Retracted Article: Ball flower like manganese, strontium substituted hydroxyapatite/cerium oxide dual coatings on the AZ91 Mg alloy with improved bioactive and corrosion resistance properties for implant applications

Abstract

Bio-degradable metals and alloys have been suggested as revolutionary potential materials for bone-related treatment. Of these materials, the AZ91 magnesium alloy (AZ91 Mg alloy) emerges as an attractive candidate due to its non-toxicity and outstanding mechanical properties. Even though magnesium alloys are widely studied as orthopedic implants for bone replacement and bone regeneration, their undesirable rapid corrosion rate under physiological conditions has limited their actual clinical applications. Therefore, increasing the corrosion resistance of the AZ91 Mg alloy is one of the key issues to address for the development of bio-degradable implants. In this study, a cerium oxide (CeO₂) coating is developed on the AZ91 Mg alloy by electrodeposition with a view of minimizing its corrosion rate during the bone healing period. Further, to improve the clinical application of AZ91 Mg alloy, manganese (Mn) and strontium (Sr) substituted hydroxyapatite (Mn, Sr-HAP) coatings were developed on the CeO₂ coated AZ91 Mg alloy. Hence, this study reports on the development of a Mn, Sr-HAP/CeO₂ dual coating on the AZ91 Mg alloy to make it a suitable alternative material for orthopedic implants.

---

1 Introduction

Orthopaedic devices essential for the fixation of bone fractures are conventional metallic materials as they need to maintain mechanical integrity and biocompatibility for the duration of the bone healing period. Metallic implants such as titanium and its alloys, stainless steel, cobalt–chromium, magnesium and nickel based alloys are utilized for orthopedic applications.^1–4 Though they are used as implant materials, a closer inspection reveals that there are many disadvantages with regard to their uses. The durability of these metallic materials is one of the key properties that add to the limitations of the material. Further, these metallic materials have demonstrated the release of toxic corrosion products and allergens.^5–10 Among the various metallic materials, the AZ91 Mg alloy is a promising and eco-friendly orthopaedic implant material because of its light weight, good biocompatibility,^11–15 bone-like mechanical properties^16–18 (reducing the risk of stress shielding) and gradual degradation in the human physiological environment.¹³ Also, AZ91 Mg alloy has the advantage of degradation, and thus if corrosion rates are controlled the material would slowly degrade, thereby decreasing health risks, costs and scarring. All these advantageous properties would offer huge benefit for human health care, by obviating the requirement for second surgeries.¹⁷

Magnesium alloys are very reactive in nature and are prone to rapid corrosion particularly in high chloride environments such as those created by human body fluids and blood plasma having pH in the range of 7.4–7.6.¹⁹ This disadvantage has seriously restricted the medical application of Mg alloys in various fields of research.^20,21 Hence, if the corrosion resistance of Mg alloy implant is enhanced, its great potential as a metallic implant material for orthopaedic applications could be enjoyed.¹⁷ In response to this, calcium phosphate based bioceramic coatings have been suggested as a mean of preventing exposure to the corrosive environment. Hydroxyapatite [Ca₁₀(PO₄)₆(OH)₂, HAP] is the most commonly used bioceramic that can accelerate the bone growth due its outstanding biocompatibility and similar composition and structure to human hard tissues.^22,23

Furthermore, addition of bioactive ions into the HAP based bioceramic material might enhance the biological properties of the implants.^17,20 In particular, the favorable effect of bioactive ions such as magnesium (Mg²⁺), strontium (Sr²⁺), manganese (Mn²⁺), silver (Ag²⁺), zinc (Zn²⁺), silicon (Si²⁺) and carbonate (CO₃²⁻) in HAP based coatings on metallic substrates has been reported.^24–30 More recently, the possibility to realize the substitution with at least two distinct elements has been put forward.^2,28–31 Among the divalent cations, Sr²⁺ is particularly attractive because of the fact that strontium ranelate is widespread for the treatment of bone defect diseases such as postmenopausal osteoporosis.³² Moreover, strontium has the property to stimulate osteoblast and to inhibit the bone resorption. There are also numerous reports which state the effects of Sr-HAP in enhancing their bioactivity and biocompatibility.^31–35

Manganese is a trace element which is essential for the growth and development of bones.³⁶ The radius of manganese ion (0.99 Å) is very close to that of calcium (0.90 Å), which enables Mn ion to enter osteoblasts through calcium ion channel. Mn²⁺ influences bone metabolism by regulating the osteoblast differentiation and bone resorption. The presence of Mn in calcium phosphate can also improve the production of osteocalcin in osteoblasts, even more effectively than strontium and magnesium.^36–42

However, these bioceramic coatings could not maintain long-term stability, and may delaminate from the surface of the implants in certain situations due to the lack of bonding strength, which in turn may lead to clinical complications and implant failure.³³ Hence, it is necessary to improve the bonding between the substrate and the coating. Moreover, Mg alloy possesses high corrosion rate in physiological medium and hence it must be protected from corrosion for its long-term application in the field of orthopaedics. To satisfy the above needs, a CeO₂ layer is developed on the Mg alloy substrate prior to the bioceramic coating. This CeO₂ layer plays a major role in improving the corrosion resistance of Mg alloy and also provides better bonding between the substrate and the HAP coating. There are few reports on CeO₂ coating on Mg alloys, for improving the corrosion resistance, mechanical properties and antibacterial properties of the coating.^43–51

A number of coating technologies are available for the development of bioceramic coatings and among them the electrochemical deposition has unique advantages due to its relatively low deposition temperature, process simplicity, capability of forming a uniform coating on porous substrates (or) complex shapes of substrate and the availability and low cost of equipments.^52,53 It is thus established as one of the most promising methods for obtaining coating on biodegradable Mg alloy.⁴¹ Recently Gopi et al., has succeeded in the electrochemical deposition of bioceramic coating on AZ91 Mg alloy.⁵⁴

The present work is aimed for the electrodeposition of Mn, Sr-HAP/CeO₂ dual-layers on Mg alloy to improve its corrosion resistance, mechanical and biological properties. The primary CeO₂ layer provides corrosion resistance to AZ91 Mg alloy and also enhances the mechanical property of the AZ91 Mg alloy. The second layer, a ball-flower structured Mn, Sr-HAP coating will certainly pave a way for the development of bone tissues through pores in between them. To the best of authors knowledge, there are no reports on the electrodeposition of Mn, Sr-HAP coating on CeO₂ coated Mg alloy.

