Background of the invention The present invention relates to the field of chemicals and more precisely to that of biocompatible alloys, as it concerns a process for covering biocompatible magnesium-based alloys, to be used for biomedical applications, with a coating which controls the corrosion and degradation thereof and makes the amount of hydrogen developed tolerable by the human body.

Background

Magnesium alloys and in particular AZ31 magnesium alloys are used for the production of biomedical devices such as prostheses and stents which are subject to corrosion phenomena inside the human body which can compromise the functioning thereof [B. Heublein, R. Rohde, V. Kaese, M. Niemeyer, W. Hartung, and A. Haverich. "Biocorrosion of magnesium alloys: a new principle in cardiovascular implant technology?" Heart 89, no. 6 (2003): 651-656; P. Zartner, R. Cesnjevar, H. Singer, and M. Weyand. "First successful implantation of a biodegradable metal stent into the left pulmonary artery of a preterm baby" Characterization and Cardiovascular Interventions 66, no. 4 (2005): 590-594.]. Said magnesium alloys have excellent mechanical properties, in particular the elastic modulus of the AZ31 magnesium alloys is comparable with that of human bone and they are biodegradable. Furthermore, they do not need to be removed because they perform their function of mechanical support for the immediate period necessary and then degrade releasing non-toxic substances, which can therefore be assimilated without problems by the human body [D.A. Robinson, R.W. Griffith, D. Shechtman, R.B. Evans, and M.G. Conzemius. "In vitro antibacterial properties of magnesium metal against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus" Acta Biomaterialia 6, no. 5 (2010): 1869-1877]. During this degradation process, hydrogen gas is developed, the quantity of which must therefore be strictly controlled [F. Witte, N. Hort, C. Vogts, S. Cohen, K.U. Gainer, R. Wollastonite, and F. Feedback. "Degradable elastomeric based on magnesium corrosion" Current Opinion in Solid State and Materials Science 12, no. 5-6 (2008): 63-72].

Surface treatments are known in the art which allow the formation of protective and biocompatible coatings [G. Song. "Control of biodegradation of biocompatible magnesium alloys" Corrosion Science 49, no. 4 (2007): 1696-1701].

Chinese patent no. CN109758605 discloses a coating of acile hydroxyapatite on the surface of a magnesium alloy and the preparation process by means of electrochemical deposition.

Chinese patent no. CN109646717 discloses a nano-hydroxyapatite coating on the surface of a magnesium alloy and the ultrasonic-based preparation process.

Chinese patent no. CN109440160 discloses a process for modifying the surface of a magnesium alloy with a composite coating of hydroxyapatite modified with dopamine.

Chinese patent no. CN109295438 discloses a process for preparing a surface micro structure hydroxyapatite coating on a magnesium alloy by means of a hydrothermal process in which the magnesium alloy is subjected to surface polishing, grinding and ultrasonic cleaning sequentially in acetone, deionized water and ethyl alcohol, thereafter drying; then the magnesium alloy thus treated is immersed in a Na Oh solution at a temperature between 60 and 90°C, then rinsed with deionized water and dried, and placed in a high pressure reactor containing the hydrothermal reaction solution. The reactor is heated up to a temperature between 100 and 140°C. After cooling, the sample is extracted and the sample is cleaned and dried; the hydroxyapatite coating consists of a two-layer structure: the upper layer is composed of a flower shaped cluster structure composed of nanobars, and through the spaces between the clusters, it can be seen that the lower layer is uniform and with compact nanobar structure.

The coating has a high resistance to corrosion and is of great commercial distribution value.

Chinese patent no. CN109183127 discloses a process for preparing a composite coating of surface hydroxyapatite on a magnesium alloy which comprises the steps in which the surface of the magnesium alloy is subjected to a treatment of honing, polishing and oxidation in sequence, subsequently immersed in the hydroxyapatite dispersion liquid modified with polydopamine at 100-120°C for 0.5-1 hours, then the surface is extracted, cleaned with distilled water, dried and immersed again in the hydroxyapatite dispersion liquid modified with polydopamine, and the operation is repeated 3-5 times.

