The role of Vitamin E in hip implant-related corrosion and toxicity: Initial outcome

Abstract

In orthopedic healthcare, Total Hip Replacement (THR) is a common and effective solution to hip-related bone and joint diseases/fracture; however, corrosion of the hip implant and the release of degradation metal ions/particles can lead to early implant failure and pose potential toxicity risk for the surrounding tissues. The main objective of this work was to investigate the potential role of Vitamin E to minimize corrosion-related concerns from CoCrMo hip implants. The study focused on two questions (i) Can Vitamin E inhibit CoCrMo corrosion? and (ii) Does Vitamin E moderate the toxicity associated with the CoCrMo implant particles?

In the study (i) the electrochemical experiments (ASTM G61) with different concentrations of Vitamin E (1, 2, 3 mg/ml against the control) were performed using normal saline and simulated synovial fluid (Bovine calf serum-BCS, 30 g/L protein, pH 7.4) as electrolytes. The polished CoCrMo disc (Ra 50 nm) was the working electrode. The findings suggested that both Vitamin E-Saline (45 ± 0.9%) and Vitamin E-BCS (91 ± 3%) solutions protected against implant corrosion at a Vitamin E concentration of 3 mg/ml, but Vitamin E-BCS showed protection at all Vitamin E (1–3 mg/ml) concentration levels. These results suggested that the Vitamin E and the protein present in the BCS imparted additive effects towards the electrochemical inhibition.

In the study (ii) the role of Vitamin E in cytotoxicity inhibition was studied using a mouse neuroblastoma cell line (N2a) for CoCrMo particles and Cr ions separately. The CoCrMo particles were generated from a custom-built hip simulator. The alamarBlue assay results suggested that Vitamin E provides significant protection (85% and 75% proliferation) to N2a cells against CoCrMo particles and Cr ions, respectively at 1 μg/ml concentration, as compared to the control group. However, the results obtained from ROS expression and DNA fiber staining suggest that Vitamin E is only effective against CoCrMo degradation particles and not against Cr ions.

In summary, the findings show that Vitamin E can minimize the corrosion processes and play a role in minimizing the potential toxicity associated with implants.

1. Introduction

Total Hip Replacement (THR) is the end-stage treatment option when all conservative treatments fail to relieve the problems of pain, stiffness, and loss of function associated with osteoarthritis, rheumatoid arthritis, osteonecrosis, or traumatic injury of the hip joint. As per the American Joint Replacement Registry (AJRR) 2019 report, around 332K primary THRs were performed annually in the United States (Stibolt et al., 2018). The significant increase in the number of THRs along with an aging population and increased prevalence of obesity, suggests an increasing future demand for this treatment (Program, n.d.; Sloan et al., 2018; Zhai et al., 2019).

Retrieval studies and case reports have shown that corrosion and/or corrosion accelerated wear (tribocorrosion/mechanically assisted crevice corrosion (MACC)) are important causes of hip implant failure (Eliaz, 2019; Mathew et al., 2010; Runa et al., 2017). In addition, tribocorrosion of the implant releases degradation particles, which include wear particles and metal ions. These particles may react with local moieties to form metal salts, colloidal organometallic complexes, or protein-coated micro- or nano-particulate debris. Previous studies have provided clear evidence of the toxicity of degradation products in the periprosthetic tissue, causing adverse local tissue reactions (ALTRs) such as necrosis, osteolysis, pseudotumor formation, and aseptic lymphocyte-dominated vasculitis-associated lesions (ALVAL) which can necessitate revision surgery (Ricciardi et al., 2016; Sansone et al., 2013). For many older patients, revision surgery is impractical, risky, and exposes them to additional morbidity, including periprosthetic joint infection; avoidance of revision surgery is a primary goal of total hip replacement.

Systemic toxicity of degradation products when they travel to remote areas of the body through the lymphatic and systemic circulatory systems is an active area of investigation. Studies on blood and tissue samples of THR patients have reported the deposition of metal particles in distal organs, such as the liver, spleen, kidneys, and heart (Bijukumar et al., 2018b; Urban et al., 2000). The primary particles associated with wear and tribocorrosion of THR are Co and Cr particles and metal ions. The particles can range from nanometers to micrometers in size. In turn, these particles can generate metal ions, which can complex with local moieties to form metal/protein complexes. These particles have been implicated in a variety of toxicities, such as polyneuropathy with progressive sensory disturbances, hypothyroidism, hearing and vision loss, cardiomyopathy, polycythemia, and fatigue (Back et al., 2005; Devlin et al., 2013; Green et al., 2017; Peters et al., 2017). The gradual increase in toxicity-related complications associated with particles in THR patients could become a significant clinical problem in orthopedics (“Biological Responses to Metal Implants,” n.d. 2019). As the number of THR patients continues to grow, and with more young people receiving implants with higher expectations for physical activity and longevity, methods for limiting corrosion and wear as well as treating local and systemic toxicity are essential in minimizing the need for revision surgeries and preventing particle-associated morbidity.

