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Fluoride Conversion Coating on Biodegradable AZ31B Magnesium Alloy
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ScienceDirect J. Mater. Sci. Technol., 2014,
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Fluoride Conversion Coating on Biodegradable AZ31B Magnesium Alloy Tingting Yan1,2)*, Lili Tan2), Bingchun Zhang2), Ke Yang2) 1) Faculty of Materials Science and Engineering, Kunming University of Science and Technology, Kunming 650093, China 2) Institute of Metal Research, Chinese Academic of Sciences, Shenyang 110016, China [Manuscript received July 29, 2013, in revised form August 27, 2013, Available online xxx]
A fluoride conversion coating was successfully prepared on AZ31B magnesium alloy by chemical reaction in hydrofluoric acid. Morphologies, composition, bonding strength, corrosion properties, in vitro cytotoxicity and antibacterial properties of the coating were investigated, respectively. The scanning electron microscopy observations revealed a dense coating with some irregular pores. The thin-film X-ray diffraction analysis indicated that the coating was mainly composed of MgO and MgF2. The electrochemical impedance spectroscopy results showed that the fluoride conversion coating significantly improved the corrosion resistance of AZ31B. The hydroxyapatite formed on the surface of the fluoride coated AZ31B after being immersed in the simulated blood plasma indicated the good bioactivity of the material. The in vitro cytotoxicity test showed that the fluoride coated AZ31B alloy was not toxic to BMMSCs (human bone marrow-derived mesenchymal stem cells). It was also found that the fluoride coated AZ31B alloy had antibacterial capability. KEY WORDS: AZ31B magnesium; Fluoride coating; Corrosion resistance; Cytotoxicity; Antibacterial
1. Introduction Recently, magnesium alloys have attracted much attention as biodegradable cardiac and orthopaedic implants due to their unique biological properties[1e9]. Biodegradable magnesium implants would provide a solution for a number of problems associated with permanent metallic implants such as permanent physical/mechanical irritation and inability to adapt to growth, and other ongoing shape changes in the human body. However, problems, such as alkalization, hydrogen release and high concentration of magnesium ions[1,10], caused by high corrosion rate of magnesium may strongly limit their clinical application. Thus, the key issue in the development of biodegradable magnesium implant is to confine the corrosion attack to a reasonably low rate. Alloying is one of the effective ways to control the degradability of magnesium. New magnesium alloys, such as MgeZneZr[11], MgeMneZn[5], MgeRE[12], MgeZne MneCa[13], MgeCa[14] etc., have been developed. However, the corrosion rate of those alloys still cannot well meet the practical demand for implants. Another effective way to reduce the corrosion rate of magnesium based materials is the surface Corresponding author. Assoc. Prof., Ph.D.; Tel.: þ86 13698721501; E-mail address: [email protected] (T. Yan). 1005-0302/$ e see front matter Copyright 2014, The editorial office of Journal of Materials Science & Technology. Published by Elsevier Limited. All rights reserved. http://dx.doi.org/10.1016/j.jmst.2013.12.015 *
treatment. As to biomaterials, surface coating is also a way of improving their bioactivity. Thus, it is possible to improve both the surface bioactivity and corrosion resistance of magnesium alloys by proper surface treatment. Many coating techniques have been developed to reduce the corrosion of magnesium alloy, such as calcium phosphate coating[15], hydrogenated amorphous silicon coating[16], polymer-based coating[17], calcium phosphate conversion coating[18], CaeMgeP containing coating[19] and so on. They could improve the surface bioactivity and corrosion resistance of magnesium alloys to different extent, which give the researchers more confidences for application of magnesium implants. Coating techniques, nevertheless, still need to be developed to meet the more serious requirements to the implants. The fluoride is essential in the human diet and is thought to be required for normal dental and skeletal growth[20]. Daily fluoride intake is suggested to be 2e5 mg[21]. It is also one of the few known agents that can stimulate the osteoblast proliferation and increase the new mineral depositions in cancellous bones. The fluoride incorporated into the bone could increase the size and thus decrease the solubility of the bone apatite crystals[22]. The therapeutic future for sodium fluoride in osteoporosis was laid with early administration and low-dose regimens in which toxic levels were avoided and mineralization was not impaired[23]. It was revealed that the SR-NaF group significantly decreased the risk for vertebral fractures and increased the spinal bone mass without reduction of bone mass at the femoral neck and total hip[24]. An experimental study in dogs indicated that the fluoride could modify the implant surface to promote osteointegration in
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the early phase of healing following the implant installation[25]. Fluoride has been used clinically for prevention and treatment of osteoporosis although some controversies still existed. In this work, a magnesium alloy of AZ31B was used as the substrate material. Though the potential harmful element, aluminium (Al), exists in the alloy with a content of 3%, the total amount of aluminium in one set of AZ31B nonbearing bone implant device (including one X-type bone plate and two bone screws) is about 12.0 mg. The release of Al for the device with complete degradation period more than 24 weeks, is much less than a provisional tolerable weekly intake (PTWI) for Al of 7.0 mg/kg body weight established by World Health Organization (WHO)[26]. In addition, AZ31B alloy is a commercial material with high mechanical property and good deformability, and is a promising biodegradable material. In this work, a compact fluoride conversion coating was prepared on the surface of an AZ31B magnesium alloy in order to control its degradation rate and further improve its biocompatibility. The morphology, composition, corrosion behaviour and biocompatibility of the coating were studied. It can be concluded that the fluoride coating can improve not only the corrosion resistance, but also the surface biocompatibility of the magnesium alloy, and the antibacterial property of the coating was revealed in this study as well. 2. Experimental 2.1. Samples preparation An extruded AZ31B magnesium alloy was used in this study, with composition of 1.2%Al, 0.74%Zn, 0.35%Mn, 0.026%Si, 0.003%Fe, 0.0028Cu, 0.0003%Ni (wt%) and balance of Mg. The alloy was cut into small plates with the size of 11 mm 11 mm 3 mm, then mechanically polished up to 2000 grit with SiC paper, and finally ultrasonically rinsed with acetone, absolute ethanol and distilled water successively.
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2.5. Electrochemical impedance spectroscopy test The corrosion resistance of bare substrate and fluoride treated AZ31B samples was evaluated by means of electrochemical impedance spectroscopy (EIS) in the simulated blood plasma, containing NaCl 6.8 g/l, MgSO4 0.1 g/l, NaHCO3 2.2 g/l, Na2HPO4 0.216 g/l, NaH2PO4 0.026 g/l, CaCl2 0.2 g/l and KCl 0.4 g/l, at 37 C. The tests were carried out with a lock-in amplifier (model 5210, EG&G PAR) coupled with a potentiostategalvanostal (model 273A, EG&G PAR), by using a typical three-electrode configuration. The cell consisted of a saturated calomel electrode as the reference, a platinum sheet as the counter electrode and the sample as the working electrode. The working electrode included an electrical connection wire, which was initially attached to the surface of samples with conductive glue, 1 cm2 surface of the sample exposed in the electrolyte and the rest of the sample sealed by epoxy resin. The samples were immersed in the electrolyte for 30 min prior to each test, allowing the system to become equilibrated with the electrolyte. The EIS measurements were carried out in the frequency range of 100 kHz to 10 mHz. The signal amplitude was 10 mV. The EIS data were presented as the Nyquist and Bode plots. 2.6. Immersion test Fluoride treated AZ31B samples were immersed in a simulated blood plasma. The ratio of the specimen area to the solution volume was 3 cm2/ml, according to ISO 10993-12[28]. All the immersion containers were kept at 37 C in a 5% CO2 incubator. The immersion solution was changed every day. At different immersion time points, samples were removed from the simulated blood plasma, gently rinsed with distilled water and dried in air. The surface and cross section morphologies and microstructures of the samples before and after immersions were characterized by scanning electron microscopy (SEM, JEOL JSM-6301F), equipped with energy-disperse spectrometer (EDS, Oxford INCA Energy 300) attachment, and an X-ray diffractometer (XRD, D/max 2500 PC).