2 Materials and methods

2.1 Mg alloy surface preparation

AZ91 Mg alloy with elemental composition: 0.59% Zn, 0.17% Mn, 8.63% Al, <0.05% Cu, <0.05% Fe and balance Mg (wt%) was used as the substrate material in this study.⁵⁴ The substrates were cut into 10 × 10 × 5 mm³ size and then embedded in epoxy resin leaving an area of 1 cm². To obtain homogeneous roughness, the samples were mechanically ground by various silicon carbide (SiC) papers of grade 800, 1000, and 1200, respectively. Before conducting the experiments, all the samples were ultrasonically cleaned in ethanol for 10 min in order to remove any surface residues. All the samples were rinsed in the deionised water and then dried and used for further experimental studies.

2.2 Preparation of electrolyte for coating

2.2.1 For Mn, Sr-HAP coating on AZ91 Mg alloy. The electrolyte for the deposition was prepared by dissolving analytical grade 0.3 M calcium nitrate dihydrate (Ca(NO₃)₂·2H₂O), 0.1 M manganese nitrate hexahydrate (Mn(NO₃)₂·6H₂O) and 0.1 M strontium nitrate dihydrate (Sr(NO₃)₂·2H₂O) in deionized water (DI) to get clear solution. To the above solution, 0.3 M diammonium hydrogen phosphate ((NH₄)₂HPO₄) solution was added drop wise under magnetic stirring for 2 h to produce the target (Ca + Mn + Sr)/P ratio of 1.67 at room temperature (28 ± 1 °C). The pH of the electrolyte was adjusted to 4.7 using ammonium hydroxide or hydrochloric acid. All the chemicals were purchased from sigma-aldrich and used without further purification.

2.2.2 For CeO₂ coating on AZ91 Mg alloy. Cerium nitrate hexahydrate (Ce(NO₃)₃·6H₂O) was taken as the precursor for the CeO₂ coating. For this purpose, different concentrations (0.05 M, 0.1 M, 0.2 M) of Ce(NO₃)₃·6H₂O was dissolved in DI water, respectively and stirred for 15 min continuously at room temperature. The obtained transparent solution was used as electrolyte for the CeO₂ coating on AZ91 Mg alloy.

2.3 Electrochemical deposition

2.3.1 Electrodeposition of CeO₂ on AZ91 Mg alloy. The electrochemical deposition of CeO₂ on AZ91 Mg alloy was carried out in the conventional three electrode system using an electrochemical instrument (CHI 760C, electrochemical workstation, USA) in which the saturated calomel electrode served as the reference electrode (SCE), platinum electrode as counter electrode and AZ91 Mg alloy as working electrode, respectively. The electrodeposition of CeO₂ with different concentrations was performed at a current density of 1 mA cm⁻² for the duration of 60 min at room temperature. After the deposition, CeO₂ coated AZ91 Mg alloy was washed with DI water and then dried in air for 24 h.

2.3.2 Mn, Sr-HAP coating on CeO₂ coated AZ91 Mg alloy. In our previous paper, the electrochemical deposition of Sr substituted HAP was achieved on 316L stainless steel at different current densities of 8, 9 and 10 mA cm⁻², respectively.⁵⁵ In the present work, we have deposited Mn, Sr-HAP on CeO₂ (0.1 M) coated AZ91 Mg alloy at constant current density of 9 mA cm⁻² for the duration of 30 min which has been proved as the most excellent current density to produce uniform coating.⁵⁵ After the electrodeposition, the specimen was gently rinsed with DI water, and then dried in desiccator for 24 h and used for further characterization.

2.4 Surface characterization of the Mn, Sr-HAP/CeO₂ coatings

The coated samples were characterized for their functional groups by Fourier transform infrared spectroscopy (FT-IR, Impact 400 D Nicholet Spectrometer) over the frequency range from 4000 to 400 cm⁻¹ with a number of 32 scans and spectral resolution of 4 cm⁻¹. The phase compositions of the coatings were identified by X-ray diffraction (XRD, Seifert, X-ray diffractometer Siemens D500 Spectrometer) in the range between 20° ≤ 2θ ≤ 60° with CuKα radiation generated at 40 kV and 30 mA with a step size of 0.02° at a scanning rate of 10 min⁻¹ with standard data compiled by the International Centre for Diffraction Data (ICDD). The surface morphology of the samples (CeO₂ coated AZ91 Mg alloy and Mn, Sr-HAP/CeO₂ dual-layer coated AZ91 Mg alloy) was studied using high resolution scanning electron microscopy (HRSEM, JEOL JSM-6400, Japan). The elemental composition of the samples was investigated by energy dispersive X-ray analysis (EDAX).