Chinese patent no. CN109161955 discloses a preparation process for the electro-deposition of a hydroxyapatite and graphene oxide coating on the surface of a magnesium alloy by immersion in a hydrolyzed solution of aminopropyl triethoxysilane to obtain the silanization of magnesium, and the electrodeposition of hydroxyapatite and graphene oxide.

Chinese patent no. CN108166036 discloses a process for preparing a nano-hydroxyapatite coating containing fluorine on the surface of a biomedical magnesium alloy.

Chinese patent no. CN108004527 discloses a process for preparing a zinc-doped hydroxyapatite coating used for a magnesium alloy material.

Chinese patent no. CN106756925 discloses a hydroxyapatite coating containing silver on the surface of a magnesium alloy.

Chinese patent no. CN105457099 discloses a two-layer fluorine- doped hydroxyapatite coating on a magnesium alloy and a microwave preparation process thereof. Chinese patent no. CN104888271 discloses a process for preparing a hydroxyapatite coating containing strontium on the surface of a biodegradable magnesium alloy.

Chinese patent no. CN104789957 discloses a microwave preparation process of a flower-shaped hydroxyapatite coating layer on the surface of the magnesium alloy.

Chinese patent no. CN104436301 discloses a process for preparing an organic-inorganic hybrid coating on the surface of a magnesium alloy.

Chinese patent no. CN104404480 discloses a process for preparing a composite coating of hydroxyapatite and bone collagen on the surface of a magnesium alloy.

Chinese patent no. CN103933611 discloses a process for preparing a composite hydroxyapatite/polylactic acid coating on the surface of the medical magnesium alloy.

Chinese patent no. CN103463681 discloses a process for preparing a biodegradable coating of fluorohydroxyapatite (FHA).

Chinese patent no. CN103446627 discloses a process for preparing a hydroxyapatite coating with a modified surface on a biodegradable magnesium alloy using heat and ultrasonic treatment.

Chinese patent no. CN101643929 discloses a preparation process by pulse electrodeposition of a hydroxyapatite coating on the surface of pure magnesium or a magnesium alloy and comprises the use of pure magnesium or a magnesium alloy as the material (substrate) for preparing the electrolyte, in which the concentration of Ca ²⁺ is 2.0-42.0 mmol * If ¹ ; the concentration of H ₂ P0 ₄ ^~ is 1.0-26.2 mmol * If ¹ ; the molar ratio of Ca/P is 1.6-2.0; the support electrolyte concentration is 0.1-1.0 mol * If ¹ ; a pH value is 4.0-6.0; using the substrate material as the cathode and graphite as the anode; heating to 50-90°C; keeping the temperature constant; electrodeposition in a one way or two-way pulse mode to control the deposition current value, where the one-way pulse electrodeposition parameters comprise: 1-40 mA/cm ² peak current density; pulse frequency 10-2000Hz; service factor 10-30 percent; the parameters of the bidirectional pulse electrodeposition comprise: peak current density 1-40 mA/cm ² ; direct pulse rate 10-500Hz; forward service factor 10-30 percent; back service factor 40-50 percent; the deposition time is 5-60 minutes.

Technical problem

In the light of what is known in the art, the inventors of the present invention have perfected a process, based on anodization, for covering magnesium alloys with a coating, in particular AZ31 magnesium alloys, to obtain a coating which has a protective function for the alloy in an aggressive environment.

In particular, the process of the present invention allows to control the morphology, the thickness and the composition of the coating by varying the parameters of the anodization process, where the morphological features are able to predetermine the useful life of the product.

With respect to the anodization treatments known in the art, the process of the present invention, thanks to the appropriate selection of the composition of the bath and of the electrical operating parameters, allows the growth of efficient coatings with an excellent corrosion resistance.