2.2. Conversion coating treatment 2.7. Cytotoxicity test The samples were immersed in a hydrofluoric acid solution with concentration of 50 wt% at 30 C for 48 h. The treated samples were then rinsed with distilled water and dried in air.
The cytotoxicity in vitro was measured by MTT assay according to ISO 10993-5[29]. The extract liquid was prepared by immersing samples in RPMI-1640 medium. The volume of
2.3. Surface characterization The morphology of the surface layer was examined by scanning electron microscopy (SEM, JEOL JSM-6301F). The composition of the layer was analysed by thin-film X-ray diffraction (TF-XRD, BRUKER AXS D8 ADVANCE). 2.4. Bonding strength test The bonding strength between fluoride coating and the AZ31B alloy substrate was measured according to ISO 14916[27] and the schematic illustration of the test is shown in Fig. 1, where one fluoride coated sample was vertically adhered to another bare sample using bisphenol-A epoxy resin (HTE-51, Shenyang Southeast Institute of Chemical Industry) with an adhesive strength of about 45 MPa. The test was performed on a universal tensile tester (AG-I 500 kN, Shimadzu) with loading rate of 1 mm/ min. Five experimental values and standard deviations were calculated from fractures of the specimens in the test.
Fig. 1 Schematic illustration of the bonding strength test.
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Table 1 Fitted data of the elements in the model of Fig. 8 Materials
Rp (U cm2)
Rc (U cm2)
Rs (U cm2)
CPEp (F cm2)
CPEc (F cm2)
Bare AZ31B Fluoride coated AZ31B
500 429640
1000 e
24 86.2
7 106 1.2794 105
0.009094 e
solution was calculated based on a volume-to-sample area ratio of 1 ml/3 cm2, which was in accord with ISO10993-12[28]. Monolayer cultures of the BMMSCs (human bone marrowderived mesenchymal stem cells) were initiated at a density of 1 103 cells per 96 well and incubated in 5% CO2 incubator at 37 C for 24 h. After the cell attachment, 50 ml extract liquid was added to each well that was further incubated in 5% CO2 incubator at 37 C for 24 h. After then, the culture fluid was taken out of the wells and 200 ml MTT was added to each well. After incubation at 37 C for 4 h, MTT fluid was taken out of the wells and 150 ml per well dimethyl sulfoxide (DMSO) was added into the wells. The cell contents of the individual cultures were measured colorimetrically. The optical density (O.D.) was recorded at 492 nm by using a micro plate reader (GF M-2000, China), and the cell proliferation rate (P) was defined P ¼
O:D:testmaterial 100% O:D:negative
(1)
The P was used to evaluate the cytotoxicity grade of each group according to the standard of GB/T 16175[30] (see the notation under Table 1). Each experiment was repeated for at least 5 times. 2.8. Antibacterial test The film attachment method[31] was adopted for the antibacterial test, which is one of the most commonly used testing methods for antibacterial properties evaluation of solid materials, quantitatively measuring the antibacterial rate of the material. Antibacterial tests were conducted against a standard Gramnegative bacterium, Escherichia coli (E. coli) ATCC25922. The culturing broth was prepared by dissolving 5 g flesh extract, 5 g NaCl and 10 g peptone into 1000 ml of distilled water and its pH was adjusted to 7.0e7.2. The culturing solution containing the bacteria was diluted to 106 cfu/ml (colony forming units/ml). 0.3 ml of this solution was homogeneously added dropwise by a dispenser onto the surface of samples, which had been horizontally placed into a sterilized Petri-dish, and then covered with sterile plastic film. The bacteria on samples were incubated at 37 C for 24 h in an incubator. After the incubation, the bacterial solution on the sample surface was diluted with 15 ml of phosphate buffer solution, and 0.1 ml of it was added into a Petri-dish. The antibacterial effect was recognized by the antibacterial rate, which was calculated as the following:
Antibacterial rate (%) ¼ 100 (A B)/A
3. Results 3.1. Coating characteristics Both the surface morphology and chemical composition of the fluoride treated samples were examined in this study. The surface morphology of the conversion coating on AZ31B presented in Fig. 2 shows that a black, dense and smooth coating with some small irregular pores scattering was formed on the surface of AZ31B sample. The irregular pores in the coating should be generated by the evolution of hydrogen and could be reduced or filled by precipitations of MgF2 and MgO particles[32] during fluoride conversion treatment. The SEM image of cross section of the fluoride treated sample shown in Fig. 3 indicates that the coating layer adhered well to the AZ31B substrate, with thickness of about 1.9 mm. The result of TF-XRD, as shown in Fig. 4, clearly indicates that the conversion coating is composed of MgF2 and MgO. Since the coating was very thin, the magnesium alloy substrate was also detected and showed high resolution in the spectrum. The result of bonding strength test indicates that the average bond strength between the fluoride coating and substrate is 43.2 5.1 MPa. Fig. 5 shows one of the test curves. However, as one can see from Fig. 6, the fluoride coating on the sample after the test still keeps the original bond with the substrate and the fracture occurs in the epoxy resin layer because the strength of epoxy resin is less than the bonding strength between the fluoride coating and the AZ31B substrate. So it can be deduced that the strength of 43.2 5.1 MPa tested in this study should be the strength of epoxy resin and the bonding strength of the fluoride coating with AZ31 substrate should be more than 43.2 MPa. Bonding strength is, quite naturally, an important property of a coating on materials. It is especially an important property of the coating used for biomaterials. Geng et al.[33] reported that the bonding strength between b-TCP coating and Mg substrate was 16.1 MPa. Lin et al.[34] studied the bonding strength between hydroxyapatite (HA) coating and 316L stainless steel substrate
(2)
where A is the number of bacteria colonies in the Petridish for the contrast stainless steel (316L stainless steel) acting with E. coli, and B is the number of bacteria colonies for the fluoride treated AZ31B acting with E. coli.
Fig. 2 Surface morphology of fluoride coating on AZ31B alloy.
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Fig. 3 SEM micrograph of cross section of fluoride coated AZ31B sample.
and the bonding strength was in a range of 22e37 MPa. Xiao et al.[35] reported that the bonding strength between HA coating and Ti substrate was between 11e22 MPa. According to ASTM 1147-F[36], the minimum bonding strength of 22 MPa is required for a coating on the medical implants. In the present study, all the tested coatings showed high bonding strength and the average bonding strength between the fluoride coating and AZ31B substrate was over 43.2 MPa and meets the requirement of the medical implants although the precise bonding strength value was not measured. The strong bonding between the fluoride coating and AZ31B substrate should come from two aspects, the mechanical interlocking and the chemical bonding, which were developed on the coating-substrate during the coating generation process. 3.2. Corrosion behaviour The EIS spectra of the samples measured in the simulated blood plasma are shown in forms of Nyquist and Bode plots, as shown in Fig. 7. It can be seen from the Nyquist plots (Fig. 7(a)), the capacitance loop diameter of the samples with fluoride conversion coating is much larger than that of the bare sample. It can also be observed that the spectra of the bare AZ31B sample have two loops and those of the fluoride coated sample have only one, the former representing the characteristics of electric double layer. The impedance modulus at different frequencies can be directly read from the Bode spectra, and the capacitive behaviour is indicated by the increase of the impedance with decreasing frequency. In the high frequency region,
Fig. 4 TF-XRD spectrum of fluoride coating on AZ31B alloy.
