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Materials Science and Engineering C 33 (2013) 4144–4154

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Materials Science and Engineering C

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Effect of sterilization process on surface characteristics and biocompatibility of pure Mg and MgCa alloys

X.L. Liu a, W.R. Zhou a, Y.H. Wu a, Y. Cheng a, Y.F. Zheng a,b,c,⁎ a Center for Biomedical Materials and Tissue Engineering, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China b State Key Laboratory for Turbulence and Complex System, College of Engineering, Peking University, Beijing 100871, China c Department of Materials Science and Engineering, College of Engineering, Peking University, Beijing 100871, China

⁎ Corresponding author at: Department of Materials S of Engineering, Peking University, Beijing 100871, China.

E-mail address: [email protected] (Y.F. Zheng).

0928-4931/$ – see front matter © 2013 Elsevier B.V. All http://dx.doi.org/10.1016/j.msec.2013.06.004

a b s t r a c t

a r t i c l e i n f o

Article history: Received 13 February 2013 Received in revised form 14 May 2013 Accepted 4 June 2013 Available online 12 June 2013

Keywords: Sterilization Magnesium alloys Surface characterization Biocompatibility Hemocompatibility

The aim of this work was to investigate the effect of various sterilization methods on surface characteristics and biocompatibility of MgCa alloy, with pure Mg as a comparison, including steam autoclave sterilization (SA), ethylene oxide steam sterilization (EO), glutaraldehyde sterilization (GD), dry heat sterilization (DH) and Co60 γ ray radiation sterilization (R) technologies. The surface characterizations were performed by environ- mental scanning electron microscopy, energy-dispersive X-ray spectroscopy, X-ray photoelectron spectroscopy, grazing incidence X-ray diffraction, water contact angle and surface free energy measurement, whereas the cytotoxicity and hemocompatibility were evaluated by cellular adhesive experiment, platelet adhesion and hemolysis test. The results showed that the five sterilization processes caused more changes on the surface of MgCa alloy than that on the surface of pureMg. The GD sterilization caused themost obvious changes on the sur- face of the pureMg, and the SA sterilizationmade the largest alteration on theMgCa alloy surface. TheGDandDH sterilization processes could cause increases on surface free energy for both pure Mg and MgCa alloys, while the other three sterilization processes reduced the surface free energy. The DH and GD sterilization processes caused the least alteration on the cell adhesion on pure Mg surface, whereas the EO sterilization performed the greatest impact on the cell adhesion on the Mg–Ca alloy surface. The hemolysis percentage of pure Mg and MgCa alloys were reduced by SA sterilization, meanwhile the other four sterilization processes increased their hemolysis percentages significantly, especially for the EO sterilization.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Sterilization is the complete elimination or destruction of all forms of microbial life and is accomplished in hospital by either physical or chemical processes [1]. There are now many different sterilization technologies widely used in hospitals, including steam autoclaving, dry heat, radiation, ethylene oxide, ultraviolet radiation and low temperature steam formaldehyde sterilization, and so on [2–5].

The biocompatibility of any biomaterial is related to the implant– body interactions and for metallic implants it is directly related to the nature of their surfaces at the beginning of and during the implantation. Since sterilization is the final step in manufacturing any implant devices, the effects of the sterilization processes on the changes of biomaterial surface must be clearly understood and characterized [6]. Taking the example of Ti and its alloys, early in 1987, Doundoulakis [7] compared the effects of five sterilization methods on the titanium surface of the implant, including endodontic glass bead sterilizer, steam treatment, dry heat sterilization, ultraviolet treatment and

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radio-frequency glow discharge. The results indicated that surface contamination imparted to materials by traditional sterilization methods could alter surface properties of materials more or less. And then the effects of sterilization on surface characteristics, me- chanical properties and biocompatibility of titanium alloys were reported [6,8–17]. There are also some investigations concerning the effects of sterilization technologies on other biomaterials such as ultra-high molecular weight polyethylene, enamel, dentin and electrostatically spun scaffolds [18–21].

Biodegradable magnesium alloys have been developed as a new kind of metallic implants recently [22]. The majority of the research works are focusing on how to enhance their corrosion resistance [23–25], mechanical properties [26,27] and biocompatibility [28,29]. To the knowledge of the present authors, only Seitz et al. [30] studied the effects of different sterilization methods on the mechanical prop- erties of magnesium and its alloys, there is no report on the effects of sterilization processes on the surface characterization and biocom- patibility for magnesium alloys.