2.5 Mechanical properties of the coatings

2.5.1 Adhesion test. The Mn, Sr-HAP, CeO₂ and Mn, Sr-HAP/CeO₂ coatings on AZ91 Mg alloy substrates, were evaluated for its adhesion strength by pull-out tests according to the American Society for Testing Materials (ASTM) international standard F 1044-05 with at least five measurements for each experiment. All the coated samples were cured in an oven at 100 °C for 50 min and then were subjected to pull-out tests at a crosshead speed of 1 mm min⁻¹, using a universal testing machine (Model 5569, Instron).⁵⁶

2.5.2 Hardness test. Vickers micro-hardness measurements were performed on the bare AZ91 Mg alloy, Mn, Sr-HAP and dual Mn, Sr-HAP/CeO₂ coated AZ91 Mg alloy samples, respectively using an Akashi AAV-500 series hardness tester (Kanagawa, Japan). The load used was 490.3 mN for a dwell time of 20 s. Each sample was subjected to at least five measurements.

2.6 MTT assay

2.6.1 Human osteosarcoma MG63 cells. Human osteosarcoma MG63 cells (HOS MG63, ATCC CRL-1427TM) supplied by National Centre for Cell Sciences (NCCS), Pune, India, were cultured in standard culture medium, Dulbecco's Modified Eagle Medium (DMEM, GIBCO), which consisted of a minimal essential medium, supplemented with 10% fetal bovine serum (FBS) and 1% non-essential amino acids (GIBCO). The medium was renewed for every 2 days and the cultures were maintained in a humidified atmosphere with 5% CO₂ and 95% humidified air, at 37 °C. The osteoblast cultures were then detached from the culture flask by incubation with 0.1% trypsin and 0.1% ethylene diamine tetraacetic acid (EDTA) for 5 min. The viability of cells colonizing on the samples were evaluated by measuring the mitochondrial dehydrogenase activity using a modified MTT (3-(4,5-dimetyl-2-tiazolyl)-2,5-diphenyl-2-tetrazolium bromide) assay. To determine the cytotoxicity of the coated samples, MG63 cells were seeded in 12-well plates at 10⁴ cells per mL. After 24 h of incubation, MTT solution in 1 mL serum free medium was added and incubated for 4 h at 37 °C in a humidified 5% CO₂ atmosphere. To determine the cell proliferation and cytotoxicity, an MTT assay was examined as a function of incubation time for 1, 4 and 7 days. The solution was then removed, dimethyl sulfoxide was added to it, and the plate was shaken for 15 min before measuring absorbance at 570 nm on an ELISA microplate reader and then % cell viability was calculated with respect to control as follows

% Cell viability = [A]_test/[A]_control × 100.

2.6.2 Fibroblast-stem cells. An MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium Bromide) assay test was used to determine the viability and proliferation of fibroblast-like stem cells on Mn, Sr-HAP/CeO₂ dual layer coated Mg alloy. The cells were cultured in Dulbecco's modified Eagle medium (DMEM, Gibco), which consisted of a minimal essential medium (Hi Media Laboratories), supplemented with 10% fetal bovine serum (FBS, Biowest, France) in 96-well tissue culture plates. The cell culture was incubated at 37 °C under a humidified atmosphere of 5% CO₂ and 95% air for one day. The dual layer coated samples were sterilized in an autoclave at 120 °C during 2 h and then placed in 96-well tissue culture plates. The cells were seeded onto the dual layer coatings and were maintained at 37 °C under 5% CO₂. The culture media in each well were replaced with immersion extracts supplemented with 10% FBS, and incubated at 37 °C in an atmosphere of 5% CO₂ and 95% every day. MTT assay was carried out as a function of incubation time for 1, 4 and 7 days. The MTT (10 mL) solution containing 5 mg of thiazolyl blue tetrazolium bromide powder was then added into each well on days 1, 4 and 7. After an incubation period, 100 mL of 10% sodium dodecyl sulphate (Sigma-Aldrich, USA) in 0.01 M HCl (Sigma-Aldrich, UK) was added into each well and incubated at 37 °C in an atmosphere of 5% CO₂ and 95% air for 24 h. To determine the cell viability of the samples, the absorbance was measured using an ELISA microplate reader at 570 nm wavelength. Cell viability (%) was calculated with respect to control wells using the following equation:

% Cell viability = ([A]_test/[A]_control) × 100.

2.7 Electrochemical studies

The electrochemical studies such as potentiodynamic polarisation and electrochemical impedance spectroscopy (EIS) were carried out in simulated body fluid (SBF) solution to analyse the corrosion protection performance of uncoated, CeO₂ coated, Mn, Sr-HAP coated and the Mn, Sr-HAP/CeO₂ dual-layer coated Mg alloy samples, respectively. The SBF composition and its ionic concentrations of human blood plasma (Table 1) were proposed by Kokubo et al.⁵⁷ and its pH value of the SBF solution was maintained at 7.4 to mimic the concentration of human blood plasma. This experiment was carried out in a SBF solution (50 mL) at a commonly accepted human body temperature (37 ± 1 °C) and the experiment solution was refreshed every day. Generally, the electrochemical experiments were conducted using a typical three-electrode system (CHI 760C, USA), using SCE as the reference electrode, AZ91 Mg alloy samples as the working electrode and platinum electrode as the counter electrode. The polarization experiments were performed from −400 mV to −2000 mV vs. SCE with a scan rate of 1 mV s⁻¹. The EIS measurements were carried out at an open circuit potential condition in the frequency range 10⁻² Hz to 10⁵ Hz with perturbation amplitude of 5 mV. All the electrochemical experiments were performed at least three times for the reproducibility and also the obtained data was recorded using internally developed software.

