Mg-Ca0.3 Electrochemical Activity Exposed to Hank’s Physiological Solution and Properties of Ag-Nano-Particles Deposits

: This work compares the degradation of Mg and Mg-Ca0.3 alloy when they are exposed for 14 days to Hank’s solution at 37 ◦ C. A combination of immersion test, electrochemical techniques (PDP, EIS, EN), and surface characterization methods (SEM-EDS, XRD, and XPS) were carried out. The pH change over time, the lower mass loss ( ≈ 20%), and the lower concentration of the released Mg 2+ ions ( ≈ 3.6 times), as well as the lower level of the surface degradation, allowed to consider the positive effect of Ca, presenting Mg-Ca0.3 alloy with lower electrochemical activity than that of Mg. The positive effect of Ca may be due to the formed layer characteristics on the alloy surface, which impedes the cathodic hydrogen evolution and Mg-ions release. The electroless deposited Ag-nano-particles (Ag-NPs) on Mg-Ca0.3 surface were characterized by SEM-EDS, XRD, UV-Vis, and contact angle. The agar-diffusion test was used to compare the growth of Staphylococcus aureus and Escherichia coli bacteria on Mg-Ca0.3 in the presence of Ag-NPs deposits in different size. Zeta-potential of the bacteria was negative, with respect to pH of the Mueller-Hinton culture broth. The greater antibacterial effect of S. aureus was attributed to its more negative zeta-potential, attracting more released Ag + ions.


