Surface & Coatings Technology 221 (2013) 1–12

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Surface & Coatings Technology

journal homepage: www.elsevier.com/locate/surfcoat

Electrochemical and chemical methods for improving surface characteristics of 316L

stainless steel for biomedical applications
Afrooz Latifi a, Mohammad Imani b,⁎, Mohammad Taghi Khorasani c, Morteza Daliri Joupari d
a
Biomedical Engineering Faculty, Science and Research Branch, Islamic Azad University, Tehran, Iran
b
Novel Drug Delivery Systems Dept., Iran Polymer and Petrochemical Institute, P.O. Box 14965/115, Tehran, Iran
c
Biomaterials Dept., Iran Polymer and Petrochemical Institute, P.O. Box 14965/159, Tehran, Iran
d
Animal and Marine Biotechnology Dept., National Institute of Genetic Engineering and Biotechnology, P.O. Box 14965/161, Tehran, Iran

a r t i c l e i n f o a b s t r a c t

Article history: Although stainless steel is a widely used material for biomedical applications, its surface properties for long term
Received 30 September 2012 application are still a serious concern. Here, chemical surface treatment by electropolishing and acid dipping is
Accepted in revised form 5 January 2013 used for improving surface properties of 316L stainless steel to provide a homogenous, smooth and corrosion re-
Available online 23 January 2013
sistant surface with high biocompatibility profile. X-ray photoelectron spectroscopy (XPS) was performed to
trace surface chemical composition before and after chemical treatment. Surface enrichment of corrosion resis-
Keywords:
316L stainless steel
tant compounds was shown by XPS. Corrosion behavior of the treated samples was evaluated by electrochemical
Electropolishing analysis. Surface energy was also studied through contact angle measurements. The roughness of the surface was
Acid dipping traced by atomic force microscopy (AFM) and scanning electron microscopy (SEM) techniques. The lowest
Surface characterization roughness (Sa: 0.96± 0.29 nm) was observed for the highest acid dipping duration (1800 s). The surface energy
Corrosion resistance was remarkably subsided by acid dipping duration. MTT assay and cell adhesion studies were conducted to
In vitro biocompatibility evaluate in vitro biocompatibility showing high cell viability percentage with proper cell adhesion on the
surface-treated samples.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction
316L stainless steel is one of the most widely used metals to fabri-
cate medical devices for orthopedic, intravascular, dental applications,
etc. just to name a few [1]. This ever increasing trend is mainly due
to its high mechanical strength, excellent corrosion resistance, good
processability, biocompatibility and low price [2,3]. Among these prop-
erties, corrosion is an important issue especially in prolonged implanta-
tion in human tissues for devices in close contact with physiological
fluids.
Corrosion is a natural process of deterioration that converts metals
into their thermodynamically stable states [4]. Occurrence of corrosion
results in the releasing of metal ions such as chromium, iron and nickel
leading to mechanical and/or clinical failure and intoxication of organs
[5–7]. To this end, a great attention is paid to electrochemical studies
during the last decades in order to determine the corrosion behavior
of different metals used for biomedical applications including stainless
steel as a function of their in vitro biocompatibility [8]. On the other
hand, metallic surface roughness is a crucial parameter in determining
its performance against the surrounding environment, thrombogenicity,
platelet adhesion and subsequent tissue reactions to the implanted ma-
terial [9–12]. Therefore, in order to overcome the adverse reactions
⁎ Corresponding author. Tel.: +98 21 4866 2456; fax: +98 21 4458 0021.
E-mail address: M.Imani@ippi.ac.ir (M. Imani).
following the use of metallic devices in the human body and increasing
the life time of devices after implantation, surface treatment of metals is
often required. Surface treatments like ultrasonic cleaning, acid dipping,
mechanical and electrochemical polishing, thermal treatment, laser sur-
face melting and plasma exposure are the most commonly used surface
treatment methods [5,9,13,14].
Air-exposed stainless steel surface is covered by thin layers of metal
oxide and hydroxide. These layers on the metal surface are 10–15 Å in
thickness and not chemically stable [13]. Removing these naturally-
occurring oxide and hydroxide layers from a metal surface by chem-
ical treatment is of great importance to leave a clean, smooth and
contamination-free surface in place.
Electropolishing is the most extensively used electrochemical treat-
ment method for metallic surfaces that results in significant improve-
ment in smoothness, hydrophilicity and corrosion resistance of the
surfaces [8,9,15,16]. This method is interesting due to its ability for
selective dissolution of metal ions and formation of passive layers on
metal surfaces, and removal of non-metallic inclusions which are re-
sponsible in promoting corrosion process and formation of new, smooth
and chemically homogenous layers on different metal substrates. This
treatment method is also applicable on metallic parts with complex ge-
ometries [9,13,14,16–19]. The flexibility of electropolishing process is
due to its performance by anodic polarization in different electrolytes
during two distinct processes i.e., anodic leveling and anodic brightening
[9,15]. Electropolishing process can be controlled by adjusting the main

