Formation of nanostructures on magnesium alloy by anodization for potential biomedical applications

Abstract

In the present work, we have investigated the formation of nanostructures on AZ31 magnesium alloy using electrochemical anodization technique. The formed nanostructures were efficiently showed bone-like apatite formation followed by its gradual increase, when immersed in simulated body fluid (SBF) and it exhibited controlled degradation in 7 days. Cell viability study was performed using MG-63 cells (human osteosarcoma cell lines) and revealed that the nanostructured surface has excellent biocompatibility by enhancing both cell adhesion and cell growth. The detailed characterization of this anodized surface was evaluated by field emission scanning electron microscopy (FESEM) and energy-dispersive X-ray spectroscopy (EDS). Furthermore, surface-corrosion before and after anodization was examined by electrochemical impedance spectroscopy (EIS) and potentiodynamic polarization studies in SBF. The in-depth studies bring out the fact that native oxide in the sample is converted to a biocompatible nanostructure, which is created due to anodization in a particular electrolyte solution containing ethylene glycol and hybrid hydrofluoric acid mixture.

1. Introduction

Biomaterials are used in various parts of the human body, including the cardiac, valves and vessels, shoulders, stents, hips, knees, elbows, dental roots, ears, etc. [1,2]. Artificial biomaterials are the solution for the arthritis problems and these artificial biomaterials are implanted through arthroplasty surgery. In the context of recent research, one of the most promising areas in implant development for biomedical applications is the development of degradable metallic implant materials, such as magnesium and its alloys. The mechanical properties of Mg and its alloys match with the natural bone, thus significantly reducing the bone mismatch, thereby the stress-shielding effect. As a result, the bone healing process may not be affected during service. A major limitation of Mg alloys is their fast degradation rate in the chloride-containing physiological environments [3]. Therefore, it is necessary to control the Mg degradation rate to facilitate the bone healing process.

Several approaches have been attempted to modify bio-metals such as acid and alkali treatment, sandblasting, electrochemical oxidation, bioactive coating of calcium phosphate, and hydroxyapatite (HAP). HAP coatings can be developed by different methods including ion implantation, plasma spraying, sol-gel, and electrophoretic deposition [4]. Conversely, delamination typically happens at the interface of implant and bone due to a lack of interactions between the substrate and coating. This leads to the elimination of the coating from the artificial implant surface [5]. Accordingly, serious efforts from biomedical researchers worldwide have been focused on improving the design and manufacture of orthopaedic implants. In that perspective, inter-disciplinary research approaches merging the efforts of bio-engineers, materials scientists, medical experts, nanoscientists, biologists, and clinicians have recently shown promising results. The development of surfaces with the nanostructures on implants is beneficial as it is inexpensive, surface chemistry can be designed, pore/tube structures can be controlled, flexible, mechanically stiff, chemically steady, more surface area, exceptional biocompatibility and more interestingly, it can be reliably formed on the surface of medical implants. In this regard, nanostructuring seems to offer a solution to this problem, leading to better performance of implants. The development of nanostructures on Mg alloy generally improves the corrosion resistance of the substrate owing to the formation of the Mg oxide/hydroxides. Also, it enhances the bioactivity in terms of accelerating the hydroxyapatite (HAP) growth. The incorporation of bioactive materials coating into the developed magnesium nanostructures could further enhance the bio-compatibility. Therefore, it is reasonable to attribute that the development of the ordered structure on Mg is one of the interesting areas for its widespread applications [4].

