Accepted Manuscript

Electrochemical Polishing as a 316L Stainless Steel Surface Treatment Method:

Towards the Improvement of Biocompatibility

Sajjad Habibzadeh, Ling Li, Elaine C. Davis, Dominique Shum-Tim, Sasha

Omanovic

PII: S0010-938X(14)00266-2
DOI: http://dx.doi.org/10.1016/j.corsci.2014.06.010
Reference: CS 5881

To appear in: Corrosion Science

Please cite this article as: S. Habibzadeh, L. Li, E.C. Davis, D. Shum-Tim, S. Omanovic, Electrochemical Polishing
as a 316L Stainless Steel Surface Treatment Method: Towards the Improvement of Biocompatibility, Corrosion
Science (2014), doi: http://dx.doi.org/10.1016/j.corsci.2014.06.010

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 Electrochemical Polishing as a 316L Stainless Steel Surface
Treatment Method: Towards the Improvement of Biocompatibility

Sajjad Habibzadeh1,*, Ling Li2, Elaine C. Davis2, Dominique Shum-Tim3 and Sasha Omanovic1

(1) Department of Chemical Engineering, (2) Department of Anatomy and Cell Biology,
(3) Divisions of Cardiac Surgery and Surgical Research, Department of Surgery
McGill University, Montreal, QC, Canada

*Author to whom correspondence should be addressed:

Dr. Sajjad Habibzadeh
Department of Chemical Engineering
McGill University
3610 University Street
Montreal, QC, H3A 0C5, Canada
Email: sajjad.habibzadeh@mail.mcgill.ca
 2

ABSTRACT

A 316L stainless steel (316L-SS) surface was electrochemically polished (EP) in an

electrolyte of a new chemical composition at different cell voltages, with the aim of improving

its corrosion resistance and biocompatibility. X-ray photoelectron spectroscopy results revealed

that the EP-formed oxide films were characterized by a significantly higher atomic Cr/Fe ratio

and film thickness, in comparison to the naturally-grown passive oxide film formed on the

untreated (control) 316L-SS surface. As a result of the increase in the oxide film thickness and

relative Cr enrichment, the EP-treated 316L-SS surfaces offered a notable improvement in

general corrosion resistance and pitting potential. In addition, the attachment of endothelial cells

(ECs) and smooth muscle cells (SMCs) to the 316L-SS surfaces revealed a positive effect of

electropolishing on the preferential attachment of ECs, thus indicating that the EP surfaces could

be endothelialized faster than the control (unmodified) 316L-SS surface. Furthermore, the EP

surfaces showed a much lower degree of thromgobenicity in experiments with the platelet-rich

plasma. Therefore, the use of the electrochemical polishing technique in treating a 316L-SS

surface, under the conditions presented in this paper, indicates a significant improvement in the

surface's performance as an implant material.

Keywords: Electrochemical polishing; 316L stainless steel; Passive oxide films; Corrosion

resistance; Biocompatibility; Blood compatibility

 3

1. Introduction

Surface properties of metallic implants play a paramount role in their biocompatibility and

thus functionality and safety [1-3]. Therefore, various surface treatment approaches have been

employed to tune the surface texture, energy and chemistry of implants [4-6]. In some cases, this

has resulted in favourable interactions of the surrounding biological environment with the

modified surface [7, 8]. Among the various surface treatments, electrochemical polishing (EP)

has been considered as a promising technique to promote corrosion resistance and

biocompatibility of the implant material in physiological conditions [5, 9-11]. This surface

treatment can be applied on complex sample shapes.

EP is a process in which anodic dissolution of metal/alloy takes place in a suitable

electrolyte [12-18]. Generally, anodic leveling and brightening are assumed to be the two

processes occurring during EP [13, 19]. Anodic leveling (i.e., macro-smoothing) is based on the

difference in dissolution rate of "peaks" and "valleys" present on a rough metal surface and refers

to the elimination of the surface roughness height greater than 1 µm [15, 20, 21]. The dissolution

rate depends on the current distribution or mass-transport conditions [20]. On the other hand,

surface brightening (i.e., micro-smoothing) refers to the exclusion of surface roughness height

lower than 1 µm, which is comparable to the wavelength of visible light, thus leading to a

reflective mirror-like surface [16, 22, 23]. Surface brightening occurs due to the suppression of

the influence of the metal/alloy microstructure, surface defects and crystallographic factors on

the dissolution process [13, 24]. As a result, random removal of atoms from the metal results in a

quite smooth surface topography.

 4

The majority of research in the area of EP has been carried out on stainless steel [2, 22],

although metals such as titanium, copper and nickel have also been studied [25-27]. The EP

technique has been used to enhance the biocompatibility of cardiovascular stents, predominantly

made of 316L stainless steel (316L-SS) [9, 11, 28-30]. Cardiovascular stents are small mesh-like

cylindrical ‘tubes’ inserted into a diseased coronary artery at the site of the artery blockage

(composed of calcified plaque build-up) in order to re-open the blocked site and allow blood to

circulate normally [31-34]. Unfortunately, although current commercial stents offer good

mechanical properties, their biocompatibility is rather poor [35-38]. This stems from neointima

hyperplasia and blood clot formation (thrombosis) after stent implantation. The former is a result

of the cellular response of smooth muscle cells (SMCs) present in the vessel wall, which are in a

healthy artery covered by a monolayer of endothelial cells (ECs) that separate SMCs from blood.