In the present paper, we selected the most widely used sterilization techniques including steam autoclave sterilization (SA), ethylene oxide steam sterilization (EO), glutaraldehyde sterilization (GD), dry heat sterilization (DH) and Co60 γ ray radiation sterilization (R) technologies,

Table 1 Description of sterilization processes and details.

Surface treatments Conditions

Mechanically polished (MP) Mechanically polished to 2000 grit + ultrasonically cleaned in absolute ethanol

MP + ethylene oxide (EO) 450 mg/L-1200 mg/L, 37 °C–63 °C, relative humidity 40%–80%, 1–6 h

MP + steam autoclave (SA) 121 °C, 102.9 kPa, 30 min MP + 2% (GD) 2%, room temperature, >10 h MP + dry heat (DH) 180 °C, 30 min MP + Co60 γ ray radiation (R) 25 KGy, room temperature

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selected pureMg andMgCa alloys as experimentalmaterials, and studied the effect of sterilization process on their surface characteristics and biocompatibility.

Fig. 1. ESEM micrographs of mechanically polished and m

2. Materials and methods

2.1. Material preparation and sterilization

Pure Mg (99.95% purity, as-cast) and MgCa alloys (Mg-1 wt.% Ca, as-extruded) were used in this investigation. The samples were cut into cylinder samples (Φ10 mm × 2 mm), then mechanically polished up to 2000 grit, ultrasonically cleaned in absolute ethanol, and finally dried in open air.

The experimental pure Mg and MgCa alloy samples were sterilized with various sterilization methods including steam autoclave steriliza- tion (SA), ethylene oxide steam sterilization (EO), glutaraldehyde ster- ilization (GD), dry heat sterilization (DH) and Co60 γ ray radiation sterilization (R), with the sterilization parameters being listed in Table 1 in detail, and the unsterilized mechanically polished counterparts being used as controls.

ultiple sterilized pure Mg (a) and MgCa alloys (b).

Fig. 2. XPS spectrograms of mechanically polished and multiple sterilized pure Mg (a) and MgCa alloys (b).

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2.2. Environmental scanning electron microscope (ESEM) and energy- dispersive X-ray spectroscopy (EDS)

An environmental scanning electron microscope (Nova™

NanoSEM430)was used to observe the surfacemorphology and potential alterations caused by different sterilization processes. The energy- disperse spectrometer (EDS) attachment was employed to detect the constitutional elemental compositions of the mechanically-polished and sterilized sample surfaces.

2.3. X-ray photoelectron spectroscopy (XPS)

X-ray photoelectron spectroscopy (Axis Ultra) was used to accom- plish the chemical analysis of the polished and sterilized sample surfaces. The target material was Al Kα, hν = 1486.7 eV, with an operating voltage of 15 kV and an emission current of 15 mA. The data were converted into VAMAS file format and imported into CasaXPS software package for manipulation and curve-fitting.

2.4. Grazing Incidence of X-ray Diffraction (GIXRD)

A polycrystalline X-ray diffraction instrument (D/max2550HB+/PC) with Cu Kα radiation and graphite monochrome filter was used to characterize the phases of the polished and sterilized sample surfaces using the tube voltage of 40 kV at the step size of 0.02° with a scanning speed of 4°/min and an incidence angle θ of 1°.

2.5. Contact angle and surface energy measurements

The surface wettability was tested by contact angle (Dataphysics Instrument, Germany) in triple for each sample. Briefly, a 1.5 μl droplet of deionizedwater or diiodomethanewas suspended from the tip of the microliter syringe supported above the sample stage. The image of the droplet was captured and the contact angle was measured using the OCA20 drop shape analysis program(Dataphysics Instrument, Germany).

The Owens–Wendt plotting procedure was used to obtain the polar, dispersive and total surface energy parameters for the polished and sterilized sample surfaces [6]. The theoretical surface energy of deionized water (γLV = 29.1(γLV

d ) + 43.7(γLV p ) = 72.8 mN/m) and

diiodomethane (γLV = 49.2(γLV d ) + 1.1(γLV

p ) = 50.3 mN/m) were employed to determine surface energy of the materials. The surface energy of thematerials (γS) can be divided into polar compo- nents (γS

p) and dispersive components (γS d). In the Owens–Wendt

plotting procedure the surface energy components of the material γS are related to the measured contact angles θ by the following equation:

γLV 1þ cosθð Þ ¼ 2 γLVdγSd � �1=2 þ γLVpγSp

� �1=2

� � :

Table 2 Elemental compositions of mechanically polished and multiple sterilized pure Mg and Mg–Ca alloy determined by EDS.