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Fig. 5 Diagram bonding strength test result.
the impedance is independent of frequency, which is the resistance of the electrolyte between the sample and the reference electrode. At the low frequency limit, the impedance is attributed to the polarization resistance (Rp) of the sample in the electrolyte. As shown in the Bode plots (Fig. 7(b)), the impedance modulus of the fluoride coated AZ31B sample is much higher than that of the bare one. In accordance with the impedance spectra, the equivalent circuits in Fig. 8 are proposed to fit the data. The fitted data are also presented in Fig. 7 and the parameters of the equivalent circuit are given in Table 1. There is a good agreement between the experimental and the fitted data. For the bare AZ31B sample, the circuit was based on the three contributions, the resistance of the electrolyte (Rs), the impedance of the electrolyteeoxide interface (the resistance of oxide layer Rp in parallel with the electrolyteeoxide interface capacitance CPEp), and at the highest frequencies a contribution associated to the oxide-substrate interface (resistance and capacitance of the interface Rc and CPEc). This equivalent circuit is consistent with the presence of two loops found in the experimental Nyquist plots. For the fluoride coated AZ31B samples, the equivalent circuit was composed of the resistance of the electrolyte (Rs) and the
Fig. 6 Photo of fluoride coated AZ31B sample after bonding strength test.
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Fig. 7 EIS spectra of bare and fluoride coated AZ31B samples in the simulated blood plasma: (a) Nyquist plots, (b) Bode plots.
impedance of the fluoride layer (resistance and capacitance of the fluoride layer Rp and CPEp). As shown in Table 1, the Rp of the fluoride coated sample is 429640 U cm2, much larger than that of the bare AZ31B sample (500 U cm2). This difference can also be seen from the Bode plots. The Rc of the substrate is about 1000 U cm2. The circuit capacitance of the fluoride coated AZ31B samples (1.2794 105 F cm2) is much lower than that of the bare AZ31B sample (sum of CPEp and CPEc). The higher Rp and lower CPE clearly demonstrate a lower corrosion rate of the fluoride coated samples than that of the bare sample. The above EIS result confirms that the fluoride coating can offer an effective protection to delay the degradation process of AZ31B magnesium alloy. Fig. 9 shows the surface and cross section morphologies of the AZ31B samples with fluoride conversion coating immersed in the simulated blood plasma for different time intervals. After 5 days immersion, white particles deposit on the surface. After 20 days, a thin deposition coating is formed on the surface and there is no severe pitting corrosion. On the 45th day, an obvious deposition coating with some cracks is found. The deposition is increased and the coating gradually grows up with increasing immersion time. On the 120th day, a thick coating with some cracks composed of spherical crystals, with thickness about 4.3 mm, is formed on the surface. The cracks in the deposition coating are possibly attributed to desorption of water from the coating during the drying process. From the cross section morphologies, it can be seen that the cracks occur in the fluoride conversion coating on the 90th day, indicating the breaking of the fluoride coating. Once the conversion coating broke, the AZ31B substrate would start to degrade. On the 120th day, the
fluoride conversion coating cannot be found. The EDS analysis on the sample immersed for 90 days, as shown in Fig. 10, reveals that the deposition contains Mg, O, F, Ca, P and C, and the Ca/P ratio is about 1.39. In order to identify the structure of the deposition, XRD was conducted on the surface of the fluoride coated sample after immersion in the simulated blood plasma for 90 days, as shown in Fig. 11 with XRD standard JCPDS patterns of Mg, HA, MgF2 and MgO for comparison. It can be found that HA phase was detected from the coating. The formation of HA illustrated the bioactivity of the coating. 3.3. Cytotoxicity test Table 2 shows the result of cytotoxicity test. The relative proliferation rates of BMMSCs on the groups of AZ31B samples with and without fluoride conversion coating were 89.7% and 36.8%, respectively, the corresponding toxicity grade being 1 and 3. The grade 1 means that the tested samples have no cytotoxicity, while the grade 3 represents moderately cytotoxic. Thus it can be concluded that the fluoride coating can eliminate the cytotoxicity of AZ31B magnesium alloy and the fluoride coating has no toxicity to the BMMSCs. 3.4. Antibacterial test Table 3 shows the antibacterial rate of the AZ31B samples with fluoride conversion coating. The antibacterial rate of the contrast 316L stainless steel is 0, while the antibacterial rate of fluoride coated AZ31B sample is over 99.99%, indicating that fluoride conversion coating can provide AZ31B an excellent antibacterial surface. Fig. 12 presents the breeding and agglomeration status of E. coli after actions with the AZ31B with fluoride conversion coating and the contrast stainless steel. It can be clearly seen that after 24 h cultivation of E. coli contacting with the samples, there is almost no bacterium in the dish where the bacteria act with the fluoride coated AZ31B (Fig. 12(a)), but large amount of bacteria in the dish where bacteria act with the contrast stainless steel (Fig. 12(b)), indicating that the bacteria cultivated on the surface of the fluoride coated AZ31B samples have almost been extinguished. 4. Discussion
Fig. 8 Equivalent electrical circuits for bare AZ31B (a) and fluoride coated AZ31B (b) samples.
The rapid corrosion rate of magnesium in the electrolytic physiological environment is one of the greatest limitations for
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Fig. 9 Surface (1) and cross section (2) morphologies of fluoride coated AZ31B immersed in the simulated blood plasma for different time intervals: (a) 5 days, (b) 20 days, (c) 45 days, (d) 90 days, (e) 120 days.
its biomedical applications. The unprotected magnesium exposed to a typical atmosphere will produce a gray oxide film of magnesium hydroxide (Mg(OH)2) which slows the corrosion[37], but the chloride ions (Cl) in physiological environment
are aggressive to magnesium, since the absorption of Cl in the oxide films of the magnesium surface can transform Mg(OH)2 to the easily soluble MgCl2[37]. The following reactions summarize the corrosion reactions to magnesium:
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Table 2 Results of in vitro cytotoxicity test
Control Fluoride coated AZ31B AZ31B
O.D. (sd)
P (%)
Toxicity grade*
0.5476 0.0162 0.4914 0.0517 0.2015 0.0400
100 89.7 36.8
/ 1 3
Notes: * Toxicity grade 0 means P 100, grade 1 means P˛½75%; 99%, grade 2 means P˛½50%; 74%, grade 3 means P˛½25%; 49%, grade 4 means P˛½1%; 24%, grade 5 means P ¼ 0[30].
Fig. 10 EDS analysis result of fluoride coated AZ31B incubated in the simulated blood plasma for 90 days.