Techniques that have been shown to be effective in reducing corrosion in complex bio-systems include implant surface chemical treatment, plasma source ion implantation (PSII), laser nitration, ion implantation, plasma ion implantation, laser melting (LSM), texturing, physical vapor deposition (PVD), and adjusting the wettability of the surface (Kumar et al., 2009; Kurella and Dahotre, 2005; Liu et al., 2016; Singh and Dahotre, 2007). One method which garnered much attention is the use of non-toxic corrosion inhibitors. Various compounds such as azoles, proteins, polyacrylamides, lipopolysaccharides, dextrose, laurate, chlorhexidine gluconate, and vitamin C have previously been studied for their anti-corrosion properties, primarily for industrial applications (Bhola et al., 2013; Faverani et al., 2014; Shibli and Saji, 2002). These anti-corrosion properties are observed mainly due to characteristic structural features and functional groups with high electro-negativity, such as those containing sulfur, oxygen, and nitrogen. Vitamin C, a potent biological antioxidant, was reported to possess strong corrosion inhibition properties (Fuchs et al., 2013). Although it was reported that corrosion inhibitors could not be used in extremely sensitive and complex bio-systems, many studies are reporting on the efficacy of corrosion inhibitors in in-vitro implant models (Manivasagam et al., 2010; Vieira et al., 2006; Winkler, 2017). The literature has also revealed that vitamin E, another powerful biological antioxidant, was also considered for its corrosion resistance activity (Al-Attar, 2011; Niki, 2015; Valko et al., 2005; Zagra and Gallazzi, 2018). There are reports that vitamin E (alone or with vitamin C) also protects against the local and systemic toxicities caused by the free radicals and reactive oxygen species (ROS) generated by particles (Birben et al., 2012; Free radicals, natural antioxidants, and their reaction mechanisms - RSC Advances (RSC Publishing) n.d.; Kurutas, 2016; Packer et al., 1979; Yan et al., 2018). In this study, the efficacy of vitamin E in protecting against corrosion and toxicity from hip implants fabricated from cobalt-chromium-molybdenum alloy (CoCrMo) was evaluated. Corrosion inhibition by vitamin E was studied using a custom-built corrosion simulator (Butt et al., 2015) in two different corrosion mediums (normal saline and simulated synovial fluid).

Along with local toxicities (ALTR), there are many reports of systemic toxicity due to different metal ions and particles generated from metal implants (Ricciardi et al., 2016; Bijukumar et al., 2018b). There are several reports of neurological symptoms in the MoM THR patients which were suspected due to systemic toxicity of cobalt and chromium ions generated from metal-on-metal hip implants (Queally et al., 2009). In this study, the N2a cell lines were used as representative cells to correlate the systemic toxicity and neurological symptoms. Hence, the toxicity protection studies were conducted on a mouse neuroblastoma cell line (N2a) using particles (CoCrMo) generated from the custom-built corrosion simulator, as well as commercially-obtained Cr ions in the form of Cr₂O₆. Thus, this study explores the possibility of using vitamin E both as a non-toxic implant corrosion inhibitor as well as a local and systemic particles toxicity inhibitor.

2. Materials and methods

2.1. Implant corrosion inhibition studies

2.1.1. Materials

In this study, a CoCrMo alloy sample in the form of a disc (11 mm diameter and 7 mm thickness) was used as the working electrode. A total of 24 discs were milled from CoCrMo alloy rods (MacMaster Carr, Elmhurst, Illinois, USA), which were then divided into 8 groups (n = 3). Each disc was first wet ground with 320–800 grit silicon carbide paper (Carbimet 2, Buehler, Lake Bluff, Illinois, USA) and then further polished with a polishing cloth, diamond paste, and lubricant (TextMet cloth and MetaDi fluids, Buehler, Illinois, USA). Mirror finishing and colloidal silica polishing (Chemomet I and MasterMed, Buehler, Illinois, USA) of the surface were done to get the final 50 ± 10 nm surface roughness. The discs were then ultrasonically cleaned (FS 20, Fisher Scientific, Pittsburg, USA) in deionized water and 70% propanol, and lastly, dried with a hot air dryer.

2.1.2. Solutions

Two solutions were used in this study; either saline (0.9% w/v NaCl solution) or simulated synovial fluid (BCS: bovine calf serum: protein concentration of 30 g/L) was used as the electrolyte solution in each experiment. Vitamin E is a fat-soluble vitamin, thus for experiments, vitamin E was emulsified using Tween 80 (15% w/v). Further dilutions of vitamin E were prepared in saline and BCS to obtain 1, 2, and 3 mg/ml concentrations.

2.1.3. Electrochemical setup

The custom-built electrochemical test setup (Fig. 1a) consists of 4 poly-sulphone chambers (10 ml capacity) able to be electrochemically connected with a three-electrode system in accordance with the American Society for Testing of Materials (ASTM) standard (G61 and G31–72). The system was placed in a hot air chamber to maintain 37 °C, mimicking in-vivo conditions. The system was immersed in either saline (0.9% w/v NaCl solution) or simulated synovial fluid (BCS: bovine calf serum: protein concentration of 30 g/L) as the electrolyte solution (corrosion medium) used to complete the electric circuit. All the electrodes were connected to an Interface 1000E potentiostat (Gamry Instruments, Warminster, Pennsylvania, USA), which in turn connected to a computer for data acquisition. The CoCrMo sample served as the working electrode (WE), graphite as the counter electrode (CE), and a saturated calomel electrode (SCE) as the reference electrode (RE) (Wimmer et al., 2015).

Fig. 1.

a. Custom made electrochemical experimental setup, b. Experimental sequence for electrochemical study in the simulated corrosion cell setup.

The electrochemical nature of the CoCrMo sample in the presence and absence of vitamin E at three different concentration levels and in two different electrolyte solutions was studied using the standard electrochemical protocol. The standard electrochemical sequence (Fig. 1b) used was as follows: Open Circuit Potential (OCP/E_oc) testing (initial and final stabilization for 5400s), Electrochemical Impedance Spectroscopy (EIS) testing (before and after CPD for 4800s) at E_oc and with an amplitude of 10 mV and frequency range from 100 kHz to 0.001Hz, and Potentiodynamic (PD) testing with a scanning rate of 2mv/s and from −0.8V to 1.8V vs. SCE. Electrochemical parameters such as the corrosion potential (${\mathrm{E}}_{\text{corr}}$) and corrosion current (${\mathrm{I}}_{\text{corr}}$) were estimated using Tafel’s curve method. EIS data were analyzed using a modified Randle’s circuit, from which polarization resistance (${\mathrm{R}}_{\mathrm{p}}$) and capacitance (${\mathrm{C}}_{\mathrm{f}}$) were determined (Mathew et al., 2012; Wimmer et al., 2013). Data were recorded and analyzed using ‘Gamry’ software by Gamry instruments. The corroded surfaces were then analyzed using SEM-EDS (JSM-IT500HR, JEOL USA Inc., Peabody, MA, USA) and White-light Interferometric (ContourGT-K 3D, Bruker, Oak Park, IL, USA) surface characterization techniques. The corrosion inhibition efficiency was calculated using Equation 1

$$\mathrm{\%}\mathrm{I}\mathrm{E}=\frac{\text{Control}\mathrm{I}\text{corr}-\text{Test}\text{Icorr}}{\text{Contro}\mathrm{l}\text{Icorr}}\times 100$$ (1)