Subject of the invention

With reference to the attached claims, the technical problem is therefore solved by providing a process for covering magnesium alloys with a coating based on magnesium phosphates, magnesium carbonates and hydroxyapatite comprising the following steps: a)anodization in galvanostatic conditions of a magnesium alloy in an electrolytic solution based on potassium dihydrogen phosphate and potassium phosphate in a non- aqueous solvent at a current density between 0.5 mA cm ^-2 and 8 mA cm ^-2 , at a temperature between 100°C and 200°C and a time between 100 seconds and 60 minutes until a magnesium alloy is obtained with a coating of magnesium oxide and phosphates, in which the magnesium alloy is the anode and the cathode is an inert electrode; b)annealing the magnesium alloy having a coating of magnesium phosphates and magnesium carbonates as obtained at the end of step a) at a temperature between 350°C and 450°C for a time between 20 and 30 hours until a magnesium alloy is obtained with a coating of magnesium phosphates and magnesium oxides; c)precipitation on the magnesium alloy having a coating of magnesium phosphates and magnesium oxides obtained at the end of step b) of an additional layer of hydroxyapatite on the coating of magnesium phosphates and magnesium carbonates crystallized by immersion in Simulated Body Fluid (SBF) at a temperature between 35°C and 38°C to obtain the final magnesium alloy product with a coating based on magnesium phosphates, magnesium carbonates and hydroxyapatite.

The object of the present invention is also the magnesium alloy with a coating based on magnesium phosphates, magnesium carbonates and hydroxyapatite directly obtained from the process of the present invention. Brief description of the figures

Figure 1 graphically shows the growth curve for anodized AZ31 at i=2mA/cm ² , t=30min by varying the temperature of the anodization bath.

Figure 2 shows the impedance spectra (Nyquist diagrams) recorded at the OCP related to the AZ31 alloy samples anodized at different temperatures.

Figure 3 graphically shows the growth curves for AZ31 anodized at T=160°C, t=30min, varying the applied current density. Figure 4 shows the impedance spectra (Nyquist diagrams) recorded at the OCP related to the AZ31 alloy samples anodized at different current densities.

Figure 5 shows the polarization curves related to the anodized AZ31 alloy samples at different current densities.

Figure 6 shows the impedance spectra (Nyquist diagrams) recorded at the OCP related to the AZ31 alloy samples anodized at different times.

Figure 7 shows the impedance spectra (Nyquist diagrams) recorded at the OCP related to the samples of uncoated AZ31 alloy, anodized alloy and anodized and heat-treated alloy.

Figure 8 shows the volume of hydrogen produced (per surface unit) as a function of the immersion time for an untreated AZ31 alloy and an anodized one.

Figure 9 shows the Raman spectrum of anodized and heat-treated AZ31 after 14 days of immersion in buffer SBF.

Detailed description of the invention

Definitions

Within the meaning of the present invention, the magnesium alloy AZ31 is defined by the following chemical composition: 2.89% wt Al, 0.92% wt Zn, 0.05% wt Mn, 0.01% wt Si, 0.002% wt Cu, 0.001% wt Ni, 0.004 % wt Fe, balance Mg.

Within the meaning of the present invention, galvanostatic conditions mean conditions of constant electric current density between the electrodes.

Within the meaning of the present invention, Simulated Body Fluid solution (SBF) or Hank's solution means a solution having the following composition: NaCl 8 g/L, KC1 0.4 g/L, NaHCOs 0.35 g/L, NaH ₂ P0 ₄ -H ₂ 0, Na ₂ HP0 ₄ -H ₂ 0, CaCl ₂ -2H ₂ 0, MgCl ₂ , MgS0 ₄ -7H ₂ 0, Glucose. Within the meaning of the present invention, inert electrode means a platinum or steel electrode.

Within the meaning of the present invention, OCP values means the recorded values of open circuit potential.

Within the meaning of the present invention, annealing means the heat treatment which can alter the micro structure of a material.

