Anomalies have been reported between biocorrosion rates of Mg alloys determined in in vitro and in vivo environments. In vivo environments contain serum biomolecules together with inorganic ions, while in vitro environments typically used for testing may or may not include serum biomolecules. In vitro corrosion testing on pure Mg in a series of electrolyte compositions has identified the influence of different biomolecular and inorganic species on the biocorrosion of pure Mg. Electrochemical analyses and electron microscopy indicate that serum biomolecules tend to accelerate the corrosion of Mg, while phosphate ions in synergy with calcium ions inhibit Mg corrosion. However, serum concentrations >30 vol% in test media lead to decreased corrosion of pure Mg. This indicates that variations in Mg biocorrosion rates seen in in vitro and in vivo media could be attributed to differences in serum concentrations in the respective media.
Magnesium (Mg) alloys are candidates for in vivo implantation owing to their biocompatibility, biodegradability, and resemblance to human bone in terms of their elastic moduli (3 GPa to 20 GPa).1 However, there have been several reports of failure of Mg implant alloys in clinical trials characterized by excessive corrosion, and hydrogen evolution.2-4 In vivo environments are highly complex, containing serum biomolecules, enzymes, and a host of organic/inorganic ions that are in flow. Such conditions are difficult to replicate in lab-scale testing, and therefore in vitro testing is typically performed in simplified media such as simulated body fluid (SBF), Hank’s balanced salt solution (HBSS), Dulbecco’s Modified Eagle Medium (DMEM), Eagle’s Minimum Essential Medium (EMEM), etc.5 Anomalies have been observed between corrosion rates measured in in vivo and in vitro conditions.6 This is particularly problematic as Mg alloys qualified for implant applications by in vitro testing, may not perform as expected under in vivo conditions.
The chemical composition of the test solutions used in in vitro studies plays a significant role in the degradation of Mg alloys. It is reported that media such as SBF, HBSS, DMEM, etc. attack pure Mg and Mg alloys at different rates. The biocorrosion rates of Mg or Mg alloys vary with media used for testing.7-8 Yang, et al.,9 studied the biocorrosion of Mg alloys at ambient temperatures in cell culture media (with and without fetal bovine serum [FBS]), 0.9 wt% NaCl solution, and DMEM (with and without FBS) and reported variations in the measured corrosion rates depending on the composition of the test media. They concluded that developing an in vitro environment to exactly mimic physiological conditions is indeed very challenging. Standard cell culture media contain a balanced salt solution, containing inorganic ions similar to physiological conditions with the ion concentrations, adjusted in such a way that the osmotic pressure is equivalent to that experienced by cells in in vivo conditions. Along with inorganic ions, essential nutrients required by mammalian cells, such as amino acids and vitamins, are added to the media. Several biochemical reactions take place in serum plasma, resulting in the buffering of pH to approximately 7.4. Both and ions can play a crucial part in these reactions and are required additions.10 Along with these ions, CO2 in serum plasma equilibrates with water, forming ions that further aid in these buffering reactions.11 Thus, a ∼5 vol% to 10 vol% CO2 atmosphere is usually used in in vitro tests to mimic these conditions.12 It has been observed that the addition of artificial buffers, such as HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) or TRIS (or trisaminomethane,(HOCH2)3CNH2), are not ideal in Mg biocorrosion, as these chemicals tend to increase the degradation rate of magnesium.13 Moreover, the effect of plasma/serum biomolecules contained in any added FBS must also be considered in the in vitro corrosion of Mg.8,14-15 FBS is a complex mix of albumin, different growth factors, and inhibitors, promoting cell growth, cell function, and also acting as a buffering agent to maintain pH in cell culture conditions.16 Several studies have considered FBS as a primary source of proteins in cell culture conditions.17-22 Usually, 5 vol% to 20 vol% of FBS is added in the media. The amount of albumin contained in such cases is only about 2 g/L to 7.2 g/L, which is much less than the concentration of albumin found in serum (∼35 g/L to 40 g/L).8,23 Besides proteins, the FBS also contains other ions, small molecules, and growth factors that could influence interfacial processes and solution chemistry.