Introduction
The magnesium and its alloys have attracted a lot of research interest as biodegradable, resorbable and biocompatible materials, when used as temporary implants in physiological media. As structural materials, they present prevalence over the non-resorbable biomaterials [1][2][3][4][5][6][7]. Mg is the lightest metal with a density of 1.738 g cm −3 which is close to the cortical bone (1.75-2.1 g cm −3 ), with an elastic modulus of 45 GPa (≈20 GPa of cortical bone), its rapidly cooled alloys can reach a strength/weight ratio of 490 kN m kg −1 [8][9][10][11], hence it is considered as a more suitable material than the biomedical titanium alloy Ti 6 Al 4 V (4.47 g cm −3 ) [12]. The released magnesium ion (Mg 2+ ) is non-toxic and is a fundamental for: the synthesis of proteins, activation of a variety of enzymes, regulation of activities of the central nervous and neuromuscular systems, regulation of blood glucose level, promotion of normal blood pressure, as well as playing an important role in the prevention and treatment of disorders of the hypertension, cardiovascular diseases, and diabetes [13][14][15]. The Mg ion is the second most abundant intracellular divalent cation, which is assimilated by the human body, and the average requirement is of~375 mg per day for a human body of approximately 70 kg of weight [16]. Unlike calcium, the clinical importance of which is indisputable, that of Mg is often underestimated, as an element necessary for the incorporation of Ca into the bone [17,18]. Magnesium has been applied as a biomaterial for cardiovascular, microsurgeries, orthopedic, and wound closures [4,[19][20][21][22].
Most metals, such as iron (Fe), chromium (Cr), titanium (Ti), nickel (Ni), cobalt (Co), tantalum (Ta), tungsten (W), niobium (Nb), and molybdenum (Mo), which are used for the manufacture of implants, present serious problems such as release of ions during the degradation process into the physiological solutions, as well as the formation of corrosion products that are not tolerable (toxic) for the body even in minute quantities [23]. Therefore, because of the many advantages over non-resorbable biomaterials, Mg and its alloys are potential materials for the design of new temporary orthopedic devices, providing mechanical properties like those of human bone, maintaining mechanical integrity on a scale of 12 to 18 weeks, while the bone tissue heals [3,[24][25][26]. Moreover, they have stimulating effects on the growth of new bone tissue, because magnesium ions (Mg 2+ ) exhibit a strong affinity to bind with phosphate ions present in the human body, influencing the mineralization of bone tissue through the formation of hydroxyapatite [Ca 10 (PO 4 ) 6 (OH) 2 ], or possible similar compounds, which can inhibit the corrosion process [27][28][29][30][31]. Nowadays there is a great demand for orthopedic implants with an increase in 2020 of~116 billion dollars in the stock market [23,24,[31][32][33].
In physiological solutions composed of a high content of NaCl (up to 3.5%), the Cl − ions penetrate the pores of the poorly soluble (0.009 g L −1 ) corrosion product of Mg(OH) 2 , causing its partial transformation to highly soluble MgCl 2 (54.3 g L −1 ) [3,20,43]: On the other hand, the biodegradation mechanism of Mg and its alloys depends strongly on the ionic composition of the surrounding solutions used for testing, whose corrosivity may change the corrosion behavior [44]. A mathematical method has been reported to predict the biodegradable corrosion mechanism and properties of commercially pure Mg-based implants in a simulated environment [45].
The mainly disadvantages that Mg may present in physiological media are: anticipated loss of mechanical integrity (implant failure); H 2 gas generated as a byproduct of corrosion (affecting the healing process); alkalinization at the surface interface (Equation (4)), which accelerates the process of Mg(OH) 2 corrosion product formation and disorder in the physiological reactions that depend on the pH [46][47][48]. The recent advancement in reducing the rapid degradation of Mg implants is linked with the introduction of alloying elements. The metal Ca (1.55 g cm −3 ) was chosen as it is the major component in the human bones, as a hydroxyapatite complex [Ca 10 (PO 4 ) 6 (OH) 2 ], which provides the rigidity and stability of the bone, as well as, the ions of Ca are essential for the chemical signals of cells [48,49]. In a solid solution its percentage can reach up to 1.34 wt.% according to the binary phase diagrams of Mg-Ca [28,50]. It has been reported that the addition of Ca in Mg alloys improves their mechanical properties and diminishes their corrosion activity [51,52]. Mg-Ca alloys, as a class of novel biodegradable magnesium alloys, have been recommended in the context of medical implants [53], with the advantage of a density like that of human bone [2,54].
The microstructure of Mg-Ca alloys systems includes α-Mg (hexagonal crystalline structure) and Mg 2 Ca phase, in which crystalline cell presents double parameter values (a = 0.623, c = 0.1012) compared to those of Mg [55][56][57][58]. The standard electrode potential of Ca is more negative (−2.87 V SHE ) than that of Mg and it may be assumed that Ca 2+ will be released, originating from the Mg 2 Ca particles, considered as an anodic active [54,[59][60][61][62][63]. However, reports suggest that in 1 M Mg(NO 3 ) 2 solution these particles present a less negative potential value (−1.54 V SCE ) and therefore, they were considered as cathodic active [64,65]. In the literature, there are controversial results for the electrochemical behavior of the Mg 2 Ca intermetallic particles present in the matrix of Mg-Ca alloys and there is not a clear justification for their biodegradation mechanism [14,24,33,[66][67][68]. It has been noted that the electrochemical activity of Mg 2 Ca particles needs to be investigated with precision to determine their effect on the biodegradation process of Mg-Ca implants [69][70][71]. Reported research concluded that the addition of a small amount of Ca (up to 2 wt.%) reduced the degradation of Mg, but a further increase of the Ca content led to an increased rate [61,70]. An interesting biological characteristic of the Mg-Ca alloys is that to provide a possible cell adhesion (osteoconductivity) and to stimulate the cell growth on the implant surface [70].
The new generation of biomaterials combines bioactivity and biodegradability of their surfaces. The increasing fight against bacterial infections during the surgery requires the development of medical devices and materials with antimicrobial properties [78]. The World Health Organization reports that every four years approximately 350 million illnesses and 190,000 deaths are caused by pathogens that proliferate on the surfaces of medical tools [79]. The bacterial proliferation and its surface adhesion on solids are crucial, caused mainly by the interaction between the solid surface potential (electric charge) and the zeta potential charge of the bacteria cellular surface [80], in an addition to other factors, such as pH of the solution and its chemical composition, the hydrophobicity of the solid surface and ionic strength.
Currently there is a consensus that indicates the importance of the zeta potential and the hydrophobicity of the substrate as physicochemical parameters that control bacterial adhesion on the solid surfaces, governed by van der Waals forces and electrostatic interactions through the double layer of substrate-electrolyte [81][82][83][84][85][86][87][88][89][90]. Results demonstrated an antibacterial effect of Mg on three common aerobic bacterial organisms, as a result of the Mg 2+ concentration increase accompanied by an increase of pH of the media [91]. Reported studies suggest that porous coatings may have a strong effect on the antibacterial properties of Mg-based alloys used as implants in orthopedics, also slowing their biodegradation rate [92].
Several studies have revealed the antimicrobial efficiency of Ag-nanoparticles (NPs), although the mechanism of their effect is not yet fully understood [93][94][95][96]. It was suggested that Ag-NPs initially bind to the bacteria cell, altering its permeability and respiration, followed by penetration into and the intracellular release of Ag-ions [97]. The silver exhibits a significant antibacterial action in the dark, where there is no light (within the human body), making this metal an attractive material for biomedical applications [98]. The introduced Ag + ions within bacteria can affect the production of various enzymes, interrupting their respiratory chain, generating stress in the reactive oxygen species (ROS), as also inhibiting cell signaling and protein synthesis [99][100][101]. The antimicrobial mechanism of Ag-NPs has been explained by the formation of Ag-free radicals, induced in the bacteria cells through perforation of its membrane, resulting in their subsequent death [93,95]. Thus, the deposition of Ag-NPs on Mg-alloys surface could be one approach to provide antimicrobial protection for the implantable medical device surface, to combat associated infections.
The aim of this work is to provide more detailed information on the effect of Ca on the electrochemical degradation process of Mg-Ca0.3 alloy, when exposed to Hank s physiological solution (at 37 • C) for up to 14 days, compared with that of pure Mg. The change of surface morphology and composition was characterized by SEM-EDS and XPS techniques. The pH of the Hank s solution and concentration of the released Mg-ions were monitored. Mass losses, potentiodynamic polarization (PDP) curves, EIS diagrams, fluctuations of corrosion current and potential (EN), revealed an additional information. Ag-NPs of different size, electroless deposited on Mg-Ca0.3 surface, were characterized by means of the UV-Vis spectroscopy, SEM-EDS, XRD and the contact angle test. The bacteria S. aureus (Staphylococcus aureus) and E. coli (Escherichia coli) were selected as models of Gram-positive and Gram-negative bacteria for testing the antibacterial properties of the Ag-NPs. The ability to prevent bacteria growth was proved by the agar disk diffusion method (Kirby-Bauer test).