0257-8972/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.surfcoat.2013.01.020
2 A. Latifi et al. / Surface & Coatings Technology 221 (2013) 1–12
parameters including anodic current density, electrolyte(s) composi-
tion, duration and temperature of electrolysis [13].
Acid dipping is another process for chemical surface treatment of
metals in which metallic contaminations on the surface are removed
using an aqueous solution of a mineral or organic acid [20]. Hence,
acid dipping can be used for natural oxide layer removal and formation
of a new, thin and homogenous layer [19,21]. Nitric acid, citric acid, and
hydrofluoric acid are effective candidates to be used in acid dipping
however, aqueous mixtures of nitric acid and hydrofluoric acid (in
different molar ratios) are commonly used for chemical treatment of
stainless steel surfaces [9,10,22,23]. Furthermore, it is shown that acid
dipping can be used as a treatment step to remove the phosphate
layer formed after electropolishing process [13].
Here, our main objective was to improve the surface properties
of 316L stainless steel parts by increasing their surface smoothness.
To this end, electropolishing and acid dipping at different durations
were performed as chemical treatments to modify surface topography
and roughness of the samples. Before any chemical treatment, a pre-
cleaning step was performed in cleanser solutions to remove grease,
oil, soil and other surface contaminations. The effect of each treatment
procedure on the surface chemical composition was investigated by
X-ray photoelectron spectroscopy (XPS). Scanning electron microscopy
(SEM) and atomic force microscopy (AFM) were used for studying the
surface topography after surface treatments. Potentiodynamic polariza-
tion test was also carried out to evaluate corrosion resistance of the
treated surfaces. Contact angle and surface energy measurements were
also performed. MTT assay was used to assess in vitro biocompatibility
of the specimens using L929 fibroblast cell line. Cell adhesion on speci-
mens was also studied using SEM technique after proper fixation.
2. Materials and methods
2.1. Materials
Medical grade 316L stainless steel (with the composition (wt.%) of
b0.03% C, 16–18% Cr, 10–14% Ni, 0.3% Mo, and Fe balance) was provid-
ed by EZM Inc. (Wetter, Germany). Phosphoric acid and sulfuric acid
were of analytical grade and purchased from Merck Chemicals Co.
(Darmstadt, Germany) and applied to make electropolish electrolyte
solutions. Nitric acid (65% w/v, Merck, Germany) and hydrofluoric
acid (48% w/v, Loba Chemie, India) were also used for preparing acid
dipping solution. Ringer solution was supplied by Darupakhsh Pharm.
Co., (Tehran, Iran). All chemicals were used as received without further
purification.
2.2. Surface treatments
Medical grade 316L stainless steel plates were cut into 10×10×1 mm
size using jigsaw. The specimens were chemically treated by two differ-
ent procedures. In the first procedure, specimens were cleaned ultrason-
ically in acetone, deionized water and isopropanol sequentially for
15 min. After cleaning, the specimens were electropolished in an elec-
trolyte solution composed of phosphoric acid (60% v/v) and sulfuric
acid (40% v/v) by an Electromet-4 electropolishing system (Buehler
Co., Illinois, USA). The resulting surface was referred to as sample 1. Elec-
trochemical polishing parameters are summarized in Table 1. After
electropolishing, the specimens were rinsed by distilled water and
Table 1
Process conditions for electropolishing.
Electropolishing Potential Current Treatment Electrolyte bath
parameter (V) density time temperature
(A/cm2) (min.) (°C)
Value 5 0.25 20 50
dried with oil-free compressed air. The second procedure consisted of
ultrasonic cleaning, electropolishing (in the same way as described
for the sample 1) and surface dipping in an acidic solution. Acid dipping
solution was composed of an optimized mixture of hydrofluoric acid
(2% v/v), nitric acid (10% v/v) and deionized water according to litera-
ture [9,10,13]. The acid solution temperature was adjusted on 45±
2 °C. Acid dipping was conducted for different durations i.e., 30, 60,
90, 600, 1200 and 1800 s. The resulting surfaces were coded to as sam-
ples 2, 3, 4, 5, 6, and 7 (corresponding to 30, 60, 90, 600 1200 and 1800 s
of acid dipping duration), respectively. After acid dipping, the speci-
mens were rinsed thoroughly using deionized water and dried using
compressed air as previously described. The samples were stored in
vacuum desiccator after each surface treatment before any further use
for analyses.
2.3. X-ray photoelectron spectroscopy (XPS) measurements
Surface chemical composition of the treated specimens was inves-
tigated by XPS using an XR3E2 (VG Microtech, UK) twin anode X-ray
source. The instrument was equipped with an Al-Kα X-ray source work-
ing at 1486.6 eV. The operation vacuum was higher than 2 ×10 −7 Pa.
Photoelectrons were detected using a hemispherical energy analyzer
at angle of 90°. The C 1s peak was used as a reference for all binding
energy calibrations. Pass energy was 97 eV for surveys and 27 eV for
high resolution spectra. Deconvolution of peaks was conducted by a
spectral data processor (SDP, version 4.1, XPS international LLC, USA)
with 80% Gaussian–20% Lorentzian peak fitting.
2.4. Surface morphology studies
Scanning electron microscopy (SEM) was performed using a Philips
XL30 (Eindhoven, The Netherlands) instrument. SEM micrographs
were taken in mixed mode (17 kV, 1000×) from the plate's surface to
evaluate surface microstructure and roughness qualitatively.
2.5. Roughness measurements
The roughness of all the neat and treated specimens was measured
quantitatively by atomic force microscopy (AFM) (Nanosurf easyscan 2,
Nanosurf Co., Liestal, Switzerland) in contact mode. Data were processed
using Nanosurf software (Version 1.3, Nanosurf Co., Liestal, Switzerland).
The AFM scanning area was chosen as 20×20 μm and the analysis was
performed to determine the arithmetic mean surface roughness (Sa)
and root mean square surface roughness (Srms). Sa and Srms were calculat-
ed according to the following equations:
M  
1 XN X
 2 
Sa ¼ η ðxi ; yi Þ ð1Þ
MN j¼1 i¼1
vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
u M  
u 1 X N X
 2 
Srms ¼t η ðxi ; yi Þ ð2Þ
MN j¼1 i¼1
