Biodegradability and antibacterial properties of MAO coatings formed on Mg-Sr-Ca alloys in an electrolyte containing Ag doped hydroxyapatite

    Biodegradability and Antibacterial Properties of MAO Coatings formed on Mg-Sr-Ca Alloys in an Electrolyte Containing Ag Doped Hydroxyapatite Mehmet Yazici, Ali Emre Gulec, Mevlut Gurbuz, Yucel Gencer, Mehmet Tarakci PII: DOI: Reference:

S0040-6090(17)30795-2 doi:10.1016/j.tsf.2017.10.033 TSF 36301

To appear in:

Thin Solid Films

Received date: Revised date: Accepted date:

9 May 2017 11 October 2017 14 October 2017

Please cite this article as: Mehmet Yazici, Ali Emre Gulec, Mevlut Gurbuz, Yucel Gencer, Mehmet Tarakci, Biodegradability and Antibacterial Properties of MAO Coatings formed on Mg-Sr-Ca Alloys in an Electrolyte Containing Ag Doped Hydroxyapatite, Thin Solid Films (2017), doi:10.1016/j.tsf.2017.10.033

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ACCEPTED MANUSCRIPT Biodegradability and Antibacterial Properties of MAO Coatings formed on Mg-Sr-Ca Alloys in an Electrolyte Containing Ag Doped Hydroxyapatite

Department of Materials Science and Engineering, Ondokuz Mayis University, 55139

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Mehmet Yazici1,2,*, Ali Emre Gulec2 , Mevlut Gurbuz3, Yucel Gencer2, Mehmet Tarakci2

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Kurupelit, Samsun, Turkey

Department of Materials Science and Engineering, Gebze Technical University, 41400

Department of Mechanical Engineering, Ondokuz Mayis University, 55139 Kurupelit,

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Gebze, Kocaeli, Turkey

Samsun, Turkey

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Abstract

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Magnesium-based alloys are promising materials as next generation biodegradable implants,

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however low corrosion resistance and inadequate mechanical properties are limiting their application as a biodegradable implant material. In this study, Mg-Sr-Ca ternary alloys were prepared in a vacuum/atmosphere controlled furnace and coated by microarc oxidation

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(MAO) process for 5 minutes to decrease the degradation rate and enhance the biocompatibility. Moreover, Ag doped Hydroxyapatite nano powder (Ag-HA) was also added to alkaline MAO solution by amount of 1 and 10 g/l to improve the antibacterial properties while enhancing their bioactivity in a one single process. XRD, SEM-EDS, FTIR spectroscopy, simulated body fluid (SBF) immersion and antibacterial tests were employed for the characterization of the coated alloys. The results showed that, the addition of more Ag-HA increased the HA formation both before and after SBF immersion test and enhanced their antibacterial properties. However, Ag-HA addition decreased the corrosion resistance of the coated alloys in SBF compared to Ag-HA free coating. The results indicated that the

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ACCEPTED MANUSCRIPT present Ag-HA nano powder added MAO coating is a good combination to enhance the corrosion resistance, bioactivity and the antibacterial properties of Mg based biodegradable alloys. oxidation,

Plasma

Electrolytic

Biodegradable,

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Hydroxyapatite, Mg alloys, Antibacterial Coatings

Oxidation,

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Microarc

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Keywords:

1. Introduction

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Biodegradable metals are gaining more and more attention as temporary medical implant materials especially in cardiovascular and musculoskeletal system of the body since they do

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not need a secondary surgical operation after completing their duty in the body thus increasing the comfort of the patients and lowering the medical costs [1]. Magnesium, the

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third most abundant element in the earth's crust, has been extensively used for the last 20

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years, mainly in automotive and aerospace industries owing to its low density and high

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specific strength [2, 3]. It is also an essential element for human metabolism and owing to its formability, biocompatibility, biodegradability and nontoxicity, magnesium based alloys are promising candidates as next generation implant materials [3, 4]. Moreover, Mg+2 ions trigger