Samples Mg (wt.%) O (wt.%) C (wt.%) P (wt.%) Ca (wt.%)

Mg-MP 98.79 1.21 – –

Mg-EO 93.10 3.06 3.84 –

Mg-SA 39.47 50.87 9.66 –

Mg-GD 42.45 24.23 31.45 1.87 Mg-DH 98.64 1.36 – –

Mg-R 97.37 2.63 – –

MgCa-MP 97.42 1.18 – – 1.40 MgCa-EO 93.06 2.02 3.91 – 1.02 MgCa-SA 62.36 24.98 10.85 – 1.81 MgCa-GD 64.48 14.41 19.41 0.92 0.77 MgCa-DH 91.85 2.22 4.26 – 1.67 MgCa-R 96.91 1.66 – – 1.43

2.6. Cell attachment

Human osteosarcoma cells (MG63)were used in the cell attachment experiments. MG63 cells were cultured in MEM, 10% FBS, 100 U·ml−1

penicillin and 100 μg·ml−1 streptomycin in a humidified atmosphere with 5% CO2 at 37 °C. Cells were retrieved from the culture by enzymat- ic dissociation with 0.25% trypsin, then cell suspension concentration was adjusted to approximately 5 × 104 cells/ml. The sterilized pure Mg and MgCa alloy samples were placed in 24 well plates and then 400 μl adjusted cell suspensions were seeded onto the sterilized sur- faces. After 6 h, 12 h and 24 h incubation, the samples were rinsed with PBS for three times and then fixed in 2.5% (v/v) glutaraldehyde solution for 1 h at 37 °C. The samples were then dehydrated in a gradient ethanol/distilled water mixture (50, 60, 70, 80, 90, 95 and 100%) for 15 min each anddried at room temperature. Themorphologies of the MG63 cells adhered to the surfaces of samples were observed by ESEM.

2.6. Hemolysis test

Healthy human blood from a volunteer containing sodium citrate (3.8 wt.%) in the ratio of 9:1 was taken and diluted with normal saline (4:5 ratio by volume). Sterilized pure Mg and MgCa alloy samples were dipped in a standard tube containing 10 ml of normal saline

Fig. 3. GIXRD patterns of mechanically polished and multiple sterilized pure Mg (a) and MgCa alloys (b) at room temperature.

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that were previously incubated at 37 °C for 30 min. Then 0.2 ml of diluted blood was added to this standard tube and the mixtures were incubated for 60 min at 37 °C. Normal saline solution was used as a negative control and deionized water as a positive control. After the incubation, all the tubes were centrifuged for 5 min at

Table 3 Elemental compositions of mechanically polished and multiple sterilized pure Mg and Mg–Ca alloys determined by XPS.

Samples Mg 2p (wt.%)

O 1s (wt.%)

C 1s (wt.%)

N 1s (wt.%)

Ca 2p (wt.%)

P 2p (wt.%)

Mg-MP 30.03 32.97 36.99 – – –

Mg-EO 21.55 29.98 48.47 – – –

Mg-SA 35.38 41.03 23.59 – – –

Mg-GD 16.12 27.26 53.4 0.87 – 2.34 Mg-DH 33.11 34.13 32.76 – – –

Mg-R 20.3 36.71 40.83 2.16 – –

MgCa-MP 25.45 32.98 40.11 1.46 – –

MgCa-EO 24.64 33.59 40.25 1.52 – –

MgCa-SA 24.24 30.80 37.75 – 7.21 –

MgCa-GD 19.37 33.76 38.56 – 5.64 2.66 MgCa-DH 31.53 34.32 34.15 – – –

MgCa-R 25.62 32.09 40.23 2.06 – –

3000 rpm and the supernatant was carefully removed and transferred to the 96-well plate for spectroscopic analysis by a microplate reader (Bio-RAD680) at 545 nm. The hemolysis was calculated based on the average of three replicates.

hemolysis ¼ OD testð Þ−OD negative controlð Þ OD positive controlð Þ−OD negative controlð Þ � 100%

2.7. Platelet adhesion

Platelet-rich plasma (PRP) was prepared by centrifuging the whole blood for 10 min at a rate of 1000 rpm/min. The PRP was over- laid to the experimental samples and incubated at 37 °C for 1 h. After the incubation, the samples were rinsed with PBS three times to re- move the nonadherent platelets. The adhered platelets were fixed in 2.5% glutaraldehyde solutions for 1 h at room temperature followed by dehydration in a gradient ethanol/distilled water mixture (50%, 60%, 70%, 80%, 90% and 100%) for 10 min each and dried at room tem- perature. The surfaces of platelet attached experimental material plates were observed by ESEM.