Where $\mathrm{I}\mathrm{E}$ is inhibition efficiency, Control ${\mathrm{I}}_{\text{corr}}$ and Test ${\mathrm{I}}_{\text{corr}}$ are the corrosion current density values obtained without and with the vitamin E treatment, respectively.

2.2. Toxicity inhibition studies

2.2.1. Cell viability studies

The Hip simulator generated DPs were characterized for particle size (DLS, TEM, SEM-EDS), size distribution in culture medium, size change due to coating, SDS-PAGE, and ICP-MS and were previously elsewhere (Bijukumar et al., 2018a). It was observed that the mean particle size of the Hip simulator generated DPs in saline solution had the size of 119 ± 138 nm with 65% below 150 nm whereas the particles processed in BCS had the size 387 ± 216 nm with 89% above 220 nm. The same was confirmed with TEM results and SDS-PAGE confirmed the presence of protein corona around the BCS processed DPs. The SEM-EDS proved the presence of Co and Cr species in both saline and BCS processed DPs. Inhibition of cytotoxicity induced by CoCrMo metal particles was studied using N2a neuroblastoma cells (ATCC CCL-131, Gaithersburg, Maryland, USA). The cells were cultured in 10% FBS (Sigma-Aldrich, USA) containing MEM (Sigma-Aldrich, USA) media (complete media). The dose-dependent cytotoxicity of CoCrMo particles (0.05 μg/ml to 100 μg/ml) and Cr ions (0.0625 μg/ml to 50 μg/ml) was evaluated using alamarBlue assay. In addition, in order to ascertain an optimal concentration of vitamin E to study its inhibitory effect, a dose-dependent toxicity study with varying concentrations (0.05 μg/ml to 1000 μg/ml) of vitamin E was performed in comparison to Tween 80 solvent. For all cytotoxicity experiments, the following standard cell culture protocol was used: Cells were plated in 96-well plates with a cell density of 10,000/cm² for 24 h. Next, the cells were incubated with complete media containing different concentrations of particles, Cr ions, and vitamin E. After 24 h of incubation with treated media, the cell media was replaced with 10%v/v AlamarBlue (Invitrogen, USA) in media and incubated for 4 h. Absorbance was measured at 570 nm with 600 nm as reference using a microplate reader (Synergy^™ 2 Multi-Mode Microplate Reader, BioTek Instruments Inc. Vermont, USA). To study the cytotoxicity inhibitory effect of vitamin E, an additional group (see Table 1) of experiments was performed consisting of incubation with media containing vitamin E (1 μg/ml, selected based on safe supplementary daily allowance and previous dose-dependent toxicity experiments of Vitamin E (Schmölz et al., 2016; Bijukumar et al., 2021)), in addition to different concentrations of either particles or Cr ions. Reactive oxygen species (ROS) production was studied using flow cytometry (BD FACS Calibur, BD Biosciences, San Jose, CA) and confocal microscopy (Leica Confocal microscope, DMI 6000 CS with Leica TCS SP5 II scanner, Leica Microsystems, USA). For flow cytometry (Dobrzynski et al., 2019; Kim et al., 2019), N2a cells were plated with a cell density of 10,000/cm² in 12-well plates. The cells were then treated for 24 h with complete media containing selected concentrations of particles (10 μg/ml) and Cr ions (1 μg/ml) with and without vitamin E (1 μg/ml). 10 μl 2′,7′–dichlorofluorescein diacetate (DCFDA) dye (Sigma-Aldrich, USA) was added to each well, and the plates were incubated for 30 min. The cells were then trypsinized, centrifuged, and carefully resuspended in the 10% FBS containing medium to assure that the cells existed in a unicellular suspension for flow cytometry. Flow cytometry data were analyzed using FACS Diva software. For confocal microscopy, after 24 h of treatment, the N2a cells were incubated with DCFDA dye for 20 min. The existing cell media was aspirated and replaced with PBS before being imaged.

Table 1.

Toxicity studies experimental groups.

Group name	Conditions
Control	Complete media
Particle treatment	Complete media + particles (10 μg/ml)
Particles + vitamin E treatment	Complete media + particles (10 μg/ml) + vitamin E (1 μg/ml)
Cr treatment	Complete media + Cr2O6 (1 μg/ml)
Cr + vitamin E treatment	Complete media + Cr2O6 (1 μg/ml) + vitamin E (1 μg/ml)
Vitamin E treatment	Complete media + vitamin E (1 μg/ml)