where, M and N are the number of samples per line and the number of
lines, respectively. η(xi, yi) is the mean height of all the points on the
surface.
2.6. Contact angle measurements
Static contact angle and surface energy were measured by the sessile
drop technique using contact angle goniometer (Kruss K10, Hamburg,
Germany) at ambient conditions. Distilled water and diiodomethane
(3 μL volume) were used to measure the surface free energy. For this
purpose, in separate tests, drops of water and diiodomethane were
placed on different locations of the samples (treated or neat) then the
 A. Latifi et al. / Surface & Coatings Technology 221 (2013) 1–12
contact angle was measured. Calculations were based on the mean
average of three independent measurements. Surface energies were
calculated according to Owens and Wendt model as:
   p p 0:5
d d 0:5
γ sl ¼ γ s þ γ l −2 γs γl −2 γ s γ l
where, γs and γl are surface free energy of solid and liquid, respectively.
γsd and γld are dispersive components of surface free energy of solid and
p
liquid, respectively. γsp and γl are polar components of surface free en-
ergy of solid and liquid, respectively [24].
2.7. Electrochemical analysis
Corrosion of all surface-treated specimens in comparison to non-
treated specimens was investigated by electrochemical analysis. Open
circuit potential (OCP) analysis was performed to accomplish the elec-
trochemical measurements. Scan rate was adjusted on 1 mV·s−1 at
37 °C in ringer solution using an IviumStat potentiostat )Ivium Technol-
ogies Inc., Eindhoven, The Netherlands) equipment which consisted of a
platinum counter electrode, a Ag/AgCl reference electrode and a work-
ing electrode. The specimen's area exposed to the electrolyte solution
was 1 cm2 and potential range was set between −0.5 and 1.5 V. Before
starting the polarization test, the specimens were immersed in ringer
solution for 30 min until the open circuit potential was stable. Data
analysis was done by using IviumSoft software (Release 1.863, Ivium
Technologies Inc., Eindhoven, The Netherlands). The corrosion potential
(Ecorr) and corrosion current density (icorr) were determined using Tafel
extrapolation method.
2.8. In vitro biocompatibility
Specimens including neat, electropolished and electropolished
plus acid dipped were chosen for in vitro biocompatibility studies by
direct contact test, cell adhesion and MTT assay according to ISO
10993-5 standard.
2.8.1. Direct contact test
Cell adhesion on the surface of specimens was assessed on days 1,
3 and 5 post-incubation of the samples in a suspension of L929 fibro-
blast cells in RPMI (Gibco Co., Eggenstein, Germany) culture medium
containing 10% (v/v) fetal bovine serum (FBS, Gibco) and 100 μg/mL
streptomycin and 100 IU/mL penicillin G (Sigma Co., Munich, Germany).
After 1 and 5 days, the cells were fixed using glutaraldehyde (2.5% v/v)
and assessed after gold sputter coating (Emitech K450×, 15 kV, Quorum
Technologies Inc., West Sussex, UK) by SEM (Tescan Vega, Brno, Czech
Republic). Cell densities and cell adhesion on day 3 were also observed
through a Nikon inverted optical microscope (Tokyo, Japan). Cell sus-
pension in culture medium was used as control for optical microscopic
observations.
2.8.2. MTT assay
The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide] assay was used to measure cell viability after incubation with the
specimens. For this purpose, all steam sterilized specimens were placed
in a 24-well culture flask. Blank culture and non-sterile natural rubber
latex (Top Glove Co., Selangor, Malaysia) were used as negative and pos-
itive controls, respectively. A suspension of 1×104 cell·mL−1 of L929 fi-
broblast cells in culture medium containing RPMI (Gibco), 10% (v/v) fetal
bovine serum (FBS, Gibco) and 100 μg/mL streptomycin, 100 IU/mL
penicillin G (Sigma) was seeded on each well. After incubating at 37 °C
in a 5% CO2 and 95% humidity incubator for 1 and 3 days, 100 μL of
MTT solution (0.5 mg·mL−1, Sigma) was added to each well and
incubated at 37 °C for 4 h. Following the removal of culture medium,
acidified isopropanol was added in order to dissolve the formazan
crystals. The optical density of formazan was measured
3
spectrophotometrically at 545 nm using an ELISA plate reader. This ab-
sorbance value is proportional to the number of viable cells. Each of the
specimens was plated in triplicate MTT assays for 1 and 3 days.
2.9. Statistical analysis
ð3Þ
Statistical analysis was performed using OriginPro70 software
(Version 7.0220, OriginLab Co., Northampton, MA, USA). Data were
reported as mean± standard deviation at significance level of p b 0.05.
Differences were considered statistically significant when the p value
was b0.05.
3. Results and discussion
3.1. XPS analysis
Fig. 1 depicts XPS survey spectra for neat sample, sample 1 and sam-
ple 7. Atomic concentration of elements on the neat and chemically-
treated specimens is tabulated in Table 2 based on the determination
of peak areas. As shown in Fig. 1a, carbon (C 1s) (80.2 at.%) and oxygen
(O 1s) (19.8 at.%) are the dominant elements present on the surface of
the neat specimens. After chemical treatment on the samples 1 and 7
i.e., electropolished specimen and electropolished plus acid dipped sam-
ples, the surface chemical composition was changed. The carbon signal
intensity was significantly decreased, whereas the oxygen signal was
enhanced (Fig. 1b, c). Reduction in carbon concentration (40.8 at.%)
on the electropolished surface was compensated by an increase in the
concentration of other elements like O 1s (49 at.%), Fe 2p (3.4 at.%),
and Cr 2p (4.2 at.%) (Table 2). Phosphorus atoms (P 2p) (2.7 at.%)
were also detected on the electropolished surface which are originated
from the electropolishing electrolyte composition. After surface treat-
ment by electropolishing plus acid dipping i.e., sample 7, reduction in
C 1s signal is also notable compared to the neat specimens. These results
indicate the efficiency of chemical methods for surface carbon decon-
tamination. A reduction in O 1s signal intensity is also observed for
sample 7. Reduction in oxygen concentration is also shown by EDX
data and can be associated with the complete removal of natural thick
oxide layer and formation of a new thin and uniform oxide layer. In-
creasing Cr 2p concentration (6 at.%) is also observed by XPS analysis