The present invention relates to a process for covering magnesium alloys with a coating based on magnesium phosphates, magnesium carbonates and hydroxyapatite comprising the following steps: a)anodization in galvanostatic conditions of a magnesium alloy in an electrolytic solution based on potassium dihydrogen phosphate and potassium phosphate in a non- aqueous solvent at a current density between 0.5 mA cm ^-2 and 8 mA cm ^-2 , at a temperature between 100°C and 200°C and a time between 100 seconds and 60 minutes until a magnesium alloy is obtained with a coating of magnesium oxide and phosphates, in which the magnesium alloy is the anode and the cathode is an inert electrode; b)annealing the magnesium alloy having a coating of magnesium phosphates and magnesium carbonates as obtained at the end of step a) at a temperature between 350°C and 450°C for a time between 20 and 30 hours until a magnesium alloy is obtained with a coating of magnesium phosphates and magnesium oxides; c)precipitation on the magnesium alloy having a coating of magnesium phosphates and magnesium oxides obtained at the end of step b) of an additional layer of hydroxyapatite on the coating of magnesium phosphates and magnesium carbonates crystallized by immersion in Simulated Body Fluid (SBF) at a temperature between 35°C and 38°C to obtain the final magnesium alloy product with a coating based on magnesium phosphates, magnesium carbonates and hydroxyapatite.

Optionally, before step a) the magnesium alloy is subjected to a cleaning step, preferably manual mechanical cleaning.

Preferably in step a) in the electrolytic solution based on potassium dihydrogen phosphate and potassium phosphate in non- aqueous solvent, K ₂ HP0 ₄ is present in a concentration between 0.3 and 0.9 M and K ₃ P0 ₄ is present in a concentration between 0.1 and 0.5 M.

More preferably in the electrolytic solution based on potassium dihydrogen phosphate and potassium phosphate in non- aqueous solvent, K ₂ HP0 ₄ is present in a concentration equal to 0.6 M and K ₃ P0 ₄ is in a concentration equal to 0.2 M.

Preferably in step a) the non-aqueous solvent is non-toxic.

Even more preferably the non-aqueous solvent is selected from the group consisting of: 1,4-butanediol, 1-decanol, dodecanol, glycerol.

More preferably the non-aqueous solvent is glycerol.

Even more preferably the electrolyte solution is 0.6 M of K ₂ HP0 ₄ and 0.2 M of K ₃ P0 ₄ in glycerol.

Preferably in step a) the current density is 2 mA cm ^-2 . Preferably in step a) the temperature is 160°C.

Preferably in step a) the time is 30 minutes.

Preferably in step b) the temperature is 400°C.

Preferably in step b) the time is 24 hours.

Preferably in step c) the simulated body fluid solution (SBF) consists of NaCl 8 g/L, KC1 0.4 g/L, NaHC0 ₃ 0.35 g/L, NaH ₂ P0 ₄ ·H ₂ 0, Na ₂ HP0 ₄ -H ₂ 0, CaCl ₂ -2H ₂ 0, MgCl ₂ , MgS0 ₄ -7H ₂ 0, Glucose.

Preferably in step c) the temperature is 37°C. Preferably the coating based on magnesium phosphates, magnesium carbonates and hydroxyapatite obtained by means of the process of the present invention has a thickness between 5 and 20 pm, more preferably 10 pm.

The magnesium alloy with a coating based on magnesium phosphates, magnesium carbonates and hydroxyapatite directly obtained from the process of the present invention can be used for the preparation of biomedical devices such as prostheses, stents.

In a preferred embodiment, an AZ31 magnesium alloy sheet was used (chemical composition: 2.89% wt Al, 0.92% wt Zn, 0.05% wt Mn, 0.01% wt Si, 0.002% wt Cu, 0.001% wt Ni, 0.004% wt Fe balance Mg) subjected to manual mechanical cleaning with the aid of a lapping machine and silicon carbide abrasive papers with decreasing grain size (P1000, P2400 and velvety cloth), then cleaned in acetone through ultrasound, rinsed with distilled water and dried with absorbent paper. In step a) the electrolyte is a 0.6 M solution of K ₂ HP0 ₄ and 0.2 M of K ₃ P0 ₄ in glycerol; the anodization is carried out in a galvanostatic regime at 160°C for 30 minutes; the electrodes are AZ31 (anode) and a Dimensionally Stable Anode (DSA) (cathode) connected to a galvanostat, the current density is 2 mA cm ^-2 . This is followed by step b) of annealing at 400°C for 24 hours and step c) is immersed in a Simulated Body Fluid solution (SBF), and in particular so-called Hank's solution (NaCl 8 g/L, KC1 0.4 g/L, NaHC0 ₃ 0.35 g/L, NaH ₂ P0 ₄ -H ₂ 0, Na ₂ HP0 ₄ -H ₂ 0, CaCl ₂ -2H ₂ 0, MgCl ₂ , MgS0 ₄ -7H ₂ 0, Glucose) thermostated at 37°C to obtain the final product having a coating thickness of about 10 pm.

