tends to form eutectic structure which caused the tensile property deteriorate.

#### **3.3 In-vitro degradation tests**

#### **3.3.1 The effects of Zn content on in-vitro degradation of the as-cast alloys**

The representative potentiodynamic polarization curves of Mg-xZn alloys in Hank's solution were shown in Fig.8, with pure Mg as contrast. As shown in Fig.8, the corrosion potential of the Mg-x Zn alloys was higher than that of pure Mg. The corrosion potential of

Research on Mg-Zn-Ca Alloy as Degradable Biomaterial 195

xCa alloys are enhanced to -1574mV, which is increased by 70 mV compared with -1646 mV of the pure Mg corrosion potential. However, it is confirmed that Mg-4.0 Zn-0.2 Ca alloy exhibits the best corrosion resistance among Mg-4.0Zn-xCa alloys, even higher than that of Mg-4.0 Zn alloy through further observation. This particular phenomenon can be explained as follows. Firstly, the addition of 4.0 wt. % Zn can cause the formation of coarse MgZn precipitate as shown in Fig.4 and 2(a), which reduces the corrosion resistance of Mg-4.0Zn alloy due to the different electrochemical behaviors between primary α-Mg and precipitate. Then, the slight addition (less than 0.5 wt. %) of Ca alloying element can cause MgZn precipitates to be effectively transform to fine ternary precipitates, which has been clearly documented in previous literature [27][28]. The refinement and homogenization of precipitate phase can improve the corrosion resistance of Mg-4.0Zn-0.2Ca alloy compared with that of Mg-4.0Zn alloy. Finally, increasing Ca content was over than 0.5wt. % cause the formation of another coarse Mg2Ca, Ca2Mg6Zn3 and Ca2Mg5Zn13 precipitates as shown in Fig.3 and 4. It is quite obvious that the precipitates increases with Ca content increasing,

Fig. 9. The potentiodynamic polarization curves of as-cast Mg-4.0 Zn-x Ca alloys in Hank's

It is well known that the cathodic polarization curves represent the cathodic hydrogen evolution through water reduction, while the anodic polarization curves do the dissolution of magnesium. That is to say, it is equivalent to that Mg-4.0 Zn-0.2 Ca alloy sample exhibits the lowest current of hydrogen evolution reaction and Mg-4.0 Zn-1.5 Ca and Mg-4.0Zn-2.0 Ca alloys samples does the highest ones, which indicates that over potential of the cathodic hydrogen evolution reaction of Mg-4.0 Zn-0.2 Ca alloy is much lower than those of Mg-4.0 Zn-1.5 Ca and Mg-4.0 Zn-2.0 Ca alloys. Therefore, the lowest cathodic hydrogen evolution

Fig.10 illustrates the pH variation of Hank's solution versus the immersion testing time for Mg-4.0Zn-xCa alloys. It could be observed that the pH variations of the alloys all obey the parabolic rate law. The pH variation rate decreases with the immersion time increasing. After 48 hrs immersion, all the pH values of the samples tend to be stable. In the early period of immersion, both pure Mg and the Mg-4.0Zn-xCa alloys acutely reacted with

reaction brings the highest corrosion resistance to the Mg-4.0 Zn-0.2 Ca alloy.

solution.

which decreases the corrosion resistance of as-cast Mg-4.0Zn-xCa alloys.

pure Mg was -1574mV. The corrosion potential was correlated with the Zn concentration. The corrosion potential of Mg-2.0Zn and Mg-3.0 Zn alloys were about -1561 and -1568 mV, respectively, which were nearly the same and about 10 mV high than that of pure Mg. The Mg-5.0 Zn and Mg-6.0 Zn alloy samples exhibit high corrosion potentials of about -1524 and -1547 mV, respectively, which were about 50 mV higher than that of pure Mg. It could be seen that the addition of Zn improved the corrosion potential of the as-cast Mg-x Zn alloys. But, the addition of elements Zn was also increased the current densities of the resulted ascast Mg alloys in Hank's solution.

Fig. 8. Potentiodynamic polarization curves of Mg-Zn alloys in SBF solution

The reason for the increase corrosion potential of Mg-x Zn alloys was that the Zn element had a high electronegative. But, when the Zn concentration increased, the corrosion resistance was decreased. The reason for the reduced corrosion resistance of Mg-x Zn alloys was that the second phase precipitated during the solid solidification processes, which accelerated the corrosion rate due to the different electrochemical behaviors of α-Mg and precipitates.