Samples and Solution Preparation
Mg (99.9 wt.%) and Mg-Ca alloy (Ca 0.3 wt.% and the balance Mg) were manufactured by an extrusion process and supplied by Helmholtz-Zentrum Hereon, Institute of Materials and Process Design (Geesthacht, Germany) as cylindrical bars (diameter = 1 cm). The nominal composition (wt.%) is presented in Table 1, according to the manufacturer. The tested materials were cut into a 3-mm thick discs; some of them were used for the immersion test, while others were prepared as electrodes for electrochemical experiments. All samples were abraded with SiC paper to 4000 grit, using ethanol as a lubricant, and then sonicated in ethanol for 5 min and dried in air at room temperature. Hank's physiological solution was prepared with the following analytical grade reagents: NaCl 8.0 g L −1 , glucose 1.0 g L −1 , NaHCO 3 0.35 g L −1 , and KCl 0.30 g L −1 ; in a low content: KH 2 PO 4 , MgSO 4 ·7H 2 O, MgCl 2 ·6H 2 O, CaCl 2 , and ultrapure deionized water (18.2 MΩ·cm) [102][103][104]. The initial pH of the newly formed solution was 7.2.

Immersion Test and Surface Characterization
The Mg and the Mg-Ca0.3 alloy samples (in triplicate) were submerged in 20 mL of Hank's physiological solution at 37 • C, in independent containers (with a daily renewal of electrolyte) for 14 days, according to the ISO 16428 [102] and ASTM G31-12a [105] standards. The residual solutions were stored to measure their pH by a pH meter and the concentration of the released Mg 2+ ions by photometry (HI83200, Hanna Instruments, Woonsocket, RI, USA). At the end of each exposure period, the samples were removed, rinsed with deionized water, and dried in air at room temperature. To evaluate the mass loss and the corrosion damage on the surface after each exposure period, the formed layer of products was chemically removed, according to the standards ASTM G1-03 [106], using the solution of 200 g L −1 CrO 3 , 10 g L −1 AgNO 3 , and 20 g L −1 Ba(NO 3 ) 2 . The values of initial mass w 0 and final mass w 1 were measured by an analytical balance (VE-204, Velab, CDMX, México) and then the mass loss was calculated (∆m = w 1 − w 0 ).
The morphology and composition of the layers formed on the Mg and Mg-Ca0.3 surfaces, before and after exposure to Hank's physiological solution, were characterized by: SEM-EDS (SEM-EDS, XL-30 ESEM-JEOL JSM-7600F, JEOL Ltd., Tokyo, Japan) and XPS (K-Alpha Surface Analyzer, Thermo Scientific, Waltham, MA, USA), after sputtering the specimens' surface with a scanning argon-ion during 15 s. The binding energies of all XPS spectra were normalized to the C1s peak at 284.8 eV. The crystalline phases of the formed products were identified by X-ray diffraction technique (Siemens, D-500, Munich, Germany) with Bragg-Brentano geometry, 25 • to 40 • 2θ angle, radiation Cukα and 34 kV/25 mA.