Yamamoto and Hiromoto18 investigated the biodegradation of pure Mg in a mixture of Earle’s balanced salt solution (EBSS), EMEM, and FBS in presence of two kinds of buffers, HEPES and NaHCO3. They proposed that protein adsorption and precipitation of insoluble salts, such as Mg3(PO4)2 and MgCO3, retard Mg degradation whereas organic compounds, such as amino acids, reduce barrier protection provided by the insoluble salt layer. Walker, et al.,24-25 examined the effect of bovine serum albumin (BSA) in EMEM and EBSS solutions on corrosion rates of pure Mg and different Mg alloys (AZ31, Mg-0.8Ca, Mg-1Zn, Mg-1Mn, and Mg-1.34Ca-3Zn). It was reported that BSA in media resulted in increased corrosion of both Mg and Mg alloys. The influence of various serum biomolecules on the corrosion of Mg/Mg alloys, and the postulated mechanisms have been collated and presented in Table 1.
Biomolecules present in plasma can indeed participate in reactions with different inorganic cations and anions. Proteins can bind with various types of inorganic ligands such as Mg2+, Ca2+, Cl−, , etc.9,26-34 Williams, et al.,35 observed that the addition of phosphates to chloride-containing solutions inhibited the corrosion of pure Mg. The inhibition efficiency of phosphates was found to increase with pH.35 Lamaka, et al.,36 tested multiple inorganic and organic compounds as potential inhibitors for Mg corrosion. They observed approximately 93% inhibition efficiency by the addition of Na3PO4 in 0.5 wt% NaCl solution signifying the importance of ions in terms of biocorrosion of Mg.
Calcium phosphates are frequently acknowledged to be adsorbed or precipitated on the implant surface owing to their low solubility.1,37 Deposition of these compounds has been linked to Ca-based species present in the test solutions, However, there are only limited studies that have examined the synergistic role of CaP compounds and serum biomolecules on in vitro corrosion of Mg.38-39 Munro and Strong40 investigated interfacial chemistry of magnesium hydroxide surfaces in aqueous phosphate solutions using in situ FTIR (Fourier-transform infrared spectroscopy). They reported that the presence of BSA accelerated the dissolution of the magnesium hydroxide layer and inhibited deposition of the calcium phosphate. The product formed on the Mg surface during immersion is highly reliant on the environment and the presence of serum biomolecules, which impacts biomineralization.49 Serum biomolecules, such as albumin, can be phosphorylated at pH above 7.4 and this is believed to occur both in in vivo and in vitro conditions.50-51 Thus, the presence of phosphate ions in bioenvironments can affect the interactions between Mg and serum. In most studies, biodegradation of Mg has been conducted in media supplemented with FBS. The role of FBS on degradation behavior of Mg and its alloys is still not well understood. In past works, FBS has been added in relatively low concentrations (∼10 vol%) to simulated body fluids (such as DMEM) in in vitro tests,52 which is significantly lower than the serum concentration (∼55 vol%) in human blood.53 In the case of pure Mg, FBS addition to testing media was found to inhibit corrosion, however, for an Mg-Y alloy, the corrosion rate was found to increase.52
Some species/compounds in FBS may accelerate corrosion (Cl−, albumin, etc.), while others serve to inhibit corrosion (phosphates, carbonates, etc.) by forming precipitated protective films on Mg surfaces.9,54-57 The synergistic effects of species found in FBS and other species, on Mg biocorrosion are not well understood.8,58 Electrochemical (EC) methods and electron microscopy have been used in this work to investigate the interaction of P ions (i.e., ) with serum biomolecules on Mg degradation under cell culture conditions. This knowledge will enable the identification of species that can either accelerate or inhibit biocorrosion of Mg, and hence guide the development of corrosion-resistant alloys and coatings.