2.8. Sterility test

All samples were tested for sterility immediately after sterilization. Briefly, the samples were glided across on the agar medium surface lightly, and then the culture dishes were incubated in a humidified atmosphere with 5% CO2 at 37 °C for 48 h. The non-sterilized samples were used as negative controls. Bacterial colonies on the agar medium surface after 48 h indicated contamination and inefficient sterilization, while a clear, uncontaminated agar medium surface indicated efficient sterilization.

3. Results

3.1. Surface morphology and surface chemical characterization

Fig. 1(a) shows the ESEMmicrographs ofmechanically polished and various sterilized pureMg samples.We can see that the GD sterilization caused themost obvious change on the surface morphology of the pure Mg. Obvious cracks and white aggregates in various sizes can be detected on the GD-sterilized pure Mg surface. The surface chemical compositions of mechanically polished and various sterilized pure Mg samples were detected by EDS and XPS, as shown in Table 2 and Fig. 2(a), respectively. The results suggest that the aggregates were composed of C, O, P and Mg elements. Homologous white aggregates can also be detected on the SA-sterilized pure Mg surface.

The surface observation of mechanically polished and various sterilized MgCa alloys shows that the SA sterilization has made the most alteration in morphology on the MgCa alloy surface, as shown in Fig. 1(b). The SA-sterilized MgCa surface possesses the highest quantities of aggregates on top, and slight surface cracks are detected. Similar aggregates with smaller size can also be detected on the GD-sterilized and DH-sterilized MgCa surfaces. The EDS and XPS spec- trogram are shown in Table 2 and Fig. 2(b), respectively, indicating that the aggregates are composed of C, O, P, Ca and Mg elements.

Fig. 3 shows the GIXRD patterns of mechanically polished and vari- ous sterilized pureMg andMgCa alloys at room temperature. Except for the SA-sterilized samples, all samples were composed of one single phase α-Mg, while the SA-sterilized samples were composed of α-Mg and Mg (OH)2.

Table 3 listed the elements of the mechanically polished and ster- ilized Mg andMgCa alloy surfaces measured by XPS. The increase of C, O element content on the sterilized pure Mg and MgCa alloy surfaces was detected by XPS. The significant difference is that P element could be detected on GD-sterilized pure Mg and MgCa alloy surfaces.

Fig. 4. Images of water contact angles of mechanically polished and multiple sterilized pure Mg and MgCa alloys.

Table 4 Contact angles of mechanically polished and multiple sterilized pure Mg and Mg–Ca alloys.

Samples Contact angle Samples Contact angle

Deionized water (°) Diiodomethane (°) Deionized water (°) Diiodomethane (°)

Mg-MP 46.55 ± 0.97 38.18 ± 0.87 MgCa-MP 34.86 ± 2.48 38.50 ± 8.40 Mg-EO 84.34 ± 0.79 64.95 ± 4.03 MgCa-EO 77.00 ± 0.47 52.69 ± 1.79 Mg-SA 59.10 ± 1.53 47.40 ± 2.55 MgCa-SA 77.56 ± 3.84 55.47 ± 1.81 Mg-GD 17.99 ± 5.65 4.64 ± 1.37 MgCa-GD 15.46 ± 4.83 10.03 ± 1.97 Mg-DH 31.53 ± 3.42 35.04 ± 7.89 MgCa-DH 31.96 ± 1.94 31.04 ± 2.83 Mg-R 82.88 ± 5.83 79.30 ± 7.43 MgCa-R 57.40 ± 3.40 49.21 ± 3.07

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3.2. Contact angle and surface free energy measurements

Water contact angles of the samples were measured to identify the surface wettability, the images were showed in Fig. 4 and the values of the water contact angles and surface free energy were listed in Tables 4 and 5, respectively. From the observations it can be seen that only the DH sterilization had no significant effect on water contact angle of pure Mg and MgCa alloy surfaces. EO sterilization, SA steriliza- tion and R sterilization caused the increases of water contact angle on the surface of pure Mg and MgCa alloys, correspondingly a reduction on the surface hydrophilicity. The GD sterilization decreased the water contact angle for pure Mg and MgCa alloy samples and produced a super-hydrophilic surface. At the same time, the GD sterilization and DH sterilization could cause increases on surface free energy of pure Mg and MgCa alloys, while the other three sterilization processes re- duced the surface free energy of pure Mg and MgCa alloys.