Electrochemical Characterization
Several electrochemical methods were carried out to study the corrosion process of Mg and Mg-Ca0.3 in Hank's solution: registration of the free corrosion potential (open circuit potential, OCP), potentiodynamic polarization curves (PDP), and electrochemical impedance spectroscopy (EIS). Mg and Mg-Ca0.3 samples were elaborated as working electrodes (0.78 cm 2 ) in a typical three-electrode cell configuration, inside a Faraday cage. A Pt mesh (Alfa Aesar, Ward Hill, MA, USA) was used as an auxiliary electrode and a saturated calomel electrode (SCE) as a reference electrode (SCE, Gamry Instruments, Philadelphia, PA, USA). The electrodes were connected to a potentiostat/galvanostat/ZRA Interface-1000E (Gamry Instruments, Philadelphia, PA, USA). The PDP curves (vs. OCP) were recorded at a scanning rate of 10 mV s −1 , in a potential range from −1 V to +1 V. The EIS spectra were acquired for 24 h of immersion in Hank's solution, employing a perturbation amplitude of ±5 mV vs. OCP (1 h to stabilize), in a frequency interval of 100 kHz to 10 mHz. The obtained EIS data were analyzed with the Gamry Echem Analyst ® software (Gamry Instruments, version 7.0.1, Philadelphia, PA, USA).
The spontaneous corrosion current fluctuations considered as electrochemical noise (EN) were associated with the corrosion phenomena and were measured according to ASTM G199-09 [107], employing two identical working electrodes and SCE reference electrode, all connected to the potentiostat in zero resistance ammeter mode (ZRA). The data were examined with Electrochemical Signal Analyzer ® (Gamry Instruments, V.7.0.1, Philadelphia, PA, USA), then transformed to the frequency domain by fast Fourier transform (FFT) in order to analyze their power spectral density (PSD) [108,109]. The values of the β-slope (an exponent of PSD plots) were used to characterize the dynamics of the corrosion process. All experiments were carried out at room temperature at 21 • C.

Electroless Deposition of Ag-NPs on Mg-Ca0.3 Surface and Characterization
The Ag-NPs were electroless deposited on Mg-Ca0.3 surface (0.78 cm 2 ), by immersion (t = 1 min, 3 min and 6 min) in solutions of AgNO 3 (10 −3 M and 10 −2 M) and ultrapure deionized water (18.2 MΩ·cm) at 21 • C. Due to the very negative potential of metallic Mg, the silver ions are spontaneously reduced during the typical process known as electroless deposition (ELD) [110,111]. The Ag-NPs were characterized by UV-Vis technique (AVASpec 2048, AVANTES, Eerbeek, The Netherlands), SEM-EDS, XRD, and contact angle test (Drop Shape Analyzer DSA25, KRÜSS, Hamburg, Germany). The antibacterial effect of Ag-NPs was evaluated with the Gram-positive bacteria S. aureus ATCC 6538 and Gram-negative bacteria E. coli ATCC 25922. Nutrient broth and Mueller-Hinton agar were used as the non-selective medium that promote bacterial growth. The antibacterial agar disk-diffusion method (Kirby-Bauer method) [112][113][114] was carried out, which consists of initial inoculation of a bacteria spread layer (100 µm) on the agar disk. The suspensions with bacteria were adjusted by optical density (spectrophotometry) at 625 nm, in order to match the turbidity equivalent to the 0.5 McFarland standard (1.5 × 10 8 CFU mL −1 ) and adjusted to 1.5 × 10 −6 CFU mL −1 by serial dilutions. All samples were sterilized with ethyl alcohol and placed in a box with ultraviolet light for 1 h, as well as all glassware were sterilized in an autoclave at 121 • C and 15 psi (for 20 min). The samples of the Mg-Ca0.3 alloy without and with deposit of Ag-NPs, were placed on the agar disk and incubated at 37 • C for 24 h. The diameter of the inhibition halo around the alloys, where the bacteria did not grow, was then measured and compared. The antimicrobial tests were performed inside an incubator, in the dark, and the results are the average of three separate tests corresponding to different days.   The EDS analysis of sites (labeled as 1, 2, 3, and 4) is presented in Figure 1C, as also the suggested compounds of MgO (site 3) and Mg 2 Ca particles (site 4), reported by metallurgical studies [61,[115][116][117]. The XRD spectra of the Mg-Ca0.3 surface is shown in Figure 1D,E, presenting the α-Mg matrix (HCP Mg-JCPDS #350821) and the intermetallic phase of Mg 2 Ca [61,62]. MgO was not detected and according to study [118], this phase appears as amorphous under environmental conditions.  Figure 2B. The pH of Hank's solution showed increasing and decreasing tendencies, which were influenced by the formation of OH − ions (Equations (2) and (3)) and the evolution of H 2 (Equation (1)) during the corrosion process. Consequently, when the pH solution was shifted to more alkaline values, the precipitation of Mg(OH) 2 corrosion product was favored. On the other hand, the glucose [CH 2 OH(CHOH) 4 CHO], which is a part of Hank's solution (1.00 g L −1 ), can be rapidly transformed into gluconic acid [CH 2 OH(CHOH) 4 COOH], which acidifies the electrolyte and causes partial destruction of the Mg(OH) 2 film [118]. It has been reported that the gluconic acid promotes the growth of the hydroxyapatite layer, due to the chelation reaction with Ca 2+ ions [117]. However, the influence of glucose has not yet been clarified. Previous works [119,120] suggested that the lowering in the pH may greatly affect the Mg degradation process under the conditions of physiological environments.  (4) and (5)). However, it may also consider the chelating effect of gluconic acid, forming compounds with the Mg 2+ ion, contributing to the reduction of MgCl 2 generation [121]. The pH change over time, the ∆m, and the concentration of the released Mg 2+ ions allowed us to consider the positive effect of Ca in the Mg-Ca0.3 alloy, presenting lower electrochemical activity than that of Mg.