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bone cell formation and accelerate the healing process [4-7]. Unlike titanium, cobalt chromium and iron based implants, magnesium has a very close elastic modulus value (≈ 45 GPa) to that of natural bone (≈20 GPa) preventing the stress-shielding problem, which may lead to loosening of the implant material and causing osteopenia in orthopedic applications [8]. However, its poor corrosion and wear resistance greatly limits the application areas of magnesium. It is well known that during the healing process, biodegradable implant material should have a similar degradation rate compared to the newly formed bone formation to preserve its integrity. However, due to its anodic nature, magnesium is very susceptible to corrosion in body fluid, degrading too early compared to the bone formation rate [5-7, 9]. Therefore, the corrosion resistance of magnesium should be increased to be able to use in the 2

ACCEPTED MANUSCRIPT body as a biodegradable implant material. Alloying and coating techniques are the primary methods to increase corrosion resistance as well as mechanical properties of magnesium alloys [10-13]. Since the alloying elements can affect the mechanical properties and

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degradation rate of magnesium as implant material, there are several studies to optimize the

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content of the alloying elements. In these studies, binary and ternary Mg based alloys were

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tailored with Ca, Sr, Sn, Zn, Mn elements. It was reported that higher corrosion resistance and mechanical performance was attained with certain amount of alloying with no significant

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toxicity [4, 13-18]. Strontium (Sr) and calcium (Ca) are important alloying elements of magnesium. Sr increases the corrosion resistance by providing a passive layer on magnesium

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and it is also consisted in human bone and promotes the osteoblast formation (precursor cell to form osteocytes) though recent studies reported that corrosion resistance was negatively

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affected with high Sr content [18-24]. Furthermore, Sr enhances strength by refining the

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grain size in magnesium alloys [20]. Ca, being a vital element in the body and the most

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abundant cation in natural bone, is essential in maintaining bone health and dental health being [25, 26]. Therefore, ternary Mg-Sr-Ca alloy is a promising candidate biomaterial due to contribution of Sr and Ca alloying properties in magnesium although high degradation rate

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was reported for Mg-Sr-Ca alloy [26]. As mentioned above, in addition to alloying, surface modification is another method to control the degradation rate of magnesium based alloys. Microarc oxidation (MAO) is a novel surface modification method applied to Mg alloys to protect its surface against corrosion. The coating properties synthetized by MAO including corrosion resistance and antibacterial properties strongly depend on electrolyte along with other process parameters. Silver ions implanted in the coatings was reported to enhance the antibacterial effect [27, 28], the cytocompatibility and cell viability [29, 30]. Therefore, in this study quaternary Mg-3Sr-6Ca alloys were prepared and then coated by MAO method in different electrolytes containing silver doped hydroxyapatite (HA) powder in 3

ACCEPTED MANUSCRIPT different amounts (0, 1 and 10 g/l) to investigate the degradation rates and the antibacterial properties of the coated samples.

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2. Experimental

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2. 1. Alloying

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Mg-3Sr-6Ca ternary alloy was prepared using high purity Mg, Sr and Ca obtained from Alfa Aesar. The alloy was prepared by induction melting under an argon controlled atmosphere

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and cast into a water-cooled copper mold. Subsequently, the alloy was cut into the samples with the dimensions of 18 mmx18 mmx5 mm. The surfaces of the samples were ground

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using SiC abrasive papers (80-1200), polished with alumina slurry and washed with ethanol in

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2. 2. MAO Coating Process

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an ultrasonic bath.