2.2.2. DNA fiber analysis studies

The effect of the DNA damage induced by CoCrMo particles on DNA replication was evaluated using DNA fiber analysis. The DNA fiber analysis was carried out as per the method prescribed by Nieminuszczy et al. (Chastain et al., 2015; Nieminuszczy et al., 2016) with slight modification. N2a cells were plated onto a 24-well plate with a density of 10,000/cm². After 24 h, the cells were treated with complete media containing 10 μg/ml particles with and without the presence of vitamin E (1 μg/ml) for 12 h. Untreated cells and vitamin E-treated cells were used as controls. After 12 h, the cells were first pulsed with 5 μM of the iododeoxyuridine (IdU) for 20 min, followed by a second pulse with 5 μM chlorodeoxyuridine (CldU) for 20 min. The cells were then washed, collected, and re-suspended in cold PBS. 2 μl of cell suspension (200 cells/μl) was then streaked horizontally onto the Superfrost Gold⁺ slides, allowed to dry to tackiness, and lysed with lysis buffer (0.5% SDS in 200 mM Tris-HCl, pH 7.4, and 50 mM EDTA) for 10 min. The slides were then tilted at a 15° angle for 15 min for the DNA to spread. These slides were fixed in a 3:1 MeOH: acetic acid solution for 10 min and then rinsed with de-ionized water (DI water). The slides were then treated with 2.5M HCl for 80 min to denature DNA, followed by treatment with 5% BSA to block non-specific binding. The slides then underwent sequential treatment with primary (Mouse Anti-BrdU (BD cat #B44), 1:300; Rat anti-BrdU (Abcam), 1:500), secondary (Alexafluor 594 rabbit anti-mouse (ThermoFisher/Invitrogen), 1:300; Alexafluor 488 chicken anti-rat (ThermoFisher/Invitrogen), 1:500), and tertiary fluorescent antibodies (Alexafluor 594 goat anti-rabbit (ThermoFisher/Invitrogen), 1:1000; Alexafluor 488 goat anti-chicken (ThermoFisher/Invitrogen), 1:1000) prepared in 5% BSA for 1 h time intervals of incubation. The incubation with antibodies was done in humidity chambers maintained at 37 °C. Images of the slides were then taken using a confocal laser scanning microscope with a 40× objective (Olympus Life Science, Massachusetts, USA) in sequential scanning mode at 488 nm and 594 nm, respectively. The resulting DNA replicons were analyzed using the CASA software (Chastain et al., 2015; Iyer and Rhind, 2017).

CASA software allows one to measure the lengths of the fiber tracts in an unbiased manner. The parameters used for DNA fiber analysis were the following: the minimum signal intensity of fiber was set to be 25 intensity units. The minimum signal-to-noise ratio (fiber intensity versus average surround intensity) was set to 2. Minimum replication intermediate-length was set to 20-pixel lengths (in our hands, non-specific background dots are typically 19 pixels or smaller). The continuity of a signal within a fiber was set to be 70%, and any signal gap within a fiber had to be less than 5 pixels in length.

3. Results

3.1. Corrosion resistance study

3.1.1. Electrochemical characterization

The electrochemical data were used to construct the potentiodynamic curves for vitamin E-saline (1, 2, and 3 mg/ml alpha-tocopherol emulsified in saline using 15% w/v Tween80) and vitamin E-BCS (1, 2, and 3 mg/ml alpha-tocopherol emulsified in saline-bovine calf serum (BCS-30 g/L) using 15% w/v Tween80) are shown in Fig. 2(a&b), respectively, and they are very distinct from each other. The I_corr values observed for CoCrMo corrosion in vitamin E-saline were in current densities ranging between 1.12 × 10–7 to 6.02 × 10–8, which were lesser compared to the Icorr values for CoCrMo corrosion in vitamin E-BCS (1.63 × 10–6 to 1.22 × 10–7). In addition to the I_corr findings, the E_corr values in vitamin E-BCS were observed to be lesser (−0.74 to −0.37) compared to vitamin E-saline (−0.13 to 0.04) (Table 2). Moreover, the presence of prominent I_pass values for 2, and 3 mg/ml of vitamin E-BCS (Fig. 2b) suggests corrosion resistance attributable to protein-vitamin E film formation on the CoCrMo surface. There was no I_pass region observed for vitamin E-saline, suggesting no film formation. However, the Icorr and E_corr values for the highest concentration (3 mg/ml) of vitamin E-saline showed some degree of protection against corrosion, which might be due to the antioxidant property of vitamin E. Statistical analysis of the data (two-way ANOVA) revealed that the protection against corrosion by vitamin E for CoCrMo was not significant (p ˃ 0.05) in saline solution, according to the E_corr and I_corr values. However, the decrease in Icorr obtained for 2 and 3 mg/ml vitamin E-BCS was significant (p ˂ 0.05), suggesting dose-dependent protection against corrosion by vitamin E on the CoCrMo surface in BCS. The CoCrMo corrosion inhibition efficiency (IE) in vitamin E-BCS (at vitamin E – 3 mg/ml) was found to be 91 ± 3%, as opposed to the 45 ± 0.9% efficiency in vitamin E-saline (at vitamin E – 3 mg/ml) compared to respective controls (only BCS and Saline respectively).

Fig. 2.

Tafel’s plots, showing the range of E_corr and I_corr values obtained for control, 1, 2, and 3 mg/ml vitamin E containing electrolyte solution (Table 2) a. Vitamin E-Saline, and b. Vitamin E-BCS.

Table 2.

Electrochemical parameters.