Over a few years, an extensive research attempt has been committed to the engineering of pioneering nanomaterials, including nanostructures prepared by anodization, hydrothermal, sol-gel, and vapors deposition techniques. Current studies have shown that TiO₂ nanotubes formed on titanium (Ti) substrates could be used as the best platform to present nanoscale structure [5–12]. The use of nanostructured materials has been proposed to resolve many of the problems presently associated with orthopedic implants [13]. The anodization parameters, viz., electrolyte concentrations, current density, and anodization time, strongly alter the quality of the oxide layer. Anodization approach is mainly used to thicken the native oxide/hydroxide films on metal surfaces, and dedicated anodization approaches have been explored to create nanoporous oxide layers [14]. The key is to use an electrolyte that leading to competition between the growth and dissolution of anodic oxide. Self-organized growth of nanoporous or nanotubular oxide layers can be achieved using optimal electrochemical parameters [15]. The applications of titania nanotube arrays (TNTA) in the area of biomedical applications are well established [7,16]. The formation of nanostructures on magnesium alloy (AZ31) by electrochemical anodization technique has not yet been reported [14,17–24]. We have fabricated the nanostructures on biodegradable magnesium alloy AZ31 by electrochemical anodization process using a hybrid electrolyte composition for potential biomedical applications. In the present study, the magnesium nanostructures formed on Mg alloys and bioactive materials (HAP + TiO₂ nanocomposites) were incorporated on nanostructures. The corrosion behavior of the nanostructured samples in simulated body fluid (SBF) was investigated by electrochemical impedance spectroscopy (EIS) and potentiodynamic polarization studies. The in-vitro biocompatibility study of the substrate and nanostructured samples using MG-63 cells is presented.

2. Materials and methods

The formation of nanostructures on AZ31 magnesium alloy was synthesized using the electrochemical anodization technique. The in-corporation/coating of bioactive materials in the prepared nanostructures was achieved by using hydroxyapatite and TiO₂ nano-composites through Electrophoretic deposition (EPD) technique.

2.1. Samples preparation

Magnesium alloy AZ31, with the composition of Mg-3Al – 1Zn (wt. %) was procured from Sysco piping solutions Inc, India in the form of a plate. Test samples with dimensions of ~ 20 × 20 × 3 mm, cut from the plate were ground with silicon carbide grits (different grades). The samples were ultrasonically cleaned using acetone and kept dried.

2.2. Electrochemical anodization

A two-electrode electrochemical anodization cell, with a platinum cathode and AZ31 anode, was used to fabricate the nanostructures on the substrates. In our study, various anodization experiments were conducted using DC power supply by varying parameters such as electrolyte, voltage, and time. The experiments were conducted at 40 and 70 V for different intervals.

2.2.1. Conventional electrolyte

The conventional electrolyte used for anodization process was obtained by mixing 0.08 (M) of 10 mL HF in 90 mL of ethylene glycol and stirred continuously using a magnetic stirrer. The distance between the electrodes was 2 cm, kept constant for all experiments. The electrolyte with a volume of 100 mL was stirred continuously using a magnetic stirrer.

2.2.2. Hybrid electrolyte

The hybrid electrolyte was prepared by mixing 1 g of synthesized TiO₂ nanopowder (particle size ~ 35 nm) with 1 (M) hydrofluoric acid with continuous stirring in magnetic stirrer in 600 RPM for 24 h. After 24 h of stirring the (TiO₂ +HF) mixture is filtered. The mixing of HF and TiO₂ leads to the formation of fluorotitanic acids “H [TiF₄ (OH) H₂O], H [TiF₅. H₂O]”, and H₂ [TiF₆] in the solution part and titanium oxyfluoride (TiOF₂ · H₂O) in the solid part [25]. From the above filtrate, 10 mL is mixed with 90 mL of ethylene glycol and stirred for 30 min with 600 RPM. This solution is used as a hybrid electrolyte for the anodization of Mg alloy (AZ31).

2.3. Electrophoretic deposition (EPD)

The synthesis procedure of hydroxyapatite and TiO₂ nanoparticles and characterizations are discussed in our previous article [4]. For the preparation of suspension, 0.25 g of nanocomposite powders (HAP + TiO₂) was added to 50 mL of acetone and sonicated for 10 min. After this, iodine (precursor), acting as a dispersant, (0.1 g) was added to stabilize the suspension [4]. The sample Mg alloy played the role of a cathode and the platinum as an anode. Both electrodes were immersed in a beaker containing the suspension. The applied voltage was fixed at 30 V and the time of deposition was 5 min. In the end, coated samples were heat-treated to enhance the adhesion strength of the coatings and reduce the porosity [4].