 Generally, the cathodic polarization curves were assumed to represent the cathodic hydrogen evolution through water reduction, while the anodic polarization curves represented the dissolution of magnesium. It could be seen that the cathodic polarization current of hydrogen evolution reaction on Mg-1.0 Zn alloy sample was much lower than that of Mg-5.0 Zn and Mg-6.0 Zn Ca alloys sample, suggesting that over potential of the cathodic hydrogen evolution reaction was lower for Mg-1.0 Zn and Mg-2.0 Zn alloys sample. As a result, the cathodic reaction was kinetically more difficult on the Mg-1.0 Zn alloy and Mg-2.0 Zn alloy sample than that on the Mg-5.0 Zn Ca alloy samples. The degradation rates of Mg-1.0Zn degraded were slower thanMg-5.0Zn, Mg-6.0Zn, which was adherence to the electrochemical results.

#### **3.3.2 The effects of Ca content on in-vitro degradation of the as-cast Mg-Zn-Ca alloys**

The representative potentiodynamic polarization curves of the pure Mg and Mg-4.0 Zn-x Ca alloys in Hank's solution were shown in Fig.9. The mean corrosion potentials of Mg-4.0Zn-

pure Mg was -1574mV. The corrosion potential was correlated with the Zn concentration. The corrosion potential of Mg-2.0Zn and Mg-3.0 Zn alloys were about -1561 and -1568 mV, respectively, which were nearly the same and about 10 mV high than that of pure Mg. The Mg-5.0 Zn and Mg-6.0 Zn alloy samples exhibit high corrosion potentials of about -1524 and -1547 mV, respectively, which were about 50 mV higher than that of pure Mg. It could be seen that the addition of Zn improved the corrosion potential of the as-cast Mg-x Zn alloys. But, the addition of elements Zn was also increased the current densities of the resulted as-

Fig. 8. Potentiodynamic polarization curves of Mg-Zn alloys in SBF solution

The reason for the increase corrosion potential of Mg-x Zn alloys was that the Zn element had a high electronegative. But, when the Zn concentration increased, the corrosion resistance was decreased. The reason for the reduced corrosion resistance of Mg-x Zn alloys was that the second phase precipitated during the solid solidification processes, which accelerated the

 Generally, the cathodic polarization curves were assumed to represent the cathodic hydrogen evolution through water reduction, while the anodic polarization curves represented the dissolution of magnesium. It could be seen that the cathodic polarization current of hydrogen evolution reaction on Mg-1.0 Zn alloy sample was much lower than that of Mg-5.0 Zn and Mg-6.0 Zn Ca alloys sample, suggesting that over potential of the cathodic hydrogen evolution reaction was lower for Mg-1.0 Zn and Mg-2.0 Zn alloys sample. As a result, the cathodic reaction was kinetically more difficult on the Mg-1.0 Zn alloy and Mg-2.0 Zn alloy sample than that on the Mg-5.0 Zn Ca alloy samples. The degradation rates of Mg-1.0Zn degraded were slower thanMg-5.0Zn, Mg-6.0Zn, which was

**3.3.2 The effects of Ca content on in-vitro degradation of the as-cast Mg-Zn-Ca alloys**  The representative potentiodynamic polarization curves of the pure Mg and Mg-4.0 Zn-x Ca alloys in Hank's solution were shown in Fig.9. The mean corrosion potentials of Mg-4.0Zn-

corrosion rate due to the different electrochemical behaviors of α-Mg and precipitates.

cast Mg alloys in Hank's solution.

adherence to the electrochemical results.