Solution	Vitamin E concentration (mg/ml)	Ecorr (mV vs. SCE)	Icorr (μA/cm2)	Rp (ohms cm2)	Cf (μF/cm2)	Implant corrosion protection (%)
Saline	0	0.04	1.12	3.90	1.29	NA
		±	×	× 106	×
		0.067	10“ 7	±	10− 5
			±	5.21	±
			3.84	× 105	5.40
			×		×
			10− 8		10− 6
	1	− 0.13	6.02	5.23	1.73	NS
		±	×	× 106	×
		0.046	10− 8	±	10− 5
			±	1.65	±
			7.33	× 106	1.93
			×		×
			10− 9		10− 6
	2	− 0.07	7.62	4.38	1.90	NS
		±	×	× 106	×
		0.134	10− 8	±	10− 5
			±	3.99	±
			3.99	× 106	6.17
			×		×
			10− 8		10− 6
	3	− 0.06	6.17	3.09	1.79	45 ± 0.9
		±	×	× 107	×
		0.004	10− 8	±	10− 5
			±	2.55	±
			4.19	× 106	4.10
			×		×
			10− 8		10− 6
BCS	0	− 0.74	1.58	4.18	2.99	NA
		±	×	× 105	×
		0.003	10− 6	±	10− 5
			±	4.71	±
			4.95	× 105	7.20
			×		×
			10− 7		10− 6
	1	− 0.74	1.63	3.64	3.21	NS
		±	×	× 105	×
		0.002	10− 6	±	10− 5
			±	4.06	±
			3.13	× 105	7.58
			×		×
			10− 7		10− 6
	2	− 0.73	*7.95	*1.25	2.23	50 ± 3
		±	×	× 106	×
		0.014	10− 7	±	10− 5
			±	4.13	±
			3.39	× 105	4.09
			×		×
			10− 7		10− 6
	3	− 0.37	*1.22	*2.64	1.88	91 ± 3
		±	×	× 106	×
		0.288	10− 7	±	10− 5
			±	3.88	±
			4.74	× 105	1.38
			×		×
			10− 8		10− 6

E_corr - corrosion potential; I_corr - corrosion current; R_p - polarization resistance; C_f - capacitance;

^*T test significant (p < 0.05); NA - control values; NS - non significant values.

The EIS data were plotted as Nyquist plots in Fig. 3a and b and as Bode plots in Fig. 3c and d for CoCrMo in vitamin E-saline and vitamin E-BCS, respectively. It is clear from the graphs that 3 mg/ml of vitamin E in the saline solution provided relatively increased corrosion resistance as compared to control saline, 1 mg/ml, and 2 mg/ml vitamin E-saline solutions. However, vitamin E-BCS offered clear dose-dependent corrosion resistance with maximum resistance observed at the 3 mg/ml concentration level, which supports the observations from the potentiodynamic curves (Fig. 2). The phase angles of the vitamin E-saline solutions showed varied time constants for all four (control, 1, 2, and 3 mg/ml Vitamin E) solutions, indicating the absence of a compact and homogeneous protective film, leading to corrosion of the CoCrMo surface.

Fig. 3.

EIS data for control, 1, 2, and 3 mg/ml vitamin E containing electrolyte solution - a and b Nyquist plot and c and d Bode plot for vitamin E in saline and vitamin E in BCS respectively, e is modified Randle’s circuit used for analysis.

In contrast, vitamin E-BCS solutions showed a one-time constant in phase angle of the Bode plots, indicating the occurrence of compact and homogeneous protective film formation on the CoCrMo surface. It was also observed that, at a higher frequency, impedance was lower than phase angle for both solution systems, but with the decrease in the frequency, impedance was stabilized and phase angles for all four solutions of vitamin E-saline were at their lowest values, indicating weak corrosion resistance properties of these solutions. In contrast, both dose-dependent impedance stabilization and phase angle widening (control ˂ 1 ˂ 2 ˂ 3 mg/ml vitamin E) were observed for vitamin E-BCS solutions, suggesting a dose-dependent corrosion resistance imparted to CoCrMo surfaces by these solutions.

A modified Randle’s circuit (Fig. 3e) comprising polarization resistance in parallel with the capacitance of the double layer and series with the resistance of solution was used for EIS experiments. The results of this study show that vitamin E-BCS solutions significantly influence both corrosion kinetics and corrosion resistance. Control BCS (only BCS) demonstrated a polarization resistance (R_p) value of 4.18 × 10⁵, whereas 1, 2 and 3 mg/ml vitamin E-BCS solutions were found to have R_p values of 3.64 × 10⁵, 1.25 × 10⁶ and 2.64 × 10⁶ (Table 2) with two-way ANOVA p-values of p = 0.629, p = 0.017, and p = 0.013, respectively. Thus, 2 and 3 mg/ml vitamin E-BCS solutions provided significant corrosion resistance (p ˂ 0.05), which corresponds with the findings of the Tafel plots. In contrast, when compared to the R_p value of control saline (only saline - 3.90 × 10⁶), neither 1, 2, nor 3 mg/ml vitamin E-saline solutions had significantly increased R_p values, providing further evidence for the formation of vitamin E-protein films in vitamin E-BCS solutions but not in vitamin E-saline solutions.

3.1.2. Surface characterization

The surface topography of all eight groups of experimental CoCrMo samples was analyzed using SEM-EDS and white light interferometry (Figs. 4 and 5). It was observed that vitamin E, in normal saline as well as BCS, assisted in the formation of protective films on the surface of the metal alloy samples (Fig. 4a–d). However, on CoCrMo samples in vitamin E-saline, Co, Cr, and Mo ions were observed in the film debris on the surface of the samples (Fig. 4e and f). These findings are reflected in the results of the white light interferometry studies, as the surface shows deposits. The saline control group samples showed relatively high average surface roughness (R_a) 0.211 μm of the study area. Whereas the R_a value for 1–3 mg/ml vitamin E – saline ranged from 0.153 to 0.217 μm of the study area. This observation suggested the formation of weak protective film over the working electrode surface and higher corrosion rates (Fig. 5a–d). In contrast, the BCS control samples showed very low 82.454 nm R_a value compared to vitamin E – saline control and samples suggesting a protective effect of protein in the electrolyte solution (Fig. 4g–j). Additionally, 1–3 mg/ml vitamin E – BCS samples showed the R_a values in the range of 69.11 nm to 0.43 μm, suggesting the formation of very thick protective film over the working electrode surface inhibiting the surface corrosion (Fig. 5e–h).

Fig. 4.

SEM and SEM-EDS images of surfaces of the CoCrMo alloy after electrochemical experiment in vitamin E-saline [SEM - a. control, b. 1 mg/ml, c. 2 mg/ml, d. 3 mg/ml at x500 magnification and SEM-EDS – e and f] and vitamin E-BCS [SEM - g. control, h. 1 mg/ml, i. 2 mg/ml, j. 3 mg/ml at x500 magnification and SEM-EDS – k and l].