SEM-EDS Analysis
After exposure to Hank's solution for 14 days, the Mg ( Figure 3A) and Mg-Ca0.3 ( Figure 3B) surfaces showed cracked layers. These cracks can connect the α-Mg with Hank's solution, improving the release of Mg 2+ ions and the evolution of the H 2 bubble. The pressure of H 2 bubbles may cause cracks or fractures in the layers. According to the EDS analysis, the main elements on Mg-Ca0.3 alloy surfaces are Mg, O, and Ca, and Cu, Al, Fe, Ni, and Zn (Table 1) appear in small amounts (below 1% wt.), which may present cathodic activity, being effective sites for hydrogen evolution [122]. After the removal of the formed layers, the cross-sectional images ( Figure 3C,D) revealed that the corrosion attacks have been less aggressive in depth,~5.5 times in Mg-Ca0.3 surface ( Figure 3D) than those in Mg ( Figure 3C). It is also evident that the localized attacks have been more pronounced on the Mg surface ( Figure 3E) than those on the Mg-Ca0.3 surface ( Figure 3F). The EDS analysis (Figure 4) indicated that the layers formed after immersion in Hank's solution for 14 days contained a very low content of Na and Cl. The presence of P element, could be considered as a part of the formed Mg 3 (PO 4 ) 2 and as well as participating in the compound Ca 10 (PO 4 ) 6 (OH) 2 (hydroxyapatite), originated from the Hank's solution [119,120].  Figure 3A,B,E,F) and suggested compounds, formed before and after removal of the surface layers.
The intermetallic Mg 2 Ca particles appeared on the surface of Mg-Ca0.3 ( Figure 3F, sites 9 and 10) even after the removal of the formed layers, suggesting their role as local cathodic sites. The obtained results allow concluding the positive effect of Ca, as a part of the Mg-Ca0.3 alloy, which contributed to its lower level of degradation.

XPS Analysis
The XPS spectrum analysis ( Figure 5) was performed as a complementary information, in order to correlate with the EDS elemental composition analysis of the layers grown on the Mg and Mg-Ca0.3 surfaces after exposure for 14 days to Hank's physiological solution. The high-resolution spectra revealed the presence of Mg, Ca, O, C, P, Cl, and Na elements. The main peaks of Mg2p (at 50.28 eV) and O1s (at 531.48 eV) were associated with Mg(OH) 2 compound [123][124][125]. The secondary peak of Mg2p (at 44.98 eV), together with the peaks of P2p (at 133.43 eV) and Ca2p (at 347.51 and 351.18 eV) were attributed to Mg 3 (PO 4 ) 2 and calcium phosphate [Ca 10 (PO 4 ) 6 (OH) 2 ] compounds. The peaks C1s and O1s were considered as a part of CaCO 3 , originated from the Hank's solution [125][126][127][128]. The intensity level of the XPS peaks, presenting the spectra of Mg-Ca0.3 surfaces, was lower than that of the peaks of XPS spectra of the Mg surface, suggesting a difference in the formed layers after exposure for 14 days to Hank s solution. The decrease in content of the compounds formed on the Mg and Mg-Ca0.3 is as follows: Ca 10 (PO 4 ) 6 (OH) 2 > Mg 3 (PO 4 ) 2 > Mg(OH) 2 > CaCO 3 . Due to the low solubility of Mg 3 (PO 4 ) 2 (1.64 × 10 −3 g L −1 ) and Ca 10 (PO 4 ) 6 (OH) 2 (4.38 × 10 −2 g L −1 ). It is considered that these compounds appeared, since they are thermodynamically more stable than other types of phosphates in aqueous solutions; these compounds are suitable for the regeneration of human bone [129]. Figure 6 shows the PDP curves after 24 h of immersion in Hank's solution. The cathodic branches have a kinetics controlled by the activation process, while the anodic branches tended to reach a limiting current value related to a barrier effect of the formed corrosion layers on the Mg and Mg-Ca0.3 surfaces. The followed strong increase in the anodic current was attributed to the chloride ions, which transformed the insoluble compound of Mg(OH) 2 to soluble MgCl 2 . The tendency to the next limiting current (at a higher anodic polarization) could be related to the formation of phosphate and carbonate salts on the surfaces, which may act as protective layers, reducing the speed of the anodic process [130][131][132]. Both limiting anodic current values of Mg-Ca0.3, presenting two surface states, seem to be lower than those of Mg, as well also the cathodic current values. The corrosion potential and current density (j corr ) were not calculated by Tafel extrapolation because the presence of precise linear Tafel regions in the PDP curves was not considered. According to the procedure of ASTM G102-89 [133], the value of the polarization resistance (R p ) was estimated from the anodic branch at a polarization of ±5 mV, as well as the respective value of corrosion current density j corr (∆E/∆I). The "apparent" Tafel constant (B') was calculated through the Stern-Geary relation: Table 2 presents the values of R p , "apparent" Tafel constant (B') and the calculated mass loss rate (MR) [133]: MR = K·j corr ·EW (6) where K is a constant (8.954 × 10 −3 ), and EW is the approximate equivalent weight of the samples (~11.17 for Mg). The higher value of Mg-Ca0.3 polarization resistance (R p ) to the corrosion process, compared to that of Mg (Table 2) has led to lower values of j corr and MR. These facts suggest the positive effect of Ca as a part of the Mg-alloy, although of a very low content (0.3 wt.%).