TF-XRD result indicates that the coating is mainly composed of MgF2 and MgO. It is well known that Mg can react with hydrofluoric acid via a replacement reaction as the following: Mg þ 2HF/MgF2 þH2 [
MgðsÞ þ 2H2 O/MgðOHÞ2 þH2 ðgÞ
(6)
(3) Simultaneously, an oxidation reaction occurs as the following:
MgðsÞ þ 2Cl ðaqÞ/MgCl2
(4)
MgðOHÞ2 ðsÞ þ 2Cl /MgCl2
(5)
One of the effective ways to reduce the corrosion of magnesium alloys is surface modification. As to biomaterials, a surface modification is also an effective way to improve its bioactivity. Therefore, it is possible to reduce the corrosion of magnesium alloy and improve the surface bioactivity by selecting a proper surface treatment to the alloy. For the above purpose, the present fluoride conversion coating is potentially applied for biomedical magnesium alloy. Li et al.[38] found that the fluoride film deposited on magnesium alloy did not improve its corrosion resistance in the 3.5% NaCl solution and they considered that the fluoride on magnesium alloy surface could only improve its reactivity at the active site. Chiu et al.[39] studied the corrosion property of MgF2 treated pure Mg in Hank’s solution and found an increase in corrosion resistance resulting from the fluoride conversion coating on Mg. In the present work, a remarkably improved corrosion resistance has been obtained for the fluoride coated fluoride AZ31B in the simulated blood plasma, shown by the electrochemical impedance spectroscopy.
Mg þ 2H2 O/MgðOHÞ2 þH2 [
(7)
Since Mg(OH)2 is not stable in the acidic solution[40], reactions should occur as the following: MgðOHÞ2 þ 2HF/MgF2 þ2H2 O
(8)
MgðOHÞ2 /MgO þ H2 O
(9)
These reactions should take place at the film/solution interface[32]. The insoluble MgF2 layer which acts as the passive films can form on the magnesium alloy surface by a chemical reaction. The MgF2 film will grow up with the immersion time, but the growth rate should slow down as the immersion continues[39]. Slow growth of the reaction layer is an indication of the barrier effect of MgF2 to the reaction. The toxicity of fluoride has been investigated. Matsui et al.[41] found that NaF could increase the intracellular Ca2þ concentration, which might be one of common features of the fluoride toxicity. Khalil et al.[42] reported that the fluoride had a sort of cytotoxicity/cytostatic effect on the cultured rat bone marrow cells. It was also found that toxicity of the fluoride was related with its concentration. Kleinsasser et al.[43] investigated the cytotoxicity extent of the fluoride using the trypan blue exclusion test. They found that non vital cells of less than 10% could be shown for the fluoride concentrations of 2 106 to 35 106 and there were 15% and 43% damaged cells after incubation with 71 106 and 213 106 fluoride, respectively.
Table 3 Antibacterial rate of fluoride coated AZ31B (E. coli, 106 cfu/ ml)
Fig. 11 XRD patterns on surface of the fluoride coated AZ31B after immersion in the simulated blood plasma for 90 days: (a) fluoride coated AZ31B, (b) Mg (JCPDS89-5003), (c) HA (JCPDS09-0432), (d) MgF2 (JCPDS 38-0882), (e) MgO (JCPDS87-0652).
Materials
Antibacterial rate
316L stainless steel as a contrast Fluoride coated AZ31B
0 > 99.99%
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Fig. 12 Photos of breeding status of E. coli after actions with fluoride coated AZ31B (a) and 316L stainless steel (b).
Meanwhile, the interaction between fluoride and magnesium has also been investigated. Colly et al.[44] found that fluoride ion affected the enamel hardening and prevented its annealing, but this effect diminished after administration of Mg. O’Dell et al.[45] studied the interaction of dietary fluoride and magnesium in guinea pigs, and they found that the fluoride in the food and water consumed by man would be particularly valuable if magnesium was limiting in the diet. One the other hand, a high intake of magnesium should be highly beneficial in areas where fluorosis prevails and the toxicity of fluoride decreased with increasing amount of magnesium in the diet of the guinea pigs[45]. In the present study, the in vitro cytotoxicity evaluation results demonstrated that AZ31B alloy samples with fluoride conversion coating were not toxic to BMMSCs, indicating the stability of compound MgF2 formed in the conversion coating with safe concentration of fluoride released into the cell culture medium. By comparing with that on fluoride conversion coating, the relative proliferation rate of BMMSCs on the groups of bare AZ31B samples was 35.8%, indicating moderately cytotoxic. It is mainly because the corrosion rate of bare AZ31B is too fast that results in the higher pH values in MTT. Groos et al.[46] indicated that colony-forming ability of RT112 cells was reduced significantly at pH values greater than 8.4 after 24 h exposure. Meanwhile, the hydrogen released during the bare AZ31B samples degrading in the solution also had some effects on cell growth[47]. All the above results proved that the fluoride conversion coating can provide AZ31B with lower biodegradation rate and decreased cytotoxicity. Surface morphologies of the samples with fluoride conversion coating after immersion in simulated blood plasma illustrated that the bioactive HA were formed on the surface of samples. For bone implants, the prerequisite to bond to living bone is the formation of biologically active apatites on their surface in the body. The apatite layer bridges chemically the bone and the implant. Thus, the formation of HA illustrates the bioactivity of the fluoride conversion coating and that is beneficial to the application of AZ31B with fluoride conversion coating as bone implants. It was reported that a bonelike apatite layer was observed after only 3 days of bare magnesium immersing in SBF, indicating the bioactive of magnesium[48]. Thus, we can deduce that the release of magnesium ions from fluoride conversion coating and AZ31B substrate is one of the contributions to the bioactivity of AZ31B with fluoride
conversion coating. Meanwhile, fluoride can enhance osteoblastic differentiation and interfacial bone formation[49]. Bernstein et al.[50] also reported that the fluoride could prevent the osteoporosis and provoke the metastatic calcification. So the fluoride in the conversion coating is a possible contribution to the bioactivity of the sample in this study. Already in 1940, it was demonstrated that the carbohydrate metabolism in pure cultures of oral streptococci and lactobacilli was inhibited by the fluoride[51]. Since then, many reports have been published about the effect of fluoride on bacteria[52e54]. Yoshinari et al.[55] demonstrated that the bacteria were injured by the pharmacological effect of the metal-fluoride complexes with their inhibition of enzymatic activity. In this work, the fluoride coated AZ31B samples well showed good antibacterial capability, which might be due to the pharmacological effect of MgeF complex (MgF2) in the conversion coating. As for biomedical implants and devices, infection caused by bacteria from peroperative contamination during the surgery is the primary cause for failure. Therefore, biomaterials with anti-microbial properties are highly desirable because the reducing risk of infection. Thus, the antibacterial property of the fluoride coated AZ31B samples is beneficial to their application as the medical implants. 5. Conclusion A compact fluoride conversion coating was successfully prepared on an AZ31B magnesium alloy. The coating was mainly composed of MgF2 and MgO. The bonding strength between the fluoride coating and AZ31B substrate was over 43.2 5.1 MPa. Electrochemical tests showed an improved corrosion resistance for the fluoride coated AZ31B alloy in the simulated blood plasma compared with that of the bare AZ31B alloy. The in vitro cytotoxicity test showed that the fluoride coated AZ31B was not toxic to BMMSCs. The fluoride coated AZ31B had antibacterial capability. To conclude, the fluoride conversion treatment is an effective way for application by using AZ31B magnesium alloy as a biomaterial. Acknowledgements The authors thank the financial support of the National Basic Research Program of China (973 Program, No. 2012CB619101) and the Basic Application Research of Yunnan Province (No. KKSA201151053).