xCa alloys are enhanced to -1574mV, which is increased by 70 mV compared with -1646 mV of the pure Mg corrosion potential. However, it is confirmed that Mg-4.0 Zn-0.2 Ca alloy exhibits the best corrosion resistance among Mg-4.0Zn-xCa alloys, even higher than that of Mg-4.0 Zn alloy through further observation. This particular phenomenon can be explained as follows. Firstly, the addition of 4.0 wt. % Zn can cause the formation of coarse MgZn precipitate as shown in Fig.4 and 2(a), which reduces the corrosion resistance of Mg-4.0Zn alloy due to the different electrochemical behaviors between primary α-Mg and precipitate. Then, the slight addition (less than 0.5 wt. %) of Ca alloying element can cause MgZn precipitates to be effectively transform to fine ternary precipitates, which has been clearly documented in previous literature [27][28]. The refinement and homogenization of precipitate phase can improve the corrosion resistance of Mg-4.0Zn-0.2Ca alloy compared with that of Mg-4.0Zn alloy. Finally, increasing Ca content was over than 0.5wt. % cause the formation of another coarse Mg2Ca, Ca2Mg6Zn3 and Ca2Mg5Zn13 precipitates as shown in Fig.3 and 4. It is quite obvious that the precipitates increases with Ca content increasing, which decreases the corrosion resistance of as-cast Mg-4.0Zn-xCa alloys.

Fig. 9. The potentiodynamic polarization curves of as-cast Mg-4.0 Zn-x Ca alloys in Hank's solution.

It is well known that the cathodic polarization curves represent the cathodic hydrogen evolution through water reduction, while the anodic polarization curves do the dissolution of magnesium. That is to say, it is equivalent to that Mg-4.0 Zn-0.2 Ca alloy sample exhibits the lowest current of hydrogen evolution reaction and Mg-4.0 Zn-1.5 Ca and Mg-4.0Zn-2.0 Ca alloys samples does the highest ones, which indicates that over potential of the cathodic hydrogen evolution reaction of Mg-4.0 Zn-0.2 Ca alloy is much lower than those of Mg-4.0 Zn-1.5 Ca and Mg-4.0 Zn-2.0 Ca alloys. Therefore, the lowest cathodic hydrogen evolution reaction brings the highest corrosion resistance to the Mg-4.0 Zn-0.2 Ca alloy.

Fig.10 illustrates the pH variation of Hank's solution versus the immersion testing time for Mg-4.0Zn-xCa alloys. It could be observed that the pH variations of the alloys all obey the parabolic rate law. The pH variation rate decreases with the immersion time increasing. After 48 hrs immersion, all the pH values of the samples tend to be stable. In the early period of immersion, both pure Mg and the Mg-4.0Zn-xCa alloys acutely reacted with

Research on Mg-Zn-Ca Alloy as Degradable Biomaterial 197

Mg and the Mg-4.0Zn-0.2Ca alloy degraded quickly, during the early stage of immersion in SBF, accompanied by the rapid formation of an insoluble protective corrosion layer, which retarded degradation. The degradation process of Mg-4.0Zn-0.2Ca alloy could be roughly summarized as follows: just after immersion in SBF solution, magnesium alloy react with fluids on the surface and get dissolved in the surrounding fluids. With the increasing time of immersion, more Mg2+, Zn2+ and Ca2+ ions were dissolved into the solution, the local pH near the surface of the Mg could be >10[30]. As a result, a magnesium-containing calcium phosphate would precipitate from the SBF solution and deposited on the surface of the

<sup>2</sup> *Mg Mg* 2*e*

2 2 22 2 *H O e H OH*

<sup>2</sup> *Mg* 2 () *OH Mg OH*

4 4 +Ca ( ) *PO Mg Mg Ca PO x y* 

Moreover, when Mg2+, Zn2+ and Ca2+ ions were dissolved into the solution, phosphatecontaining Mg/Ca insoluble protective layer was formed and tightly attached to the matrix. Previous studies [31] have shown that this corrosion layer promotes the osteo-inductivity and osteo-conductivity, predicting good biocompatibility of magnesium and retarded degradation. Therefore, it is proposed that the Mg2+, Zn2+ and Ca2+ released during degradation are safe. Hence, we come to the conclusion that the degradation of the Mg-

E(V) Current(mA/cm2) V(mm/year)

Table 5. Corrosion potential, corrosion current and corrosion rate of Mg-4.0Zn-0.2Ca alloys

The samples after electrochemical measurements were observed by SEM. The typical Surface morphology of Mg-Zn-Ca alloys after electrochemical measurements was shown in Fig.11. Corrosion attack on a large area was observed. At the same time, the filiform corrosion and pitting corrosion were found on the Mg-Zn-Ca alloys sample's surface after electrochemical measurements. The former mainly distributed on the grain boundary, and

XRD patterns of the corrosion products on the surface of Mg-Zn-Ca alloys immersed in Hank's solution were presented in Fig.12. The XRD results suggest that magnesium hydroxide [Mg (OH) 2], other phosphates and hydroxyapatite (HA) were precipitated on the

As-cast -1.60 2.67 2.05 Extruded -1.57 2.43 1.98

2

3- 2+ 2

4.0Zn-0.2Ca alloy was harmless and has good biocompatibility.