on sample 7. Moreover, after acid dipping the peak related to P 2p dis-
appeared in the XPS survey spectrum. Haidopoulos et al. also reported
that the phosphate layer formed after electropolishing in phosphorus
solution can be removed by acid dipping [13]. After electropolishing
and acid dipping of sample 7, fluorine (9.2 at.%) was detected on the
surface. The presence of this element on the surface can be due to dip-
ping of the sample in the acidic solution containing hydrofluoric acid.
The high-resolution XPS spectra of C 1s, O 1s, Fe 2p3/2, Cr 2p3/2 and
F 1s are shown in Fig. 2. The binding energies and FWHMs of high-
resolution XPS spectra of C 1s, O 1s, Fe 2p3/2, Cr 2p3/2 and F 1s are
shown in Table 3. After deconvolution of C 1s, three peaks at 284.98 eV
for carbon and C\H, 286.58 eV for C\O and 288.7 eV for COO species
were fitted to carbon signal detected on neat sample (Fig. 2a and
Table 3). These peaks are approximately close to energy values reported
in the previous literature for C 1s [25]. The high-resolution spectrum of
O 1s (as shown in Fig. 2b) is curve-fitted with three different peaks at
530.5 eV, 532.1 eV and 533.1 eV for the neat sample (Table 3). The
first peak can be assigned to iron and chromium oxides [10] and the sec-
ond peak can be assigned to hydroxides [10]. The peak at 533.1 eV can
be due to organic oxygen impurities [26]. Similar curves with energy
values close to the above values were fitted for O 1s signal detected
on sample 1 and sample 7 (Fig. 2c, d and Table 3). Considering the
high-resolution spectrum of O 1s, the dominant oxide compounds on
the surface after electropolishing and acid dipping are evident in
Fig. 2c, d. The hydroxide species of 70.1 at.% concentration, iron and
chromium oxides of 14.9 at.% concentration and other impurities are
the surface compounds produced after electropolishing. While, the
4 A. Latifi et al. / Surface & Coatings Technology 221 (2013) 1–12
Fig. 1. XPS survey spectra of (a) neat, (b) sample 1 and (c) sample 7.
hydroxide of 44.3 at.% concentration, iron and chromium oxides of
23.9 at.% concentration and other impurities are the surface com-
pounds produced after electropolishing plus acid dipping. Hence,
these results show that the acid dipping process which modifies the
surface oxide layer composition would lead to an improved passive sur-
face layer. The Cr 2p3/2 peaks are curve-fitted with four different peaks
Table 2
Chemical composition of the neat and chemical treated specimens (at.%) according to
XPS measurements.
Sample code Composition (at.%)
C O Fe Cr P F
Neat 80.2 19.8 – – – –
Sample 1 40.8 49 3.4 4.2 2.7 –
Sample 7 37 44.2 3.2 6 – 9.2
at about 574.30 eV, 576.80 eV, 577.30 eV and 578.30 eV for sample 1
and sample 7 (Fig. 2e, f and Table 3). These curves correspond to
Cr(0), Cr2O3, Cr(OH)3 and CrO3 species, respectively [27]. As shown in
Fig. 2f, Cr2O3 of 56.5 at.% concentration present on the metal surface is
a more dominant species than other chromium compounds after acid
dipping. Also, chromium oxide concentration on the acid-dipped sur-
face i.e., sample 7 increased by approximately three times higher than
the same oxide formed on the electropolished surface i.e., sample 1.
Fig. 2g shows Fe 2p3/2 signal fitted with distinct peaks at 708.2 eV,
710.87 eV, and 711.8 eV for sample 1. These peaks can be attributed
to Fe3O4, Fe2o3 and FeOOH, respectively (Table 3) [27]. Three distinct
peaks were fitted to Fe 2p3/2 at 708.33 eV, 711 eV and 711.8 eV for sam-
ple 7 (Fig. 2h and Table 3). These peaks can be attributed to Fe3O4, Fe2o3
and FeOOH, respectively [28]. Fluorine (F 1s) peak was deconvoluted to
three different peaks at 685 eV, 686.74 eV and 688.69 eV in Fig. 2i and
Table 3. The first peak can be attributed to iron-fluoride and chromium-
fluoride bonding [10]. The second and third peaks at 686.74 and
688.69 eV can be attributed to carbon-fluoride bonding [28]. Since the
metal fluoride complexes exhibit high water contact angle [28], the
presence of fluorine on the acid-dipped surface i.e., sample 7 can result
in changes in the surface wettability as presented in Section 3.4.
3.2. SEM/EDX analysis
Fig. 3 shows SEM micrographs of neat and chemically treated spec-
imens. As observed in the SEM micrographs, different surface topogra-
phies distinctly emerged which are related to the treatment type.
Neat specimen (Fig. 3a), shows a very rough porous oxide-layer coated
surface topography with deep fissures and scratches. Electropolishing
has resulted in a smooth with less peaks and troughs, defect free and
uniform surface as can be observed in Fig. 3b.
Acid dipping process removes most of the remaining organic con-
taminations and phosphate layer formed during electropolishing and
finishes the surface treatment performed during the electropolishing
step [13]. Thus a very smooth surface and visible bulk structure with
distinct grain boundaries showing different crystallographic orienta-
tions can be distinguished following the acid dipping performed at
different durations (Fig. 3c–h). According to the EDX analysis results
(Table 4), the oxygen on acid dipped surfaces is reduced by increasing
the acid dipping duration. This conclusion is supported by a reduction
in the relative intensity of atomic oxygen signal observed between
sample 1, samples 2–3, sample 4 and samples 5–7. The XPS results
also verified the oxygen reduction after acid dipping. According to
X-ray attenuation length of iron, the distance that X-ray photons
travels in a material before the energy intensity falls to 1/e of its orig-
inal value at the surface, oxygen with low photon energy i.e., ≅ 525 eV
can be detected from a depth lower than 200 nm [29]. Hence, it can
be considered that the observed oxygen EDX results show the oxygen
concentration of the samples' surface.
Finally, according to the roughness results, it can be concluded that
the removal of native oxide layer and carbon contaminated layer is effec-
tively achieved during the two chemical treatment methods applied in
this research and a new smooth layer resistant to corrosion is formed.
Providing smooth surfaces for metallic biomaterials can improve their
hemocompatibility profile, prevent them from platelet aggregation, and
decrease neointimal hyperplasia in cardiovascular devices [9]. Hence,
 A. Latifi et al. / Surface & Coatings Technology 221 (2013) 1–12 5