Examples

AZ31 samples were used (chemical composition: 2.89% wt Al, 0.92% wt Zn, 0.05% wt Mn, 0.01% wt Si, 0.002% wt Cu, 0.001% wt Ni, 0.004 % wt Fe, balance Mg). This magnesium alloy combines high hot ductility with high level mechanical performance. The samples are represented by thin sheets whose area was carefully measured before the tests were carried out. The sheets were subjected to manual mechanical cleaning with the aid of a lapping machine and silicon carbide abrasive papers with decreasing grain size (P1000, P2400 and velvety cloth), then they were cleaned in acetone through ultrasound, rinsed with distilled water and dried with absorbent paper. The electrolyte used for the growth of the protective coatings is a 0.6 M solution of K ₂ HP0 ₄ and 0.2 M of K ₃ P0 ₄ in glycerol suitably stirred and thermostated with the aid of a heating plate provided with a thermocouple. The anodizations were carried out in a galvanostatic regime; the electrodes, having the AZ31 sample as anode and a Dimensionally Stable Anode (DSA) as cathode, were connected to a galvanostat. An electrochemical characterization was carried out to study the performance of the coatings produced. In detail, the following were carried out: measurements of open circuit potential over time (OCP), measurements of Electrochemical Impedance Spectroscopy (EIS, in order to evaluate the polarization resistance of the coatings) and polarization curves. All the tests were carried out inside a solution of Simulated Body Fluid (SBF), and in particular so-called Hank's solution (NaCl 8 g/L, KC10.4 g/L, NaHC0 ₃ 0.35 g/L, NaH ₂ P0 ₄ -H ₂ 0, Na ₂ HP0 ₄ -H ₂ 0, CaCl ₂ -2H ₂ 0, MgCl ₂ , MgS0 ₄ -7H ₂ 0, Glucose) thermostated at 37°C. The impedance spectra were recorded in a frequency range between 100 kHz and 100 mHz, with an AC potential signal of amplitude 10 mV, at the previously measured OCP potential. The electrochemical cell used for the characterization includes a three-electrode configuration in which the AZ31 sheet constitutes the working electrode, the counter electrode (used exclusively to close the circuit) is formed by a platinum retina and an Ag/AgCl electrode was used (0.2 V vs. Standard Hydrogen Electrode) as reference electrode. Finally, the electrodes were connected to a PARSTAT 2263 potentiostat. The polarization curves were recorded in a ± 250 mV interval with respect to the OCP measured with an electrode potential scan rate of 1 mV/s. The electrolytic anodization solution consists of a bath of 0.6 M K ₂ HP0 ₄ and 0.2 M of K ₃ P0 ₄ in glycerol.

It is known that the use of a water-free electrolyte is able to form a porous oxide with a thin barrier layer at the metal/electrolyte interface [Q.Lu, T. Hashimoto, P. Skeldon, G.E. Thompson, H. Habazaki, and K. Shimizu. "Nanoporous anodic niobium oxide formed in phosphate/glycerol electrolyte" Electrochemical and Solid-State Letters 8, no. 5 (2005): B17- B20; Q. Lu, G. Alcala, P. Skeldon, G. E. Thompson, M. J. Graham, D. Masheder, K. Shimizu, and H. Habazaki. "Porous tantala and alumina films from non-thickness limited anodising in phosphate/glycerol electrolyte" Electrochimica Acta 48, no. 1 (2002): 37-42].

