In vitro biocompatibility and antibacterial behavior of anodic coatings fabricated in an organic phosphate containing solution on Mg–1.0Ca alloys
Introduction
Magnesium alloys have been suggested as a revolutionary biodegradable metal with the following considerations [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11]. (1) Magnesium alloys have similar density, elastic modules and compressive yield strength to natural bone. (2) Magnesium alloys possess antibacterial ability due to the rapid increase of pH values of the bacterial suspensions [7], [8]. (3) As a natural ionic presence with significant functional roles in biological systems, magnesium is necessary for the calcium incorporation into the bone and therefore can stimulate the growth of new bone tissue [1], [3], [5]. (4) The cytotoxicity of Mg–xZn–1.0Ca alloys (x = 1, 2 or 3) is Grade 0–1 [5], meeting the biocompatibility requirement used as biomaterials. (5) Calcium is a major component in human bone and can stimulate bone growth [9]. (6) Alloying with calcium can improve the corrosion resistance and mechanical properties of pure magnesium [9], [10], [11]. However, the high degradation rates of magnesium alloys in a physiological environment inhibit their clinical applications as biodegradable implants. Among Mg–1.0Ca, Mg–2.0Ca and Mg–3.0Ca alloys, the Mg–1.0Ca alloy achieves the best corrosion resistance, but its in vivo corrosion rate is still too high to match the bone healing process [1].
Micro arc oxidation (MAO) is a widely used method and can effectively improve the corrosion resistance of magnesium alloys [2], [3], [8], [12], [13], [14], [15], [16], [17], [18], [19], [20]. In addition, the porous structure of the MAO coatings is helpful to cell adhesion [2] and shows antibacterial ability with a mild increase of the pH value [8]. The coating properties such as surface roughness and chemical composition are considered as the most important parameters for altering cell behavior. For example, cell attachment increases with the increase of coating thickness and roughness, and number and size of micropores, which can be regulated by selecting electrolyte compositions and electric parameters [21]. Phosphorus is a main element in bone materials and can increase the apatite layer formation rate [22]. Therefore, phosphates are usually used as the main electrolytes of MAO [3], [12], [13], [14], [15], [16], [17], [18]. However, anodic coatings obtained in an inorganic phosphate solution achieve worse corrosion resistance than those formed in a silicate containing solution [12], [13], [14]. Therefore, to meet the demand for magnesium alloys used as orthopedic biomaterials, selecting and investigating the performance of highly efficient and environmentally friendly organic phosphates is necessary.
1,2,3,4,5,6 hexakis (di-hydrogen phosphate) myo-inositol (C6H6(H2PO4)6, abbreviated in this work as H12Phy), generally known as phytic acid, is deposited as mixed ‘phytin’ salts of mineral cations such as calcium, magnesium and potassium and widely present in nature (plants, animals and soils) [23], [24], [25], [26]. H12Phy consists of 24 oxygen atoms, 12 hydroxyl groups and 6 phosphate carboxyl groups (as shown in Fig. 1) and has a powerful chelating capacity with di- and trivalent cations such as Mg2 +, Ca2 + and Fe3 + ions [23], [24], [25], [26]. Compared with H12Phy, sodium phytate (Na12Phy), an alkaline metal complex of H12Phy, has some more advantageous properties. For example, Na12Phy was listed as a “generally recognized as safe (GRAS) substance by the Food and Drug Administration of the United States by 1997 and has been used as a preservative for baked goods in the U.S. [25], while H12Phy was not given the status in 1995.
H12Phy can develop conversion coatings on magnesium alloys and improve the corrosion resistance of conversion coatings [27], [28], [29], [30]. The main product obtained by conversion coatings is magnesium phytate (abbreviated as Mg6Phy). As a natural organic substance containing phosphorus and Mg elements, Mg6Phy is biocompatible [24] and has protective effects against osteoporosis [31]. Na12Phy has been used as an inhibitor of copper and the result show that it can promote the formation of a passive film [32]. Recently, Na12Phy was used as a main MAO electrolyte on the Mg–1.0Ca alloy and it can considerably improve the coating corrosion resistance by developing a thick coating with stable constituents [20].