When a stent is deployed inside an artery, disruption (removal) of the EC monolayer occurs,

which subsequently leaves the underlying SMCs directly exposed to the blood and the stent

surface. The SMCs then undergo rapid proliferation which, in turn, results in re-narrowing of the

artery or in-stent restenosis (ISR) [39-44]. On the other hand, thrombosis is induced by the

activation of the intrinsic coagulation system and formation of blood clots on the stent surface,

when plasma proteins and platelets adhere to the surface in the early period after stent

deployment [45-47]. Therefore, fast EC-over-SMC attachment and proliferation, or fast stent

endothelialization and damaged tissue re-endothelialization, combined with minimum platelet

activation and aggregation on the stent wall, are highly desirable in order to render the stent more

bio-/hemocompatible.

Furthermore, blood induces corrosion of the stent, leading to degradation of its mechanical

properties, which may result in mechanical failure [5, 48, 49]. The corrosion of 316L-SS stents
 5

causes the release of cytotoxic constituents such as nickel and chromium ions [50, 51]. It was

previously shown that these potentially toxic compounds are stocked in the tissues surrounding

the stent and can migrate through the blood, to be accumulated in vital organs such as kidney,

spleen and liver [2, 52-54]. In addition, Shih et al reported that the corrosion products can even

alter the rat aortic SMC morphology [50].

In the present study, electropolishing (EP) of 316L-SS was employed as a surface

treatment method in an attempt to improve the surface biocompatibility of the material. EP of

316L-SS results in the removal of microstructure irregularities and non-metallic inclusions

related to initialization of the corrosion processes, particularly localized corrosion, and can thus

contribute to the enhancement of the biocompatibility of the material and its

functionality/longevity [10, 55, 56]. EP also removes a native (naturally-grown) surface passive

oxide film on the 316L-SS surface, and forms a new one, which is believed to be more compact

and chemically more homogeneous [4, 57]. This stable passive layer acts as a barrier to the

release of cytotoxic ions from the materials’ surface [2, 58].

In this paper, outcomes of the influence of EP cell voltage on the resulting physico-

chemical properties (chemical composition, corrosion behaviour) of the 316L-SS surface and

surface passive oxide layer are presented. In addition, the interaction of ECs, SMCs and platelets

with the EP surfaces is also presented.

2. Materials and Methods

2.1. Preparation of samples

 6

316L stainless steel substrates were 12.7 mm-diameter discs machined to a thickness of 2

mm. Before the electrochemical polishing, the substrates were first wet smoothed successively

with 600 to 1200-grit abrasive sandpaper (5 min for each sample on each abrasive sand paper).

Samples were then further polished using a 1 µm alumina suspension to obtain a mirror-like

surface finish, followed by rinsing sequentially with acetone, isopropanol and deionized (DI)

water. EP was performed in a batch electrochemical cell, in which a 316L-SS sample served as

an anode, and a graphite rod served as a cathode. The separation between the electrodes was 5

mm. The EP electrolyte of a new solution composition was developed: 60% (v:v) phosphoric

acid (85 wt%), 20% (v:v) sulfuric acid (95-97 wt%), 10% (v:v) glycerol (99.5 wt%) and 10%

(v:v) DI water. The following potential differences (cell voltages) were applied between the

electrodes during a period of 3 min: 2.5, 4 and 10 V. The corresponding samples are in the text

named as EP-2.5V, EP-4V, and EP-10V, respectively. The temperature of electrolyte during the

EP process was kept between 65-70oC by immersing the EP cell in a water-bath.

2.2. Sample characterization

Electron micrographs of sample surfaces were produced using a Philips XL-30 field

emission scanning electron microscope (FE-SEM). An atomic force microscope (AFM) was used

to assess the average surface roughness (Ra) of 316L-SS (control) and EP surfaces. AFM

examinations were performed in ambient air in the semi-contact mode using a SOL VER

P47HPRO scanning probe microscope (SPM, NT-MDT). Different zones and scanning sizes (1–

10 µm) were swept on each sample surface in order to perform a statistical analysis of the

surface roughness.

X-ray photoelectron spectroscopy (XPS) measurements were performed using a VG

instrument Escalab 220i XL equipped with an argon ion gun. The X-ray monochromatic source
 7

was Al (1486.6 eV). The ion etching beam was used at 3 keV with a magnification of ten, which

provided an etching area of 1.5×1.5 mm2. The etching rate was 2 nm min−1 (calibrated using a

SiO2 sample) and the pressure during the etching was kept at 10−8 mbar. The reference energies

used for calibration of the binding energies were the Ag3d5/2 signal at 367.9 eV and the Cu2p3/2

signal at 932.7 eV. The analyzer was fixed at normal position (90°) to the surface. A survey

spectrum was first recorded to identify all elements present on the sample surface, followed by

recording high resolution spectra. The spectra were fitted using the CasaXPS software package

(version 2.13.16) employing Shirley background subtraction and a combination of Gaussian and

Lorentzian line shapes with addition of an asymmetry factor for the metal peaks. In order to

perform a quantitative analysis, 2p spectra of the selected elements were recorded. Next, the

calculated area under the deconvoluted peak, after background subtraction, was correlated to the

atomic concentration of the corresponding element using the corresponding correction factor.