For the magnesium, using an aqueous solution as an electrolyte, an inhomogeneous surface coating is obtained with the presence of numerous defects since a layer of MgO will be formed having a so-called "Pilling-Bedworth ratio" (PBR) of 0.81, which leads to tensile stresses capable of destroying the oxide formed. To increase a film with good covering properties it is therefore necessary to form an oxide with PBR between 1 and 2, since a PBR >2 leads to compressive stresses, and therefore, to a non-protective oxide. Furthermore, the electrolyte and the dissociation products thereof must be biocompatible. For these reasons, a non-aqueous solvent was selected, in order to limit the formation of MgO.

The use of phosphate salts provides species which incorporate into the protective film and coat the Mg alloy in a compact manner. The magnesium phosphate, Mg ₃₍ P0 ₄₎₂ , has a PBR of 2.29 [R.-C. Zeng, L. Sun, Y.-F. Zheng, H.-Z. Cui, and E.-H. Han. "Corrosion and characterisation of dual phase Mg-Li-Ca alloy in Hank's solution: The influence of microstructural features." Corrosion Science 79 (2014): 69-82].

To optimize the coating manufacturing process in terms of its performance, the parameters of the anodization process such as electrolyte temperature, anodization current density and anodization time have been varied.

Figure 1 shows the growth curves, cell voltage with respect to time, related to the anodization process with an imposed current density, i, equal to 2 mA cm ^-2 and for an anodization time of 30 minutes, by varying the temperature of the anodization bath (100°C, 160°C and 200°C).

Regardless of the bath temperature, two zones can be distinguished in the growth curves of the coatings on AZ31 alloy: the first zone (up to about 200 s) in which the cell voltage varies linearly with time, and the second in which the cell voltage remains constant (on average). A linear variation of the voltage with time indicates the increase of a barrier film [M. Santamaria, F. Di Quarto, S. Zanna, and P. Marcus. "The influence of surface treatment on the anodizing of magnesium in alkaline solution." Electrochimica Acta 56, (2011): 10533-10542.], i.e., a film with uniform thickness and composition. The different slope dV/dt of the initial section of the three curves obtained at different temperatures therefore indicates a different increase of the barrier film in the three conditions. The curve recorded at 200°C differs, compared to the other two, also in the section in which the cell voltage remains constant on average, with a decrease in the recorded voltage at about 700 s. This second part of the growth curve is instead usually indicative of the formation of a porous film, due to "sparking" phenomena, i.e., damage and consequent reconstitutions of the film, above the barrier film which, in the case of the growth of the coating at 200°C, breaks causing a sudden decrease in the cell voltage.

The quality of the increased coatings was also evaluated through OCP measurements and the recording of impedance spectra. Table 1 shows the OCP values measured after the anodization process. Table 1

The most anodic OCP value is that related to the sample anodized at 160°C while the OCP values of the anodized samples at the other two temperatures were found to be coincident and more cathodic. Figure 2 instead shows the Nyquist diagrams related to the impedance spectra recorded at the OCP after the anodization process.

The graph shows how the global impedance of the system comprising the anodized alloy at 160°C is the highest, thus showing how the coating has a higher protective action against the underlying alloy. Hereinafter, therefore, all the tests will concern coatings increased by anodization conducted at 160°C.

Once the anodization temperature was fixed, the effect of the current density on the features of the increased coating was then studied, keeping the total process time fixed at 30 min. Figure 3 shows the growth curves recorded at three different current densities, 0.5 mA cm-2, 2 mA cm-2 and 8 mA cm-2 respectively.

The slopes, dV/dt, of the initial section of the three curves are different, ranging from 5.1 V s-1 (growth conducted at 8 mA cm-2) and 0.3 V s-1 (growth conducted at 0.5 mA cm-2). From this parameter, it is possible to derive the electric field, Ed, which is applied on the coating increased enhanced by Faraday's law, expressed as: dV i E _d PM

— = ?j - dt z F p where dV/dt is that measured during the growth curve (in the linear section), PM is the molecular weight and p the density of the increased coating, which strictly depend on the composition of the coating itself. From the electric field, it is possible to derive the so-called anodization ratio, AR, which gives an indication of how much the coating increases per volt of voltage applied during growth, and therefore of the thickness of the increased layer. This only applies to the barrier layer which increases during the first step, i.e., until the voltage varies linearly over time.

