**2.4.1 Electrochemical measurements**

A typical three-electrode system consisting of graphite rod as counter electrode, saturated calomel electrode (SCE) as a reference electrode and specimen (1cm2 exposed areas) as a working electrode was used. Potentiodynamic polarization experiments were carried out at a scan rate of 0.5 mV/s. The electrochemical measurements of specimens with thickness of 4 mm and a gauge diameter of 15mm were machined from the ingot and ground with 2000 grit SiC paper, and they were rinsed with distilled water and dried by hot air.

#### **2.4.2 Immersion tests**

The immersion tests were carried out in Hank's solution according to ASTM-G31-72 [21]. Samples were removed after 30 days of immersion, rinsed with distilled water, and were cleaned with chromic acid to remove the corrosion products. The degradation rates (in units of mm year-) were obtained according to ASTM-G31-72. An average of five measurements was taken for each group. The pH value of the solution was also recorded in the immersion tests at absolute group for 144 hours.

#### **2.5 Cytotoxicity assessments**

L-929 cells were adopted to evaluate the cytotoxicity of Mg-Zn-Ca alloys. The cells were cultured in Dulbecco's modied Eagle's medium (DMEM), 10% fetal bovine serum (FBS), 100 Uml-1penicillin and 100 mg ml-1 streptomycin at 37 oC in a humidied atmosphere of 5% CO2. The cytotoxicity tests were carried out by indirect contact. Extracts were prepared using DMEM serum free medium as the extraction medium with the surface area of extraction medium ratio 1.25 ml/cm2 in a humidified atmosphere with 5% CO2 at 37 oC for

Research on Mg-Zn-Ca Alloy as Degradable Biomaterial 187

The films were then stained with Hematoxylin and eosin. Histological images were

A t-test was used to determine whether any significant differences existed between the mean values of the cytotoxicity and animal tests of the experiment. The statistical

**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** 

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.

Mg-1.0Zn 0.023 0.976 0.058 0.031 0.004 Balance Mg-2.0Zn 0.033 1.852 0.030 0.039 0.007 Balance Mg-3.0Zn 0.029 2.732 0.022 0.036 0.007 Balance Mg-4.0Zn 0.019 3.925 0.021 0.032 0.008 Balance Mg-5.0Zn 0.027 5.223 0.031 0.034 0.009 Balance Mg-6.0Zn 0.024 5.977 0.019 0.033 0.012 Balance

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,

Al Zn Mn Si Fe Mg

Materials Chemical composition (wt.%)

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

observed on an optical microscope.

significance was defined as P < 0.05.

**3. Results and discussion** 

**2.7 Statistical analysis** 

**as-cast alloys** 

72 h. The supernatant fluid was withdrawn and centrifuged to prepare the extraction medium, then refrigerated at 4 oC before the cytotoxicity test. The control groups involved the use of DMEM medium as negative controls. Cells were incubated in 96-well cell culture plates at 5×104 cells/ml medium in each well and incubated for 24 h to allow attachment. The medium was then replaced with 100μl of extracts. After incubating the cells in a humidified atmosphere with 5% CO2 at 37 oC for 2, 4 and 7 days, respectively, cell morphology was observed by optical microscopy (Nikon ELWD 0.3 inverted microscope).The neutral red viability assay was performed according to published procedures. A stock solution of neutral red (Beyotime, China) was prepared in water (1%). The stock solution was diluted to 50 μg/ml in complete culture medium and 200μl of the staining solution were added to each well after removal of the exposure medium. The cells were incubated for 3 h at 37°C, The cells were then fixed with 200μl formaldehyde/CaCl2(3.7%/l%) and destained with 200μl methanol/glacial acetic acid (50%/l%), The plates were shaken for 60 min at room temperature using a plate shaker. Optical densities were measured at 540 nm in a multiwell spectrophotometer (Bio-RAD 680).The cell relative growth rate (RGR) was calculated according to the following formula:

RGR= ODtest / ODnegative × 100%

#### **2.6 Animal test 2.6.1 Surgery**

Animal tests were approved by the Ethnics Committee of the First Affiliated Hospital of Harbin Medical University. The in-vivo degradation experiments were performed in the animal laboratory of the hospital. A total of 15 adult New Zealand rabbits (6 females), 2.0~2.5kg in weight, were used. In the experimental group, sodium pentobarbital (30mg kg-1) was administered to perform anesthesia by intravenous injection. The sterile Mg-Zn-Ca alloy rod sample was implanted into the femora of the rabbit.