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Fluoride Conversion Coating on Biodegradable AZ31B Magnesium Alloy Tingting Yan1,2)*, Lili Tan2), Bingchun Zhang2), Ke Yang2) 1) Faculty of Materials Science and Engineering, Kunming University of Science and Technology, Kunming 650093, China 2) Institute of Metal Research, Chinese Academic of Sciences, Shenyang 110016, China [Manuscript received July 29, 2013, in revised form August 27, 2013, Available online xxx]
A fluoride conversion coating was successfully prepared on AZ31B magnesium alloy by chemical reaction in hydrofluoric acid. Morphologies, composition, bonding strength, corrosion properties, in vitro cytotoxicity and antibacterial properties of the coating were investigated, respectively. The scanning electron microscopy observations revealed a dense coating with some irregular pores. The thin-film X-ray diffraction analysis indicated that the coating was mainly composed of MgO and MgF2. The electrochemical impedance spectroscopy results showed that the fluoride conversion coating significantly improved the corrosion resistance of AZ31B. The hydroxyapatite formed on the surface of the fluoride coated AZ31B after being immersed in the simulated blood plasma indicated the good bioactivity of the material. The in vitro cytotoxicity test showed that the fluoride coated AZ31B alloy was not toxic to BMMSCs (human bone marrow-derived mesenchymal stem cells). It was also found that the fluoride coated AZ31B alloy had antibacterial capability. KEY WORDS: AZ31B magnesium; Fluoride coating; Corrosion resistance; Cytotoxicity; Antibacterial
1. Introduction Recently, magnesium alloys have attracted much attention as biodegradable cardiac and orthopaedic implants due to their unique biological properties[1e9]. Biodegradable magnesium implants would provide a solution for a number of problems associated with permanent metallic implants such as permanent physical/mechanical irritation and inability to adapt to growth, and other ongoing shape changes in the human body. However, problems, such as alkalization, hydrogen release and high concentration of magnesium ions[1,10], caused by high corrosion rate of magnesium may strongly limit their clinical application. Thus, the key issue in the development of biodegradable magnesium implant is to confine the corrosion attack to a reasonably low rate. Alloying is one of the effective ways to control the degradability of magnesium. New magnesium alloys, such as MgeZneZr[11], MgeMneZn[5], MgeRE[12], MgeZne MneCa[13], MgeCa[14] etc., have been developed. However, the corrosion rate of those alloys still cannot well meet the practical demand for implants. Another effective way to reduce the corrosion rate of magnesium based materials is the surface Corresponding author. Assoc. Prof., Ph.D.; Tel.: þ86 13698721501; E-mail address: [email protected] (T. Yan). 1005-0302/$ e see front matter Copyright 2014, The editorial office of Journal of Materials Science & Technology. Published by Elsevier Limited. All rights reserved. http://dx.doi.org/10.1016/j.jmst.2013.12.015 *
treatment. As to biomaterials, surface coating is also a way of improving their bioactivity. Thus, it is possible to improve both the surface bioactivity and corrosion resistance of magnesium alloys by proper surface treatment. Many coating techniques have been developed to reduce the corrosion of magnesium alloy, such as calcium phosphate coating[15], hydrogenated amorphous silicon coating[16], polymer-based coating[17], calcium phosphate conversion coating[18], CaeMgeP containing coating[19] and so on. They could improve the surface bioactivity and corrosion resistance of magnesium alloys to different extent, which give the researchers more confidences for application of magnesium implants. Coating techniques, nevertheless, still need to be developed to meet the more serious requirements to the implants. The fluoride is essential in the human diet and is thought to be required for normal dental and skeletal growth[20]. Daily fluoride intake is suggested to be 2e5 mg[21]. It is also one of the few known agents that can stimulate the osteoblast proliferation and increase the new mineral depositions in cancellous bones. The fluoride incorporated into the bone could increase the size and thus decrease the solubility of the bone apatite crystals[22]. The therapeutic future for sodium fluoride in osteoporosis was laid with early administration and low-dose regimens in which toxic levels were avoided and mineralization was not impaired[23]. It was revealed that the SR-NaF group significantly decreased the risk for vertebral fractures and increased the spinal bone mass without reduction of bone mass at the femoral neck and total hip[24]. An experimental study in dogs indicated that the fluoride could modify the implant surface to promote osteointegration in
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the early phase of healing following the implant installation[25]. Fluoride has been used clinically for prevention and treatment of osteoporosis although some controversies still existed. In this work, a magnesium alloy of AZ31B was used as the substrate material. Though the potential harmful element, aluminium (Al), exists in the alloy with a content of 3%, the total amount of aluminium in one set of AZ31B nonbearing bone implant device (including one X-type bone plate and two bone screws) is about 12.0 mg. The release of Al for the device with complete degradation period more than 24 weeks, is much less than a provisional tolerable weekly intake (PTWI) for Al of 7.0 mg/kg body weight established by World Health Organization (WHO)[26]. In addition, AZ31B alloy is a commercial material with high mechanical property and good deformability, and is a promising biodegradable material. In this work, a compact fluoride conversion coating was prepared on the surface of an AZ31B magnesium alloy in order to control its degradation rate and further improve its biocompatibility. The morphology, composition, corrosion behaviour and biocompatibility of the coating were studied. It can be concluded that the fluoride coating can improve not only the corrosion resistance, but also the surface biocompatibility of the magnesium alloy, and the antibacterial property of the coating was revealed in this study as well. 2. Experimental 2.1. Samples preparation An extruded AZ31B magnesium alloy was used in this study, with composition of 1.2%Al, 0.74%Zn, 0.35%Mn, 0.026%Si, 0.003%Fe, 0.0028Cu, 0.0003%Ni (wt%) and balance of Mg. The alloy was cut into small plates with the size of 11 mm 11 mm 3 mm, then mechanically polished up to 2000 grit with SiC paper, and finally ultrasonically rinsed with acetone, absolute ethanol and distilled water successively.
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2.5. Electrochemical impedance spectroscopy test The corrosion resistance of bare substrate and fluoride treated AZ31B samples was evaluated by means of electrochemical impedance spectroscopy (EIS) in the simulated blood plasma, containing NaCl 6.8 g/l, MgSO4 0.1 g/l, NaHCO3 2.2 g/l, Na2HPO4 0.216 g/l, NaH2PO4 0.026 g/l, CaCl2 0.2 g/l and KCl 0.4 g/l, at 37 C. The tests were carried out with a lock-in amplifier (model 5210, EG&G PAR) coupled with a potentiostategalvanostal (model 273A, EG&G PAR), by using a typical three-electrode configuration. The cell consisted of a saturated calomel electrode as the reference, a platinum sheet as the counter electrode and the sample as the working electrode. The working electrode included an electrical connection wire, which was initially attached to the surface of samples with conductive glue, 1 cm2 surface of the sample exposed in the electrolyte and the rest of the sample sealed by epoxy resin. The samples were immersed in the electrolyte for 30 min prior to each test, allowing the system to become equilibrated with the electrolyte. The EIS measurements were carried out in the frequency range of 100 kHz to 10 mHz. The signal amplitude was 10 mV. The EIS data were presented as the Nyquist and Bode plots. 2.6. Immersion test Fluoride treated AZ31B samples were immersed in a simulated blood plasma. The ratio of the specimen area to the solution volume was 3 cm2/ml, according to ISO 10993-12[28]. All the immersion containers were kept at 37 C in a 5% CO2 incubator. The immersion solution was changed every day. At different immersion time points, samples were removed from the simulated blood plasma, gently rinsed with distilled water and dried in air. The surface and cross section morphologies and microstructures of the samples before and after immersions were characterized by scanning electron microscopy (SEM, JEOL JSM-6301F), equipped with energy-disperse spectrometer (EDS, Oxford INCA Energy 300) attachment, and an X-ray diffractometer (XRD, D/max 2500 PC).