MATERIALS AND METHODS
Material and Test Solutions
Commercially pure Mg (Purity ∼99.95%) (Fe ≤ 40 ppm) (AMAC, Australia) was used for all testing.59-60 In vitro tests were conducted in six different solutions (listed in Table 2). The NaCl (Sigma-Aldrich) solution with [Cl−] analogous to blood plasma approximately 0.103 M was selected as the base solution. Na3PO4 (Merck) was added to the solution as a source of P (phosphates) ions (i.e., ions). The amount of Na3PO4 was added with [P ions] approximately 1.45 mM/L (equivalent to [P ions] in human blood plasma).61 The synergistic interaction of P ions with biomolecules was simulated by adding about 20 vol% FBS (Gibco, Thermofisher) (as a source of serum biomolecules) to the solutions. The complex physiological environment was simulated using RPMI-1640 (Roswell Park Memorial Institute Medium) (Source-Gibco, without L-Glutamine and HEPES/TRIS) media.14 RPMI-1640 is a general-purpose media with a broad range of applications for mammalian cells, specifically hematopoietic cells.14,62 It is a modification of McCoy’s 5A medium14,63 and was developed for the long-term culturing of peripheral blood lymphocytes.62 It has similarities with DMEM. RPMI-1640 uses a bicarbonate buffering system, whereas media like DMEM or EMEM use TRIS or HEPES to achieve pH buffering.14
The effect of serum biomolecules was simulated by adding about 20 vol% FBS (Gibco) in the RPMI-1640 media. The composition of test solutions was listed in Table 2. Detailed composition of RPMI-1640 and FBS was listed in Tables 3 and 4.14,51,62
EC viz. open-circuit potential (OCP), electrochemical impedance spectroscopy (EIS), and potentiodynamic polarization (PDP) tests were performed. All tests were performed in a three-electrode EC cell with the specimen as the working electrode, the standard Ag/AgCl electrode as reference electrode, and a Pt-mesh counter electrode, using a potentiostat. The tests were performed at 37°C under cell culture conditions (∼95% humidity and ∼5% CO2) using an incubator. The polarization resistance (RP), double-layer capacitance (CDL), and solution resistances (RS) were estimated from the equivalent circuits and fitting of EIS data (discussed later). The detailed parameters of all EC tests are provided in the Supplemental Material.
The interaction between serum biomolecules and Mg surfaces was investigated using PDP and EIS by varying the FBS concentration in 0.103 M NaCl solution from 2 vol% to 80 vol%. The dissolution behavior was further investigated using PDP and EIS to examine the chelation of Mg with serum biomolecules. The measurements were taken while varying the concentration of FBS from 0 to 80 vol% in 0.103 M NaCl solution in cell culture conditions for approximately 48 h. All of the EC measurements were repeated at least three times to ensure reproducibility.
Scanning Electron Microscopy and Energy Dispersive X-Ray Spectroscopy
The surface morphologies of the corroded samples after 24 h of immersion were characterized using a JEOL JSM 7001F† FEG (Field Emission Gun)-scanning electron microscope equipped with a field emission gun. A 10 keV to 30 keV voltage was used for imaging with a working distance of 10 mm to 12 mm to obtain a clear depth of field due to the roughness of the corrosion layer. The chemical composition of the surface was analyzed using an energy dispersive x-ray detector (Oxford Instruments Aztec† x-ray analysis system and 80 mm2 SDD [Silicon drift detector]) attached to the microscope. A dead time of less than 30 s and counts more than 10,000 were maintained during the scans. Spot and line energy dispersive x-ray spectroscopy (EDS) scans were performed to investigate the chemical composition of the corrosion layer.
Focussed Ion Beam Cross-Sectional Milling
The cross section of the corroded samples after 24 h of immersion was analyzed using a focused ion beam (FIB) coupled dual beam scanning electron microscopy (SEM) (FEI Quanta† 3D-FEG-SEM). Samples were cleaned with ethanol and dried (with an air gun) before placing them in the microscope. A Pt layer (2 µm to 3 µm) was deposited to protect the corrosion layer from ion damage. Cross-sectional milling was performed using a voltage of 30 kV with the current range of 3 nA to 5 nA (for regular cross sectioning) and 0.5 nA to 1 nA (for cleaning the cross section) with a Ga-ion FIB Gun. Spot and line EDS scans were performed to obtain chemical information on the corrosion layer using an EDAX Pegasus† detector attached using an accelerating voltage of 10 keV to 15 keV.
Open-Circuit Potential in Different Test Solutions
Electrochemical Impedance Spectroscopy
The double-layer capacitance increases linearly with time in 0.103 M NaCl solution. The addition of FBS in 0.103 M NaCl solution elevated the CDL, as seen in Figure 3(a). The addition of P ions (i.e., ions) in 0.103 M NaCl solution dropped the CDL after 4 h and this decrease was more pronounced when both P ions and FBS were added together in NaCl solution. The CDL increased in P ions and FBS-containing solutions after 24 h of immersion.