Electrochemical Noise (EN) Analysis
The spontaneous fluctuations of the current density of Mg and Mg-Ca0.3 at low amplidute, during 24 h of exposure to Hank's solution, are presented in Figure 7. They may be considered as an electrochemical noise (EN), resulting in variations in the kinetics of the electrochemical corrosion process [134][135][136]. The EN resistance (R n ) values were estimated, suggesting as similar to the resistance to polarization (R p ) and inversely proportional to the corrosion rate (controlled by charge transfer) [136][137][138]. The R n value of Mg-Ca0.3 was 3.42 kΩ cm 2 , which is 5.5 times greater than that of Mg (0.62 kΩ cm 2 ), indicating that the product layer formed on Mg-Ca0.3 is more protective against corrosion after 24 h explosion in Hank's physiological solution at 21 • C. The pitting index (PI) was calculated (Equation (7)) to compare the localized corrosion susceptibility of the samples: where σ i and i RNC are the standard deviation and the root mean square of current noise, respectively. The PI was 0.06 for Mg-Ca0.3 and 0.11 for Mg, both materials do not present localized corrosion (<0.06), according to ASTM G199-09 [107]. However, the cross-sectional SEM images ( Figure 3C,D) and those of the surfaces after the formed layer removal ( Figure 3E,F), showed that the corrosion attacks are more localized on the surface of the Mg and more uniform on Mg-Ca0.3. The EN data were analyzed in the domain of time and frequency, in order to characterize the dynamin of the corrosion process. The Figure 8 shows the power spectral density (PDS) of the corrosion current as a function of low frequencies (10 −2 Hz-1 Hz), obtained by the fast Fourier transform (FFT) method. The extracted exponent (β slope) values were 0.36 for Mg and 0.32 for Mg-Ca0.3, which correspond to the fractional Gaussian noise (fGn: −1 < β < 1), classifying the corrosion process as persistent processes [139][140][141].  Figure 9A shows the Nyquist diagrams for Mg and Mg-Ca0.3 exposed to Hank's physiological solution for 24 h at 21 • C, as well as the equivalent electrical circuit used to fit the experimental EIS data ( Figure 9B) [142]. The Bode diagrams are presented in Figure 9C. The Mg Nyquist diagram reveals two capacitive loops, representing the two surface states during the anodic process of degradation of Mg in Hank's solution (see Figure 6); for Mg-Ca0.3 the effect of two loops is not sufficiently observable. According to the literature, the capacitive loop at high frequencies represents the charge transfer resistance (Rt) [143][144][145][146][147][148][149][150], while the second loop at medium frequencies can be associated with the mass transport process, including the Mg 2+ release [144,147,148,150]. The Bode diagrams are congruent with the Nyquist diagrams and help to appreciate the existence of two interfaces.

Electrochemical Impedance Spectroscopy (EIS)
The suggested circuit includes as main components: the solution resistance (Rs), a constant phase element (CPE1) and a resistor (R1), both associated with the double layer and charge transfer resistor (substrate/electrolyte interface). The (CPE2) and the resistance (R2) are characteristic of the corrosion process at the metal surface through the corrosion layer and the release of Mg 2+ ions. The constant phase elements (CPE) have been used instead of a capacitance, due to the possible microscopic roughness or the heterogeneities of the Mg and Mg-Ca0.3 surfaces [143][144][145]. Table 3 presents the estimated fitted parameters of the circuit elements from the EIS diagrams, in which fitting is of 10 −4 . The values of the total polarization resistance (R p ) were calculated (Equation (8)) and they indicate ( Table 3) that the R p for Mg-Ca0.3 was~three times higher than that of Mg, suggesting that the formed layer in the presence of Ca was more protective.