Fig. 2. High-resolution spectra of (a) C 1s for neat, (b) O 1s for neat, (c) O 1s for sample 1, (d) O 1s for sample 7, (e) Cr 2p3/2 for sample 1, (f) Cr 2p3/2 for sample 7 (g) Fe 2p3/2 for
sample 1 (h) Fe 2p3/2 for sample 7 and (i) F 1s for sample 7.
6 A. Latifi et al. / Surface & Coatings Technology 221 (2013) 1–12

Table 3
Binding energies and FWHMs of high-resolution XPS spectra of C 1s, O 1s, Fe 2p3/2, Cr 2p3/2 and F 1s.

Sample Element Deconvoluted peaks FWHM

no. (binding energy) (eV)

Neat C 1s C\(C,H) C\O C\OO 1.83

(284.98) (286.58) (288.7)
Neat O 1s O\(Fe, Cr) Hydroxides \CO3, \COH 2.29
(530.5) (532.1) (533.1)
1 O 1s O\(Fe, Cr) Hydroxides \CO3, \COH 2.27
(530.50) (531.90) (533.20)
1 Cr 2p3/2 Cr(0): Cr2O3 Cr(OH)3 CrO3 3.35
(574.30) (576.80) (577.30) (578.30)
1 Fe 2p3/2 Fe3O4 Fe2O3 FeOOH 3.96
(708.2) (710.87) (711.8)
7 O 1s O\(Fe, Cr) Hydroxides \CO3,\COH 1.76
(530.5) (532) (533.1)
7 Cr 2p3/2 Cr(0) Cr2O3 Cr(OH)3: CrO3 2.92
574.30 576.90 577.30 578.34
7 Fe 2p3/2 Fe3O4 Fe2O3 FeOOH 4.05
(708.33) (711) (711.8)
7 F 1s Fe\F, Cr\F C\F 2.30
(685) (686.74, 688.69)