The electrolyte was prepared by dissolving 2 g/l KOH and 10 g/l Na3PO4 in distilled water for

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the MAO process. Furthermore, in addition to this solution, Ag doped HA powder was added to solution by amounts of 1 g/l (AgHA-1) and 10 g/l (AgHA-10) respectively. The particle

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size distribution was very narrow and measured mean particle size used in this study was approximately 50-60 nm. Wet chemical method was used to synthesize silver ion containing HA antibacterial ceramic powders. Detailed synthesis procedure and antibacterial efficiency were given in our previous work [31]. Thus, three different electrolytes were used for MAO coating process. The electrolyte was continuously mixed during the coating with air to keep the electrolyte homogeneous and the temperature constant between 11-13°C. The positive and negative current density values were set to 3 A/cm2, and the pulsing frequency selected as 85 Hz. The anodic and cathodic pulse values were both set to 500 μs and pulse break duration was 250 μs for all coatings. The samples were coated with the same parameters in three different electrolytes for the duration of 5 min. 4

ACCEPTED MANUSCRIPT 2. 3. Characterization of MAO Coating XRD analysis of the bare substrate and MAO coated alloys were carried out with monochromatic CuKα radiation using Rigaku X-Ray Diffractometer (40 kW, 40 mA) over a

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2θ range of 10° to 90° with a scanning rate of 1 °/min. The surfaces of the coated samples

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were examined by Philips XL30 FEG SEM upon gold coating to prevent charging on the surface. Furthermore, elemental compositions of coatings were investigated using SEM–EDS.

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2. 4. Simulated Body Fluid (SBF) Test

All the MAO coatings were immersed in SBF at 37.0 °C ± 0.5 °C for 5 days, 10 days and 15

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days. The SBF solution was prepared by following the procedure suggested by Tas et al. [32]. The chemical composition of SBF suggested by Tas and human plasma were given in Table

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1.

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During the immersion test, the SBF solution was refreshed in every 2 days. After the

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immersion process, the samples were washed with deionized water, air-dried. The mass change calculations, XRD and SEM-EDS analysis were performed to determine weight loss/gain, morphological and structural changes on the surface of immersed samples. Average

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weight loss rate ΔW (mg/cm2/d) was calculated using the equation (1) [33]. W  Wb  Wa  / St

(1)

Wa and Wb are the weights after and before the SBF test respectively, S is the surface area and t is the immersion duration as days. ΔW values were converted to corrosion rate (CRw) rate [mm/y] using the equation (2) [33-35].

CRw  2.1W

(2)

where 2.1 is the constant for magnesium based materials to convert the average weight loss rate ΔW (mg/cm2/d) to the average penetration rate (mm/y).

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ACCEPTED MANUSCRIPT 2. 5. FTIR In order to determine the chemical structure and functional groups of the coatings FTIR spectroscopy was employed (FT-IR, Perkin Elmer Spectrum 100) in the spectral range of 800-

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4000 cm-1. The same process was repeated after the coated alloys were immersed in the SBF

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for 15 days to investigate the variations in the binding structure. 2. 6. Antibacterial Test

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Antibacterial Test was carried to observe the antibacterial activity of the MAO-coated alloys. Before carrying out the tests, the samples were sterilized at 120°C for 15 min. 105 Escherichia

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coli bacteria colonies were poured into the petri dish to surround the alloys and the alloys were incubated for 24 hours at 37°C. After the incubation process the samples were sterilized

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again at 120 °C for 15 min. The determine whether the bacteria colonies have multiplied on

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the surface SEM (Philips XL30 FEG SEM, secondary electron mode). The samples were

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coated by gold again before carrying out the SEM analysis to prevent charging. 3. Results and Discussion

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Macro photos of the MAO coated surfaces in Ag-HA free (AgHA-0), AgHA-1 and AgHA-10 were illustrated in Fig 1. There is a clear color difference among the samples according to the Ag-HA content in the electrolyte. Uniform coatings on the all three samples are evident according to the macrographs. The yellow color is attributed to the silver presence in HA nanoparticles doped into the coating surfaces homogenously as reported in the literature [36]. Figure 2 shows the FTIR results of the MAO coated surfaces together with Ag-HA. The peak located at 1027 cm-1 marked as PO43- is known to belong to the HA phase as it was proved in FTIR analysis of the Ag-HA [50]. Moreover, this peak has intensified with the increasing amount of Ag-HA content in the electrolyte. Although there are no HA peaks on the XRD