2.3. Electrochemical measurement

Electrochemical measurements were carried out using an Ecochemie Autolab PGSTAT30

potentiostat/galvanostat, equipped with the FRA2 electrochemical impedance spectroscopy (EIS)

module. The GPES/FRA v.4.9.7 software was employed to control the instrument, as well as for

data collection and treatment. A three-electrode electrochemical cell was used in all

electrochemical characterization measurements. A graphite rod was used as the counter electrode

(CE) and the reference electrode (RE) was a saturated calomel electrode. Electropolished 316L-

SS samples were used as the working electrode (WE). The WE sample was placed in a specially

constructed electrochemical cell, exposing 0.5 cm2 of the electropolished side of the WE to the

electrolyte. As electrolyte, aqueous 0.16 mol l-1 NaCl solution was used. This concentration was

selected since it corresponds to the chloride concentration in human body fluids. In addition,
 8

only NaCl was used as a salt to prepare the electrolyte since it is more corrosion aggressive than

the corresponding NaCl-containing phosphate buffer, Ringer's or Hank's solutions, commonly

used to simulate body fluids.

Corrosion measurements were performed in the following order. The WE was first kept at

open circuit potential (OCP) until the potential change rate decreased to less than 2 mV min−1

(up to one hour). This step was followed by an electrochemical impedance spectroscopy (EIS)

measurement at OCP, over a frequency range from 10 mHz to 100 kHz. The AC voltage

amplitude was set to ±10 mV. Next, linear anodic polarization of the WE was conducted from

OCP to a potential at which a current density of 1 mA cm-2 was reached, at which point the

polarization direction was reversed. All the corrosion experiments were performed in an oxygen-

free electrolyte and at 22 ± 1oC, which was acquired by continuously purging the electrolyte with

argon, starting 30 min prior to the measurement and continuing during the measurement.

2.4. Platelet adhesion

Venous blood (50–100 ml) was obtained from healthy volunteers (free from medications

known to interfere with platelet function for at least 10 days before sampling) who gave

informed consent in accordance with the policies of the ethics committee of the Montreal Heart

Institute. Blood samples were anti-coagulated with acid citrate dextrose and processed to yield

platelet rich plasma (PRP) [59, 60]. The PRP was then centrifuged and the platelet pellet was re-

suspended in Hanks’ balanced salt solution (HBSS)–HEPES buffer free from Ca2+ and Mg2+,

with 0.4 mmol l-1 EDTA and 1 µg ml-1 prostacyclin (PGl2, pH 6.5). Any remaining red blood

cells were removed by centrifugation. The platelet pellet was then re-suspended in (HBSS)-

HEPES/PGl2 and EDTA-free buffer (pH 7.4), with Ca2+ (1.3 mmol l-1 CaCl2) and Mg2+ (0.81
 9

mmol l-1 MgSO4), and adjusted to a physiological concentration of 2.5 × 108 ml-1 using an

automated cell counter (Microdriff 16, Beckman Counter, Inc).

For adhesion studies, PRP was added on top of samples in 24-well plates until the entire

surface was covered. Samples were incubated for 60 min at 37o C in a static system. They were

then rinsed with phosphate buffer saline (PBS) and fixed with 2.5% glutaraldehyde, followed by

dehydration in ethanol–water baths of gradually increasing concentrations to 100% ethanol, and

in amyl acetate–ethanol baths of gradually increasing concentrations to 100% amyl acetate.

Samples were dried using critical point drying (CPD, Ladd Research Industries, South

Burlington, VT, USA), sputter coated with gold–palladium and imaged using a Philips XL30

FEG field emission gun scanning electron microscope. The number of attached platelets was

then determined using NIH imageJ, version 1.46r (open source software available at

http://rsbweb.nib.gov/ij).

2.5. Cell attachment

To determine cell attachment, human umbilical vascular endothelial cells (HUVECs)

(Lonza, Walkersville, MD) or human coronary artery smooth muscle cells (hCASMCs) (Lonza),

between passage 4-6, were plated at a density of 50,000 cells cm-2 and allowed to attach to

untreated 316L-SS (control) or electropolished 316L-SS surfaces in 24-well plates, at 37 °C for 4

hr. HUVECs were grown in endothelial cell growth media reconstituted with EGMTM-2

BulletKit (Lonza) and hCASMCs were grown in EBM-2 (endothelial basal medium)

reconstituted with SmGM-2 BulletKit (Lonza). The cells were then washed with PBS and fixed

with 2% (v/v) paraformaldehyde in PBS for 15 min. After incubation with PBS containing 1

wt% BSA and 0.1 wt% saponin for 15 min, the cells were stained for nuclei with 4,6-diamidino-

2-phenylindole (DAPI) (1:5,000) for 3 min. Images were recorded using a Zeiss digital camera
 10

mounted on a Zeiss fluorescence microscope and the numbers of nuclei were counted using NIH

imageJ version 1.46r.

3. Results and discussions:

3.1. Surface topography/morphology

Figure 1 displays scanning electron micrographs of an untreated 316L-SS surface

(control) and surfaces electrochemically-polished at voltages of 2.5, 4 and 10 V. Scratch marks

resulting from mechanical polishing are clearly seen on the untreated rough surface (Figure 1a).

However, a surface of different morphology was created by electrochemical polishing of the

316L-SS sample at 2.5 V (Figure 1b), on which grain boundaries can partially be distinguished.