The colouring of the coating increased at a lower current density is similar to that of the alloy without coating synonymous with a thin and not very protective coating, instead the coating increased at 2 mA cm-2 is clearer and more homogeneous with respect to that increased at 8 mA cm-2. Table 2 shows the anodization ratios for the three different coatings, considering the formation of MgO and Mg3(P04)2 respectively.

Table 2 Figure 4 shows the Nyquist diagrams related to the impedance spectra recorded at the OCP after the anodization process at different current densities. It can be seen from the graph that the global impedance of the system comprising the alloy anodized at 2 mA cm-2 is the highest, thus showing how the coating has a higher protective action against the underlying alloy than the coatings increased at 0.5 mA cm-2 and 8 mA cm- 2. Finally, the polarization curves related to the three coatings (figure 5) were recorded. Although the corrosion potential recorded for the coating increased at 0.5 mA cm-2 is the most anodic (-1.34 V vs. Ag/AgCl), the lower current density related to the cathode process (i.e., the development of hydrogen gas) was recorded for the coating increased at 2 mA cm-2.

The third parameter which was optimized is the anodization time. The results obtained, in terms of global impedance and cathode current density, show that the coating increased at 160°C and 2 mA cm-2 offers the best performance. With reference to the latter, an attempt was made to optimize the process also as a function of the time which was thus set at 100 seconds, 30 minutes and 60 minutes, maintaining temperature (160°C) and current density (2 mA cm-2) constant. The 100-second test aims to show the quality of the barrier film while the 60-minute test aims to thicken the porous film as much as possible. The overall impedance is higher in the case of coating increased for 30 minutes (figure 6).

The presence of a porous layer is necessary to ensure a good quality of the film (which does not occur for the coating increased for 100 seconds) while, after a certain growth time, the breakdown phenomena tend to destroy the film and therefore reduce the protective capacity thereof. The anodization process was therefore optimized as a function of the current density, time and temperature of the electrolyte. The process parameters for the growth of the coating will therefore be set as follows: i = 2 mA cm-2, t = 30 minutes and T = 160°C. With the aim of stabilizing and making the increased coating less soluble, as well as increasing its resistance to corrosion, a post anodization heat treatment was carried out capable of inducing the crystallization of the layer itself. In particular, the anodized samples were placed in an oven at a temperature of 400°C for a period of 24 hours. From the images acquired by SEM microscopy of the anodized sample and the anodized and heat-treated sample, it was possible to notice how the heat-treated coating has a more compact structure and provides greater protection to the metal substrate.

The Nyquist diagrams related to the impedance spectra acquired at the open circuit potential value for a non-anodized AZ31 alloy, for an anodized alloy and finally for an anodized and heat-treated alloy were then compared (Figure 7). The anodized and heat-treated sample shows an overall impedance one order of magnitude greater than the simply anodized sample, and two orders of magnitude greater than the "bare" AZ31 alloy.

The possible production of hydrogen induced by the corrosion of the alloy was also investigated. In fact, the production of hydrogen gas is to be avoided inside the human body and leads to the local basification of the environment through the consumption of H+ ions. To quantify the amount of H2 gas produced by the corrosion of the alloy, a gravimetric method was used through the use of an analytical scale instead of an electrochemical method, which suffers from the presence, due to the corrosion of Mg, of the "Negative Difference Effect" [ S. Fajardo, and G.S. Frankel. "A kinetic model explaining the enhanced rates of hydrogen evolution on anodically polarized magnesium in aqueous environments." Electrochemistry Communications 84 (2017): 36-39; M. Curioni, L. Salamone, F. Scenini, M. Santamaria, and M. Di Natale. "A mathematical description accounting for the superfluous hydrogen evolution and the inductive behaviour observed during electrochemical measurements on magnesium." Electrochimica Acta 274 (2018): 343-352]. The gravimetric method is based on the quantification of the hydrostatic force produced by the volume of electrolyte replaced by the hydrogen produced by the corrosion of the analysed sample.