UV-Vis Spectrophotometry and SEM Micrograph
The electroless deposited (ELD) Ag-NPs (zero-valent Ag) on Mg-Ca0.3 were initially characterized by the time evolution of the UV-Vis absorption spectra ( Figure 10A) and change in color of the MgCa0.3 surface ( Figure 10B). The absorption band with a maximum at around 290 nm that decreases up to 320 nm ( Figure 10A) is attributed to electronic interband transition between electrons in s-orbitals and p-empty orbitals of metallic silver [146,147]. The minimum observed at 320 nm in all samples is related to the well-known decrease of the imaginary part (k) of the refraction index characteristic of metallic silver [148]. This wavelength outlines the limits between interband transition, from 320 nm toward lower wavelengths. A maximum of the adsorbance near to 400 nm is a result of the electron density resonance phenomena (surface plasmon resonant band). The bandwidth, asymmetry, and position depend on the Ag-NPs particle size and size distribution. The interaction of the particles with the substrate also affects the optical response due to the localization of the plasmon fluctuations (localized surface plasmon resonance, LSPR).
According to study [149], the Ag-NPs may exhibit various colors depending on their localized surface plasmon resonance (LSPR) and consequently they can represent different optical states (black, magenta, cyan, yellow, silver mirror, and transparent). The change in color is influenced by the LSPR band, when the Ag-NPs show difference in morphology, size, and shape. The SEM images ( Figure 10C) reveal that the Ag-NPs are well-isolated and in a very low size ( Figure 10D) by using the electrolyte of low concentration of Ag + (10 −3 M, 1 min), because of the slow growth rate. However, at one order higher concentration of Ag + (10 −2 M, 1 min), the better connection between Ag-particles leads to the formation of agglomerates, as the diffusion of the Ag-ions is more facilitated to the substrate. It is considered that this effect of short-range interaction induces the LSPR band in the long wavelength region [146,147,149], that is why the photograph of the surface becomes darker at higher concentration of Ag-NPS agglomerates. The Ag-NPs and agglomerate size distribution on the Mg-Ca03 surface is presented in Figure 10D.
The Figure 11 presents SEM images (10,000×) and EDS elemental analysis, which compare the surface of Mg-Ca0.3 before ( Figure 11A) and after the deposit of Ag-NPs (10 −3 M, t = 3 min) ( Figure 11B). EDS analysis suggests that the Ag-NPs are deposited in the vicinity of the intermetallic phases of Mg 2 Ca ( Figure 11B).

X-ray Diffraction (XRD)
In order to characterize the Ag-NPs a complementary analysis by XRD was carried out ( Figure 12). The spectra indicated the pattern of the hexagonal Mg-metal matrix and the presence of nanostructured Ag crystals of FCC cell structure. The calculated average size of Ag (T) nanocrystallites was 10 nm, according to the Debye-Scherrer's equation [150][151][152]: where K is a dimensionless factor (0.9); λ is the wavelength of the X-rays (1.5406 Å); B is the mean width of the peak at a half of the height (fhwm), considered for the calculation; and θ B is the angle corresponding to the maximum of the reflection.

Contact Angle and Surface Free Energy (SFE)
The contact angle ( Figure 13A) was measured following the ASTM D 5725-99 methodology for the drop shape analysis [153]. The surface free energy (SFE) was calculated based on the contact angle value ( Figure 13B). The results revealed that the contact angle depends on the concentration (molarity) of the solution for Ag-NPs electroless deposition, as well as, on the time for deposition (quantity of Ag-NPs on the Mg-Ca0.3 alloy, Figure 10C). The angle value was greater than 120 • on the B surfaces (substrate Mg-Ca0.3), as well as on all Ag-NPs deposits formed by 10 −3 M and 10 −2 M AgNO 3 , at different times. Therefore, these surfaces are considered as superhydrophobic [154][155][156][157]. According to studies [158][159][160][161][162][163][164], the superhydrophobic surfaces may exhibit an antibacterial behavior, as also a reduction of a fluidic drag and an anticorrosive protection for metal surfaces.  On the other hand ( Figure 13B), the calculated surface free energy (SFE) showed a decrease in the positive values of B, M1, and M2 surfaces, while the M3 y M4 surfaces presented very negative values of the SFE. The last fact suggests that these surfaces will exhibit a poor cell bacterial adhesion, as well as wettability properties [154][155][156][157][158][159][160][161][162]. To measure the zeta-potential of the bacteria, pH titration was carried out, in order to understand the electrostatic interactions between bacteria and Ag-NPs deposits on the Mg-Ca0.3 surface. The data in Figure 14 showed that zeta potential of E. coli and S. aureus tended to be more negative values with respect to the pH of the Mueller-Hinton culture broth (pH ≈ 7.0 and up to 11), with a bacteria concentration of 10 8 CFU mL −1 . The pH of the culture broth (≈7.0 and up to 11) was chosen to be similar to that of the pH range of change of Hank s solution during the exposure of Mg-Ca0 (Figure 2A). At the initial pH of 7.0 the zeta potential was more negative for S. aureus (−5.67 mV) than that of E. coli (−3.16 mV), followed by a sudden shift at pH ≈ 7.25 to more negative values close to each other and ending at ≈−11 mV at pH = 11.