Further increase in applied voltage to 4 V resulted in diminishing the grain boundary grooves,

and decreasing the surface roughness (see Table 1). However, the surface roughness did not

further diminish notably upon increasing the electropolishing voltage to 10 V (Figure 1c), but

rather hemispherical pits appeared. Note that the circumference/edge of the pits appears to be of

a regular hemispherical shape and smooth, indicating that the formed pits were also

electrochemically-polished (smoothed). These hemispherical pits were formed likely due to the

removal of inclusions in the alloy during the surface electropolishing.

3.2. XPS measurements

To assess the chemical composition of a naturally-grown passive oxide film on the

untreated 316L-SS surface (control) and the electrochemically-formed oxide film on the EP

surfaces, XPS analysis was carried out. Figure 2 shows raw (dots) and deconvoluted (lines) XPS
 11

spectra recorded on the control and electrochemically-polished (at 10 V) passive film-covered

surfaces. The solid lines represent the sum of deconvoluted contributions (dashed lines). A very

good agreement between the modeled (solid lines) and experimental spectra (dots) was obtained

in all cases.

The chromium spectra of the control and EP-10V surfaces (Figures 2a and 2c,

respectively) indicate that the contribution of metallic chromium component in the Cr2P3/2

spectra was decreased for EP-10V surface (i.e. the outer part of the passive film) in comparison

to the control surface. This might be attributed to the thicker passive film formed on the EP-10V

during electropolishing process where oxidation of Cr metal occurs. The major oxidative state of

Cr on the surface of passive film was Cr(III) in Cr(OH)3 and Cr2O3. In addition, the content of

Cr(OH)3 on the outermost layer of EP-10V oxide film was greater than that in the film naturally

grown on the control surface (compare the areas under the corresponding peaks), while the

opposite is true for Cr2O3. This indicates that the surface of the EP-10V film is enriched with a

hydrous Cr(III)-oxide film, relative to the naturally-grown passive film. This is to expect

considering that the former was formed in an aqueous solution under high anodic bias, and thus

rapidly, while the latter was formed at open-circuit potential (OCP) and rather slowly, thus

having time to form a more thermodynamically stable (anhydrous) structure (Cr2O3). Further,

Cr(VI) species, which was identified in the passive film electrochemically formed on EP-10V

surface, was not detected in the passive film naturally grown on the control 316L-SS surface (the

origin of this behaviour will be discussed later in the text, in relation to Figure 4).

The peaks at binding energy of 709 and 710.9 eV on the Fe spectra of passive films in

Figures 2b and 2d are assigned to Fe(II) in FeO [61, 62] and Fe(II) + Fe(III) in Fe3O4 [63, 64].

One can find that the electrochemical polishing of the 316L-SS surface resulted in a rise of Fe3O4
 12

content in the outmost part of the passive oxide film (surface) formed on the EP-10V substrate.

This is due to the electrochemical oxidation of Fe and Fe(II) at high anodic potentials during the

electropolishing process. The peak situated at higher binding energy of 714.9 eV is so-called a

satellite peak and can be attributed to Fe(II) in FeO [65]. No shake-up satellite structures are

visible on the chromium spectra.

Further analysis of Cr and Fe spectra was performed to determine the depth distribution of

their oxidation states and formed species in the passive film. Figure 3a demonstrates that the

atomic Cr/Fe ratio in the outermost layer of the three EP passive oxide films is considerably

greater than that in the film naturally grown on the control surface (ca. 2.2 vs. 0.4, respectively).

This indicates that the passive oxide film formed on the 316L surface by electropolishing is

enriched with chromium species relative to both the control surface and the bulk 316L-SS

composition, suggesting the enhancement in preferential oxidation of Cr. Note that the Cr/Fe

ratio in the outer part of the passive film formed on all three EP surfaces is similar, despite the

difference in the thickness of these three films (Figure 3a).

Figure 3b shows the oxygen profile in the naturally-grown passive film (control surface)

and in the three EP passive films. The relative oxygen content declines going from the outmost

surface of the passive films, to the substrate/passive film (metal/oxide) interface, where no

oxygen species were detected. Hence, from the results in Figure 3b, it is possible to approximate

the thickness of the passive film formed on the four 316L surfaces. Thus, the oxide film formed

by electropolishing of the 316L-SS substrate at the cell voltage of 2.5 V gives the largest

thickness, ca. 27 nm. By increasing the cell voltage to 4 V, the film thickness decreases to ca. 18

nm, and then down to ca. 14 nm on the surface electropolished at 10 V. Nevertheless, all three

EP films are thicker than the passive film naturally-grown on the control surface (ca. 10.5 nm),
 13

indicating that the EP films should, thus, be more corrosion protective (which will be discuses

later in the text). The thickness of the passive film on the control surface is close to values

reported in other papers [66-69]. On the other hand, the EP films are thinner than the passive

oxide film formed on a 316LVM-SS surface employing an electrochemical cyclic polarization

(ca. 35 nm) [70].

The trend of oxygen distribution through the oxide film in Figure 3b is similar to the

distribution of oxidized Cr species, Figure 4. Namely, the relative atomic fraction of the oxidized

chromium species decreases going from the outmost surface of the passive oxide film down to

the metal/oxide interface, where it drops to zero, as expected. An interesting observation in

Figure 4a is that no Cr(VI) species were detected anywhere in the naturally-grown passive film,

while, on the other hand, the species were presented in the three EP films (Figures 4b-c), with an

increasing relative atomic fraction going towards the outer part of the passive oxide film. The

origin in the difference is related to the type of formation and growth of the passive oxide films.