2.4. Immersion studies

Immersion studies were performed on the bare substrate and nanostructured samples by immersing them in freshly prepared acellular SBF (simulated body fluid) Hanks’ solution continuously for 7 days at 37 °C. “This SBF solution contains ion concentrations that are nearly equal to that of human blood plasma (Na+ = 42.0, K⁺ = 5.0, Mg²⁺ = 1.5. Ca²⁺ = 2.5, HCO₃ ^– = 4.2, HPO₄ ^2- = 1.0, SO₄ ^2- = 0.50 and Cl⁻ = 147.96 mM). The chemical composition of this solution is as follows: 0.185 g CaCl₂, 0.4 g KCl, 0.06 g KH₂PO₄, 0.1 g MgCl₂·6H₂O, 0.1 g MgSO₄·7H₂O, 8.0 g NaCl, 0.35 g NaHCO₃, 0.48 g Na₂HPO₄ and 1.00 g d-glucose in 1 L of ultrapure water. The pH of the solution was adjusted to 7.2–7.6 with 1 M HCl” [26–28].

2.5. Electrochemical measurements

All the electrochemical measurements including the substrate and anodized samples were performed in the CH 604D Electrochemical Workstation (CH Instruments, USA) by using conventional three-electrode glass cell. Hanks’ solution with a volume of 200 mL was used for required testing. “A Pt foil was used as a counter, the sample as a working electrode, and a saturated calomel electrode (SCE) as a reference electrode. The reference electrode and the working electrode were placed very close to each other. To establish an open circuit potential (EOCP) or the steady-state potential, the sample was immersed in Hanks’ solution for an hour before the application of alternating sinusoidal potential with an amplitude of 10 mV. Electrochemical Impedance Spectroscopy (EIS) measurement studied at the frequency ranges between 10 mHz to 100 kHz. After finishing each experiment, the impedance data were recorded as Bode plots i.e., the plot of log |Z| as a function of log f and log f as a function of phase angle (θ), where |Z| is the absolute impedance and f is the frequency. To extract suitable equivalent circuit parameters, the data were analyzed by using the ZSimpwin program (Princeton Applied Research, USA), where the fitting quality was standardized by the χ2 value. After EIS measurements, potentiodynamic polarization studies were carried out in a potential range of 200 mV below and above OCP values with a scan rate of 1 mV/ s. The corrosion potential (E_corr) and corrosion current (I_corr) were calculated from the Tafel plot (potential vs. log (i)). The corrosion current was determined by using the Stern-Geary equation [29],

$$I_{\text{corr}}=\frac{{\text{β}}_{\text{a}}\times {\text{β}}_{\text{c}}}{2.3R_{\text{p}}\left({\text{β}}_{\text{a}}+{\text{β}}_{\text{c}}\right)},$$

Where β_a and β_c denote the Tafel slopes of the anodic and cathodic part of the Tafel plot, and R_p is polarization resistance” [27,30–32].

2.6. Cell growth studies

2.6.1. Cell viability and cell staining

To check the biocompatibility of the samples, cell viability tests were performed on MG-63 (human osteosarcoma cell lines), obtained from “NCCS (National Centre for Cell Science), Pune, India”. Cells were maintained in the MEM solution (Modified Eagle’s Medium, purchased from Himedia, India) containing 10 % FBS (Fetal Bovine Serum from Gibco, USA) and 1% antibiotic solution (Penicillin streptomycin from Himedia). The cells were incubated at a temperature of 37 °C with 5% CO₂ in a humidified atmosphere. The indirect procedure as per “ISO 10993 – 1” with slight alteration as described earlier [33] was followed for cytocompatibility measurements. Before conducting the cell culture studies, the samples (substrate, Mg nanostructures, Mg nanostructures coated with HAP + TiO₂) were sterilized with ethanol and UV treatment for 1 h. Then they were washed with PBS and incubated for 72 h in MEM media to obtain its extract. These extract with media is used for the studies to estimate the cell adhesion, cell viability, and proliferation capacity. The above extracts (500 μl) were added to a 30 mm cell culture plate and MG 63 cells were seeded at a density of 6 × 10⁵ cells/mL. All the culture plates were added with different sample extracts, excluding in the control group. After 24 h of further incubation, the nuclei and cytoskeleton (live cells) were stained with Hoechst (33342, Thermo Fischer, India) dye and Calcein AM, respectively. Propidium iodide (PI) dye was used to stain the nucleus of the dead cells and observed with a fluorescence microscope (Olympus).