**3.3.3 Corrosion morphology and products** 

the latter mostly occurred in second phase location.

Mg-Zn-Ca alloys surface.

magnesium samples, per the following equation:

Anodic reaction:

Cathodic reaction:

Hank's solutions and rapidly generated bubbles. And these reactions of Mg and H2O in Hank's solution generated a large amount of OH- and leaded to the pH values of the solutions be obviously increased. Comparing the pH values of Mg-4.0Zn-xCa alloys, it can be found that the pH variations of pure Mg and Mg-4.0Zn-0.2 Ca alloy are much lower than those of Mg-4.0Zn-1.5Ca and Mg-4.0Zn-2.0Ca alloys. The pH values of Mg-4.0Zn-1.5Ca and Mg-4.0Zn-2.0Ca alloys are remarkably increased to 8.2 from 7.4 after 12hrs immersion tests, which is even equal to those of pure Mg and Mg-4.0Zn-0.2Ca alloy after 96hrs immersion tests. At the end of the immersion tests,the pH values are increased to 8.22 and 8.32 for pure Mg and Mg-4.0 Zn-0.2 Ca alloy, respectively. In particular, the pH value is elevated to 11 from 7.4 for Mg-4.0Zn-2.0Ca alloy. This phenomenon can be explained as follows. The standard potential of coarse second phases of Mg-4.0Zn-1.5Ca and Mg-4.0Zn-2.0Ca alloys is higher than that of the pure Mg. Therefore, the selective attack occurred between α-Mg and the second phase, and the reaction in Hank's solutions is acute. Thus, the pH values of Mg-4.0Zn-1.5Ca and Mg-4.0Zn-2.0Ca alloys are rapidly increased. However, the uniform microstructure and lower reaction rates of pure Mg, Mg-4.0Zn-0.2Ca and Mg-4.0Zn-0.5Ca alloys cause a slow increase of pH values. After 48hrs immersion, bubbles are quite decreased which corresponds a slow reaction rate and leads a slow increase of the pH values for the samples. In addition, the increasing corrosion films including HA and other phosphates formed by the reaction during the immersion test can further reduce the reaction rates or degradation of the alloys [29].

Fig. 10. pH variation of Hank's solution versus the immersion testing time for as-cast Mg-4.0Zn-xCa alloys.

According to the above-mentioned search, we found that Mg-4.0Zn-0.2Ca alloy have an excellent corrosion resistance among the Mg-Zn and the Mg-Zn-Ca alloys. Thus, the immersion test was only performance on the Mg-4.0Zn-0.2Ca alloy. The degradation rates of the alloy after 30-day immersion were listed in Table 5. The degradation rates of Mg-4.0Zn-0.2Ca alloy degraded were slower than pure Mg, which was adherent to the electrochemical results.

Mg and the Mg-4.0Zn-0.2Ca alloy degraded quickly, during the early stage of immersion in SBF, accompanied by the rapid formation of an insoluble protective corrosion layer, which retarded degradation. The degradation process of Mg-4.0Zn-0.2Ca alloy could be roughly summarized as follows: just after immersion in SBF solution, magnesium alloy react with fluids on the surface and get dissolved in the surrounding fluids. With the increasing time of immersion, more Mg2+, Zn2+ and Ca2+ ions were dissolved into the solution, the local pH near the surface of the Mg could be >10[30]. As a result, a magnesium-containing calcium phosphate would precipitate from the SBF solution and deposited on the surface of the magnesium samples, per the following equation:

Anodic reaction:

196 Biomaterials – Physics and Chemistry

Hank's solutions and rapidly generated bubbles. And these reactions of Mg and H2O in Hank's solution generated a large amount of OH- and leaded to the pH values of the solutions be obviously increased. Comparing the pH values of Mg-4.0Zn-xCa alloys, it can be found that the pH variations of pure Mg and Mg-4.0Zn-0.2 Ca alloy are much lower than those of Mg-4.0Zn-1.5Ca and Mg-4.0Zn-2.0Ca alloys. The pH values of Mg-4.0Zn-1.5Ca and Mg-4.0Zn-2.0Ca alloys are remarkably increased to 8.2 from 7.4 after 12hrs immersion tests, which is even equal to those of pure Mg and Mg-4.0Zn-0.2Ca alloy after 96hrs immersion tests. At the end of the immersion tests,the pH values are increased to 8.22 and 8.32 for pure Mg and Mg-4.0 Zn-0.2 Ca alloy, respectively. In particular, the pH value is elevated to 11 from 7.4 for Mg-4.0Zn-2.0Ca alloy. This phenomenon can be explained as follows. The standard potential of coarse second phases of Mg-4.0Zn-1.5Ca and Mg-4.0Zn-2.0Ca alloys is higher than that of the pure Mg. Therefore, the selective attack occurred between α-Mg and the second phase, and the reaction in Hank's solutions is acute. Thus, the pH values of Mg-4.0Zn-1.5Ca and Mg-4.0Zn-2.0Ca alloys are rapidly increased. However, the uniform microstructure and lower reaction rates of pure Mg, Mg-4.0Zn-0.2Ca and Mg-4.0Zn-0.5Ca alloys cause a slow increase of pH values. After 48hrs immersion, bubbles are quite decreased which corresponds a slow reaction rate and leads a slow increase of the pH values for the samples. In addition, the increasing corrosion films including HA and other phosphates formed by the reaction during the immersion test can further reduce the

Fig. 10. pH variation of Hank's solution versus the immersion testing time for as-cast Mg-

According to the above-mentioned search, we found that Mg-4.0Zn-0.2Ca alloy have an excellent corrosion resistance among the Mg-Zn and the Mg-Zn-Ca alloys. Thus, the immersion test was only performance on the Mg-4.0Zn-0.2Ca alloy. The degradation rates of the alloy after 30-day immersion were listed in Table 5. The degradation rates of Mg-4.0Zn-0.2Ca alloy degraded were slower than pure Mg, which was adherent to the electrochemical

reaction rates or degradation of the alloys [29].

4.0Zn-xCa alloys.

results.

$$\text{Mg} \rightarrow \text{Mg}^{+2} + 2e^-$$

Cathodic reaction:

$$2H\_2O + 2e \rightarrow H\_2 + 2OH^-$$

$$Mg^{\cdot 2} + 2OH^- \rightarrow Mg(OH)\_2$$

$$PO\_4^{3-} + \text{Ca}^{2+} + Mg^{2+} \rightarrow Mg\_xCa\_y(PO\_4)\_2$$

Moreover, when Mg2+, Zn2+ and Ca2+ ions were dissolved into the solution, phosphatecontaining Mg/Ca insoluble protective layer was formed and tightly attached to the matrix. Previous studies [31] have shown that this corrosion layer promotes the osteo-inductivity and osteo-conductivity, predicting good biocompatibility of magnesium and retarded degradation. Therefore, it is proposed that the Mg2+, Zn2+ and Ca2+ released during degradation are safe. Hence, we come to the conclusion that the degradation of the Mg-4.0Zn-0.2Ca alloy was harmless and has good biocompatibility.


Table 5. Corrosion potential, corrosion current and corrosion rate of Mg-4.0Zn-0.2Ca alloys

#### **3.3.3 Corrosion morphology and products**

The samples after electrochemical measurements were observed by SEM. The typical Surface morphology of Mg-Zn-Ca alloys after electrochemical measurements was shown in Fig.11. Corrosion attack on a large area was observed. At the same time, the filiform corrosion and pitting corrosion were found on the Mg-Zn-Ca alloys sample's surface after electrochemical measurements. The former mainly distributed on the grain boundary, and the latter mostly occurred in second phase location.

XRD patterns of the corrosion products on the surface of Mg-Zn-Ca alloys immersed in Hank's solution were presented in Fig.12. The XRD results suggest that magnesium hydroxide [Mg (OH) 2], other phosphates and hydroxyapatite (HA) were precipitated on the Mg-Zn-Ca alloys surface.