Table 5 Surface free energy of mechanically polished and multiple sterilized pure Mg and Mg–Ca a

Samples Surface free energy (mN/m)

γS d γS

p γS = γS d + γS

p

Mg-MP 29.57 ± 0.35 24.42 ± 0.58 54.00 ± 0.67 Mg-EO 21.67 ± 2.64 6.73 ± 5.15 28.41 ± 4.33 Mg-SA 27.00 ± 1.56 17.85 ± 1.53 44.85 ± 0.93 Mg-GD 36.00 ± 0.59 34.69 ± 2.29 70.69 ± 1.70 Mg-DH 28.63 ± 3.23 34.01 ± 1.23 62.64 ± 2.60 Mg-R 13.14 ± 3.84 11.14 ± 4.07 24.28 ± 3.95

3.3. Cell attachment

The results of cell attachment studies were presented in Figs. 5 and 6. With the extension of the incubation time of theMG63 cells, the numbers of the adhered cells gradually increased onMgCa alloy surfaces. However there were few cells adhered on the pure Mg surfaces except for the DH-sterilized and GD-sterilized pure Mg surfaces. Meanwhile a more significant corrosion could be observed on the samples' surface that prolonged the incubation time of the MG63 cells.

Fig. 5 presented the micrographs of the MG63 cells on sterilized pure Mg surface incubated for 6 h, 12 h and 24 h. Although the DH-sterilized and GD-sterilized pure Mg surfaces gained more attached MG63 cells than the other three sterilized sample surfaces, the morphology of the cells were not normally fusiform or polygonous but spheroidal. The corrosion on the EO-sterilized and R-sterilized pure Mg surfaces was more evident and acicular corrosion products could be detected.

lloys.

Samples Surface free energy (mN/m)

γS d γS

p γS = γS d + γS

p

MgCa-MP 27.43 ± 3.77 33.06 ± 1.52 60.50 ± 2.40 MgCa-EO 27.74 ± 1.18 7.39 ± 0.57 35.13 ± 0.66 MgCa-SA 26.13 ± 0.94 7.75 ± 1.87 33.88 ± 1.74 MgCa-GD 35.17 ± 0.41 36.05 ± 1.40 71.22 ± 1.11 MgCa-DH 30.58 ± 0.97 32.41 ± 0.62 62.99 ± 1.31 MgCa-R 25.63 ± 1.14 19.69 ± 1.65 45.32 ± 2.70

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Fig. 6 presented the micrograph of the MG63 cells on sterilized MgCa alloy surface incubated for 6 h, 12 h and 24 h.With the extension of the incubation time of the MG63 cells, the numbers of the adhered cells gradually increased on MgCa alloy surfaces. The SA-sterilized and R-sterilized MgCa alloy surfaces gained more attached spheroidal MG63 cells than the other three sterilized sample surfaces, whereas acicular corrosion products were detected on the EO-sterilized MgCa alloy surface.

3.4. Hemocompatibility

Fig. 7 showed the hemolysis percentage of mechanically polished and various sterilized pure Mg and MgCa alloys The hemolysis percentage of pureMg andMgCa alloys were reduced after SA steriliza- tion, meanwhile the other four sterilization processes increased the hemolysis percentage significantly, especially for the EO sterilization.

Fig. 5. Micrographs of the MG63 cells on sterilized pure Mg

Fig. 8 illustrated the morphologies of human platelets adhering to mechanically polished and various sterilized experimental materials after incubation in PRP for 1 h. It could be seen that platelets adhering to the sample surfaces were nearly round with only one or two short pseudopodia, implying a negative activation. The EO sterilized and SA sterilized experimental materials got the similar number of platelets with the mechanically polished control group, whereas the number of platelets adhering to the other samples was much less. In addition, more corrosion products were observed on the surfaces of DH steril- ized Mg and GD sterilized MgCa samples.