considering the obtained surface roughness properties (as evident in
SEM results), it can be concluded that a combination of electropolishing
plus acid dipping method is an effective surface finishing for cardiovascu-
lar devices.
3.3. Roughness measurements
Fig. 4 shows 3-dimentional AFM micrographs captured from the
surface of neat and chemically-treated specimens. Fig. 4a shows a
laminated microstructure with deep irregularities for neat samples
mainly composed of oxide layer [13]. As shown in Fig. 4b, surface ap-
pearance becomes mirror like, much more uniform in orientation of
peaks and valleys and smooth after electropolishing that turns to a
bright surface to the naked eye after chemical treatment. Presence
of hills and valleys with different heights is the structural characteris-
tics of electropolished surfaces [15]. Smoother surfaces with disap-
pearance of much of the irregularities, and reduction in peak and
valley heights are shown in AFM micrographs for samples treated
by acid dipping (Fig. 4c–h). However, in this study, smooth surface
with hills and valleys in nanometer scale was observed for all treated
specimens. Sa and Srms values, presented in Table 5, reveal a significant
reduction in roughness after different treatment methods including
electropolishing and acid dipping process (p b 0.05). Diminishing sur-
face roughness after electropolishing can be attributed to the widely
suggested mechanism of electropolishing i.e., macro-smoothing and
micro-smoothing during anodic dissolution and brightening, respec-
tively. The metal ions of 316L stainless steel dissolve in electropolishing
solution with their highest valence and create a smooth and defect-free
surface according to the following reactions [15]:
−
Fe→FeðIIIÞ þ 3e
−
Ni→NiðIIÞ þ 2e
− therefore, the electropolished surfaces, have as expected shown higher
Cr→CrðVIÞ þ 6e :
The mass of material removed from the metal surfaces during
electropolishing process is expressed by Faraday's law of electrolysis
[30]:
. imens. Increase in the surface energy can be explained according to the
ItM
W loss ¼
nF
where, t and I are time and current consumed during electropolishing
process, respectively. M is anodic molecular weight. F and n are Faraday's
constant and valence of metal ions, respectively.
According to the Sa and Srms results (Table 5), it can be concluded
that the specimens treated by electropolishing plus acid dipping pos-
sess significantly lower roughness as compared to those specimens
that were only electropolished (p b 0.05). Furthermore, while surface
roughness is significantly reduced after electropolishing plus acid
dipping treatments in variable lengths of time compared to those just
only electropolished specimen, a slight decrement in Sa and Srms was
observed by increasing in acid dipping time. Average roughness of
acid-dipped surfaces at the longest acid dipping duration (sample 7)
was significantly (p b 0.05) lower than samples treated for shorter dura-
tions e.g., sample 2. The same significant differences were observed for
root mean square roughness between samples 3 and 5 (p b 0.05). SEM
micrographs showed similar topography for all acid-dipped samples
treated in different time intervals. Nevertheless, findings based on sam-
ples roughness obtained from AFM results, imply that by longer dura-
tion of acid dipping (e.g., sample 7) the smoother (Sa: 0.96 ± 0.29 nm,
Srms: 1.71± 0.78nm for the same sample) the surface becomes. Based
on a published report, excessive surface removal and dimensional
changes occur during increased immersion time of metallic biomate-
rials in acid etching solutions. In contrast, insufficient contamination re-
moval occurs following insufficient immersion time and hence, it would
be highly crucial to optimize the immersion time in acidic solutions [9].
3.4. Contact angle measurements
Contact angle is an essential method to assess the cleanliness of solid
surfaces and surface treatment efficacy [31]. Fig. 5 represents contact
angle and surface energy results found for neat and electropolished
specimens. As shown in Fig. 5, water and diiodomethane contact angles
of 316L stainless steel are significantly lowered after electropolishing
ð4Þ (p b 0.05). Water contact angle of neat stainless steel was 83.6° but de-
creased to 56.5° after electropolishing (Fig. 5). Since the surface energy
ð5Þ increases with decreases in water and diiodomethane contact angles
ð6Þ surface energy (Fig. 5). In electropolished samples, the observed de-
crease in contact angle of water and diiodomethane indicates signifi-
cant (p b 0.05) increment of surface energy which affects the surface
wettability characteristics of 316L stainless steel hence, the surface
can be made wet by wetting liquid easily compared with the neat spec-
ð7Þ reduced surface roughness and complete removal of surface contami-
nations like hydrocarbons by applying an efficacious electropolishing
process. The reduced carbon content in the electropolished surfaces is
already verified by XPS examination. Decreased protein adsorption
capability on the electropolished surfaces with higher wettability and
 A. Latifi et al. / Surface & Coatings Technology 221 (2013) 1–12 7

Fig. 3. SEM micrographs of 316L stainless steel surfaces: (a) neat, (b) sample 1, (c) sample 2 (d) sample 3 (e) sample 4, (f) sample 5, (g) sample 6 and (h) sample 7.

Table 4 smoother surfaces is generally observed which in turn causes lower

Oxygen concentration on the neat and chemical treated
specimens.
thrombogenicity [9]. Significant increases in water contact angle
(p b 0.05) and lowered surface energy of all chemically-treated speci-
Surface Oxygen conc. mens (Fig. 5) were observed after extensive acid dipping e.g., for
treatment (at.%)
1200 s. After acid dipping for 1200 s and 1800 s, the water contact
Neat 21.35 angle reached its highest observed value around 98° and surface energy
Sample 1 22.79
reached its lowest value at around 21 to 25 mN/m. The significant re-
Sample 2 18.05
Sample 3 18.05 duction in surface energy can be explained by the presence of fluorine
Sample 4 13.30 which is already shown by XPS analysis. Fluorine, as an electronegative
Sample 5 8.12 element with high reactivity, reacts extensively with positive ions like
Sample 6 8.86 Fe, Cr and Ni ions after acid dipping in hydrofluoric acid ending to
Sample 7 9.59
fluoride-based coordination compounds [32]. Since these complexes
8 A. Latifi et al. / Surface & Coatings Technology 221 (2013) 1–12

Fig. 4. AFM micrographs of 316L stainless steel surfaces: (a) neat (b) sample 1, (c) sample 2, (d) sample 3 (e) sample 4, (f) sample 5, (g) sample 6 and (h) sample 7.

are known due to their water repellent characteristics, increase in the of 316L stainless steel. The anodic potentiodynamic polarization curve
contact angle and decrease in the surface energy at higher acid dipping of the neat specimens shows a typical corrosion behavior of a passive
durations can be attributed to the presence of these complexes on the material subjected to pitting corrosion (Epit) when the potential is
surface.

3.5. Corrosion resistance

Fig. 6 shows potentiodynamic polarization curve for neat and

chemically treated stainless steel specimens. It is clearly evident
that chemical treatment improves the electrochemical characteristics

Table 5
Roughness parameters, Sa and Srms, of 316L stainless steel surfaces after chemical sur-
face treatment.