2.6.2. MTT assay

MTT assay “(3- [4,5-dimethylthiozol-2-yl]-2,5 diphenyl tetrazolium bromide)” (Analytical grade, HiMedia, Mumbai, India) was performed using MG63 cells to assess the cell viability on all the prepared samples following the method defined in earlier studies [34–36]. Briefly, each sample extracts (obtained as described in section 2.6.1) was added in a 30 mm cell culture plate and MG63 cells were seeded at a density of 0.5 × 10⁵ cells/mL. The viability tests were performed after 3 days of cell culture. 50 μL of 5 mg/mL MTT (stock) was included in each plate and then incubated at a temperature of 37 °C in a CO₂ incubator for 4 h. After 4 h, the cell culture medium containing MTT was removed from the culture plate by aspiration. Thereafter, the formed formazan crystals were dissolved by adding 500 μL dimethyl sulfoxide (DMSO, Merck). Then, the 200 μL of DMSO dissolved formazan crystals aliquots were transferred into a 96-well plate for reading the optical density at 570 nm through an “enzyme-linked immunosorbent assay (ELISA)” plate reader (Biorad) [37]. All the implants were assayed in triplicates.

2.7. Characterization techniques

Quanta 400 FESEM was used to evaluate the surface morphology of the samples. The 3D and 2D profiles of FESEM images were acquired via the scanning probe image processor WSxM 5.0 develop 7.0 software [38]. To measure the water contact angles of the samples, the sessile drop method was followed by using surface electro-optics phoenix contact angle meter (South Korea). Water droplets (around 8·0 μL each) were carefully dropped onto the samples using a syringe. The approximate value of contact angle was obtained by averaging the values measured at different positions on each sample.

3. Results and discussion

FESEM images of the polished AZ31 substrate are shown in Fig. 1 (a) at lower magnification and Fig. 1 (b) at higher magnification. Similarly, Fig. 1 (c and d) shows the 3D and 2 D profiles of the substrate generated from FESEM image at higher magnification. As can be seen in the images, the surface appears to be smooth which confirms that the substrate had lower surface roughness values.

Fig. 1. (a and b) FESEM images of the AZ31 substrate, (c and d) 3D and 2D profiles obtained from FESEM image.

FESEM images of the anodized samples using conventional electrolyte preparation methods with lower and higher magnifications are given in Fig. 2 (a and b), respectively. Fig. 2 (c and d) shows the corresponding 3D and 2D profiles. All these results reveal the degraded and/ or corroded morphology of the surface. The EDS measurements provide the wt.% of different elements as listed in Table 1 before and after anodization of the AZ31 substrate. EDS results confirm that the wt. % of the procured alloy is same as that of the standard composition of AZ31. Aluminum is around 3 wt% and Zinc ~ 1 wt% and rest is magnesium. In contrast, anodized samples show an increase of oxygen wt.% which affirms the formation of the oxide layer after anodization.

Fig. 2. (a and b) FESEM images of anodized AZ31 using HF and ethylene glycol by a conventional electrolyte (c and d) 3D and 2D profiles obtained from FESEM image.

Table 1. EDS of AZ31 substrate and anodized AZ31 (conventional electrolyte).