Research on Mg-Zn-Ca Alloy as Degradable Biomaterial 199

Fig. 13. Morphologies of L-929 cells cultured for 7 day in different extraction media:(a)

Fig. 14. Cell viability cultured in 100% extraction medium for 1, 2, 4 and 7 days.

Negative control, (b) as-cast, (c) extruded.

Fig. 11. The typical surface morphology of Mg-4.0 Zn-0.2 Ca alloy after electrochemical measurements: (a) macrostructure (b) microstructure

Fig. 12. XRD patterns of the corrosion products of Mg-4.0 Zn-0.2 Ca alloy immersed in Hank's solution.

#### **3.4 Cytotoxicity assessments**

The pH values of the extraction medium were measured, and only the values of Mg-1.0 Zn, Mg-2.0 Zn, Mg-3.0 Zn, Mg-4.0 Zn, Mg-4.0 Zn-0.2 Ca and Mg-4.0 Zn-0.5 Ca alloys were below than 8.0. That's mean all of these alloys have a potential probability used as the biomaterials. As the economic reason, only Mg-4.0 Zn-0.2 Ca was selected to evaluate the cytotoxicity through examining both the viability and morphology of L-929 cells in this study.

The morphologies of L-929 cells cultured in different extracts after 7 day incubation were shown in Fig.13. It could be seen that the cell morphologies in different extracts were normal and healthy, which was similar to that of the negative control. Fig.14 shows the RGR of L-929 cells after 2, 4 and 7 days of incubation. There was no significant difference between the RGR of cells in the extracts and those in the negative control. According to standard ISO 10993-5: 1999 [32], the cytotoxicity of these extracts was Grade 0-1. In other words, the Mg-4.0Zn-0.2Ca alloy has a level of biosafety suitable for in cellular applications.

Fig. 11. The typical surface morphology of Mg-4.0 Zn-0.2 Ca alloy after electrochemical

Fig. 12. XRD patterns of the corrosion products of Mg-4.0 Zn-0.2 Ca alloy immersed in

through examining both the viability and morphology of L-929 cells in this study.

4.0Zn-0.2Ca alloy has a level of biosafety suitable for in cellular applications.

The pH values of the extraction medium were measured, and only the values of Mg-1.0 Zn, Mg-2.0 Zn, Mg-3.0 Zn, Mg-4.0 Zn, Mg-4.0 Zn-0.2 Ca and Mg-4.0 Zn-0.5 Ca alloys were below than 8.0. That's mean all of these alloys have a potential probability used as the biomaterials. As the economic reason, only Mg-4.0 Zn-0.2 Ca was selected to evaluate the cytotoxicity

The morphologies of L-929 cells cultured in different extracts after 7 day incubation were shown in Fig.13. It could be seen that the cell morphologies in different extracts were normal and healthy, which was similar to that of the negative control. Fig.14 shows the RGR of L-929 cells after 2, 4 and 7 days of incubation. There was no significant difference between the RGR of cells in the extracts and those in the negative control. According to standard ISO 10993-5: 1999 [32], the cytotoxicity of these extracts was Grade 0-1. In other words, the Mg-

measurements: (a) macrostructure (b) microstructure

Hank's solution.

**3.4 Cytotoxicity assessments** 

Fig. 13. Morphologies of L-929 cells cultured for 7 day in different extraction media:(a) Negative control, (b) as-cast, (c) extruded.

Fig. 14. Cell viability cultured in 100% extraction medium for 1, 2, 4 and 7 days.

Research on Mg-Zn-Ca Alloy as Degradable Biomaterial 201

Fig.16 showed a high magnification microstructure of the bone implant interface after 3 months implantation by SEM. It could be clearly seen that the degradation layer was not dense, and many cracks were found. In order to reveal the chemical composition of the degradation layer, EDS was used to analyze the chemical composition of interface. The results were shown in Fig.16 (b). From the analysis results, it could be figured out that the degradation layer was mainly composed of carbon, oxygen, magnesium, calcium and phosphorous. However, the chemical composition was not homogeneous through the whole layer. At the position close to the Mg implant side, higher calcium content and higher Ca/P ratio were found. At the position close to the bone side, the calcium content was still high, but the Ca/P ratio became much smaller compared with at the position close to the Mg

implant side. However, there was a sharp change in Mg content at the interface.