Surface treatment Sa (nm) ± SD Srms (nm) ± SD

Neat 161.34 ± 57.15a,b,c,d,e 206.58 ± 70.06a,b,c,d,e

Sample 1 5.05 ± 0.28a,b,c,d,e 8.43 ± 0.40a,b,c,d,e
Sample 2 1.54 ± 0.22a 2.89 ± 0.86a
Sample 3 1.40 ± 0.24b 2.73 ± 0.16b
Sample 4 1.43 ± 0.25c 2.19 ± 0.38c
Sample 5 1.23 ± 0.24d 1.76 ± 0.45b
Sample 6 1.28 ± 0.63e 2.14 ± 1.35d
Sample 7 0.96 ± 0.29a 1.71 ± 0.78e

Letters show statistically significant differences between values (p b 0.05). Fig. 5. Contact angle and surface energy of neat, sample 1, and samples 2–7.
 A. Latifi et al. / Surface & Coatings Technology 221 (2013) 1–12
Fig. 6. Cyclic polarization curves for neat and chemical treated specimens in ringer's
solution at 37 °C.
higher than a threshold value i.e., ≅600 mV for this specimen. The
electropolished specimen also shows a passive behavior with the pitting
potential (about 700 mV) higher than what it is observed in the neat
specimens indicating the formation of a more uniform and compact pas-
sive layer. Improved corrosion resistance of the electropolished surfaces
is due to the several inter-related events during electropolishing process
like changes in the surface chemical composition, heterogeneity and
roughness [14,18]. Surface composition of the specimens changes dur-
ing the electropolishing process. These changes include formation of dif-
ferent metal oxides on the metal substrate which inhibits the diffusion
of corrosion process initiators. Among the metal oxides which can be
formed on the surface of 316L stainless steel, chromium oxide is respon-
sible for corrosion resistance of the material due to its low diffusion con-
stant [33]. Here, formation of Cr(III) oxidized compounds on the surface
of electropolished specimens was confirmed by XPS analysis. Formation
of iron oxide was also revealed by XPS analyses, but the atomic concen-
tration ratio of Cr/Fe was calculated at 1.2. This result indicates
Cr-enrichment in the surface due to capability of electropolishing for se-
lective dissolution of elements leading to passive layer formation. In-
creasing of Cr/Fe mass ratio after electropolishing process is reported in
previous studies [10,13,15,22,23]. Corrosion potential (Ecorr) of the
electropolished specimens shifts towards higher Ecorr values as compared
with the neat specimen, according to the results shown in Table 6. Fur-
thermore, the corrosion current density (icorr) calculated by Tafel extrap-
olation method shows smaller value for electropolished specimens when
compared with the neat specimens. In our experience, pitting potential
was not observed for specimens treated by electropolishing plus acid
dipping; even though the potential increased up to 1 V. Moreover, the
results indicate that the corrosion potential of samples treated by
electropolishing plus acid dipping is more noble than what was observed
for neat specimens and samples treated only by electropolishing. This re-
sult can be associated with the surface Cr enrichment phenomenon and
reduction in carbon contamination after acid dipping [13]. Nitric acid is
a strong oxidizing agent to form the passive layer while hydrofluoric
acid is a weak but extremely corrosive compound. Mixtures of these
Table 6
Corrosion behavior of neat and chemical treated specimens in ringer's solution.
Surface treatment icorr (μA/cm2) Ecorr (mV)
Neat 0.921 −343
Sample 1 0.61 −292
Sample 2 0.0170 −227
Sample 3 0.0070 −52
Sample 4 0.0185 −25
Sample 5 0.0043 −30
Sample 6 0.0061 25
Sample 7 0.0066 −16
9
solutions at different ratios yield different Cr/Fe ratios on the acid dipped
surfaces and lead to surface passivity. XPS results showed significant in-
crease in atomic concentration ratio of Cr/Fe after acid dipping. The
value of Cr/Fe ratio was calculated at 1.87. Furthermore, the surface
chemical composition changed after acid dipping and the amount of
chromium oxide increased more than three times compared to speci-
mens which had only been electropolished. Since the chromium oxide
is believed to impart corrosion resistance in metals, acid dipped sample
i.e., sample 7 can exhibit better performance in corrosive environment.
Corrosion behavior of the electropolished plus acid dipped speci-
mens was strongly affected by acid dipping duration. Current density
of the acid dipped specimens, at higher dipping durations, decreases
and corrosion potential shifts to more noble potential. With a few
exceptions, higher potential value (25 mV) and lower anodic current
value (0.006 μA/cm 2) were observed for specimens acid dipped for
1200 s (sample 6). Complete removal of non-metallic inclusions
by acid dipping result in optimized corrosion resistance. Also, the
smoother surface can lead to greater resistance to corrosive factors
[14]. Hence, higher smoothness of the electropolished plus acid dipped
specimens compared to the only electropolished specimens is another
reason for better corrosion behavior.
3.6. In vitro biocompatibility
Virgin, electropolished and electropolished plus acid dipped (1800 s)
specimens were studied regarding their in vitro biocompatibility profile.
Fig. 7 shows cell viability of the chosen specimens measured quantita-
tively by MTT assay on days 1 and 3 post incubation. Based on the results
provided for the first and third days, it is evident that chemical treatment
did not adversely affect cell growth. In fact, the cell growth is improved
and it is comparable with control group after chemical treatment.
According to the results, the number of viable cells adjacent to the neat
specimens is significantly lower than what was observed for control
group on the first day (pb 0.05), while there is no significant difference
(pb 0.05) in the number of viable cells exposed to the chemically-
treated surfaces in comparison to the control group. Significant differ-
ences in the number of viable fibroblasts on the surface of neat speci-
mens on day 1 are the direct consequence of surface roughness on cell
growth. After 3 days incubation in the cell culture medium, no statisti-
cally significant difference was observed in the cell viability of all the
specimens. Formation of cell monolayer on the surfaces after 3 days
improved the smoothness of all specimens and created a proper surface
for cell growth. The number of viable cells after 3 days significantly
Fig. 7. Cell viability of neat, sample 1 and sample 7.
10 A. Latifi et al. / Surface & Coatings Technology 221 (2013) 1–12