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ACCEPTED MANUSCRIPT (Fig. 3) results, FTIR spectrum verifies the HA is included on the coated surface. It is possible that the concentration of HA phase in the coatings may be below the detection limit of XRD or may be present in amorphous form. In addition, broad spectral peaks between 1000 cm−1

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and 1030 cm−1 supports the amorphous or nano-crystalline HA formation owing to the rapid

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quenching of oxide melt on the surface [37]. On the other hand, since the used powder has a

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particle size of 50-60 nm, it may have resulted in precipitate formation with nano particle size on the coating surface preventing the detection by XRD analysis.

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SEM micrographs of MAO coated samples before and after antibacterial tests were presented in Fig 4. The typical surfaces with various sized pores, cracks, deposited particles and

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relatively smooth areas were observed on the MAO coating synthesized on Mg-3Sr-6Ca alloy. The coatings with glassy appearance were obtained using HA free and low amount of

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Ag+ doped HA powder (Ag-HA) containing electrolyte while relatively rough surfaces were

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obtained using the electrolyte containing 10 g/l HA. Although the coating parameters were

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the same, there are considerable differences on the surface morphology of the coating with an increase of the HA content in the electrolyte. Especially, the high content of HA in the

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electrolyte increased the number of discharge channels and decreased the opening size of discharge channels. This may be related to decrease in dielectric breakdown voltage of local regions in the coatings [38] due to the chemical constituents of the coatings. The decrease in breakdown voltage may be either because of more HA nanoparticles with Ag resulted in less breakdown voltage because of higher content of Ag in the coating or because of the evaporation of Ca and P contained in HA which partially decomposed under plasma conditions resulted in more porous coating with lower dielectric breakdown voltage. HA precipitates were observed on the coating surface by the addition of HA into the electrolyte. The precipitates were only present near the discharge channels coated in the electrolyte containing 1 g/l HA while they were dispersed on the whole surface when the HA content in 7

ACCEPTED MANUSCRIPT the electrolyte has increased to 10 g/l. The transfer and incorporation of HA on the coating surface from the electrolyte to the coating attributed to two possible mechanisms. Due to the high temperature and pressure (103-104 K, 100 MPa) in the discharge channels, the substrate

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alloy melts and mixes with the previously formed coating material and transfers through the

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channel where it reacts with the ions of the electrolyte. Thus, the formed molten oxide is

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pushed out of the discharge channel to the surface and contacts with the cold HA containing electrolyte. The contact between molten oxide and cold electrolyte may encapsulate HA Second possible mechanism may be the discharge

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nano-particles on the coating surface.

channels filled with electrolyte containing high nano sized HA particles and spark formation

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evaporates the electrolytes and the HA residues encapsulated in the coating. The successive spark formation result in complete incorporation and homogeneous distribution of the

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encapsulated HA constituents by mentioned two mechanisms into the coatings. Therefore, it

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can be concluded that increasing the HA powder in the electrolyte promotes the incorporation

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of more HA on the surface of the coatings. It should be also noted that cracks, observed on the coating surface was a direct result of thermal shocks by means of rapid cooling of the oxide

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melt by the cold electrolyte [39]. Fig. 4 also shows the residues of spongy shaped E-coli colonies that could reproduce on the MAO surface. The viability of the bacteria was determined qualitatively on the surface by the Imagej software and shown on the right hand corners of Figs 4 (a-1, b-1, c-1). The bright regions represent the bacteria residues on the figure. The values for viable E-coli bacteria on MAO coating surfaces using AgHA-0, AgHA-1 and AgHA-10 electrolytes were determined as approximately 49 %, 20 % and almost 0 %, respectively. It is clearly seen that viability rate decreases as Ag-HA content increases on the surface.