Element	AZ31 Substrate (Wt%)	Anodized AZ31 (conventional electrolyte) (Wt %)
Mg	96.69	04.17
Al	2.60	93.19
Zn	0.71	01.92
O	–	00.72

To ensure the electrolyte mechanism, anodization was also carried out on a titanium alloy, Ti-6Al-4 V with the same conventional electrolyte containing ethylene glycol and HF and anodization parameters. Fig. 3 (a and b) shows FESEM images of TiO₂ nanotube array attained after anodization of Ti–6Al–4 V (1 h at 30 V). Similarly, Fig. 3 (c, d, and e) shows the 3D image of the TiO₂ nanotube array generated from FESEM image at higher magnification. In these images, the high degree of ordering of the tubes is seen. Fig. 3(f and g) shows the 2D profiles of the TiO₂ nanotubes array obtained from FESEM image.

Fig. 3. (a and b) FESEM images of anodized Ti-6Al-4 V using HF and ethylene glycol by conventional electrolyte preparation method (c -e) 3D and (f and g) 2D profiles obtained from FESEM images.

These results indicate that the conventional electrolyte can develop nanostructures on titanium alloy but not on the magnesium alloys (AZ31). Therefore, a new hybrid electrolyte preparation method is necessary to develop nanostructures on Mg alloys. For this purpose, we aimed at a new hybrid electrolyte preparation in order to anodize the Mg alloy AZ31.

After several trial and error experiments, we found a hybrid electrolyte that is efficient for achieving anodization of the Mg alloy. The hybrid electrolyte was prepared by mixing 1 g of synthesized TiO₂ nanopowder (particle size ~ 35 nm) with 1 (M) hydrofluoric acid continuously stirred at 600 RPM for 24 h. After 24 h, the mixture (TiO₂ + HF) was filtered. From the above filtrate, 10 mL is mixed with 90 mL of ethylene glycol and stirred at 600 RPM for 30 min. The obtained mixture is the desired solution that was used as a hybrid electrolyte for the anodization of AZ31 Mg alloy. Ethylene glycol, fluorotitanic acids “H [TiF₄ (OH) H₂O], H [TiF₅. H₂O], and H₂ [TiF₆]” in this hybrid electrolyte enhances field-assisted oxidation of the Mg alloy accompanying chemical engraving process which creates the ordered nanostructures on AZ31 alloy. FESEM images of the acquired surface morphologies for the samples anodized at 40 V for 20 min are shown in Fig. 4 (a and b) at lower and higher magnification. Fig. 5 (a-c) shows similar images for the samples anodized at 70 V for 1 h. The 3D and 2 D profiles of these surface morphologies are shown in Fig. 4 (c–g) for 40 V and Fig. 5 (d and e) for 70 V. In these images, a high degree of ordering of the nanostructures having increased roughness profiles is observed. It is worth noting that the anodized samples show a porous structure with a diameter of ~60 nm at 40 V and 80 nm at 70 V. No inter-tubular space and wall separation were observed in the anodized samples. The results of 3D and 2D profiles confirm the same. The wt.% of elements attained by EDS is given in Table 2 for the AZ31 substrate after anodization at 40 V and 70 V. EDS results showed a wt.% increase in oxygen which confirms the formation of the oxide layer after anodization.

Fig. 4. (a and b) FESEM images of anodized AZ31 (40 V, 20 min) using HF and ethylene glycol by hybrid electrolyte preparation (c - g) 3D and 2D profiles of FESEM image (a).

Fig. 5. (a-c) FESEM images of anodized AZ31 (70 V 1 h) using HF and ethylene glycol by hybrid electrolyte preparation (d and e) 3D and 2D profiles of FESEM image (c).

Table 2. EDS of anodized AZ31 substrate (hybrid electrolyte) at 40 V and 70 V.

Element	40 V (Wt%)	70 V (Wt%)
Mg	90.39	92.33
Al	3.79	01.38
Zn	0.50	00.20
O	5.32	6.09

To examine the wettability of the substrate and anodized samples, the water contact angle was measured as shown in Fig. 6. The water contact angle is ~ 60.2° ± 1° on the AZ31 substrate before anodization. In contrast, the water contact angle is ~ 130.6° ± 1° after anodization. The formation of nanostructures increases the surface roughness on the anodized samples compared to that of the substrate. This is further substantiated from 3D and 2D profiles of the substrate and anodized samples shown in Figs. 1, 4, and 5, respectively. Therefore, after anodization, the formed nanostructures are hydrophobic compared to the substrate. No significant change was found in water contact angles due to the increase of anodization voltage from 40 V to 70 V. Samples anodized at 40 V were selected for conducting further studies.