Potentiodynamic Polarization Tests
The results from potentiodynamic polarization tests closely align with those from EIS. The presence of a pseudo-passive layer is evident both in polarization curves and EIS plots (indicated by the presence of a second capacitive loop and higher impedance values66,69 ) for 0.103 M NaCl solution with both P ions and serum biomolecules. A very high corrosion rate was observed in 0.103 M NaCl solution with serum biomolecules, indicated by an increase in anodic activity. It is likely that anodic activity had increased due to the affinity of the serum biomolecules for Mg through complexation.
Serum also contains some free P ions (i.e., ) and Ca2+ ions.71-72 To investigate the effect of P ions () and Ca2+ ions on the corrosion rates, the number of free P ions () and Ca2+ ions present in each solution were calculated (Table 5). The free Ca2+ ion concentration is calculated from the concentration of calcium nitrate in RPMI-1640 (Table 2) and Ca in FBS (Table 3). The state of Ca in these two media is expected to be in the form of free Ca2+ ions, or else it would precipitate in solution. The chelation of Ca by proteins is not considered while calculating the concentration of Ca. The free phosphate (P) ions in RPMI are predominantly in the form of from the dissociation of sodium phosphate (dibasic) (Table 2). However, due to the pH being neutral to slightly alkaline, phosphates also can be in the form of ions. Thus, free P includes phosphate ions in the form of both and .
The values of icorr and RP changed by a factor of 10 even in the presence of small concentrations of Ca2+ and P ions in solutions containing serum molecules. The increased [P ions] indicated a decrease in icorr and increase in RP values. It is evident that these ions alter the chemical reactivity of serum biomolecules toward the Mg surface.
Interaction of Serum Biomolecules with Mg Surface
Corrosion Morphology of the Surface and Corroded Cross-Section Interface
Samples immersed in 0.103 M NaCl solution for 24 h under cell culture (Figures 12[a] and [b]) revealed a very porous surface layer that was mainly composed of Mg and O from EDS data (Figures 12[d] through [f]). The corrosion morphologies of samples immersed for 24 h in 0.103 M NaCl + 20 vol% FBS (Figures 12[e] and [f]) revealed “noodle-like” structures with bigger pores compared with those formed in 0.103 M NaCl solution. The surface was found to be rich in Mg, C, and O as indicated by the EDS (Figure 12[h]). The presence of some agglomerated Ca- and P-rich particles, sparsely distributed over the surface was also observed (Figure 12[e]) and in the EDS plot (Figure 12[g]). These Ca- and P-rich particles might come from the FBS as predicted based on EC data.
With RPMI-1640 (Figure 16[a]), the formation of a thick dense layer explains its higher corrosion resistance when compared to 0.103 M NaCl + 20 vol% FBS-treated samples. EDS plots (Figures 16[b] and [c]) showed the presence of Ca and P at the corrosion interface, indicating the formation of Ca- and P-based layers. The amount of P and Ca at the interface is very high and, as observed in the EDS spectrum of spot 1, Figure 16(b) and is quite uniform from the line spectrum in Figure 16(c).
The interface of the sample with the lowest corrosion rate (sample immersed in RPMI-1640 + 20 vol% FBS) is shown in Figure 16(d), where the corrosion layer appeared to be very thick and uniform, with little observable porosity. EDS plots (Figures 16[e] and [f]) showed the presence of high amounts of Ca and P in the layer, where the protection is coming from the formation of the Ca/P phase.
The thickness of the corrosion layer varied widely with treatment conditions, with the 0.103 M, NaCl + 20 vol% treated sample having the lowest average thickness of 1.4 µm, in comparison to the RPMI-1640 treated sample, with an average thickness of approximately 3.5 µm.
The EC results reported in this study indicated that corrosion rates increase in presence of FBS in 0.103 M NaCl, whereas the addition of P ions in the NaCl + FBS solution decreased the rate. The results also indicated the overall [Ca2+] and [P ions] present in the solutions play a significant role in determining the corrosion rate of Mg samples. The increased cathodic activity in the presence of serum biomolecules also increases the dissolution rate. The EC tests viz. EIS and CA results with the variation of FBS show similar results. At a low concentration, FBS accelerates corrosion, as the [Ca2+] and [P ions] are insufficient to provide an inhibitory effect. However, as the percentage of FBS increases, the overall [Ca2+] and [P ions] increase, thus decreasing corrosion, through the formation of a dense layer of Ca/Mg phosphate phases. In RPMI-1640, both with and without FBS, the high concentrations of Ca2+ and P ions block the acceleration of corrosion by serum biomolecules. The corrosion rate further decreases in RPMI-1640 with FBS possibly due to the adsorption of serum biomolecules on the phosphate-based layer, decreasing the porosity further as observed by SEM.