It is well known that Ag

suppresses bacterial activities, so the effect is rather because of the Ag content in Ag-HA [27,

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ACCEPTED MANUSCRIPT 40]. This result also confirms that Ag-HA certainly incorporated on MAO coatings using AgHA containing electrolyte. Fig. 5 shows the degradation rates (CRW) of all MAO coated samples immersed in SBF for 5,

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10 and 15 days. It is clearly seen that, the addition of Ag-HA into the electrolyte of MAO

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coating decreases the degradation resistance, though with the increase in the immersion duration, the degradation rate has increased in all coatings. The addition of Ag-HA may have affected the compactness of the coating [41] and increased the pore concentration as

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explained in Fig. 4. The increase in pore concentration may have decreased the corrosion rates as it increases the total defective surface area where degradation process took place.

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Fig. 6 presents the XRD patterns of the coatings formed with different Ag-HA concentration and immersed in SBF for 15 days. Strong HA peaks were detected clearly for AgHA-1 and

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AgHA-10. Intensity of HA peaks increases with increase in HA content.

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Fig. 7 reveals the surface morphology of all MAO coated samples in the electrolyte containing different Ag-HA after 15-day SBF immersion test. The SEM images clearly show

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that the deposited reaction products covered all surfaces through SBF immersion.

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considering XRD results and the similar studies reported in the literature these reaction products are HA called as secondary HA (S-HA) [38].

There is clear crack formation

throughout the HA covered surface after removal from SBF. Although the low amount of AgHA is not very effective on width of the cracks, it is considerably effective with higher addition of Ag-HA into the MAO electrolyte. The cracks formation was due to the drying effect which results in tensional stress on the S-HA coating though it is very small for 10 g/l Ag-HA containing. This is related to the MAO coating surface morphology before SBF immersion and formed S-HA thickness (Fig. 4a-c).

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ACCEPTED MANUSCRIPT Fig. 8 shows surface SEM micrograph of the MAO coated sample in AgHA-10 electrolyte and then immersed in SBF for 5 and 15 days. The figure shows that the precipitates were distributed more homogenously. By considering FTIR and XRD results these precipitates

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were S-HA formed by growth on Ag-HA precipitates which behave as nucleation sites (Fig.

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7). The increase of immersion duration to 15 days result in further growth of the S-HA

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precipitates and eventually covering up whole surface of the coating with S-HA as a layer. Before the immersion test, increased Ca/P ratio verified that HA powder precipitation is

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directly proportional with HA content in the electrolyte, according to SEM-EDS results (Fig 9) [41]. After SBF test, HA, Calcium Strontium Oxide (Ca0.7 OSr0.3) and Ca(OH)2 phases were

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detected by the XRD on the coatings increased the concentrations of calcium ions so the total Ca/P dramatically increased compared to the as coated specimens. These Ca and P based

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as coated samples.

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phases covered the entire surface and increased the Ca and P concentrations compared to the

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Fig. 10 shows the FTIR spectrum for characterizing the precipitates formed on the coatings in electrolyte with different content of Ag-HA after the immersion test of 15 days. The PO43-

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peak located at 1027 cm-1, CO32- peaks located at 1415 cm-1 and 865 cm-1 are proving the presence of S-HA formation [34, 42, 43]. Intensifying of peaks for the coating with Ag-HA content shows the crystallinity of S-HA. 4. Conclusions In this study, MAO coatings were fabricated on the Mg-Sr-Ca alloys in electrolyte containing 2 g/l KOH and 10 g/l Na3PO4 with addition of antibacterial Ag-HA nano particles by amount of 0, 1, 10 g/l concentration. The results showed that, with the increase of the Ag-HA, more Ag-HA particles precipitated into the coating. The addition of more Ag-HA increases the SHA formation during the immersion test but also decreases the corrosion resistance of the alloys in SBF. Furthermore, S-HA morphologies formed during the SBF test is directly 10

ACCEPTED MANUSCRIPT related to Ag-HA concentration in the electrolyte. Although there was a considerable bacteria proliferation for AgHA-0, antibacterial properties increased with the addition of AgHA-1 into the electrolyte and almost no proliferation was observed with the addition of AgHA-10. The

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ratio of Ca/P values tend to theoretical values of HA (1.67) with the increasing of Ag-HA

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concentration in the electrolyte.