2.2. Conversion coating treatment 2.7. Cytotoxicity test The samples were immersed in a hydrofluoric acid solution with concentration of 50 wt% at 30 C for 48 h. The treated samples were then rinsed with distilled water and dried in air.
The cytotoxicity in vitro was measured by MTT assay according to ISO 10993-5[29]. The extract liquid was prepared by immersing samples in RPMI-1640 medium. The volume of
2.3. Surface characterization The morphology of the surface layer was examined by scanning electron microscopy (SEM, JEOL JSM-6301F). The composition of the layer was analysed by thin-film X-ray diffraction (TF-XRD, BRUKER AXS D8 ADVANCE). 2.4. Bonding strength test The bonding strength between fluoride coating and the AZ31B alloy substrate was measured according to ISO 14916[27] and the schematic illustration of the test is shown in Fig. 1, where one fluoride coated sample was vertically adhered to another bare sample using bisphenol-A epoxy resin (HTE-51, Shenyang Southeast Institute of Chemical Industry) with an adhesive strength of about 45 MPa. The test was performed on a universal tensile tester (AG-I 500 kN, Shimadzu) with loading rate of 1 mm/ min. Five experimental values and standard deviations were calculated from fractures of the specimens in the test.
Fig. 1 Schematic illustration of the bonding strength test.
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Table 1 Fitted data of the elements in the model of Fig. 8 Materials
Rp (U cm2)
Rc (U cm2)
Rs (U cm2)
CPEp (F cm2)
CPEc (F cm2)
Bare AZ31B Fluoride coated AZ31B
500 429640
1000 e
24 86.2
7 106 1.2794 105
0.009094 e
solution was calculated based on a volume-to-sample area ratio of 1 ml/3 cm2, which was in accord with ISO10993-12[28]. Monolayer cultures of the BMMSCs (human bone marrowderived mesenchymal stem cells) were initiated at a density of 1 103 cells per 96 well and incubated in 5% CO2 incubator at 37 C for 24 h. After the cell attachment, 50 ml extract liquid was added to each well that was further incubated in 5% CO2 incubator at 37 C for 24 h. After then, the culture fluid was taken out of the wells and 200 ml MTT was added to each well. After incubation at 37 C for 4 h, MTT fluid was taken out of the wells and 150 ml per well dimethyl sulfoxide (DMSO) was added into the wells. The cell contents of the individual cultures were measured colorimetrically. The optical density (O.D.) was recorded at 492 nm by using a micro plate reader (GF M-2000, China), and the cell proliferation rate (P) was defined P ¼
O:D:testmaterial 100% O:D:negative
(1)
The P was used to evaluate the cytotoxicity grade of each group according to the standard of GB/T 16175[30] (see the notation under Table 1). Each experiment was repeated for at least 5 times. 2.8. Antibacterial test The film attachment method[31] was adopted for the antibacterial test, which is one of the most commonly used testing methods for antibacterial properties evaluation of solid materials, quantitatively measuring the antibacterial rate of the material. Antibacterial tests were conducted against a standard Gramnegative bacterium, Escherichia coli (E. coli) ATCC25922. The culturing broth was prepared by dissolving 5 g flesh extract, 5 g NaCl and 10 g peptone into 1000 ml of distilled water and its pH was adjusted to 7.0e7.2. The culturing solution containing the bacteria was diluted to 106 cfu/ml (colony forming units/ml). 0.3 ml of this solution was homogeneously added dropwise by a dispenser onto the surface of samples, which had been horizontally placed into a sterilized Petri-dish, and then covered with sterile plastic film. The bacteria on samples were incubated at 37 C for 24 h in an incubator. After the incubation, the bacterial solution on the sample surface was diluted with 15 ml of phosphate buffer solution, and 0.1 ml of it was added into a Petri-dish. The antibacterial effect was recognized by the antibacterial rate, which was calculated as the following:
Antibacterial rate (%) ¼ 100 (A B)/A
3. Results 3.1. Coating characteristics Both the surface morphology and chemical composition of the fluoride treated samples were examined in this study. The surface morphology of the conversion coating on AZ31B presented in Fig. 2 shows that a black, dense and smooth coating with some small irregular pores scattering was formed on the surface of AZ31B sample. The irregular pores in the coating should be generated by the evolution of hydrogen and could be reduced or filled by precipitations of MgF2 and MgO particles[32] during fluoride conversion treatment. The SEM image of cross section of the fluoride treated sample shown in Fig. 3 indicates that the coating layer adhered well to the AZ31B substrate, with thickness of about 1.9 mm. The result of TF-XRD, as shown in Fig. 4, clearly indicates that the conversion coating is composed of MgF2 and MgO. Since the coating was very thin, the magnesium alloy substrate was also detected and showed high resolution in the spectrum. The result of bonding strength test indicates that the average bond strength between the fluoride coating and substrate is 43.2 5.1 MPa. Fig. 5 shows one of the test curves. However, as one can see from Fig. 6, the fluoride coating on the sample after the test still keeps the original bond with the substrate and the fracture occurs in the epoxy resin layer because the strength of epoxy resin is less than the bonding strength between the fluoride coating and the AZ31B substrate. So it can be deduced that the strength of 43.2 5.1 MPa tested in this study should be the strength of epoxy resin and the bonding strength of the fluoride coating with AZ31 substrate should be more than 43.2 MPa. Bonding strength is, quite naturally, an important property of a coating on materials. It is especially an important property of the coating used for biomaterials. Geng et al.[33] reported that the bonding strength between b-TCP coating and Mg substrate was 16.1 MPa. Lin et al.[34] studied the bonding strength between hydroxyapatite (HA) coating and 316L stainless steel substrate
(2)
where A is the number of bacteria colonies in the Petridish for the contrast stainless steel (316L stainless steel) acting with E. coli, and B is the number of bacteria colonies for the fluoride treated AZ31B acting with E. coli.
Fig. 2 Surface morphology of fluoride coating on AZ31B alloy.
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Fig. 3 SEM micrograph of cross section of fluoride coated AZ31B sample.
and the bonding strength was in a range of 22e37 MPa. Xiao et al.[35] reported that the bonding strength between HA coating and Ti substrate was between 11e22 MPa. According to ASTM 1147-F[36], the minimum bonding strength of 22 MPa is required for a coating on the medical implants. In the present study, all the tested coatings showed high bonding strength and the average bonding strength between the fluoride coating and AZ31B substrate was over 43.2 MPa and meets the requirement of the medical implants although the precise bonding strength value was not measured. The strong bonding between the fluoride coating and AZ31B substrate should come from two aspects, the mechanical interlocking and the chemical bonding, which were developed on the coating-substrate during the coating generation process. 3.2. Corrosion behaviour The EIS spectra of the samples measured in the simulated blood plasma are shown in forms of Nyquist and Bode plots, as shown in Fig. 7. It can be seen from the Nyquist plots (Fig. 7(a)), the capacitance loop diameter of the samples with fluoride conversion coating is much larger than that of the bare sample. It can also be observed that the spectra of the bare AZ31B sample have two loops and those of the fluoride coated sample have only one, the former representing the characteristics of electric double layer. The impedance modulus at different frequencies can be directly read from the Bode spectra, and the capacitive behaviour is indicated by the increase of the impedance with decreasing frequency. In the high frequency region,
Fig. 4 TF-XRD spectrum of fluoride coating on AZ31B alloy.