After operation, all animals received a subcutaneous injection of penicillin to avoid a wound contamination and were allowed to move freely in their cages without external support. After operation, five rabbits were sacrificed randomly at 1, 2 and 3 months, respectively.

### **2.6.2 Degradation and histological analysis**

The bone samples with magnesium implants were fixed in 2.5% glutaraldehyde solution and then embedded in epoxy resin for microstructure analysis. The samples were sliced by hard tissue slicer (ZJXL-ZY-200814-1). Samples were made perpendicular to the long axis of the implant to get a cross-section of the implant and surrounding bone tissue. The crosssection microstructure was observed by an optical microscope (Nikon ELWD 0.3 inverted microscope) and a scanning electronic microscope (Hitachi S-5500). The residual implant areas were measured on the cross-section images using analysis software. The ratio of the residual cross-section area of implants to the original cross-section area (residual area/implant area×100%) was used to assess the in vivo degradation rate of magnesium alloys. The element distributions in the residual implants and the degradation layer after 3 months implantation were analyzed.

For histological analysis, the bone samples with magnesium implants were fixed in 4% formaldehyde solution, dehydrated, and then decalcified in ethylene diamine tetra acetate. Then, the specimens were embedded in paraffin and cut into films with 5μm in thickness. The films were then stained with Hematoxylin and eosin. Histological images were observed on an optical microscope.

### **2.7 Statistical analysis**

186 Biomaterials – Physics and Chemistry

72 h. The supernatant fluid was withdrawn and centrifuged to prepare the extraction medium, then refrigerated at 4 oC before the cytotoxicity test. The control groups involved the use of DMEM medium as negative controls. Cells were incubated in 96-well cell culture plates at 5×104 cells/ml medium in each well and incubated for 24 h to allow attachment. The medium was then replaced with 100μl of extracts. After incubating the cells in a humidified atmosphere with 5% CO2 at 37 oC for 2, 4 and 7 days, respectively, cell morphology was observed by optical microscopy (Nikon ELWD 0.3 inverted microscope).The neutral red viability assay was performed according to published procedures. A stock solution of neutral red (Beyotime, China) was prepared in water (1%). The stock solution was diluted to 50 μg/ml in complete culture medium and 200μl of the staining solution were added to each well after removal of the exposure medium. The cells were incubated for 3 h at 37°C, The cells were then fixed with 200μl formaldehyde/CaCl2(3.7%/l%) and destained with 200μl methanol/glacial acetic acid (50%/l%), The plates were shaken for 60 min at room temperature using a plate shaker. Optical densities were measured at 540 nm in a multiwell spectrophotometer (Bio-RAD 680).The cell relative

RGR= ODtest / ODnegative × 100%

Animal tests were approved by the Ethnics Committee of the First Affiliated Hospital of Harbin Medical University. The in-vivo degradation experiments were performed in the animal laboratory of the hospital. A total of 15 adult New Zealand rabbits (6 females), 2.0~2.5kg in weight, were used. In the experimental group, sodium pentobarbital (30mg kg-1) was administered to perform anesthesia by intravenous injection. The sterile Mg-Zn-Ca

After operation, all animals received a subcutaneous injection of penicillin to avoid a wound contamination and were allowed to move freely in their cages without external support. After operation, five rabbits were sacrificed randomly at 1, 2 and 3 months, respectively.

The bone samples with magnesium implants were fixed in 2.5% glutaraldehyde solution and then embedded in epoxy resin for microstructure analysis. The samples were sliced by hard tissue slicer (ZJXL-ZY-200814-1). Samples were made perpendicular to the long axis of the implant to get a cross-section of the implant and surrounding bone tissue. The crosssection microstructure was observed by an optical microscope (Nikon ELWD 0.3 inverted microscope) and a scanning electronic microscope (Hitachi S-5500). The residual implant areas were measured on the cross-section images using analysis software. The ratio of the residual cross-section area of implants to the original cross-section area (residual area/implant area×100%) was used to assess the in vivo degradation rate of magnesium alloys. The element distributions in the residual implants and the degradation layer after 3

For histological analysis, the bone samples with magnesium implants were fixed in 4% formaldehyde solution, dehydrated, and then decalcified in ethylene diamine tetra acetate. Then, the specimens were embedded in paraffin and cut into films with 5μm in thickness.

growth rate (RGR) was calculated according to the following formula:

alloy rod sample was implanted into the femora of the rabbit.

**2.6.2 Degradation and histological analysis** 

months implantation were analyzed.

**2.6 Animal test 2.6.1 Surgery** 

A t-test was used to determine whether any significant differences existed between the mean values of the cytotoxicity and animal tests of the experiment. The statistical significance was defined as P < 0.05.