Fig. 6. Water contact angle (a) substrate and (b) anodized AZ31 samples.

Potentiodynamic polarization graphs of the bare substrate and anodized AZ31 in the Hanks’ solution (SBF) are shown in Fig. 7 (left). The corrosion current (I_corr) was acquired by extrapolation of the cathodic and anodic branches of the polarization graphs to the corrosion potential. The corrosion potential, corrosion current density, and polarization resistance are listed in Table 3. The corrosion current density (I_corr) is 3.135 μA/cm² for the AZ31 substrate decreases to 1.173 μA/ cm² in anodized AZ31 is (Table 3). Thus, the corrosion resistance of the substrate is significantly increased by anodization. It can be seen from Fig. 7 that the formation of a passive film reduces the current by almost an order of magnitude in the anodized samples. Furthermore, the current in the passivation region is almost constant up to the voltage of -1.5 V which indicates that the stability of the passive layer is better in the anodized sample compared to the substrate. All these results, indeed, establish the fact that the anodized samples have better corrosion resistance and passivation characteristics compared to the bare substrate.

Fig. 7.

(left) Tafel plots of the substrate and anodized AZ31 samples. (Right) Macrographs of the substrate and anodized AZ31 samples (a) as-received AZ31 substrate, (b) anodized AZ31, (c) AZ31 substrate after corrosion, (d) anodized AZ31 after corrosion.

Table 3. Results of potentiodynamic polarization studies.

S.No	Sample	Ecorr (V)	icorr (μA/cm2)	Rp (Ohm cm2)
1.	AZ31 substrate	–1.481	3.135	9973
2.	Anodized AZ31	–1.614	1.173	11424

Surface micrographs before and after corrosion for bare substrate as well as anodized samples are shown in Fig.7 (Right). From the micrographs, it can be seen that in the case of the bare substrate, the surface has more corrosion spots and the material is highly degraded. In the case of the sample after anodization, the surface has fewer corrosion spots compared to the substrate. These results suggest that after anodization the corrosion resistance increased almost one-fold.

The results of electrochemical impedance test in the Hanks’ solution are obtainable in the form of Nyquist and Bode plots in Fig. 8. The Nyquist plots in Fig. 8 (a) are semi-circular, with a larger diameter with the anodized sample, which indicates higher impedance. In Fig. 8 (b and c), the phase angle changes rapidly from – 50o to – 60o to high-frequency range for the bare substrate and anodized samples. The limiting impedance at the high-frequency end corresponds to the solution resistance, R_e. The phase angle is nearly 0 in the frequency range from 0.01 Hz to 100 Hz. Fig. 8 (d) shows the equivalent circuit containing the two-layer model used for fitting the substrate and the anodized samples EIS data. The equivalent electrochemical circuit is containing CPE (constant phase element) and resistances. The resistive components R₂, R₁, R_e and are associated with the inner layer, outer layer, and solution resistance, respectively. The Q symbol signifies the probability of a non-ideal capacitance, called a CPE (constant phase element), whose impedance is defined as “ZCPE = [Q (jωn)] – 1 with ‘n’ is less than 1; for an ideal capacitance n = 1”. In this case, Q₂ signifies the capacitance of the inner layer, Q₁ is the capacitance of the outer layer.

Fig. 8.

Electrochemical impedance plots obtained in Hanks’ solution for AZ31 substrate and anodized samples (a) Nyquist plot, (b and c) bode plots, and (d) Equivalent circuit (EC) diagram used for fitting EIS data of the AZ31 substrate and anodized samples.