 Fig. 16. (a) SEM microstructure of the interface between magnesium implant and bone interface after3 month post implantation, (b) EDS analysis patterns of implant and bone

Fig.17 showed the tissue response to the Mg-4.0Zn-0.2Ca alloy pins implantation at 1, 2 and 3 months. It could be clearly seen that some lymphocytes were identified in histological tissue in 1 month after operation, but there was no visible evidence of multinucleated giant cells. After 2 months implantation, there was an active bone formation, which was evident by large number of new disorganized trabeculae. After 3 months implantation, new bone tissue was formed around the magnesium implant. In comparison with the histological microstructure obtained at the cortical bone near the implantation site, as shown in Fig.17, no difference could be found in the histological microstructure between the new bone and

Magnesium alloys have attracted much attention as potential biodegradable bone implant materials due to their biodegradability in the bioenvironmental as well as their excellent mechanical properties such as high strength and an elastic modulus close to that of bone. In this paper, the in-vitro cytotoxicity and in-vivo biocompatibility of new kind of Mg-4.0Zn-0.2Ca alloy was studied. The cytotoxicity test indicated that the Mg-4.0Zn-0.2Ca alloy had no cytotoxicity. Rabbit implantation indicated that the Mg-4.0Zn-0.2Ca alloy did not cause any inflammation reaction. One month after operation, all magnesium implants were fixed tightly. There was no gap between the bone and the residual implant. Optical images from Fig.15 and SEM microstructure from Fig.16 showed clearly that there was a degradation layer formed on the surface of the magnesium implants. Histological images showed that new bone tissue was in contact with the magnesium implant through this degradation layer.

interface after 3 months post implantation.

**3.6 Histological analysis** 

the cortical bone.

The MTT tests were widely used cytotoxicity tests because they are easy, fast and cheap. But, in the case of Mg materials, the use of these test kits leads to false positive or false negative results. It is conceivable that Mg in the highly alkaline environment may be able to open the ring form of the tetrazolium salt and bind to it, which could lead to a change in colors similar to the formation of formazan in the case of the MTT tests with cells [20]. Thus the results of cytotoxicity tests conducted by MTT test will be higher than the true story. It is well known that the neutral red assay and the MTT assay exhibited almost identical reaction patterns for most test materials in L929 cells [33]. Thus, in this study, the neutral red kits were used to assay the cytotoxicity. Its measurement principle is based on the uptake of the vital dye neutral red into lysosomes of viable cells. Neutral red is accumulated because of the low intravesicular pH value. Lysosomes are, however, only one type of subcellular compartments which are acidified by ATP-driven proton pumps (V-type ATPase) and related low intravesicular pH value across vesicle membranes [34]. Accumulation of neutral red in acidic intracellular vesicles needs both ATP as a universal metabolic energy source for proton translocation against an electrochemical H+-gradient and tightly sealed vesicle membrane to maintain potential differences. In an alkaline environment with Mg2+, neutral red could lead to a change in color to yellow. But, the uptake of the vital dye neutral red into lysosomes was red color. The preliminary results of this test show that it seems not to be influenced by corroding Mg. Therefore, the neutral red assay may be regarded as a valid alternative method to determine cell viability, as it shows no interference with the corroding materials. The in-vitro cytotoxicity of Mg-4.0Zn-0.2Ca alloy was found to be Grade 0-1, indicating that the alloy was bio-safe.

#### **3.5 In-vivo degradation**

Furthermore, in order to further study the biocompatibility of Mg-4.0Zn-0.2Ca alloy, the invivo test was conducted on this new type magnesium alloy. Fig.15 showed the optical images of the cross-section of bone and magnesium implants after 3 months implantation. It could be seen that all the shapes of the magnesium implant had been changed from rod shape to irregular shape, indicating the implant was corroded by the body fluid, or the implant degraded in the body fluid. Meanwhile, a degradation layer or a reaction layer could be clearly found on the surface of the alloy implant, as indicated by D in Fig.15. In addition, newly formed bone was observed between the degradation layer and bone tissue around the magnesium alloy implants, as shown by N in Fig.15. The degradation rate was calculated according to the ratios of the cross section area of the residual implant to the original implant. After 3 months implantation, about 35-38% Mg-4.0Zn-0.2Ca alloy implant was degraded. Significant difference (p < 0.05) in the in-vivo degradation rates was observed.