Table 1 Ionic composition of human blood plasma and SBF⁵⁷

Ions	Na^+	K^+	Ca^2+	Mg^2+	Cl^−	HCO3^−	HPO4^2−	SO4^2−
Human blood plasma (mM)	142	5	2.5	1.5	103	27	1	0.5
SBF (mM)	142	5	2.5	1.5	147.8	4.2	1	0.5

---

3 Results and discussion

3.1 Surface characterization of the Mn, Sr-HAP/CeO₂ dual layer coating

3.1.1 FT-IR analysis. The FT-IR spectrum of Mn, Sr-HAP/CeO₂ dual-layer coated AZ91 Mg alloy (Fig. 1), revealed the formation of both Mn, Sr-HAP as well as CeO₂ coating are evident. The strong absorption bands found at 3610 and 1639 cm⁻¹ are attributed to the stretching and bending mode of water molecules in the Mn, Sr-HAP, respectively. Further, the characteristic peaks appeared at 1013 cm⁻¹ (ν₃) and 594 cm⁻¹ & 547 cm⁻¹ (ν₄) as well as the peaks observed at 1087 cm⁻¹ (ν₃) and 978 cm⁻¹ (ν₁) are assigned to the phosphate groups of Mn, Sr-HAP. Whereas, the OH⁻ stretching and bending bands for Mn, Sr-HAP are shifted to frequencies around 3445 and 645 cm⁻¹, respectively.⁵⁶ Besides, the peak in the region of 445 cm⁻¹ is attributed to the stretching of Ce–O on the surface of the CeO₂.⁵⁸ As a result, the spectrum strongly demonstrated the formation of Mn, Sr-HAP/CeO₂ dual coating on AZ91 Mg alloy.

Fig. 1 FT-IR spectrum of Mn, Sr-HAP/CeO₂ dual coating on AZ91 Mg alloy.

3.1.2 XRD analysis. Fig. 2 shows the XRD patterns of pure AZ91 Mg alloy (as control) and Mn, Sr-HAP/CeO₂ dual coated AZ91 Mg alloy, respectively. For the XRD pattern of pure AZ91 Mg alloy (Fig. 2(a)), the phases of Mg and Al were observed at 2θ values of 32.2°, 34.6°, 36.0°, 36.9°, 43.7°, 48.0° and 57.5°, respectively.⁵⁹ Whereas, for the dual coated AZ91 Mg alloy, the major peaks corresponding to Mn, Sr-HAP were observed at 2θ values of 25.19°, 31.8°, 32.2°, 45.3°, 49.1° and 52.6° and no other secondary peaks were found.⁵⁴ In the case of Mn, Sr-HAP/CeO₂ coated sample, the diffraction peak positions shifted towards the lower 2θ values (Fig. 2(b)) from the standard XRD patterns for HAP that indicate the substitution of Mn and Sr into the HAP lattices as reported by Yong et al.⁶⁰ Similarly, the diffraction peaks of CeO₂ are located at 2θ values of 28.9°, 56.41° and 59.7° which are well evident from the JCPDS card no. 43-1002.⁶¹ As it can be seen from Fig. 2(b), the characteristic peaks of both Mn, Sr-HAP and CeO₂ are found in the dual coated AZ91 Mg alloy. Thus, all these peaks (Fig. 2(b)) confirm the formation of the Mn, Sr-HAP/CeO₂ dual coating on AZ91 Mg alloy.

Fig. 2 XRD pattern of (a) Pure AZ91 Mg alloy and (b) Mn, Sr-HAP/CeO₂ dual coating on AZ91 Mg alloy.

3.1.3 SEM and EDAX analysis. HRSEM micrographs of the CeO₂ (at three different concentrations of 0.05 M, 0.1 M, and 0.2 M) and Mn, Sr-HAP/CeO₂ dual coating on AZ91 Mg alloy, respectively are shown in Fig. 3. The surface morphological evaluation of the CeO₂ coatings on AZ91 Mg alloy at 1 mA cm⁻² with three different concentrations for 60 min reveals that among the coatings, the coating obtained for 0.1 M CeO₂ (Fig. 3(b)) consisted of uniform and completely covered microhexagonal structure and hence it is considered as optimum. Fig. 3(d) represents the surface morphology of Mn, Sr-HAP/CeO₂ dual coating on AZ91 Mg alloy which exhibits an uniform and ball-flower like microstructure. Ball flower like dual coating is composed of fine interconnected flakes with pores in between them which can be seen in Fig. 3(e). The formation of ball-flower morphology of the dual coating depends strongly on the electrochemical conditions that initiate nucleation process. Higher current density may generate hydrogen bubbles and result in the porous ball-flower like structure. The nucleation process is the most important step in the formation of a new structure via self-assembly or self organization. Nucleation provides a favourable environment for compact uniform ball-flower structure formation. The interconnected flakes with pores in the dual coating can allow the attachment and proliferation of diverse cell types responsible for bone tissue growth and bone formation.

Fig. 3 HRSEM micrographs of CeO₂ coatings on AZ91 Mg alloy at three different concentrations of (a) 0.05 M (b) 0.1 M and (c) 0.2 M, (d) Mn, Sr-HAP coating on the CeO₂ coated AZ91 Mg alloy at 9 mA cm⁻², (e) higher-magnification view of the Mn, Sr-HAP/CeO₂ dual-layer coated AZ91 Mg alloy and (f) EDAX spectrum of Mn, Sr-HAP/CeO₂ dual coating on AZ91 Mg alloy.

Fig. 3(f) shows the EDAX spectrum of the Mn, Sr-HAP/CeO₂ dual coating on AZ91 Mg alloy specimen which indicates the presence of Ca, Mn, Sr, Ce, O and P groups in the relative coating. This result supports for the formation of Mn, Sr-HAP/CeO₂ dual coating on AZ91 Mg alloy.