increased compared with the viable cells observed on day 1 (p b 0.05).
An increase in the cell viability percentage with an increase in the
cell culture duration indicates that the chemical treatments provide
a biocompatible surface with no cytotoxic effect on L929 fibroblast
cells over three day test period while promoting cell proliferation. Cell
behavior including cell morphology, adhesion and proliferation on all
specimens were assessed by inverted optical microscopy (magnification
400×) on day 3 and SEM method after fixing the cells with glutaralde-
hyde on days 1 and 5. Cells with spindle-shaped, flattened and well
spread morphology were observed on all specimens in Figs. 8 and 9.

Fig. 8. SEM micrographs of cell adhesion on days 1 and 5: (a) neat (day 1), (b) sample 1 (day 1), (c) sample 7 (day 1), (d) neat (day 5), (e) sample 1 (day 5) and (f) sample 7 (day 5).
 A. Latifi et al. / Surface & Coatings Technology 221 (2013) 1–12
Fig. 9. Optical images of cell adhesion on day 3 with magnification 400× (a) blank, (b) neat, (c) sample 1 and (d) sample 7.
These morphologies suggesting normal morphology for cells which are
reported in literature [34,35]. The morphology of cells attached on all
groups is very close to each other, indicating no obvious difference in
cell morphology of treated and non-treated specimens and also control
group. Normal morphology with no cell detachment following different
culture duration i.e., days 1, 3 and 5 was observed by microscopic exam-
inations that can be considered as an evidence for nontoxic effects.
Furthermore, microscopic examinations revealed different cellular pop-
ulation over different culture durations. These results are associated
with excellent cell growth and cell proliferation findings. However,
higher rate of proliferation occurred for all specimens at higher culture
duration and the cells proliferate on all surfaces equally well with no ob-
vious differences. Comparing the cell proliferation results on days 1 and
5, it can be concluded that monocells at lower scale (20 μm) are clearly
distinguishable on first day of post-incubation while monocells cannot
be observed after 5 days indicating the completely covered surfaces by
cell monolayers. However, the greatest cell confluency was observed
for specimens incubated for 5 days, providing no free space for more
cell proliferation.
4. Conclusion
Chemical treatment including electropolishing and acid dipping
at different durations was conducted to improve the surface rough-
ness, topography and corrosion resistance of 316L stainless steel
samples. According to the results, electropolishing process has effec-
tively improved the surface quality. The experimental conditions of
electropolishing process applied in this study (combination of phos-
phoric acid and sulfuric acid as electropolishing electrolytes, current
density i.e., 0.25 A·cm −2, electrolyte bath temperature i.e., 50 °C)
resulted in an ultraclean, very smooth and corrosion resistant surface.
The surface chemical composition was affected by two different kinds
of treatment. Hydroxide entities were dominant on the surface after
11
electropolishing. More corrosion resistant species, chromium oxides,
appeared on the surface of specimens after electropolishing plus
acid dipping in the presence of hydrofluoric acid. Surface roughness
of the electropolished plus acid dipped specimens was decreased by
more than fifty-fold as compared to an only electropolished surface.
Increase in acid dipping duration up to 1800 s resulted in a signifi-
cantly lower surface roughness i.e., Sa ≅ 0.96 ± 0.29. The surface contact
angle was significantly decreased after electropolishing and surface
energy was correspondingly increased. The presence of F on the surface
of 316L stainless steel, after acid dipping, resulted in a significant
reduction in the surface energy. The highest acid dipping duration
i.e., 1800 s showed the lowest surface energy i.e., ≅21.16 mN/m. Corro-
sion behavior of the electropolished and electropolished plus acid
dipped specimens showed significant improvement over neat or only
electropolished samples according to the potentiodynamic polarization
curves indicating specimens’ stability in the corrosive medium. Addi-
tionally, it was found that acid dipping at higher duration i.e., 1200 s
provides lower corrosion current density and higher corrosion poten-
tial. In vitro biocompatibility of the chemically treated specimens
met the international standard requirements (ISO 10993-5) and no
sign of toxicity was observed after 1, 3 and 5 days post incubation.
Cell viability percentage more than 80% was measured for all speci-
mens. Excellent cell adherence and proliferation was observed on the
chemically-treated specimens during cell culture experiments for 1, 3
and 5 days.
Acknowledgments
The authors would like to thank Atila Ortoped Co. for their kind
supporting to provide the 316L stainless steel plates. The assistance of
Laser and Plasma Research Institute of Shahid Beheshti University for
AFM analysis is greatly appreciated. Technical assistance of Mr. Salehi
is also acknowledged.
12 A. Latifi et al. / Surface & Coatings Technology 221 (2013) 1–12
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