Besides proper corrosion resistance, good biocompatibility and antibacterial performance are also necessary for surface modified magnesium alloys used as biomaterials [4], [8], [17], [18]. According to previous results, magnesium alloys possess an antibacterial function due to their corrosion in the bacterial solution, which results in rapid increases of pH values [7], [8]. When magnesium alloys are immersed into the bacterial solution for a short time or covered with dense coatings such as fluoride and silicon coatings fabricated by chemical conversion, they loose antibacterial functions [4], [8]. Therefore, preparation of antibacterial coatings on magnesium alloys is meaningful.
As an environmentally friendly organic substance, Na12Phy takes part in the MAO coating formation in a way that is different from conversational inorganic electrolytes. In addition, the biocompatibility and the antibacterial ability of the MAO coatings developed in a solution containing H12Phy or Na12Phy should be further investigated in order to verify whether these properties meet the demands for use as biomaterials. Based on our previous research results that Na12Phy can considerably improve the corrosion resistance of MAO treated magnesium alloys [20], in this paper, the formation process of MAO coatings in a solution containing 3 g/L NaOH and 15 g/L Na12Phy on the Mg–1.0Ca alloy were investigated by field emission scanning electron microscope (FE-SEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). The biocompatibility and antibacterial ability of the MAO treated Mg–1.0Ca alloy were tested by the MTT assay and pellicle sticking method. In addition, the antibacterial ability of pure Na12Phy and Mg6Phy powder was tested in order to clarify the antibacterial mechanism of MAO coatings.
Section snippets
Materials and MAO treatment
An ingot of Mg–1.0Ca alloy with nominal 1 wt.% calcium and balance Mg was provided by Jiangxi Ketai Advanced Material Co., Ltd. Before MAO treatment, the Mg–1.0Ca alloy was cut into 5 cm × 5 cm × 1 cm and successively ground on SiC paper up to 1000 grit finish, immediately washed with distilled water and dried in a cool air stream. In the experiment, the anodizing solution consisted of 3 g/L NaOH (AR.) and 15 g/L Na12Phy (purity ≥ 98%, Danyang Xu Sheng Industrial Co., Ltd.) (pH = 12.94), which can achieve
Surface and cross-sectional morphologies of anodic coatings
Fig. 2 shows surface and cross-sectional morphologies of anodic coatings obtained by MAO treatment on the Mg–1.0Ca alloy.
As depicted in Fig. 2a, the obtained MAO coating is smooth and uniform. The pore size is generally 1 μm and the largest pore is 2 μm in diameter. The distance between two adjacent pores is 0.6–2 μm. According to Fig. 2b, the coating is formed on the substrate and is about 6 μm thick.
EDS analysis shows that the as-developed coatings contain 10.73% C (in at.%, the same below),
Effects of Na12Phy on the coating composition
As an organic macromolecule compound, H12Phy or Na12Phy has a powerful chelating capability with metal ions. Alkali metal phytates are completely soluble in aqueous solution, while metal–phytate complexes with the di- and trivalent cations such as Ca2 +, Mg2 +, Zn2 +, Cu2 + and Fe3 + are insoluble at neutral pH [25], [32], [36]. Under low temperature, H12Phy or Na12Phy is stable. Therefore, the conversion coatings containing phytates can be developed on magnesium alloys in the solutions containing H2
Conclusions
Anodic coatings fabricated in the alkaline solution containing Na12Phy by MAO treatment on Mg–1.0Ca alloy are mainly composed of MgO by XRD analysis. Amorphous magnesium phosphate is developed in the MAO coatings, suggesting that Na12Phy is decomposed into inorganic phosphates and lower myo-inositol phosphate esters due to spark discharge. The MAO samples achieve excellent antibacterial effects against S. aureus and E. coli due to their corrosion in the tested bacterial suspensions. In vitro
Acknowledgements
The authors thank the support of the National Natural Science Foundation of China (Nos. 51061007 and 51361011), the Natural Science Foundation of Jiangxi Province, China (No. 20132BAB206011) and 2011 in vitro diagnostic reagents and instruments from the Collaborative Innovation Center.
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