Namely, the naturally-grown passive oxide film on 316L-SS forms at open-circuit potential, at

which the formation of Cr(VI) species is not thermodynamically possible [64]. However, when

voltage of 2.5 V (and larger) is applied between the 316L-SS working electrode sample and the

counter electrode in the electropolishing experiments, some Cr(III) species are oxidized to

soluble Cr(VI) species, forming mostly Cr2O72−, which remain 'arrested' in the growing oxide

film [64]. Unlike Cr species, the depth analysis of Fe in the naturally-grown and EP passive

oxide films revealed no distinct differences in the distribution of Fe(0), Fe(II) and Fe(III) species

through the films among the three EP samples (see Figure 5). However, unlike on EP samples, it

appears that the highest relative atomic fraction of Fe(II) species in the naturally-grown passive
 14

film is located in the middle of the passive film (Figure 5a), rather than at the outer side of the

passive film (Figures 5b-d).

3.3. General corrosion resistance

The first step in characterizing the general corrosion behaviour of the EP and control

samples was to perform open-cicruit measurements. Figure 6 shows the corresponding evolution

of OCP with time. For all the samples, the trend in OCP is very similar; with time, OCP shifts

gradually to more negative potentials. In addition, after ca. 25 minutes, OCP on the three EP

samples is slightly lower than that on the control sample. The behaviour in Figure 6 can be

explained in the following manner; XPS results clearly demonstrated that the three EP films are

enriched with Cr(VI) species, and that the relative Cr(VI) content increases going towards the

oxide film/electrolyte interface (Figure 4). Our previous studies [64] have shown that Cr(VI)

species contribute to the p-type semiconductivity. Thus, the electron transfer through the oxide

film for the partial cathodic corrosion reaction (in the current case, hydrogen evolution) is

kinetically inhibited on the EP oxide films characterized by a higher relative p-type

semiconductivity contribution. Consequently, the hydrogen evolution overpotential increases

relative to that on the native passive film (i.e. the control surface, characterized by the absence of

Cr(VI) species, Figure 4a). Consequently, the open circuit potential is more negative on the three

EP surfaces than on the control surface.

As mentioned in the introduction, electrochemical polishing (EP) has been considered as a

promising technique to promote corrosion resistance and biocompatibility of metallic implants

[5, 9-11]. Based on the above-presented XPS results, one could expect to see an increase in

general corrosion resistance of the EP samples, in comparison to the control surface. In order to
 15

investigate this, EIS measurements were performed on the four surfaces at OCP, and the results

are presented in Figure 7. Visual analysis of the results reveals that the impedance responses of

the EP surfaces are different than that of the control surface. In order to obtain a physical picture

of the electrode/electrolyte interface and the processes occurring at the electrode surface as well

as in the oxide film, experimental EIS data were modeled using non-linear least square fit

analysis software and an electrical equivalent circuit (EEC).

The impedance spectra presented in Figure 7b show a clear evidence of the existence of

two time constants. Therefore, a two-time-constant EEC presented in the inset to Figure 7a was

employed in the modeling procedure. Here, Rel represents the electrolyte resistance, while a

higher-frequency part of the spectrum can be associated with the response of the electrochemical

double-layer capacitance (CPE1) and charge-transfer resistance (R1). A response recorded at

lower frequencies can be prescribed to the response of a slower process, i.e. to the transport of

charged species (metal ions or oxygen-containing species, i.e., oxygen and metal vacancies,

respectively) through the passive-oxide film, characterized by its capacitance CPE2 and

resistance R2 [70-73]. A frequency-dependent constant phase element, CPE (Ω−1 sn cm−2 or

Fsn−1cm−2) with exponent n, was used instead of pure capacitance (C) to obtain a better

agreement between theoretical and experimental data in the fitting procedure. In general, a CPE

is employed due to a relaxation time induced by the inhomogeneities, such as the surface

roughness/porosity, adsorption, or diffusion on the micro and atomic/molecular level at the

electrode/electrolyte interface or through a surface film [74-76]. Figure 1 shows that the

morphology of the four 316L-SS surfaces investigated in this work is highly heterogeneous. This

results in a heterogeneous distribution of (surface) charge. Consequently, neither the 316L-

 16

SS/electrolyte interface (CPE1) nor the distribution of charge through the oxide film (CPE2), can

be described using pure capacitance – hence the use of CPE.

EEC parameter values obtained by fitting the experimental data in Figure 7, employing the

EEC in the inset to Figure 7a, are presented in Table 2. In addition to the above-defined

parameters, the table also lists total resistance values (Rtotal = R1+R2), which represent the

corrosion resistance. Table 2 shows that the time constant for both the high frequency (CPE1-R1)

and low frequency (CPE2-R2) processes was larger for the three EP oxide films, in comparison to

the naturally-grown oxide film (control), indicating better corrosion resistance of the EP films.

Indeed, the total resistance values demonstrate a significantly higher resistance of the EP oxide

films towards corrosion, especially the oxide film formed at a cell voltage of 2.5 V. The

increased corrosion resistance is due to both the increased thickness of the EP passive oxide

films and a greater atomic Cr/Fe ratio in the film (Figure 3). The former is, most likely,

responsible for the differences in the corrosion resistance among the EP films, since the Cr/Fe

ratio in the three EP-formed films is similar. Several investigations also concluded that the

thickness of passive film is a property that is directly related to the resulting general corrosion

resistance [70, 77, 78].