3.5. Sterility test

Sterilization effectiveness was studied by sterility test, there was no presence of bacterial colony except for the Mg-MP agar medium

surfaces incubated for 6 h (a), 12 h (b) and 24 h (c).

Fig. 5 (continued).

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surface after 48 h, indicating that all the sterilization processes employed in the present work were effective.

4. Discussion

There are now many different sterilization processes widely used in hospitals and each process has its advantages and disadvantages. (i) SA sterilization is the most widely used process in hospitals and laboratories, whose advantages are non-toxic, non-corrosive, highly effective and inexpensive, but it is unsuitable for heat-labile items. (ii) DH sterilization can be used to sterilize heat-stable materials, but is not recommended for use in hospitals because it is inefficient com- paredwith autoclaves andmany devices cannot withstand the high tem- peratures involved [31]. (iii) Compared with DH and SA sterilization processes, the compatibility of EO sterilization processes with a wide range of materials and its chemical molecule penetration properties in not so aggressive environments make it the most suitable sterilization process for themajority of heat-sensitive andmoisture-sensitivemedical products, however the disadvantages of EO sterilization processes are its toxicity and high cost [7]. (iv) Irradiation uses γ rays from cobalt-60 at over 25,000 Gy to produce sterility. Co60 γ ray radiation sterilization is ideal for pre-packed heat-labile single-use items and is widely used in industry [31]. (v) Glutaraldehyde is widely used as a cold sterilant to disinfect a variety of heat-sensitive instruments, such as endoscopes, bronchoscopes, and dialysis equipment. But exposure to glutaraldehyde would bring about respiratory effects and anaphylactic reaction [32].

In the present study, the effectiveness of the above five sterilization processes was confirmed by sterility test, but the effect of each ster- ilization processes on surface characteristics and biocompatibility of pure Mg and Mg–Ca alloys are apparently different. The changed microstructure after the Ca addition may be the main reason for the different performance of Mg–1Ca. Instead of a single α phase of pure Mg, Mg–1Ca possesses a second phase of Mg2Ca, although it is not detectable in the present study due to the preferred ori- entation of GIXRD [33].Wan [34] has revealed thatMg2Ca is associated with mechanical property and corrosion property of Mg, and Kirkland [35] has also proved that Mg2Ca intermetallic particles generated by

the alloying with Ca result in systematically enhanced dissolution kinetics.

(1) The change of surface chemistry; Sterilization processes led to a more obvious alteration on morphology of the MgCa alloy sur- faces than the pure Mg surfaces, as shown in Fig. 1. The most plentiful white aggregates were observed on GD-sterilized pure Mg surface and SA-sterilized MgCa alloy surface, followed by EO-sterilized and DH-sterilized samples. This may be the result of different sterilization environments involved (Table 1). Both SA and GD sterilization processes require environments with elevated temperature, pressure and high humidity. In contrast, the Co60 γ ray radiation sterilization process is processed at room temperature and ordinary pressure and has little potential for surface change. As determined by XPS, atomic percentage of carbon and oxygen was increased on the sterilized surfaces, and P element could be detected on GD-sterilized pure Mg and MgCa alloy surfaces (Table 3). The carbon and oxygen composi- tions presented on surfaces may be attributed to the compounds adsorbed from the air and the contaminations we detected in SEM photographs. We tried to characterize effect of sterilization processes on the phases ofmagnesiumalloys byGIXRD, the results showed that the SA-sterilized samples were composed of α-Mg and Mg (OH)2 (Fig. 3). The result was in complete agreement with the data we observed by XPS and EDS, and indicated that the white aggregates which appeared on the sterilized surfaces were the corrosion products of pure Mg and MgCa alloys. According to the data we achieved (Fig. 4 and Table 4), the GD sterilization processes decreased the water contact angles and produced the super-hydrophilic surfaces, in contrast to the other four sterilization processes which increased the water contact angles and made more hydrophobic surfaces. Table 5 listed the surface free energy of the samples, the GD-sterilized and DH-sterilized samples got higher surface free energy, while the other three sterilization processes led to the reduction of surface free energy. The alteration could be ascribed to the contaminations on the sterilized surfaces. As reported bymany au- thors, inorganic contaminants and smoother surfaces are expected

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to decrease the surface energy of metallic surfaces [23]. Surface wettability is one of the most important parameters affecting the biological response to an implanted material. Wettability affects protein adsorption, platelet adhesion/activation, blood coagulation and cell/bacterial adhesion. Generally hydrophilic surfaces are considered to be more protein-adsorbent and suitable for cells [36], thus the decrease of surface hydrophilicity could have a pernicious effect on the biocompatibility of sterilizedmagnesium alloys.