The decreased corrosion rates of Mg in presence of P (i.e., ) ions could also be attributed to competitive adsorption between P ions and serum biomolecules on metal surfaces.70,74 Human serum albumin has an iso-electric point of 4.9,70,75 which implies that serum molecules have a negative charge at neutral pH. This can favor their adsorption on Mg surfaces, and thus promote chelation/attack of Mg by such molecules. The inhibitory effects of adsorbed P ions on corrosion of other alloys (namely a CrCoMo alloy) have been previously reported by Muñoz and Mischler74 Similarly, it is postulated that competitive adsorption of P ions, as opposed to serum biomolecules, can serve to inhibit Mg corrosion.
There is clear evidence of the same effect in the EC results and is also reflected in the SEM micrographs. The P ions chemically bind with the surface Mg2+ ions forming an insoluble Mg phosphate layer, preventing the penetration of serum biomolecules. The presence of Ca2+ ions in the solution increases the density of the phosphate surface layer, as Ca2+ has a high affinity toward P ions and forms dense and insoluble CaP-based phases which further inhibit corrosion better, as they are immune to attack by Cl− unlike Mg phases, such as Mg3(PO4)2.55,76
The results also indicated that the addition of FBS up to approximately 30 vol% increased the icorr and further addition of FBS in 0.103 M NaCl solution decreased the icorr. The decrease in corrosion rate can be related to [Ca2+] and [P ions] in the FBS. The solubility product of dibasic calcium phosphate salts (CaHPO4) (10−6) is significantly higher when compared to that of hydroxyapatite (10−58) and tricalcium phosphate (10−25).77 It is therefore unlikely that precipitation of CaHPO4 leads to the inhibition of Mg corrosion as seen in the current study. Rather, corrosion inhibition could be attributed precipitation of hydroxyapatite or Ca3(PO4)2-based compounds.
With time, the passivation layer changes as high-affinity species begin to displace the initially adsorbed species. These changes lead to a breakdown of the protective layer, as evidenced by the deterioration of the impedance response with time in all of the solutions. Such results highlight the importance of [Ca2+] and [P ions] in test solutions. The concentration of FBS thus needs to be considered carefully in in vitro testing as it can alter the overall [Ca2+] and [P ions] in the solution.
The following conclusions can be drawn from this study:
The EC tests conducted in deconstructed solutions indicated that the addition of serum biomolecules accelerates the corrosion of Mg in NaCl solutions. However, the addition of P ions into such solutions serves to inhibit corrosion.
One reason for the anomalies observed in previous in vitro and in vivo biocorrosion studies may be partly ascribed to differences in the concentration of serum biomolecules used in such studies. The [Ca2+] and [P ions] present in serum also play a significant role in corrosion of Mg, and interaction of serum biomolecules with the Mg surface.
P ions affect cathodic activity. The chelating/binding capacity of serum biomolecules changes in presence of P ions. Additionally, the synergy between Ca2+, P ions and serum biomolecules reduces the corrosion rate by altering the adsorption ability of the serum biomolecules.
SEM, EDS, and FIB/SEM characterization show that Ca2+ and P ions produce dense CaP surface layers that inhibit Mg corrosion under in vitro conditions.
With RPMI-1640, the addition of serum improved the corrosion resistance of Mg. This is due to formation of a highly dense surface rich in Ca and P, because of high [Ca2+] and [P] in the solution. These surface layers inhibit the attack of Mg by serum biomolecules.
The authors gratefully acknowledge the support from the staff of the Department of Material Science and Engineering, Monash University, Australia. The authors also acknowledge all of the staff of Monash Centre for electron microscopy (especially Dr. Xiya Fang and Dr. Yang Liu). The authors also gratefully acknowledge the funding support from MIPRS and MGS scholarship. S.T. and S.M. would like to acknowledge Prof. Nick Birbilis and Dr. Katherine Nairn.
Sanjay Krishna Mohan: Conceptualization, Methodology, Formal analysis, Investigation, Data curation, Writing—original draft, Visualization.
Terence Turney: Conceptualization, Methodology, Writing—review and editing, Supervision.
Sebastian Thomas: Conceptualization, Methodology, Resources, Writing—review and editing, Visualization, Supervision, Funding acquisition.