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Fig. 5 Diagram bonding strength test result.
the impedance is independent of frequency, which is the resistance of the electrolyte between the sample and the reference electrode. At the low frequency limit, the impedance is attributed to the polarization resistance (Rp) of the sample in the electrolyte. As shown in the Bode plots (Fig. 7(b)), the impedance modulus of the fluoride coated AZ31B sample is much higher than that of the bare one. In accordance with the impedance spectra, the equivalent circuits in Fig. 8 are proposed to fit the data. The fitted data are also presented in Fig. 7 and the parameters of the equivalent circuit are given in Table 1. There is a good agreement between the experimental and the fitted data. For the bare AZ31B sample, the circuit was based on the three contributions, the resistance of the electrolyte (Rs), the impedance of the electrolyteeoxide interface (the resistance of oxide layer Rp in parallel with the electrolyteeoxide interface capacitance CPEp), and at the highest frequencies a contribution associated to the oxide-substrate interface (resistance and capacitance of the interface Rc and CPEc). This equivalent circuit is consistent with the presence of two loops found in the experimental Nyquist plots. For the fluoride coated AZ31B samples, the equivalent circuit was composed of the resistance of the electrolyte (Rs) and the
Fig. 6 Photo of fluoride coated AZ31B sample after bonding strength test.
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Fig. 7 EIS spectra of bare and fluoride coated AZ31B samples in the simulated blood plasma: (a) Nyquist plots, (b) Bode plots.
impedance of the fluoride layer (resistance and capacitance of the fluoride layer Rp and CPEp). As shown in Table 1, the Rp of the fluoride coated sample is 429640 U cm2, much larger than that of the bare AZ31B sample (500 U cm2). This difference can also be seen from the Bode plots. The Rc of the substrate is about 1000 U cm2. The circuit capacitance of the fluoride coated AZ31B samples (1.2794 105 F cm2) is much lower than that of the bare AZ31B sample (sum of CPEp and CPEc). The higher Rp and lower CPE clearly demonstrate a lower corrosion rate of the fluoride coated samples than that of the bare sample. The above EIS result confirms that the fluoride coating can offer an effective protection to delay the degradation process of AZ31B magnesium alloy. Fig. 9 shows the surface and cross section morphologies of the AZ31B samples with fluoride conversion coating immersed in the simulated blood plasma for different time intervals. After 5 days immersion, white particles deposit on the surface. After 20 days, a thin deposition coating is formed on the surface and there is no severe pitting corrosion. On the 45th day, an obvious deposition coating with some cracks is found. The deposition is increased and the coating gradually grows up with increasing immersion time. On the 120th day, a thick coating with some cracks composed of spherical crystals, with thickness about 4.3 mm, is formed on the surface. The cracks in the deposition coating are possibly attributed to desorption of water from the coating during the drying process. From the cross section morphologies, it can be seen that the cracks occur in the fluoride conversion coating on the 90th day, indicating the breaking of the fluoride coating. Once the conversion coating broke, the AZ31B substrate would start to degrade. On the 120th day, the
fluoride conversion coating cannot be found. The EDS analysis on the sample immersed for 90 days, as shown in Fig. 10, reveals that the deposition contains Mg, O, F, Ca, P and C, and the Ca/P ratio is about 1.39. In order to identify the structure of the deposition, XRD was conducted on the surface of the fluoride coated sample after immersion in the simulated blood plasma for 90 days, as shown in Fig. 11 with XRD standard JCPDS patterns of Mg, HA, MgF2 and MgO for comparison. It can be found that HA phase was detected from the coating. The formation of HA illustrated the bioactivity of the coating. 3.3. Cytotoxicity test Table 2 shows the result of cytotoxicity test. The relative proliferation rates of BMMSCs on the groups of AZ31B samples with and without fluoride conversion coating were 89.7% and 36.8%, respectively, the corresponding toxicity grade being 1 and 3. The grade 1 means that the tested samples have no cytotoxicity, while the grade 3 represents moderately cytotoxic. Thus it can be concluded that the fluoride coating can eliminate the cytotoxicity of AZ31B magnesium alloy and the fluoride coating has no toxicity to the BMMSCs. 3.4. Antibacterial test Table 3 shows the antibacterial rate of the AZ31B samples with fluoride conversion coating. The antibacterial rate of the contrast 316L stainless steel is 0, while the antibacterial rate of fluoride coated AZ31B sample is over 99.99%, indicating that fluoride conversion coating can provide AZ31B an excellent antibacterial surface. Fig. 12 presents the breeding and agglomeration status of E. coli after actions with the AZ31B with fluoride conversion coating and the contrast stainless steel. It can be clearly seen that after 24 h cultivation of E. coli contacting with the samples, there is almost no bacterium in the dish where the bacteria act with the fluoride coated AZ31B (Fig. 12(a)), but large amount of bacteria in the dish where bacteria act with the contrast stainless steel (Fig. 12(b)), indicating that the bacteria cultivated on the surface of the fluoride coated AZ31B samples have almost been extinguished. 4. Discussion
Fig. 8 Equivalent electrical circuits for bare AZ31B (a) and fluoride coated AZ31B (b) samples.
The rapid corrosion rate of magnesium in the electrolytic physiological environment is one of the greatest limitations for
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Fig. 9 Surface (1) and cross section (2) morphologies of fluoride coated AZ31B immersed in the simulated blood plasma for different time intervals: (a) 5 days, (b) 20 days, (c) 45 days, (d) 90 days, (e) 120 days.
its biomedical applications. The unprotected magnesium exposed to a typical atmosphere will produce a gray oxide film of magnesium hydroxide (Mg(OH)2) which slows the corrosion[37], but the chloride ions (Cl) in physiological environment
are aggressive to magnesium, since the absorption of Cl in the oxide films of the magnesium surface can transform Mg(OH)2 to the easily soluble MgCl2[37]. The following reactions summarize the corrosion reactions to magnesium:
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Table 2 Results of in vitro cytotoxicity test
Control Fluoride coated AZ31B AZ31B
O.D. (sd)
P (%)
Toxicity grade*
0.5476 0.0162 0.4914 0.0517 0.2015 0.0400
100 89.7 36.8
/ 1 3
Notes: * Toxicity grade 0 means P 100, grade 1 means P˛½75%; 99%, grade 2 means P˛½50%; 74%, grade 3 means P˛½25%; 49%, grade 4 means P˛½1%; 24%, grade 5 means P ¼ 0[30].
Fig. 10 EDS analysis result of fluoride coated AZ31B incubated in the simulated blood plasma for 90 days.