Fig. 5.

White light interferometer 3D surface topography of working electrode metal alloy pin surfaces after corrosion using vitamin E-saline [a. control, b. 1 mg/ml, c. 2 mg/ml, d. 3 mg/ml at 2 mm² magnification] and vitamin E-BCS [e. control, f. 1 mg/ml, g. 2 mg/ml, h. 3 mg/ml at 2 mm² magnification].

3.2. Toxicity inhibition study

3.2.1. Cytotoxicity studies of CoCrMo particles and Cr ions

The cell viability of the N2a cells after 24 h treatment with different concentrations of CoCrMo particles and Cr ions in the presence and absence of vitamin E was measured using alamarBlue assay (Fig. 6a&b). It was observed that the cytotoxicity of particles at concentrations ranging from 100 to 1 μg/ml was significantly greater (p ˂ 0.05) compared to the control (Fig. 6a). Additionally, Cr ion concentrations from 50 to 0.5 μg/ml showed significant cytotoxicity (p ˂ 0.05) compared to the control (Fig. 6b). Investigation of the cytotoxicity of vitamin E (performed in order to optimize the dosage) showed no significant toxicity induced by vitamin E from 0.05 μg/ml to 10 μg/ml concentrations (Fig. 6c). Hence, in the current study, the cytotoxicity inhibitory effect of vitamin E was studied using 1 μg/ml of vitamin E, as this was found to be the highest tolerated concentration of vitamin E that was not on the same order of magnitude as a toxic dose.

Fig. 6.

AlamarBlue assay. Graph showing cytotoxicity of (a) CoCr particles, (b) Cr ions at varying concentrations. (c) Optimization of vitamin E concentration and (d) showing cytotoxicity inhibiting effect of vitamin E on particles and Cr ions to N2a cells.

The results of the particles + vitamin E treatment and Cr + vitamin E treatment groups are shown in Fig. 6d. Treatment with vitamin E inhibited the cytotoxic effect induced by particles at all concentrations tested (p < 0.005). At a concentration of 100 μg/ml particles, approximately 85% cell viability was observed in the presence of vitamin E compared to ~20% viability in the presence of particles alone. Similar results were observed for both 10 and 1 μg/ml particles, where percent viability improved to 90–95% in the presence of vitamin E. However, the presence of vitamin E did not induced any significant difference in cell viability in the presence of Cr ions at the 10, 1, and 0.1 μg/ml concentrations selected for this test. Based on the results of the cytotoxicity studies, particles and Cr ion concentrations of 10 and 1 μg/ml, respectively, were chosen for future experiments to visualize the effect of vitamin E.

3.2.2. ROS production studies

The ROS intensity curves and fluorescent imaging after 24 h treatment (Fig. 7a–c) for N2a cells suggested that vitamin E (1 μg/ml) inhibited the ROS generation due to particles, even at a concentration of 10 μg/ml (p ˂ 0.0005). However, vitamin E did not inhibited the ROS generation induced by 1 μg/ml Cr ions. These observations were supported by the confocal microscopy fluorescent image analysis of all six groups, as shown in Fig. 7c.

Fig. 7.

ROS production assay. (a) Flow cytogram of ROS production by the cells after treatment with particles and Cr ions in the presence and absence of vitamin E (b) Graphical representation of ROS intensities and (c) Confocal microscopy fluorescence images of ROS expression.

3.2.3. DNA fiber analysis studies

The fluorescently labeled DNA fiber slides (DNA from cells treated with 10 μg/ml particles, in the presence and absence of vitamin E) were imaged using a confocal microscope with an oil immersion 40x lens (Fig. 8d). Interpretations of the possible fiber tract patterns, replication intermediates are shown in Fig. 8a. RG/GR replication intermediate track lengths and the proportions of the different replication intermediates were compared with untreated control. RG/GR tract lengths were taken as a measure of the amount of DNA replicated from a single replication fork during the nucleotide pulses. There was no significant difference found in the average RG/GR tract length between the control group and vitamin E treatment group, with average lengths of 63 ± 2 and 73 ± 4, respectively (Fig. 8b). Both Particles and Cr treated groups were found to have significant (p < 0.005) reductions in this measurement of 49 ± 2 and 47 ± 3, respectively. However, with the additional presence of vitamin E, the particle + vitamin E treatment group saw a significant (p < 0.005) decrease in this effect with 10% reduction compared to only particle treatment group, resulting in RG/GR tract lengths that were statistically comparable to the control group (59 ± 2 vs. 63 ± 2, respectively). This effect was not observed in the Cr + vitamin E treatment group (47 ± 3 vs. 63 ± 2 for the control group) (Table 3).

Fig. 8.

a. DNA replication fork patterns b. RG/GR track length comparisons c. The relative percentage of replication intermediate population.

Table 3.

The percent distribution of the red and green fluorescent DNA fragments.

	Red Only	Green Only	RG/GR	RGR	GRG	Number Scored
Control	32.4	37.4	63.1	75.6	76.4	1418
particle treatment	29.8	25.0	48.9	71.8	83.3	899
particles + vitamin E treatment	32.6	38.8	58.6	65.8	76.8	1063
Cr treatment	23.8	37.9	47.2	97.7	60.5	140
Cr & vitamin E treatment	34.5	46.1	49.8	50.7	65.1	166
Vitamin E treatment	47.5	53.3	75.0	68.3	84.5	193

In measuring the relative percentage of the different replication intermediate patterns, changes in their relative frequencies were understood to reflect altered replication dynamics. Neither the particle treatment nor particle + vitamin E treatment groups were found to differ significantly in the proportion of tract patterns from the control (untreated) group. In contrast, compared to control, both Cr treatment and Cr + vitamin E treatment groups saw significant (p < 0.0029, p < 0.0001) changes in this proportion.