Agar Disk-Diffusion Test
This test was used to compare the ability to inhibit bacteria growth of E. coli and S. aureus on different substrates: bare surface of Mg-Ca0.3 (B) and in the presence of Ag-NPs, deposited from lowest to highest concentration of AgNO 3 at different times (M1 to M4). The agar disk-diffusion test was carried out in an incubator at 37 • C after 24 h in the dark ( Figure 15). The histogram in Figure 16A (agar disk-diffusion test) compares the results of the diameters (halos) of zone of inhibition that the bacteria presented, and the corresponding images are presented in Figure 15. On the bare of Mg-Ca0.3 substrate, zone of inhibition was not observed. It was evident that a greater antibacterial activity was observed on the substrate M4, which surface is covered by agglomerates of Ag-NPs in an extended area ( Figure 10C,D). For all the samples, S. aureus presented a larger zone of inhibition than E. coli, which indicated that S. aureus is more sensitive to the Ag-NPs than E. coli. After measuring the diameters of the inhibition halos, the percent of diameter of inhibition growth (PIDG) was determined ( Figure 16B) according to the following equation [165]: Diameter of sample − Diameter of control Diameter of control ·(100) (10) where diameter of control is the diameter of the Mg-Ca0.3 samples (1 cm), and the diameter of sample is the average diameter of the zone of inhibition after agar disk-diffusion test of three replicates. The results revealed that the zone of inhibition ( Figure 16B) depends on the Ag-NPs content and their size deposited on the Mg-Ca0.3 surface: a higher content (surface of available area) of Ag-NPs gives a possibility for more binding capacity of the particle interaction with the bacterial cell membrane. The higher inhibition percentage reached was 83% for S. aureus and~64% for E. coli.
The exact antimicrobial mechanism of Ag-NPs is still not completely known; however, it is suggested that it depends on chemisorbed Ag + ions, released from the silver partial oxidation as a consequence of its sensitivity to oxygen [166]. Studies report that the Ag-NPs can generate long-lasting antibacterial activity due to the consequent release of Ag + ions which kill the bacteria [167][168][169][170]. On the other hand, considering that the surface charge of Ag-NPs is not actually measured [171], their interaction with bacteria cell is still open to discussion.

Conclusions
The electrochemical degradation process of Mg-Ca0.3 alloy, exposed for 14 days to Hank's physiological solution (at 37 • C), was compared with that of pure Mg, in order to provide more detailed information on the effect of Ca. The intermetallic Mg 2 Ca particles appeared on the surface of Mg-Ca0.3 even after the removal of the formed layers, suggesting their role as local cathodic sites. According to the analysis of XPS spectra, the decrease in the content of the compounds formed on the Mg-Ca0.3 and Mg surfaces was as follows: Ca 10 (PO 4 ) 6 (OH) 2 > Mg 3 (PO 4 ) 2 > Mg(OH) 2 > CaCO 3 .

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The values of the EN-resistance (R n ) of Mg-Ca0.3 (3.42 kΩ cm 2 ) was 5.5 times greater than that of Mg (0.62 kΩ cm 2 ), indicating that the product layer formed on Mg-Ca0.3 is more protective.

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The total polarization resistance (R p ), estimated from the EIS data indicated that R p for Mg-Ca0.3 was~three times higher than that of Mg, suggesting the Ca positive effect.

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The electroless deposited Ag-nanoparticles (Ag-NPs) on Mg-Ca0.3 were characterized by the time evolution of the UV-Vis absorption spectra and change in color of the Mg-Ca0.3 substrate. The XRD spectra indicated the presence of nanostructured Ag crystals of FCC cell, in which the calculated average size was 10 nm.

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The measured contact angle of the Ag-NPs (>120 • ) indicated that their surfaces may be considered as superhydrophobic, and thus, they may exhibit an antibacterial behavior. The calculated surface free energy (SFE) showed a tendency to very negative values, which suggest that the Ag-NPs exhibit a poor cell bacteria adhesion, as well as poor wettability properties.

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The ability to prevent growth of S. aureus (Staphylococcus aureus) and E. coli (Escherichia coli) bacteria, in the presence of Ag-NPs, was proved by the agar disk diffusion method (Kirby-Bauer test). The greater antibacterial effect of S. aureus was attributed to its more negative zeta-potential, attracting more the released Ag + ions from the Ag-NPs. Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: Data presented in this study are available on request from the corresponding author. The data are not public, due to privacy issues.