3.2 Mechanical characterization

3.2.1 Adhesion strength. The adhesion strength of Mn, Sr-HAP/CeO₂ dual coating on AZ91 Mg alloy specimen is one of the most important properties for in vivo implantation. Fig. 4(a) shows the adhesion strength of CeO₂, Mn, Sr-HAP and Mn, Sr-HAP/CeO₂ dual coatings on AZ91 Mg alloy, respectively. The adhesion strength of CeO₂, Mn, Sr-HAP and Mn, Sr-HAP/CeO₂ dual coatings on AZ91 Mg alloy was of 14.3 ± 1.0, 10.1 ± 0.4 and 12.9 ± 1.1 (Mpa), respectively. The as-developed Mn, Sr-HAP/CeO₂ dual coating with adhesion strength of 12.9 ± 1.1 (Mpa) will be suitable for orthopaedic applications.

Fig. 4 (a) Adhesion strength and (b) microhardness of uncoated, CeO₂ coating, Mn, Sr-HAP coating and Mn, Sr-HAP/CeO₂ dual coating on AZ91 Mg alloy specimens.

3.2.2 Hardness. The Vickers micro hardness (Hv) results of the uncoated, Mn, Sr-HAP and Mn, Sr-HAP/CeO₂ dual-layer coated AZ91 Mg alloy specimens, respectively are shown in Fig. 4(b) For the bare and Mn, Sr-HAP coated AZ91 Mg alloy samples, the Hv value was found to be 89.1 ± 5.2 and 325.5 ± 11.1(Hv), respectively. The micro-hardness value obtained for the Mn, Sr-HAP/CeO₂ dual coatings on AZ91 Mg alloy was higher 386.7 ± 12.8 (Hv) than that of the Mn, Sr-HAP and bare Mg alloy samples.

3.3 In vitro cytotoxicity study

3.3.1 HOS MG63 cells. The MTT assay was used to determine the HOS MG63 cell viability on the Mn, Sr-HAP/CeO₂ dual coating. The absorbance at 570 nm wavelength is directly proportional to the number of living cells in the culture medium. The % cell viability was calculated for the Mn, Sr-HAP/CeO₂ dual-layer coated AZ91 Mg alloy and compared with the control at 1, 4 and 7 days of culture. The % cell viability results are shown as bar diagram in Fig. 5. The dual coatings exhibited extensive cell viability which is similar to that of the control group for 1, 4 and 7 days of culture. The superior cell viability of the dual coatings is mainly due to the presence of Mn, Sr-HAP and CeO₂. The osteoblast proliferation is also found to improve on increasing the number of days from 1 to 7 days of culture. Accordingly, the viability results are in good agreement as can be observed from the optical microscopic images. Fig. 6(a)–(f) shows the optical microscopic results of control (a, c, e) and Mn, Sr-HAP/CeO₂ dual-layer coated AZ91 Mg alloy sample (b, d, f) obtained in 24-well tissue culture plates at 1, 4 and 7 days of incubation. The Mn, Sr-HAP/CeO₂ dual coatings at 7 days of culture showed the presence of more viable cells similar to that of control which clearly evidences that the biocompatibility of the Mn, Sr-HAP/CeO₂ dual coatings has not been affected by the presence of CeO₂ layer. Thus, the MTT assay test clearly shows that the Mn, Sr-HAP/CeO₂ dual coatings widely improved the viability of cells (99.3%) which encourages the dual layer coated Mg alloy for the orthopaedic applications.

Fig. 5 Bar diagram showing the % viability of HOS MG63 cells on Mn, Sr-HAP/CeO₂ dual-layer coated AZ91 Mg alloy at 1, 4 and 7 days of incubation.

Fig. 6 Optical microscopic images showing the % viability of HOS MG63 cells on control (a, c, e) and Mn, Sr-HAP/CeO₂ dual-layer coated AZ91 Mg alloy (b, d, f) at 1 day, 4 days and 7 days of incubation.

3.3.2 Fibroblast stem cells. The cell viability of fibroblast stem cells on dual layer (Mn, Sr-HAP/CeO₂) coated AZ91 Mg alloy was determined by MTT assay. The absorbance at a wavelength of 570 nm is directly proportional to the number of living cells in fibroblast stem cells culture medium. The percentage cell viability of 125 μg mL⁻¹ dual-layer coating was calculated with respect to the control for 1, 4 and 7 days and the results are shown in Fig. 9. From the figure it is well evident that the % cell viability of fibroblast stem cells gradually increased from 1 to 7 days of incubation and in particular, the dual layer coating showed higher/greater cell viability (99.5%) at 7 days of incubation which may be due to the presence of mineral ions in HAP. This result is further substantiated by the optical microscopic images (Fig. 10) for control and dual layer coated AZ91 Mg alloy at 1,4 and 7 days of incubation. It is well evident from the figure that the number of cells was found to be viable in the dual coating. The dual layer coating exhibited cell morphology similar to that of control group and the cells were completely spread out, proving the biocompatibility of the dual layer coated AZ91 Mg alloy. This confirms the biocompatible nature of the dual layer. Thus, from our findings it is well proved that the dual layer coating did not affect the bioactivity but instead promoted the growth of cells and hence the dual layer coated AZ91 Mg alloy can be used as an orthopedic implant.

Fig. 7 Potentiodynamic polarisation curves of uncoated, Mn, Sr-HAP coated, CeO₂ coated and Mn, Sr-HAP/CeO₂ dual-layer coated AZ91 Mg alloy specimens in SBF solution.

Fig. 8 (a) Nyquist, (b) bode and (c) phase plots of uncoated, Mn, Sr-HAP coated, CeO₂ coated and Mn, Sr-HAP/CeO₂ dual-layer coated AZ91 Mg alloy specimens in SBF solution.