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5. Acknowledgment

We would like to special thanks to Nanotech Company (Eskisehir-Turkey) and Prof. Aydın

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Dogan for allowing us to prepare antibacterial powder and use the powder. The authors acknowledge the help of technicians Ahmet Nazim and Adem Sen in performing the SEM

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and XRD measurements. In addition, the authors also express their thanks to the Zeynep Girgin Ersoy for performing the antibacterial test.

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[38] S. Cengiz, Y. Azakli, M. Tarakci, L. Stanciu, Y. Gencer, Microarc oxidation discharge types and bio properties of the coating synthesized on zirconium, Materials Science and Engineering: C, 77 (2017) 374-383. [39] H.F. Guo, M.Z. An, S. Xu, H.B. Huo, Formation of oxygen bubbles and its influence on current efficiency in micro-arc oxidation process of AZ91D magnesium alloy, Thin Solid Films, 485 (2005) 53-58. [40] W.-H. Song, H.S. Ryu, S.-H. Hong, Antibacterial properties of Ag (or Pt)-containing calcium phosphate coatings formed by micro-arc oxidation, Journal of Biomedical Materials Research Part A, 88A (2009) 246-254.

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ACCEPTED MANUSCRIPT [41] X. Lin, X. Wang, L. Tan, P. Wan, X. Yu, Q. Li, K. Yang, Effect of preparation parameters on the properties of hydroxyapatite containing micro-arc oxidation coating on biodegradable ZK60 magnesium alloy, Ceramics International, 40 (2014) 10043-10051.

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[42] S. Meejoo, W. Maneeprakorn, P. Winotai, Phase and thermal stability of nanocrystalline

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hydroxyapatite prepared via microwave heating, Thermochim Acta, 447 (2006) 115-120.

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[43] L. Berzina-Cimdina, N. Borodajenko, Research of Calcium Phosphates Using Fourier Transform Infrared Spectroscopy, Infrared Spectroscopy - Materials Science, Engineering and

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ACCEPTED MANUSCRIPT List of Figure Captions Fig. 1. Macroscopic photos of the coating surfaces in different electrolytes.

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Fig. 3. XRD patterns obtained from the MAO coated surfaces.

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Fig. 2. FTIR results obtained from the MAO coated surfaces together with Ag-HA.

Fig. 4. Surface SEM micrographs of as MAO coated; (a) AgHA-0, (b) AgHA-1, (c) AgHA-10 and as MAO coated and exposed to antibacterial test; (a-1) AgHA-0, (b-1) AgHA-1, (c-1)

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Fig. 5. Degradation rates of all MAO coated samples immersed in SBF for 5, 10 and 15 days. Fig 6. XRD patterns after 15 days of SBF immersion test.

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(b) AgHA-1, (c) AgHA-10

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Fig 7. The surface morphology of all samples after 15-day SBF immersion; (a) AgHA-0,

Fig. 8. The surface morphology of before and after immersion test for AgHA-10 containing

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Fig 9. Ca/P atomic ratio of the samples before and after SBF test. Fig. 10. FTIR spectrums of the samples after 15-day SBF immersion; (a) AgHA-0, (b) AgHA-1, (c) AgHA-10

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142

142

Cl-

125

103

HCO-3

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K+

5

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1.5

1.5

Ca+2

2.5

2.5

1

1

0.5

0.5

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Ion

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2

19

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20

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4a-1 21

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26

7c

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ACCEPTED MANUSCRIPT Highlights Silver doped Hydroxyapatite (Ag-HA) addition improved antibacterial properties.

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HA morphology is related to Ag-HA content in the electrolyte.

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The increase of Ag-HA content in the electrolyte increased the HA formation.

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Increasing the Ag-HA concentration resulted in the Ca/P ratio approaching to 1.67.

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