Table 4 listed the values of the electrical constraints acquired by fitting the impedance data of the bare substrate and anodized samples. The electrochemical behavior of Mg alloys is modeled with the duplex structure of the native oxide layer on the surface. The inner layer is related to the high corrosion resistance of the substrate, which is 3.91 × 10⁴ Ωcm² in the current case. The Q₂ component is related to the capacitance of this inner barrier layer. The resistance of the outer porous layer is 577 Ω cm², which is very low compared to the barrier layer. The non-ideal value for the constant phase element representing the outer layer is 0.70 and the inner layer is 0.80. The results for anodized samples in Table 4 shows that the resistance R₁ of the outer layer is about 2357 Ωcm² and that of the inner barrier layer is 4.02 × 10⁴ Ωcm². The non-ideal value for the CPE representing the outer layer is 0.87 and the inner layer is 0.90, which is much closer to unity compared to the substrate. These results show that anodization substitutes the native oxide layer with the structure of the porous outer layer by forming a denser oxide layer. The new oxide layer formed after anodization is responsible for the better passivation and electrochemical behavior in the electrochemical studies [31]. Potentiodynamic polarization data also backing this inference.

Table 4. Electrochemical impedance parameters obtained by fitting equivalent circuit model for substrate and anodized samples.

S.No	Samples	Re (Ω cm2)	Q1 (S sn cm –2)	n1	R1 (Ω cm2)	Q2 (S sn cm –2)	n2	R2 (Ω cm2)
1	AZ31 substrate	16	4.56 × 10−6	0.70	577	3.79 × 10−6	0.80	3.91 × 104
2	Anodized AZ31	14	3.62 × 10−7	0.87	2357	4.28 × 10−6	0.90	4.02 × 104

The FESEM images of the bare substrate and anodized sample surface before and after 7 days’ immersion in SBF are given in Fig. 9. In these images, apatite appears to be a white deposit. As can be seen in the images, the apatite deposits are more in both samples and the size of the deposits is larger. After 7 days’ immersion in the Hanks’ solution, it is evident that the formation of apatite was initiating from sides of the nanostructure as wall thickness increases due to the formation of apatite on the walls of the nanostructures. The Ca/P ratio is found to be 1.65 for the 7 days immersed samples, which is ~1.66 for hydroxyapatite [Ca10·(PO₄)₆·(OH)₂], the principal component of bone material. Therefore, the apatite formed might be composed of hydroxyapatite and calcium phosphate (CP) phases. The stable phases of calcium phosphate depend upon temperature and the presence of water that is either during processing or in the environment. The formation of the CP phases interacts with water or body fluids to form hydroxyapatite [28,31,39]. These results confirm that the development of apatite growth on the nanostructures gradually upturns after immersion in the Hanks’ solution. The above observations indicate that anodized sample has a higher potential to induce the development of well-adhered apatite on its surface as the growth begins from inside and walls, of the nanostructures.

Fig. 9.

(a) AZ31substrate as received, (b and c) AZ31substrate after 7 days’ immersion in Hanks’ solution and (d) anodized AZ31 substrate (e and f) anodized AZ31substrate after 7 days’ immersion in Hanks’ solution.

Fig. 10 shows the graphical representation of all the results obtained from various characterizations of the AZ31 substrate and anodized samples. It can be seen from the graphs that after anodization of AZ31 Mg alloy; oxygen wt%, roughness, contact angle, polarization resistance, corrosion resistance (decrease in corrosion current) are enhanced with decreased Mg degradation. Inductively coupled plasma optical emission spectroscopic (ICP-OES) studies were performed on the AZ31 substrate and anodized samples to analyze Mg release from the metal. The concentration of Mg (mg/liter) released in the substrate is higher compared to the anodized samples. Similarly, the release of Al and Zn (alloying elements of AZ31) reduced after anodization. These results confirm that the anodization/ nanostructuring of Mg alloy reduces the release of metals, by increasing the lifetime of the implants by preventing them from faster biodegradation.

Fig. 10. Graphical representation of the results obtained from various characterizations of the AZ31 substrate and anodized samples.