Fig. 9 Bar diagram showing the % viability of fibroblast stem cells on Mn, Sr-HAP/CeO₂ dual-layer coated AZ91 Mg alloy at 1, 4 and 7 days of incubation.

Fig. 10 Optical microscopic images showing the viability of fibroblast stem cells on control (a, c, e) and Mn, Sr-HAP/CeO₂ dual-layer coated AZ91 Mg alloy (b, d, f) at 1 day, 4 days and 7 days of incubation.

3.4 Electrochemical studies

3.4.1 Potentiodynamic polarization measurements. Fig. 7 shows the potentiodynamic polarization curves of the uncoated, CeO₂ coated, Mn, Sr-HAP coated and Mn, Sr-HAP/CeO₂ dual-layer coated AZ91 Mg alloy samples, respectively in SBF solution. The electrochemical polarization parameters such as corrosion potential (E_corr) and corrosion current density (I_corr) that are obtained from the polarization curves are presented in Table 2. The polarization results of the uncoated AZ91 Mg alloy specimen revealed the E_corr and I_corr values as −1510 mV vs. SCE and 9.1 A cm⁻², respectively. While the polarization curves of the Mn, Sr-HAP coating on AZ91 Mg alloy sample shows the E_corr and I_corr values as −1350 mV vs. SCE and 5.6 A cm⁻², respectively. The potentiodynamic polarisation curve recorded for CeO₂ coated AZ91 Mg alloy sample at 1 mA cm⁻² showed the E_corr and I_corr values as −1300 mV vs. SCE and 1.4 A cm⁻², respectively. As it can be seen from the figure, the polarization values obtained for CeO₂ coated AZ91 Mg alloy sample were found to be nobler than that of the Mn, Sr-HAP coated and uncoated AZ91 Mg alloy specimens which is owing to the formation of compact CeO₂ coating on the AZ91 Mg alloy sample. The lower E_corr and higher I_corr values of the Mn, Sr-HAP coated AZ91 Mg alloy is due to its ball flower like structure with pores in between them. So, the polarization values of CeO₂ coated AZ91 Mg alloy sample reveals that it can be developed as a primary layer prior to Mn, Sr-HAP coating on AZ91 Mg alloy so as to act as a corrosion protection barrier in the SBF solution. The E_corr and I_corr values corresponding to the Mn, Sr-HAP/CeO₂ dual-layer coated AZ91 Mg alloy sample are observed as −1250 mV vs. SCE and 1.1 A cm⁻², respectively (Fig. 7). The maximum shift of E_corr (Fig. 7) towards the less negative direction (noble direction) is an indication that the Mn, Sr-HAP/CeO₂ dual coating possessed higher corrosion protection resistance in SBF solution, when compared to that of the uncoated, Mn, Sr-HAP and the CeO₂ coated AZ91 Mg alloy samples. The enhanced corrosion protection performance observed for Mn, Sr-HAP/CeO₂ dual-layer coated AZ91 Mg alloy sample is supposed to be owing to the uniform and compact surface coverage of the CeO₂ that serves as a protective barrier layer between the top layer (Mn, Sr-HAP) and the AZ91 Mg alloy.

Table 2 Electrochemical parameters of the uncoated, Mn, Sr-HAP coated, CeO₂ coated and Mn, Sr-HAP/CeO₂ dual-layer coated AZ91 Mg alloy specimens in SBF solution

Sample condition	Polarisation parameters		Impedance parameters
	Ecorr (mV vs. SCE)	Icorr × 10^−5(A cm^−2)	Rp (Ω cm^2)	|Z| (Ω cm^2)
Uncoated	−1510	9.1	1020	1036
Mn, Sr-HAP	−1350	5.6	3060	3095
CeO2	−1300	1.4	3250	3392
Mn, Sr-HAP/CeO2	−1250	1.1	3695	4192

---

3.4.2 Electrochemical impedance spectroscopic studies. A comparison of the polarization studies with EIS experiments can give more valuable information on the corrosion resistance mechanism. Especially, EIS is the most influential technique which can offer the useful information on both resistive and capacitive behaviour of all the as-coated AZ91 Mg alloy specimens in the SBF solution. Fig. 8(a)–(c) shows the Nyquist, bode and phase plots of uncoated, CeO₂ coated, Mn, Sr-HAP coated and Mn, Sr-HAP/CeO₂ dual-layer coated AZ91 Mg alloy samples, respectively in SBF solution. The polarisation resistance (R_p) and the total impedance (|Z|) values for the uncoated AZ91 Mg alloy sample are obtained as 1020 and 1036 Ω cm², respectively. The R_p and |Z| values obtained for the Mn, Sr-HAP coated AZ91 Mg alloy sample are found to be 3060 and 3095 Ω cm², respectively. As seen from the Fig. 8(a)–(c), the R_p and |Z| values obtained for the CeO₂ coated AZ91 Mg alloy sample was found to be 3250 and 3392 Ω cm², respectively which are greater than that of the Mn, Sr-HAP coated AZ91 Mg alloy (3060 and 3095 Ω cm²) specimens, respectively. The R_p and |Z| values for Mn, Sr-HAP/CeO₂ dual-layer coated AZ91 Mg alloy sample are found to be 3695 and 4192 Ω cm², respectively, which are greater than that of the CeO₂ coated AZ91 Mg alloy specimen. For the dual coated AZ91 Mg alloy sample, the Nyquist plot exhibits two capacitive semicircles and out of the them, the first semicircle at higher frequencies can be ascribed to the Mn, Sr-HAP (ball flower like layer) and the second semicircle at low frequencies corresponds to the CeO₂ layer (uniform and compact). The greater R_p and |Z| values of the Mn, Sr-HAP/CeO₂ dual coatings are due to the superior and effective barrier of compact CeO₂ layer formed the AZ91 Mg alloy substrate prior to the Mn, Sr-HAP porous coating. This indicates that the dual-layer of Mn, Sr-HAP/CeO₂ on AZ91 Mg alloy possessed enhanced corrosion protection performance in physiological fluid.