(2) i; Since different sterilization processes result in the change of the pure Mg and MgCa alloy surfaces to a different degree, the biocompatibility of pure Mg and MgCa alloys would be altered correspondingly. With the extension of the incubation time of

Fig. 6. Micrographs of the MG63 cells on sterilized MgCa allo

the MG63 cells, the numbers of the adhered cells gradually in- creased on MgCa alloy surfaces. However there were few cells adhered on the pure Mg surfaces except for the DH-sterilized and GD-sterilized pure Mg surfaces (Figs. 5 and 6). The EO steril- ization process was proved to be the most pernicious one in terms of the MG63 cell adhesion, which may be due to the toxicity of the ethylene oxide. The GD and DH sterilization pro- cesses brought the best biocompatibility for the pureMg sample, whereas the SA and R sterilization processes obtained best biocompatibility for MgCa alloy samples. The hemocompatibility of multiple sterilized pure Mg and MgCa alloys were studied by hemolysis test and platelet adhesion. Fig. 7 shows the hemolysis of mechanically polished and

y surfaces incubated for 6 h (a), 12 h (b) and 24 h (c).

Fig. 6 (continued).

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multiple sterilized pureMg andMgCa alloys, and Fig. 8 illustrates the morphologies of human platelets adhering to mechanically polished and consequently sterilized Mg alloys with various processes after incubation in PRP for 1 h. Because of the toxicity of ethylene oxide, the EO-sterilized pureMg andMgCa alloys got the highest hemolysis percentage and the most platelet adhesion. The other four sterilization processes increased the hemolysis percentage of pure Mg and MgCa alloys at different levels, this may be attributed to the large pH variation after 1 h incubation in saline solution [37]. For platelet adhesion, all of the experimen- tal samples presented negative activation. Especially for DH steril- ized Mg and GD sterilized MgCa, severely corroded morphologies were observed. A thick layer of corrosion products with needle- shaped crystallization were detected, which may be attributed to the more hydrophilic surface after sterilization. To sum up, the Co60 γ ray radiation sterilization comprehensively minimize the effects of the sterilization process on the surface

Fig. 7. Hemolysis percentage of mechanically polished and multiple sterilized pure Mg and MgCa alloys.

chemistry and consequent biocompatibility, and is believed to be the optimal sterilizationmethod for future biomedicalmagnesium alloys.

5. Conclusions

As the final step in manufacturing any implant device, sterilization processes could alter surface characteristics of magnesium alloys, consequently the cytotoxicity and hemocompatibility were changed by various sterilization processes to a different degree. The GD steril- ization caused the most obvious changes on the surface morphology of the pure Mg, while the SA sterilization made the most alteration on the MgCa alloy surface. The GD sterilization and DH sterilization could cause the increases on surface free energy for pure Mg and MgCa alloys, while the other three sterilization methods reduced the surface free energy of pureMg andMgCa alloys. The DH sterilization and GD sterilization caused the least alteration on the cell adhesion on pureMg surface, and the EO sterilization performed the greatest impact on the cell adhesion on the MgCa alloy surface. The hemolysis percent- age of pureMg andMgCa alloyswere reduced by SA sterilization,mean- while the other four sterilization processes increased the hemolysis percentage significantly, especially for the EO sterilization. From what has been discussed above, we could reasonably arrive at the conclusion that the Co60 γ ray radiation sterilization may be the most appropriate sterilization process for biomedical magnesium alloys.

Acknowledgments

Thisworkwas supported byNational Basic Research Programof China (973 Program) (GrantNos. 2012CB619102 and2012CB619100), National High Technology Research and Development Program of China (863 Program) under grant numbers 2011AA030101 and 2011AA030103, National Natural Science Foundation of China (No. 31170909), National Science Fund for Distinguished Young Scholars (Grant No. 51225101), and Research Fund for the Doctoral Program of Higher Education under Grant No. 20100001110011.

Fig. 8. ESEM images of platelets adhering to mechanically polished and multiple sterilized pure Mg (a) and MgCa alloys (b).

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