3.4. Pitting corrosion resistance

Pitting corrosion is one of the most severe types of localized attack on 316L-SS, which

significantly limits its application as a body implant material. Pitting corrosion can adversely

influence both biocompatibility and mechanical strength of the implant. This also can lead to a

complete mechanical failure of an implant, such in the case of stents [48]. Thus, it is also

important to evaluate the effect of electropolishing of 316L-SS on the resulting stability of the

surface, not only in terms of its resistance to general corrosion, but also to pitting corrosion.
 17

Figure 8 displays anodic polarization curves of the control and EP 316L-SS surfaces

recorded in 0.16 M NaCl. The pitting potential of the control surface is at 0.1 V. However, when

the 316L-SS surface is electropolished at 10 V, the onset of pitting shifted to 0.34 V. Even more,

the difference between the pitting and OCP increased from 0.25 to 0.59 V, respectively. Figure 8

also shows that EP-2.5V and EP-4V surfaces display higher pitting corrosion potential than the

control, albeit lower than the EP-10V surface (The onset of pitting potential was found to be 0.29

V and 0.18 V for EP-2.5V and EP-4V surfaces, respectively). This implies that the film thickness

is not responsible for the observed increase in pitting potential of the three EP surfaces (the trend

in pitting potential change, Figure 8, is opposite to that of the oxide film thickness, Figure 3), but

some other effects should also be considered. Referring to our previous work [64], we could state

that the increase in the pitting potential of the EP surfaces (Figure 8) can be attributed to the

presence of Cr(VI) species in the corresponding passive oxide films, while no Cr(VI) was

detected in the passive film naturally-grown on the control surface (Figure 4).

It is worth mentioning, however, that literature reports controversial results on the

influence of 316L-SS surface modification on the resulting pitting corrosion potential. Thus,

Hryniewicz et al reported that electrochemical polishing of 316L-SS resulted in a shift in the

pitting potential by about 0.15 V in the Ringer’s solution [56]. No improvement in pitting

corrosion resistance was noted in Ringer's solution when a 316L-SS surface was thermally

modified [5]. Anodic polarization of a 316L-SS surface at a constant potential and UV

illumination for five hours yielded a positive shift in the pitting potential (by ca. 0.17 V) in 0.1 M

chloride solution [79]. Our previous work has demonstrated that the pitting resistance of

316LVM stainless steel (a higher grade of 316 stainless steel than the "L" grade used in the

current work) can significantly be increased by electrochemically forming a surface passive

 18

oxide film under cyclic potentiodynamic polarizaiton conditions; a shift in pitting potential by ca.

0.8 V, in comparison to the surface on which a naturally-grown film was formed, was obtained

[64]. Although the increase in pitting corrosion potential obtained by electropolishing of 316L-

SS (Figure 8) is smaller than in the latter work (partially due to the lower grade of stainless steel

used), one should notice that it is still larger than (or at least comparable to) those reported in the

other works mentioned above. In addition, the application of the EP procedure offers some other

improvements in the materials' biocompatibility, as evidenced further in the text.

3.5. Platelet adhesion

The three electropolished 316L-SS surfaces are potentially good candidates for implant

biomaterials due to their superior corrosion resistance in comparison to the control sample (non-

treated 316L-SS surface). However, although high corrosion resistance of a metallic implant is

an important requirement that needs to be considered, positive (i.e. desirable) interactions of the

surrounding tissue with the implant surface is another key requirement for the implant material.

For example, adhesion and aggregation of platelets on an implant surface would play a critical

role in the process of thrombus formation, which would then influence the thrombogenicity and,

potentially, the functionality of implant surface [80, 81]. In order to compare the

thrombogenicity of the three EP surfaces to that of the control surface, interactions between

blood and the surfaces were assessed in vitro in platelet adhesion experiments.

Figure 9 shows the number of platelets attached on the control and EP surfaces after 60

min of static incubation in PRP. Platelet attachment on the three EP surfaces was significantly

lower than that on the control 316L-SS surface (p 0.01). Fewer adherent platelets on the EP

surfaces demonstrate a lower degree of activation compared with the control 316L-SS. A

decrease in platelet attachment by ca. 71%, 89% and 93% on EP-2.5, EP-4V and EP-10V
 19

surfaces, respectively, with respect to the control surface, indicates a considerable improvement

in blood-compatibility of the EP surfaces. However, there was no statistically significant

difference (p > 0.05) between platelets attached on the EP-4V and EP-10V surfaces.

SEM images of platelets attached on the control and EP surfaces after 60 min of

incubation in PRP are shown in Figure 10. One can observe that the platelet distribution on the

control and the EP surfaces is quite different. Namely, large quantities of platelets aggregated on

the control surface, with well-developed pseudopodia (Figure 10a). This is in agreement with

previous observations of high thrombogenicity of a non-modified 316L-SS surface [45, 82-84].

However, there are no signs of agglomeration of platelets on the EP-4V and EP-10V surfaces

(Figures 10c and 10d, respectively), showing well-preserved and isolated platelet morphology.

On the other hand, most of the adherent platelets on the EP-2.5V surface (Figure 10b) re-

interconnected by pseudopods. Nevertheless, in spite of the spreading pseudopodium, the EP-

2.5V surface still clearly shows better hemocompatibility than the control surface.