The downward force measured by the scale is the result of various forces: the total mass of the beaker, sample and wire, mbeak, is assumed to be constant; the other force directed downwards is the one acting due to the solution which is located above the beaker containing the sample. The upward force, on the other hand, is that of buoyancy resulting from the accumulation of hydrogen in the inverted beaker. The net weight force measured by the scale can be expressed using the following formula: where pe and pH2 are the densities of the electrolyte and hydrogen respectively, g is the gravitational acceleration, A is the section of the inverted beaker, h2 and hi are the heights of the free-surface beaker and of the H2/solution interface respectively. Assuming constant mbeak and hi, and that the density variation of the electrolyte due to the hydrogen produced is negligible, the mass variation measured by the mnet scale will be equal to peAAh = peVH2 where Ah and VH2 represent the height and volume of hydrogen produced in the beaker. The flow rate of hydrogen produced can therefore be expressed through the following relationship: The advantage of this technique derives from the fact that, ideally, all the hydrogen produced is detected, comprising the bubbles which remain attached to the sample surface [Fajardo, and G.S. Frankel. "Gravimetric method for hydrogen evolution measurements on dissolving magnesium" Journal of The Electrochemical Society 162, no. 14 (2015): C693-C701; M. Curioni, "The behaviour of magnesium during free corrosion and potentiodynamic polarization investigated by real-time hydrogen measurement and optical imaging" Electrochimica Acta 120 (2014): 284-292]. On the other hand, however, the quantity of hydrogen which is solubilized in the electrolytic solution is not considered. Figure 8 shows the experimental curves related to the produced hydrogen resulting from the corrosion of a "bare" AZ31 alloy and an anodized and heat-treated AZ31 alloy, immersed in Hank's solution.

The slopes of the linear sections of the curves shown in Figure 8 are 0.189 and 0.0056 ml cm-2 day-1 for an AZ31 alloy without coating and for an anodized AZ31 alloy, respectively. The results obtained show a notable decrease in hydrogen produced in the case of the anodized and heat-treated alloy. The value of 0.0056 ml cm-2 day-1 appears to be lower than the tolerable limits for the in vivo applications tested on guinea pigs for biodegradable orthopaedic implants (0.01 ml cm-2 day- 1) [G. Song. "Control of biodegradation of biocompatible magnesium alloys" Corrosion Science 49, no. 4 (2007): 1696- 1701].

Given the possible application of these anodized and treated alloys inside the human body, it is also essential to understand if hydroxyapatite precipitates on the sample once immersed in a solution which simulates body fluid (SBF), given that it is reported in the literature that the precipitation of hydroxyapatite on the surface of a structure (scaffold) by simple immersion in SBF improves cell engraftment [J.-H. Jo, J.-Y. Hong, K.-S. Shin, H.-E. Kim, and Y.-H. Koh. "Enhancing biocompatibility and corrosion resistance of Mg implants via surface treatments" Journal of Biomaterials Applications 27, no. 4 (2012): 469-476]. The anodized and heat-treated AZ31 alloy samples were then immersed in SBF thermostatic buffer at 37°C for different immersion times (3, 7 and 14 days) to evaluate the bioactivity thereof and the consequent increase in biocompatibility. 10 ml of buffer solution was added per surface cm2 and it was replaced on a daily basis in order to avoid excessive increases in pH. Table 3 shows the compositions of the samples, obtained by EDX survey, for different immersion times.

Table 3

The percentages of P and Ca (constituent elements of hydroxyapatite) present in the samples increase with increasing immersion time. In the Raman spectrum of the sample immersed for 14 days, the peak present at 950 cm-1 confirms the presence of hydroxyapatite on the sample surface, thus increasing the biocompatibility of the anodized and heat- treated AZ31 alloy sample (figure 9).