Fig. 15. Optical images of the cross-sections of Mg-Zn-Ca implants and bones after 3 months post implantation (M, metal; D, degradation layer; N, new bone; B,bone).

The MTT tests were widely used cytotoxicity tests because they are easy, fast and cheap. But, in the case of Mg materials, the use of these test kits leads to false positive or false negative results. It is conceivable that Mg in the highly alkaline environment may be able to open the ring form of the tetrazolium salt and bind to it, which could lead to a change in colors similar to the formation of formazan in the case of the MTT tests with cells [20]. Thus the results of cytotoxicity tests conducted by MTT test will be higher than the true story. It is well known that the neutral red assay and the MTT assay exhibited almost identical reaction patterns for most test materials in L929 cells [33]. Thus, in this study, the neutral red kits were used to assay the cytotoxicity. Its measurement principle is based on the uptake of the vital dye neutral red into lysosomes of viable cells. Neutral red is accumulated because of the low intravesicular pH value. Lysosomes are, however, only one type of subcellular compartments which are acidified by ATP-driven proton pumps (V-type ATPase) and related low intravesicular pH value across vesicle membranes [34]. Accumulation of neutral red in acidic intracellular vesicles needs both ATP as a universal metabolic energy source for proton translocation against an electrochemical H+-gradient and tightly sealed vesicle membrane to maintain potential differences. In an alkaline environment with Mg2+, neutral red could lead to a change in color to yellow. But, the uptake of the vital dye neutral red into lysosomes was red color. The preliminary results of this test show that it seems not to be influenced by corroding Mg. Therefore, the neutral red assay may be regarded as a valid alternative method to determine cell viability, as it shows no interference with the corroding materials. The in-vitro cytotoxicity of Mg-4.0Zn-0.2Ca alloy was found to be Grade 0-1, indicating that the alloy was bio-safe.

Furthermore, in order to further study the biocompatibility of Mg-4.0Zn-0.2Ca alloy, the invivo test was conducted on this new type magnesium alloy. Fig.15 showed the optical images of the cross-section of bone and magnesium implants after 3 months implantation. It could be seen that all the shapes of the magnesium implant had been changed from rod shape to irregular shape, indicating the implant was corroded by the body fluid, or the implant degraded in the body fluid. Meanwhile, a degradation layer or a reaction layer could be clearly found on the surface of the alloy implant, as indicated by D in Fig.15. In addition, newly formed bone was observed between the degradation layer and bone tissue around the magnesium alloy implants, as shown by N in Fig.15. The degradation rate was calculated according to the ratios of the cross section area of the residual implant to the original implant. After 3 months implantation, about 35-38% Mg-4.0Zn-0.2Ca alloy implant was degraded.

Fig. 15. Optical images of the cross-sections of Mg-Zn-Ca implants and bones after 3 months

Significant difference (p < 0.05) in the in-vivo degradation rates was observed.

post implantation (M, metal; D, degradation layer; N, new bone; B,bone).

**3.5 In-vivo degradation** 

Fig.16 showed a high magnification microstructure of the bone implant interface after 3 months implantation by SEM. It could be clearly seen that the degradation layer was not dense, and many cracks were found. In order to reveal the chemical composition of the degradation layer, EDS was used to analyze the chemical composition of interface. The results were shown in Fig.16 (b). From the analysis results, it could be figured out that the degradation layer was mainly composed of carbon, oxygen, magnesium, calcium and phosphorous. However, the chemical composition was not homogeneous through the whole layer. At the position close to the Mg implant side, higher calcium content and higher Ca/P ratio were found. At the position close to the bone side, the calcium content was still high, but the Ca/P ratio became much smaller compared with at the position close to the Mg implant side. However, there was a sharp change in Mg content at the interface.

Fig. 16. (a) SEM microstructure of the interface between magnesium implant and bone interface after3 month post implantation, (b) EDS analysis patterns of implant and bone interface after 3 months post implantation.