4. Discussion

This study was intended to explore the role of vitamin E in both the corrosion inhibition of THR implants as well as protection against cell toxicity related to the degradation particles generated from implant corrosion/tribocorrosion. Both electrochemical studies and surface topography studies provided evidence that vitamin E in the presence of proteins in the electrolyte generates a protective layer on the implant surface, which reduces the rate of corrosion. It was also observed that vitamin E provides suitable protection against particle-induced cellular toxicity, even at the highest tested concentration of particles (100 μg/ml). However, this protection was not observed specifically against Cr ion-induced toxicity, which requires further investigation. These results are reflected in the findings of the ROS production studies, in which vitamin E was found to significantly reduce ROS production in cells exposed to particles but not in cells exposed to Cr ions. These findings are in accordance with the results of the DNA fiber analysis studies, which found further differences in the behavior of particles and Cr ions. More details are included below.

4.1. Vitamin E as a nontoxic corrosion inhibitor

In this work, the electrochemical behavior of CoCrMo alloy was studied in two types of electrolytes; saline, and bovine calf serum. In our previous study, the corrosion inhibition effect of proteins in the electrolyte solutions was reported (Mathew et al., 2012). The results obtained in this study are in accordance with our previous findings. In this study, in addition to the effect of proteins, we evaluated the effect of vitamin E on corrosion. Vitamin E is a well-known and potent lipid-soluble antioxidant, which is transported while bound to alpha1 and beta lipoproteins in serum (Stocker and Azzi, 2000; Takahashi et al., 1977; Voegele et al., 2002). The possible interaction of vitamin E with proteins and further enhancement of corrosion inhibition by this interaction was the basis of our experimental design studying the effect of vitamin E in different electrolyte solutions (saline and BCS). The potentiodynamic curves suggest the formation of a dose-dependent protective film on the CoCrMo alloy surface, as evidenced by multiple delayed de-passivation and re-passivation curves observed for experiments with 2 and 3 μg/ml vitamin E-BCS solutions (Fig. 2b). This observation indicates that the initial metal oxide film on the surface of the CoCrMo alloy disc was replaced by a protective film in the presence of protein-vitamin E (Fig. 9a and b) (Williams et al., 1988; Wimmer et al, 2003, 2010).

Fig. 9.

Proposed mechanism of corrosion inhibition and reduction in cellular toxicity [Lower part of figure represents the implant surface corrosion mechanism and upper part of the figure represents the cell and toxicity mechanism] a) Without vitamin E (red arrow represents increase in ROS production and more toxicity) b) With protein-vitamin E complex (green arrow represents decrease in ROS production as the metal nanoparticles are surrounded by protein-vitamin E complex, providing less cellular damage in terms of ROS generation and DNA damage). M ⁺ - metal ions, M^np - metal nanoparticle and e⁻ electrons.

Although the underlying mechanisms of the corrosion inhibition of vitamin E are not yet known, it was observed that the chemical nature of vitamin E and its interaction with protein and metal surfaces might enhance the insulating barrier at the metal solution interface. The effect of vitamin E, in addition to the effect of proteins on corrosion was further confirmed using EIS studies (Liao et al., 2011; Scholes and Unsworth, 2006). The EIS Bode plots (Fig. 3d) indicated a constant phase angle at higher frequencies and increased at low frequencies. However, impedance exhibited gradual increase at lower frequencies increasing concentration (control, 1, 2, and 3 mg/ml) of vitamin E-BCS solution. These results indicate the role of corrosion protection effect of vitamin E (Lovander et al., 2018; Stango and Vijayalakshmi, 2018). Moreover, the slightly increased impedance of the 3 mg/ml vitamin E-saline solution compared to that of other concentrations of vitamin E in-saline suggests corrosion protection based on the antioxidant effect of vitamin E alone (Fuchs-Godec and Zerjav, 2015). The increased polarization resistance (R_p) and decreased capacitance (C_f) with increasing concentrations of vitamin E in BCS (Table 2) suggest a dose-dependent corrosion resistance provided by vitamin E-BCS solutions which were not seen with vitamin E-saline solutions, where only the 3 mg/ml vitamin E concentration showed a slight non-significant increase in resistance and decrease in capacitance (Barão et al., 2012). The surface characterization of the test surfaces provided clear evidence of protective film formation in vitamin E-BCS solutions compared to vitamin E-saline solution. Thus, the results unmistakably show that as the concentration of vitamin E increases, the corrosion rate decreases. In this study, we observed the corrosion inhibition efficiency of vitamin E with proteins from BCS was approximately 91 ± 3% in comparison to vitamin E alone in saline solution, which was around 45 ± 0.9%.

4.2. Vitamin E as an inhibitor of particles-induced toxicity

The toxicity caused by particles (CoCrMo and Cr ions) generated in the process of CoCrMo corrosion is of primary concern due to the resultant local and systemic complications that can be a source of morbidity in THR patients. The deposition of particles has been reported in organs such as the liver, spleen, kidneys, and heart, leading to conditions such as cardiomyopathy, polycythemia, and hypothyroidism (Bijukumar et al., 2018b; Urban et al., 2000). The present study provided clear evidence for CoCrMo particles-induced cellular toxicity, as well as for the efficacy of vitamin E in preventing particle-induced toxicity. Cell viability assays showed that vitamin E provided protection against particle-induced cytotoxicity in N2a cells, even at the highest concentration tested (100 μg/ml) (Fig. 6a) (Dobrzynski et al., 2019; Kim et al., 2019). However, vitamin E failed to prevent cytotoxicity induced by Cr ions, even at the lowest concentration tested (0.1 μg/ml). This difference may be due to differences in the cell entry mechanisms that metal nanoparticles and Cr ions employ, and in the pathways through which particles and Cr ions induce cellular damage.