4 Conclusions

In this paper, we have demonstrated the effectiveness of bioactive and corrosion protection behaviours of electrodeposited Mn, Sr-HAP/CeO₂ dual coating on AZ91 Mg alloy. The surface morphological images of the as-formed dual coating on AZ91 Mg alloy reveals an uniform ball-flower like Mn, Sr-HAP coating on CeO₂ coated AZ91 Mg alloy. The cell viability results on HOS MG63, fibroblast stem cell cultures revealed non-toxic effect Mn, Sr-HAP/CeO₂ dual coating on AZ91 Mg alloy. Having investigation on the corrosion protection performance of all the as formed coatings, we found that the dual coating plays an effective role in improving the corrosion resistance of AZ91 Mg alloy. Thus, the Mn, Sr-HAP/CeO₂ dual coating on AZ91 Mg alloy will be more effective in implant applications.

Acknowledgements

One of the authors D. Gopi acknowledges the major financial support from the Department of Science and Technology, New Delhi, India (DST-TSD, Ref. no.: DST/TSG/NTS/2011/73), DST-EMEQ, Ref. no.:SB/EMEQ-185/2013) and Council of Scientific and Industrial Research (CSIR, Ref. no: 01(2547)/11/EMR-II, Dated:12.12.2011). Also, D. Gopi and L. Kavitha acknowledge the University Grants Commission (Ref. no. F. 30-1/2013 (SA-II)/RA-2012-14-NEW-SC-TAM-3240 and Ref. no. F. 30-1/2013(SA-II)/RA-2012-14-NEW-GE-TAM-3228) for the Research Awards.