3.6. Endothelial and smooth muscle cell attachment

Adhesion of cells to an implant surface precedes other cellular process, such as spreading,

proliferation and differentiation. Accordingly, cell attachment is the initial step in a cascade of

cell-biomaterial interactions and can determine the respective biocompatibility of the implant.

With respect to coronary stents, their biocompatibility can be assessed by investigating the

interaction of ECs and SMCs with the stent surfaces. As already explained in the introduction,

following EC injury after stent implantation, SMCs start growing towards the intimal layer of

blood vessels [85]. This SMC proliferation leads to intimal hyperplasia, which compromises

vascular function. Therefore, fast EC over SMC attachment and proliferation, i.e. fast stent

endothelialization and damaged tissue re-endothelialization, combined with minimum platelet

 20

activation and aggregation on the stent’s surface, are highly desirable in order to render the stent

more biocompatible. The potential for endothlialization of the stent surface can be estimated by

determining the EC-to-SMC count ratio on the stent surface [85-88]. It should be noted that

short-term interactions of ECs and SMCs with the stent surface predominantly contribute to their

competitive behaviour on the stent surface [45, 89, 90]. Consequently, the initial attachment

behavior, rather than the proliferative response of both types of cells, is considered to be

important for the assessment of the endothelialization potential of the stent surface.

Figure 11 shows the EC-to-SMC attachment ratio on the control and EP surfaces after

incubation period of 4 hr. The EC/SMC ratio of the three EP surfaces was statistically

significantly higher than that of the control 316L-SS surface (p < 0.01). However, no statistically

significant difference of this ratio was observed among the three EP surfaces. The EC/SMC ratio

on the control surface is ca. 1.25, indicating a slightly higher affinity of ECs to attach to the

surface. However, on the EP-10V surface, the ratio increased by ca. 40%, to ca. 1.74, which

indicates that the EP surface is much better substrate for the attachment of ECs than for the

attachment of SMCs. Similarly, the other two EP surfaces, EP-4V and EP-2.5V, appear to be

better substrates for the EC attachment, yielding a 30% and 20% higher EC/SMC attachment

ratio over the control surface, respectively. Hence, it is possible that SMC proliferation could be

inhibited in a competitive environment due to the higher affinity of ECs towards the EP surfaces,

indicating that the EP surfaces would be endothelialized faster than the control 316L-SS surface.

4. Conclusion:

In this work, 316L stainless steel was electrochemically polished at an electropolishing cell

voltage difference of 2.5 V, 4 V and 10 V. An electrolyte of a new solution composition for the
 21

electrochemical polishing (EP) of 316L stainless steel was developed. It was found that the

passive oxide film formed on the electropolished (EP) surfaces was enriched with chromium

during the EP process relative to the passive film naturally grown on the untreated (control)

surface and the bulk material phase. In addition, the EP-formed passive films were found to be

thicker than the naturally-grown passive film. As the combined result of these two effects, the EP

passive oxide films offered better general corrosion resistance than the naturally-grown passive

film and the onset of pitting was shifted to higher anodic potentials.

Further, the electropolishing of the 316L-SS surface was found to improve the surface

biocompatibility and hemocompatibility, as evidenced by a significant increase in the attachment

of ECs in comparison to SMCs, and a significant decrease in the platelet adhesion and activation,

respectively.

In conclusion, the use of the electrochemical polishing technique in treating a 316L-SS

surface, under the conditions presented in this paper, indicates a significant improvement in the

surface's performance as an implant material, most notably a coronary stent material. The EP-

10V surface seems to offer largest improvements: highest pitting corrosion potential, lowest

thrombogenicity, and highest potential for endothelization.

However, in-vivo experiments are needed in order to verify this conclusion and determine

which of the three EP-cell voltages actually offers the greatest benefits in treating the surface of a

commercial 316L-SS coronary stent with respect to restenosis.

Acknowledgments:
 22

The authors gratefully acknowledge the financial support from the Natural Science and
Engineering Research Council of Canada (NSERC), the Canadian Institutes of Health Research
(CIHR), and the Fonds de recherche du Québec - Nature et technologies (FQRNT).
 23

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 29

Table caption:

Table 1. Surface roughness of control and electropolished 316L-SS measured by AFM. A scan

area was 10μm×10μm.

Table 2. EEC parameter values obtained by modeling of the EIS spectra in Figure 7 using the
EEC presented in the inset to Figure 7a. The values represents mean values of three
measurements.

Figure captions:

Figure 1. SEM micrographs of (a) untreated 316L-SS surface (control), and 316L-SS surfaces
electrochemically polished at cell voltages of (b) 2.5 V, (c) 4 V, and (d) 10 V.
Figure 2. Representative XPS spectra recorded on the control 316L-SS surface (a, b), and on the
316L-SS surface electrochemically polished at 10 V (c, d). All the components in the graphs are
labeled based on binding energies of the peaks. Dots represent the experimental spectrum and the
solid line represents the corresponding simulated spectrum, which is the sum of denconvoluted
contributions (dashed lines).
Figure 3. (a) Cr/Fe atomic ratio, and (b) oxygen depth profile in the naturally-grown passive
film on 316L-SS (control), and passive films formed on 316L-SS by electrochemical polishing at
cell voltages of 2.5, 4 and 10 V.
Figure 4. Depth profile of different oxidation states of Cr in the passive film formed (a) naturally
on the control 316L-SS surface, and by electropolishing of the 316L-SS surface at cell voltages
of (b) 2.5 V, (c) 4 V and (d) 10 V. The data were obtained by modeling XPS spectra recorded at
different depths of the passive films.
Figure 5. Depth profile of different oxidation states of Fe in the passive film formed (a) naturally
on the control 316L-SS surface, and by electropolishing of the 316L-SS surface at cell voltages
of (b) 2.5 V, (c) 4 V and (d) 10 V. The data were obtained by modeling XPS spectra recorded at
different depths of the passive films.
Figure 6. Time evolution of open circuit potential (OCP) of (1) control, (2) EP-2.5V, (3) EP-4V
and (4) EP-10V 316L-SS surfaces in 0.16 M NaCl solution.
Figure 7. (a) Nyquist and (b) Bode representation of electrochemical impedance response of ()
the control, () EP-2.5V, () EP-4V and () EP-10V 316L-SS surfaces recorded at OCP in
0.16 M NaCl after one hour of stabilization of the surfaces at OCP (Figure 6). The symbols
represent the experimental data while the lines represent the modeled data obtained by
employing the EEC in the inset.
 30

Figure 8. Anodic polarization curves of (1) control, (2) EP-2.5V, (3) EP-4V and (4) EP-10V
316L-SS surfaces. Scan rate = 1 mV s-1. The curves were recorded in 0.16 M NaCl.
Figure 9. Adhesion of platelets on the control and electrochemically-polished 316L-SS surfaces
measured after 60 min of incubation in PRP. A significant decrease in the number of adhered
platelets is seen on all EP surfaces in comparison to the control (** p < 0.01). Results are
expressed as mean value ± SD of three samples averaged over the entire sample surface. Note
that the asterisks above each bar represent a significant difference relative to the control whereas
asterisks over the bracket indicate a significant difference between EP-2.5 V and the other EP
surfaces.
Figure 10. SEM images of platelets attached to the (a) control and 316L-SS surfaces
electrochemically-polished (EP) at cell voltages of (b) 2.5 V, (c) 4 V and (d) 10 V. The images
were taken after 60 min of static incubation in PRP.
Figure 11. The count ratio of the endothelial to smooth muscle cells (EC/SMC) attached on the
control and the 316L-SS surfaces electrochemically-polished at cell voltages of 2.5 V, 4 V and
10 V. The cell attachment period was 4 hr. (**) p < 0.01. Note that the asterisks above each bar
represent a significant difference relative to the control.
 31

TABLES AND FIGURES

Table 1.

Surface
Sample
roughness (nm)

316L-SS 188 ± 9

EP-2.5V 107 ± 6

EP-4V 77 ± 4
EP-10V 97 ± 11
 32

Table 2.

EEC Surfaces
parameters Control EP-2.5V EP-4 V EP-10 V
Rel
127 ± 0.4 124 ± 0.3 137 ± 0.2 168 ± 0.4
(Ω)
CPE1 × 105
4.9 ± 0.3 5.2 ± 0.1 7.5 ± 0.4 9.1 ± 0.4
(Ω−1 sn cm−2)
n1 0.89 ± 0.05 0.84 ± 0.03 0.83 ± 0.01 0.82 ± 0.03
R1
1.4 ± 0.2 3.3 ± 0.1 1.6 ± 0.1 1.5 ± 0.2
(kΩ cm2)
CPE2 × 105
2.9 ± 0.3 3.4 ± 0.4 4.3 ± 0.2 4.2 ± 0.2
(Ω−1 sn cm−2)
n2 0.72 ± 0.03 0.81 ± 0.02 0.91± 0.02 0.92 ± 0.01
R2
147 ± 2 804 ± 11 464 ± 2 399 ± 1
(kΩ cm2)
Rtotal
148 807 465 401
(kΩ cm2)
 33

Figure 1.

(a) (b)

(c) (d)
 34

Figure 2
 35

Figure 3
 36

Figure 4
 37

Figure 5
 38

Figure 6
 39

Figure 7

80

70
(a)

60 10 mHz

50
-Z'' (kΩ cm2)

40

30

20

10 mHz
10

0
0 10 20 30 40 50 60 70 80
2
Z' (kΩ cm )
80

70 (b)
60
-Phase angle (degree)

50

40

30

20

10

0
-2.5 -1.5 -0.5 0.5 1.5 2.5 3.5 4.5
log [f (Hz)]
 40

Figure 8
 41

Figure 9
 42

Figure 10

(a) (b)

(c) (d)
 43

Figure 11
 44

Electrochemical Polishing as a 316L Stainless Steel Surface

Treatment Method: Towards the Improvement of Biocompatibility

Sajjad Habibzadeh1, Ling Li2, Elaine C. Davis2, Dominique Shum-Tim3 and Sasha Omanovic1,*

(2) Department of Chemical Engineering, (2) Department of Anatomy and Cell Biology,
(3) Divisions of Cardiac Surgery and Surgical Research, Department of Surgery
McGill University, Montreal, QC, Canada

HIGHLISHTS

• Electropolishing of 316L stainless steel increases its corrosion resistance

• New electropolishing electrolyte composition is suggested
• Larger thickness and chromium enrichment of the passive film is obtained
• Electropolishing improves the surface biocompatibility and hemocompatibility