It has been reported that vitamin E, a lipophilic molecule, localizes to the cell membrane where it inhibits lipid peroxidation due to ROS production (Packer et al., 1979, 2001, 2001; Rao et al., 2006; Sahiti et al., 2019). Metal nanoparticles, such as the particles investigated in the present study, enter cells via phagocytosis/pinocytosis, bringing the nanoparticles in direct contact with the cell membrane. Furthermore, biologically active particles may induce oxidative damage upon interaction with the cell membrane, where vitamin E is localized. In contrast, Cr(VI) ions can only be taken up by cells through nonspecific transmembrane ion channels, bypassing direct interaction with the cell membrane. Cr is able to induce cellular damage through various mechanisms, including the formation of complexes with DNA and cellular proteins. These diverse modes of damage may lead to cell death, with or without significant interference in DNA replication.

With these differences in cellular entry mechanisms and the pathways employed for inducing cellular damage, one would expect a significant difference in the efficacy of vitamin E in protecting against cytotoxicity and reducing the production of ROS by particles versus Cr ions. Both particles and Cr ions were found to cause significant cytotoxicity and significantly increase the production of ROS in N2a cells. However, vitamin E was only able to protect the toxicity of particles, which may be attributed to its localization at the cell membrane, where it is able to directly contact and counter the primary mode of damage induced by particles. On the other hand, Cr ions are able to bypass interaction at the cell membrane inducing damage through a greater range of mechanisms than those against which vitamin E is able to protect.

Thus, the probable cellular toxicity reduction mechanism suggests that the electrolyte (NS) interact with the implant surface and generates more number of metal ions and metal particles. Hence showing less corrosion inhibition as well as increased cellular toxicity due to ROS generation and DNA damage (even in presence of vitamin E). Whereas, in the presence of protein from the serum (newborn calf serum in NS), it is possible that vitamin E supports the formation of a protective layer on the implant surface enhancing corrosion inhibition. Similarly, the metal nanoparticles can also be surrounded by a protective layer of protein-vitamin E complex, providing less cellular damage in terms of ROS generation and DNA damage. However, there might be a lack of interaction of metal ions with the protein-vitamin E complex, which might show the ROS production in the presence of metal ions (like Cr ions) and vitamin E (Fig. 9a and b).

This proposed explanation is in accordance with the results of the DNA fiber analysis studies, in which particles and Cr ions were again found to behave differently. Both particles and Cr ions were found to decrease the amount of DNA replicated over 40 min. However, vitamin E was found to significantly reduce this effect only in the particle treatment group and not the Cr ion treatment group. This finding may reflect vitamin E’s ability to protect against oxidative stress, but not the greater range of damage modes that Cr ions are able to induce. In contrast, replication dynamics were only found to be significantly altered in response to Cr ions but not particles. This may reflect a greater ability by Cr ions to effect DNA replication dynamics than particles, possibly also attributable to their greater range of damage modes. Vitamin E was found to reduce this effect to levels statistically comparable to the control group, suggesting that vitamin E may play some role in protecting against Cr ion induced toxicity. The significant differences in the amount of DNA replicated and the replication dynamics between control and vitamin E treated groups likely reflects a decrease in the baseline amount of ROS production from control levels, though this was not observed in our ROS studies.

4.3. Limitations of the study

The authors acknowledge that there are certain limitations to the current study. Our report is only focused on the importance of understanding the mechanisms of interaction of vitamin E with metal surfaces and metal particles (nano/micro) and ions in detail. The particles which are generated from the corrosion simulator are mainly from the CoCrMo surface and the elemental and structural characterizations are not yet known. It was also understood from the study that increasing corrosion protection was observed with increasing concentration of vitamin E, and current studies are now determining the best vitamin E concentration to optimize corrosion resistance. One major limitation of this study is that, the presence of Vitamin E on the surface of the materials is not analyzed. From the results reported here, a possible mechanism of vitamin E-protein film formation was proposed. The chemical characterization of the same will be taken up to provide more insight and better correlation with the EIS and other electrochemical data. The authors also understand that there are limitations in this study towards the prediction of failure of Cr ion toxicity inhibition and ROS-related toxicity due to the metal ion concentration inside the cell. In the discussion, the authors proposed multiple mechanisms working collectively to provide the obtained results. However, further experiments will be conducted for the quantification of the metal ions in solution with ICP and ROS generation due to ions and particles in the acellular assay to predict the exact mechanisms for the results as a future prospective study.

5. Conclusions

With the limited experimental results from this study, the following conclusions can be derived. This study demonstrated the role of vitamin E in protecting against CoCrMo corrosion in the custom-built corrosion simulator as well as in inhibiting cell toxicity. From the electrochemical results obtained, it is evident that vitamin E, in combination with proteins in BCS, supports a protective film formation on the CoCrMo surface and inhibits corrosion. Because its interaction with proteins appears essential in the formation of this protective film, the effect should not be expected when used in other electrolyte solutions like normal saline. Vitamin E also protected cells against damage caused by particulate corrosion products but was ineffective against Cr ion-mediated toxicity. The ROS generation studies supported the above claim as the particle-induced ROS generation in vitamin E-treated cells were significantly decreased. The inhibition to replication fork rates was abrogated for cells treated with particles and vitamin E, but not for cells treated with Cr and vitamin E. Together, these findings suggest that vitamin E with proteins in serum plays a significant role in inhibiting CoCrMo corrosion as well as particle-induced tissue toxicity.

Driven by our findings in this study, the work will be extended to understand the variation in the chemical interaction of vitamin E with metal particles and ions. Further acute and chronic implant metal particle toxicity (local and systemic) inhibitory effects of vitamin E using different types of cells and micro-fluidic approaches will be employed. The new techniques will be explored to get the implant metal corrosion resistance advantage of vitamin E in actual in-vivo conditions to ultimately benefit patients undergoing THR with CoCrMo alloy implants.