TF-XRD result indicates that the coating is mainly composed of MgF2 and MgO. It is well known that Mg can react with hydrofluoric acid via a replacement reaction as the following: Mg þ 2HF/MgF2 þH2 [
MgðsÞ þ 2H2 O/MgðOHÞ2 þH2 ðgÞ
(6)
(3) Simultaneously, an oxidation reaction occurs as the following:
MgðsÞ þ 2Cl ðaqÞ/MgCl2
(4)
MgðOHÞ2 ðsÞ þ 2Cl /MgCl2
(5)
One of the effective ways to reduce the corrosion of magnesium alloys is surface modification. As to biomaterials, a surface modification is also an effective way to improve its bioactivity. Therefore, it is possible to reduce the corrosion of magnesium alloy and improve the surface bioactivity by selecting a proper surface treatment to the alloy. For the above purpose, the present fluoride conversion coating is potentially applied for biomedical magnesium alloy. Li et al.[38] found that the fluoride film deposited on magnesium alloy did not improve its corrosion resistance in the 3.5% NaCl solution and they considered that the fluoride on magnesium alloy surface could only improve its reactivity at the active site. Chiu et al.[39] studied the corrosion property of MgF2 treated pure Mg in Hank’s solution and found an increase in corrosion resistance resulting from the fluoride conversion coating on Mg. In the present work, a remarkably improved corrosion resistance has been obtained for the fluoride coated fluoride AZ31B in the simulated blood plasma, shown by the electrochemical impedance spectroscopy.
Mg þ 2H2 O/MgðOHÞ2 þH2 [
(7)
Since Mg(OH)2 is not stable in the acidic solution[40], reactions should occur as the following: MgðOHÞ2 þ 2HF/MgF2 þ2H2 O
(8)
MgðOHÞ2 /MgO þ H2 O
(9)
These reactions should take place at the film/solution interface[32]. The insoluble MgF2 layer which acts as the passive films can form on the magnesium alloy surface by a chemical reaction. The MgF2 film will grow up with the immersion time, but the growth rate should slow down as the immersion continues[39]. Slow growth of the reaction layer is an indication of the barrier effect of MgF2 to the reaction. The toxicity of fluoride has been investigated. Matsui et al.[41] found that NaF could increase the intracellular Ca2þ concentration, which might be one of common features of the fluoride toxicity. Khalil et al.[42] reported that the fluoride had a sort of cytotoxicity/cytostatic effect on the cultured rat bone marrow cells. It was also found that toxicity of the fluoride was related with its concentration. Kleinsasser et al.[43] investigated the cytotoxicity extent of the fluoride using the trypan blue exclusion test. They found that non vital cells of less than 10% could be shown for the fluoride concentrations of 2 106 to 35 106 and there were 15% and 43% damaged cells after incubation with 71 106 and 213 106 fluoride, respectively.
Table 3 Antibacterial rate of fluoride coated AZ31B (E. coli, 106 cfu/ ml)
Fig. 11 XRD patterns on surface of the fluoride coated AZ31B after immersion in the simulated blood plasma for 90 days: (a) fluoride coated AZ31B, (b) Mg (JCPDS89-5003), (c) HA (JCPDS09-0432), (d) MgF2 (JCPDS 38-0882), (e) MgO (JCPDS87-0652).
Materials
Antibacterial rate
316L stainless steel as a contrast Fluoride coated AZ31B
0 > 99.99%
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T. Yan et al.: J. Mater. Sci. Technol., 2014,
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Fig. 12 Photos of breeding status of E. coli after actions with fluoride coated AZ31B (a) and 316L stainless steel (b).
Meanwhile, the interaction between fluoride and magnesium has also been investigated. Colly et al.[44] found that fluoride ion affected the enamel hardening and prevented its annealing, but this effect diminished after administration of Mg. O’Dell et al.[45] studied the interaction of dietary fluoride and magnesium in guinea pigs, and they found that the fluoride in the food and water consumed by man would be particularly valuable if magnesium was limiting in the diet. One the other hand, a high intake of magnesium should be highly beneficial in areas where fluorosis prevails and the toxicity of fluoride decreased with increasing amount of magnesium in the diet of the guinea pigs[45]. In the present study, the in vitro cytotoxicity evaluation results demonstrated that AZ31B alloy samples with fluoride conversion coating were not toxic to BMMSCs, indicating the stability of compound MgF2 formed in the conversion coating with safe concentration of fluoride released into the cell culture medium. By comparing with that on fluoride conversion coating, the relative proliferation rate of BMMSCs on the groups of bare AZ31B samples was 35.8%, indicating moderately cytotoxic. It is mainly because the corrosion rate of bare AZ31B is too fast that results in the higher pH values in MTT. Groos et al.[46] indicated that colony-forming ability of RT112 cells was reduced significantly at pH values greater than 8.4 after 24 h exposure. Meanwhile, the hydrogen released during the bare AZ31B samples degrading in the solution also had some effects on cell growth[47]. All the above results proved that the fluoride conversion coating can provide AZ31B with lower biodegradation rate and decreased cytotoxicity. Surface morphologies of the samples with fluoride conversion coating after immersion in simulated blood plasma illustrated that the bioactive HA were formed on the surface of samples. For bone implants, the prerequisite to bond to living bone is the formation of biologically active apatites on their surface in the body. The apatite layer bridges chemically the bone and the implant. Thus, the formation of HA illustrates the bioactivity of the fluoride conversion coating and that is beneficial to the application of AZ31B with fluoride conversion coating as bone implants. It was reported that a bonelike apatite layer was observed after only 3 days of bare magnesium immersing in SBF, indicating the bioactive of magnesium[48]. Thus, we can deduce that the release of magnesium ions from fluoride conversion coating and AZ31B substrate is one of the contributions to the bioactivity of AZ31B with fluoride
conversion coating. Meanwhile, fluoride can enhance osteoblastic differentiation and interfacial bone formation[49]. Bernstein et al.[50] also reported that the fluoride could prevent the osteoporosis and provoke the metastatic calcification. So the fluoride in the conversion coating is a possible contribution to the bioactivity of the sample in this study. Already in 1940, it was demonstrated that the carbohydrate metabolism in pure cultures of oral streptococci and lactobacilli was inhibited by the fluoride[51]. Since then, many reports have been published about the effect of fluoride on bacteria[52e54]. Yoshinari et al.[55] demonstrated that the bacteria were injured by the pharmacological effect of the metal-fluoride complexes with their inhibition of enzymatic activity. In this work, the fluoride coated AZ31B samples well showed good antibacterial capability, which might be due to the pharmacological effect of MgeF complex (MgF2) in the conversion coating. As for biomedical implants and devices, infection caused by bacteria from peroperative contamination during the surgery is the primary cause for failure. Therefore, biomaterials with anti-microbial properties are highly desirable because the reducing risk of infection. Thus, the antibacterial property of the fluoride coated AZ31B samples is beneficial to their application as the medical implants. 5. Conclusion A compact fluoride conversion coating was successfully prepared on an AZ31B magnesium alloy. The coating was mainly composed of MgF2 and MgO. The bonding strength between the fluoride coating and AZ31B substrate was over 43.2 5.1 MPa. Electrochemical tests showed an improved corrosion resistance for the fluoride coated AZ31B alloy in the simulated blood plasma compared with that of the bare AZ31B alloy. The in vitro cytotoxicity test showed that the fluoride coated AZ31B was not toxic to BMMSCs. The fluoride coated AZ31B had antibacterial capability. To conclude, the fluoride conversion treatment is an effective way for application by using AZ31B magnesium alloy as a biomaterial. Acknowledgements The authors thank the financial support of the National Basic Research Program of China (973 Program, No. 2012CB619101) and the Basic Application Research of Yunnan Province (No. KKSA201151053).
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T. Yan et al.: J. Mater. Sci. Technol., 2014,
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