Notes and references

1. D. Gopi, A. Karthika, M. Sekar, L. Kavitha, R. Pramod and J. Dwivedi, Mater. Lett., 2013, 105, 216–219.
2. D. Gopi, A. Karthika, S. Nithiya and L. Kavitha, Mater. Chem. Phys., 2014, 144, 75–85.
3. D. Gopi, V. Collins Arun Prakash, L. Kavitha, S. Kannan, P. R. Bhalaji, E. Shinyjoy and J. M. F. Ferreira, Corros. Sci., 2011, 53, 2328–2334.
4. D. Gopi, J. Indira, L. Kavitha and J. M. F. Ferreira, J. Appl. Electrochem., 2013, 43, 331–345.
5. J. Black, E. Maitin, H. Gelman and D. Morris, Biomaterials, 1983, 4, 160–164.
6. K. Merritt and S. Brown, J. Biomed. Mater. Res., 1995, 29, 627–633.
7. J. J. Yang and K. Merritt, J. Biomed. Mater. Res., 1994, 28, 1249–1258.
8. J. Woodman, J. Black and D. Nunamaker, J. Biomed. Mater. Res., 1983, 17, 655–668.
9. G. Almpanis, G. Tsigkas, C. Koutsojannis, A. Mazarakis, G. Kounis and N. Kounis, Int. J. Cardiol., 2010, 145, 364–365.
10. K. Dietrich, F. Mazoochian, B. Summer, M. Reinert, T. Ruzicka and P. Thomas, J. Dtsch. Dermatol. Ges., 2009, 7, 410–412.
11. X. Gu, Y. Zheng, S. Zhong, T. Xi, J. Wang and W. Wang, Biomaterials, 2010, 31, 1093–1103.
12. Z. Li, X. Gu, S. Lou and Y. Zheng, Biomaterials, 2008, 29, 1329–1344.
13. M. P. Staiger, A. M. Pietak, J. Huadmai and G. Dias, Biomaterials, 2006, 27, 1728–1734.
14. F. Witte, J. Fischer, J. Nellesen, H.-A. Crostack, V. Kaese and A. Pisch, Biomaterials, 2006, 27, 1013–1018.
15. F. Witte, V. Kaese, H. Haferkamp, E. Switzer, A. Meyer-Lindenberg and C. J. Wirth, Biomaterials, 2005, 26, 3557–3563.
16. Z. Rongchan, D. Wolfgang, W. Frank, H. Norbert and B. Carsten, Adv. Biomater., 2008, 10, 3–14.
17. C. Shuai, G. Shaokang, C. Bin, L. Wen, W. Jun and W. Ligou, Appl. Surf. Sci., 2011, 257, 4464–4467.
18. L. Longchuan, G. Jiacheng and W. Yong, Surf. Coat. Technol., 2004, 185, 92–98.
19. J. E. Gray-Munro, S. Christine and S. Michael, J. Biomed. Mater. Res., Part A, 2009, 91, 221–230.
20. H. Huo and F. Wang, J. Mater. Sci. Technol., 2007, 23, 379–382.
21. D. Gopi, P. R. Bhalaji, S. Ramya and L. Kavitha, J. Ind. Eng. Chem., 2014 DOI:10.1016/j.jiec.2014.08.019.
22. K. J. L. Burg, S. Porter and J. F. Kellam, Biomaterials, 2000, 21, 2347–2359.
23. R. Narayanan, S. K. Seshadri, T. Y. Kwon and K. H. Kim, Scr. Mater., 2007, 56, 229–232.
24. B. Bracci, P. Torricelli, S. Panzavolta, E. Boanini, R. Giardino and A. Bigi, J. Inorg. Biochem., 2009, 103, 1666–1674.
25. E. Boanini, M. Gazzano and A. Bigi, Acta Biomater., 2010, 6, 1882–1894.
26. Y. Tang, H. F. Chappell, M. T. Dove, R. J. Reeder and Y. J. Lee, Biomaterials, 2009, 30, 2864–2872.
27. I. A. Cacciotti, M. Bianco and L. Lombardi, J. Eur. Ceram. Soc., 2009, 29, 2969–2978.
28. D. Gopi, S. Nithiya, E. Shinyjoy and L. Kavitha, Spectrochim. Acta, Part A, 2012, 92, 194–200.
29. D. Gopi, S. Nithiya, E. Shinyjoy, D. Rajeswari and L. Kavitha, Ind. Eng. Chem. Res., 2014, 53, 7660–7669.
30. D. Gopi, S. Ramya, D. Rajeswari, P. Karthikeyan and L. Kavitha, Colloids Surf., A, 2014, 451, 177–186.
31. S. G. Dahl, P. Allain, P. J. Marie, Y. Mauras, G. Boivin and P. Ammann, Bone, 2001, 28, 446–453.
32. S. C. Skoryna, Trace Subst. Environ. Health, 1984, 18, 3–23.
33. E. Bonnelye, A. Chabadel, F. Saltel and P. Jurdic, Bone, 2008, 42, 129–138.
34. S. P. Nielsen, Bone, 2004, 35, 583–588.
35. R. V. Suganthi, K. Elayaraja, M. I. A. Joshy, V. S. Chandra, E. K. Girija and S. N. Kalkura, Mater. Sci. Eng., C, 2011, 31, 593–599.
36. F. Sima, G. Socol, E. Axente, I. N. Mihailescu, L. Zdrentu, S. M. Petrescu and I. Mayer, Appl. Surf. Sci., 2007, 254, 155–1159.
37. J. H. Beattie and A. Avenell, Nutr. Res. Rev., 1992, 5, 167–188.
38. A. S. Hazell, P. Desjardins and R. F. Butterworth, Neurochem. Int., 1999, 35, 11–17.
39. Y. J. Bae and M. H. Kim, Biol. Trace Elem. Res., 2008, 124, 28–34.
40. R. M. Leach Jr and A. M. Muenster, J. Nutr., 1962, 78, 51–56.
41. C. Paluszkiewicz, A. Slosarczyk, D. Pijocha, M. Sitarz, M. Bućko, A. Zima, A. Chróścicka and M. J. Lewandowska-Szumieł, J. Mol. Struct., 2010, 976, 301–309.
42. B. Bracci, P. Torricelli, S. Panzavolta, E. Boanini, R. Giardino and A. Bigi, J. Inorg. Biochem., 2009, 103, 1666–1674.
43. X. Cuia, Y. Yang, E. Liu, G. Jin, J. Zhong and Q. Li, Appl. Surf. Sci., 2011, 11, 395–404.
44. H. Hasannejad, T. Shahrabi and M. Jafarian, Mater. Corros., 2013, 64, 9999–10009.
45. Y. J. Xue, J. S. Li, W. Ma, M. D. Duan and M. M. Lan, Int. J. Surf. Sci. Eng., 2010, 4, 202–213.
46. H. Hasannejad, T. Shahrabi, M. Jafarian and A. Rouhaghdam, J. Alloys Compd., 2011, 509, 1924–1930.
47. S. T. Aruna, V. K. William Grips, V. Ezhil Selvi and K. S. Rajam, J. Appl. Electrochem., 2007, 37, 991–1000.
48. S. T. Aruna, C. N. Bindu, V. Ezhil Selvi, V. K. William Grips and K. S. Rajam, Surf. Coat. Technol., 2006, 200, 6871–6880.
49. T. S. Lim, H. S. Ryu and S. H. Hong, Corros. Sci., 2012, 62, 104–111.
50. W. F. Ng, M. H. Wong and F. T. Cheng, Mater. Chem. Phys., 2010, 119, 384–388.
51. O. Gunduz, C. Gode, Z. Ahmadd, H. Gokce, M. Yetmez, C. Kalkandelenh, Y. M. Sahin and F. N. Oktar, J. Mech. Behav. Biomed. Mater., 2014, 35, 70–76.
52. C. L. Wen, S. K. Guan, L. Peng, C. X. Ren, X. Wang and Z. H. Hu, Appl. Surf. Sci., 2009, 255, 6433–6438.
53. Y. W. Song, D. Y. Shan and E. H. Han, Mater. Lett., 2008, 62, 3276–3279.
54. D. Gopi, N. Murugan, S. Ramya and L. Kavitha, J. Mater. Chem. B, 2014, 2, 5531–5540.
55. D. Gopi, S. Ramya, D. Rajeswari and L. Kavitha, Colloids Surf., B, 2013, 107, 130–136.
56. D. Gopi, S. Ramya, D. Rajeswari, M. Surendiran and L. Kavitha, Colloids Surf., B, 2014, 114, 234–240.
57. T. Kokubo and H. Takafama, Biomaterials, 2006, 27, 2907–2915.
58. D. Girija, S. Halehatty, B. Naik, B. N. Sudhamani and B. Vinay Kumar, Arch. Appl. Sci. Res., 2011, 3, 373–382.
59. R. Rojaee, M. Fathi and K. Raeissi, Appl. Surf. Sci., 2013, 285, 664–673.
60. H. Yong, D. Qiongqiong, H. Shuguang, Y. Yajing and P. Xiaofeng, J. Mater. Sci.: Mater. Med., 2013, 24, 1853–1864.
61. O. Zuas, H. H. Abimanyu and W. Wibowo, Process. Appl. Ceram., 2014, 8(1), 39–46.

---