The electrophoretic deposition (EPD) of HAP + TiO₂ was carried out on the nanostructured samples as a procedure discussed in section 2.3. Fig. 11 (a and b) shows the FESEM images (lower and higher magnifications) of electrophoretic deposition (EPD) of nanocomposites (HAP + TiO₂) on the nanostructured substrate (anodized substrate). Similarly, Fig. 11 (c and d) shows the FESEM images (lower and higher magnifications) after 7 days’ immersion in the Hanks’ solution. In these images, apatite seems to be a white deposit. As can be seen in the images, the apatite deposits are high on the sample and the size of the deposits is larger. After 7 days of immersion in the Hanks’ solution, it is evident that the growth of new apatite originated from the existing nanocomposite coatings. Similarly, the EDS results also indicate an increase in Ca, P, and O wt. % after 7 days of immersion. These results suggest that the formation of apatite growth after immersion in the Hanks’ solution begins in the HAP + TiO₂ nanocomposite and is gradually increased by the increase in immersion days. The above interpretations indicate that the HAP + TiO₂ coating on the nanostructure has the higher potential to induce the growth of well-adhered apatite on its surface.

Fig. 11.

Anodized AZ31 substrate after EPD (left) (a) at lower magnification and (b) at higher magnification, (c and d) after 7 days’ immersion in Hanks’ solution, (right) EDS before and after 7 days of immersion in Hanks’ solution.

Fig. 12 shows the fluorescence images of MG-63 (human osteosarcoma cell lines) on the (1) control (without sample extract), (2) Mg substrate extract, (3) Mg nanostructures extract, (4) HAP + TiO₂ incorporated Mg nanostructures extract. The samples were stained with Calcein AM to check the cell viability (green), Hoechst stain (blue) stains the cell nucleus, and Propidium Iodide (PI) dye stains the dead cells (red). To understand the difference between dead and live cells all three images (a, b, and c) are merged into a single image (d). The cells show the usual shape and look healthy with stretched filopodia and better spreading and proliferation. The number of cells was found to be higher in HAP + TiO₂ incorporated nanostructures extracts than the other extracts. 3-(4,5 Dimethyl thiazol-2 yl)-2,5 diphenyltetrazolium bromide assay (MTT assay (Fig. 13)) has been used to detect the cytotoxicity of Mg substrate, nanostructures, and nanostructures with HAP + TiO₂ on osteoblast-like cells (MG-63). After 72 h of incubation, the number of viable cells in the nanostructures and HAP + TiO₂ is further increased. No apparent cytotoxicity was observed after 72 h incubation; all samples maintained good cell growth signifying that they are not cytotoxic.

Fig. 12.

Fluorescence images of MG-63 (human osteosarcoma cell lines) on extracts of (1) Control (without extract), (2) Mg substrate, (3) Mg nanostructures, (4) Mg nanostructures coated with HAP + TiO₂ stained with (a) Calcein AM (viable cells), (b) Hoechst (cell nucleus) (c) Propidium Iodide (PI) (dead cells) and (d) merge images of a, b and c.

Fig. 13. The viability of MG63 cells incubated with extracts for a duration of 72 h.

4. Conclusions

In the present study, nanostructures with pore diameters ~ 60 to 80 nm have been successfully formed on AZ31 magnesium alloy at different voltages with hybrid electrolyte composition. The ethylene glycol with fluorotitanic acids “H [TiF₄ (OH) H₂O], H [TiF₅.H₂O], and H₂ [TiF₆]” in the hybrid electrolyte, enhances field-assisted oxidation of AZ31 Mg alloy and uniform chemical engraving process, respectively. Potentiodynamic polarization and electrochemical impedance studies in the SBF show that the anodized samples have enhanced passivation behavior, which can be comparable to the substrate. The progress of the apatite alongside the nanostructure and at the bottom shows that the developed apatite forms a well-adhered layer among the implant and the bone. In-vitro cell culture studies revealed that the cells exhibit usual shape and look healthy with prolonged filopodia and improved proliferation and spreading on surface-modified Mg substrates. Furthermore, to the above, it is an eminent fact that the adhesion of cells on metal surfaces can be improved by using nanostructured surfaces, which, indeed, would enhance their bone-forming ability.