Electrochemical and chemical methods for improving surface characteristics of 316L stainless steel for biomedical applications

Surface & Coatings Technology 221 (2013) 1–12

Contents lists available at SciVerse ScienceDirect

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 Novel Drug Delivery Systems Dept., Iran Polymer and Petrochemical Institute, P.O. Box 14965/115, Tehran, Iran 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 b c

a r t i c l e

i n f o

Article history: Received 30 September 2012 Accepted in revised form 5 January 2013 Available online 23 January 2013 Keywords: 316L stainless steel Electropolishing Acid dipping Surface characterization Corrosion resistance In vitro biocompatibility

a b s t r a c t Although stainless steel is a widely used material for biomedical applications, its surface properties for long term application are still a serious concern. Here, chemical surface treatment by electropolishing and acid dipping is used for improving surface properties of 316L stainless steel to provide a homogenous, smooth and corrosion resistant 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 resistant compounds was shown by XPS. Corrosion behavior of the treated samples was evaluated by electrochemical analysis. Surface energy was also studied through contact angle measurements. The roughness of the surface was traced by atomic force microscopy (AFM) and scanning electron microscopy (SEM) techniques. The lowest roughness (Sa: 0.96± 0.29 nm) was observed for the highest acid dipping duration (1800 s). The surface energy was remarkably subsided by acid dipping duration. MTT assay and cell adhesion studies were conducted to 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 fabricate 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 properties, corrosion is an important issue especially in prolonged implantation 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 material [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: [email protected] (M. Imani). 0257-8972/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.surfcoat.2013.01.020

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 surface 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 naturallyoccurring oxide and hydroxide layers from a metal surface by chemical treatment is of great importance to leave a clean, smooth and contamination-free surface in place. Electropolishing is the most extensively used electrochemical treatment method for metallic surfaces that results in significant improvement 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 responsible 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 geometries [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

2

A. Latifi et al. / Surface & Coatings Technology 221 (2013) 1–12

parameters including anodic current density, electrolyte(s) composition, 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 precleaning 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 polarization 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 specimens was also studied using SEM technique after proper fixation.

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 literature [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 samples 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 specimens 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. Materials and methods

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.

2.1. 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 provided 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.

2.3. X-ray photoelectron spectroscopy (XPS) measurements Surface chemical composition of the treated specimens was investigated by XPS using an XR3E2 (VG Microtech, UK) twin anode X-ray source. The instrument was equipped with an Al-Kα X-ray source working 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

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 calculated according to the following equations:

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 different procedures. In the first procedure, specimens were cleaned ultrasonically in acetone, deionized water and isopropanol sequentially for 15 min. After cleaning, the specimens were electropolished in an electrolyte 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. Electrochemical polishing parameters are summarized in Table 1. After electropolishing, the specimens were rinsed by distilled water and

Sa ¼

Srms

N X M   1 X  2  η ðxi ; yi Þ MN j¼1 i¼1

vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi u N X M   u 1 X  2  ¼t η ðxi ; yi Þ MN j¼1 i¼1

ð1Þ

ð2Þ

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

Table 1 Process conditions for electropolishing. Electropolishing parameter

Potential (V)

Current density (A/cm2)

Treatment time (min.)

Electrolyte bath temperature (°C)

Value

5

0.25

20

50

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

3

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:

spectrophotometrically at 545 nm using an ELISA plate reader. This absorbance value is proportional to the number of viable cells. Each of the specimens was plated in triplicate MTT assays for 1 and 3 days.

   p p 0:5 d d 0:5 γ sl ¼ γ s þ γ l −2 γs γl −2 γ s γ l

2.9. Statistical analysis

ð3Þ

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 energy of solid and liquid, respectively [24].

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.

2.7. Electrochemical analysis

3. Results and discussion

Corrosion of all surface-treated specimens in comparison to nontreated specimens was investigated by electrochemical analysis. Open circuit potential (OCP) analysis was performed to accomplish the electrochemical measurements. Scan rate was adjusted on 1 mV·s−1 at 37 °C in ringer solution using an IviumStat potentiostat )Ivium Technologies Inc., Eindhoven, The Netherlands) equipment which consisted of a platinum counter electrode, a Ag/AgCl reference electrode and a working 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.

3.1. XPS analysis

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 fibroblast 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 suspension 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 bromide] 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 positive controls, respectively. A suspension of 1×104 cell·mL−1 of L929 fibroblast 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

Fig. 1 depicts XPS survey spectra for neat sample, sample 1 and sample 7. Atomic concentration of elements on the neat and chemicallytreated 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 samples, 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 treatment 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 decontamination. 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. Increasing Cr 2p concentration (6 at.%) is also observed by XPS analysis on sample 7. Moreover, after acid dipping the peak related to P 2p disappeared 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 dipping 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 highresolution 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 second 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 Table 2 Chemical composition of the neat and chemical treated specimens (at.%) according to XPS measurements. Sample code

Neat Sample 1 Sample 7

Composition (at.%) C

O

Fe

Cr

P

F

80.2 40.8 37

19.8 49 44.2

– 3.4 3.2

– 4.2 6

– 2.7 –

– – 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 surface 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 sample 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 chromiumfluoride 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. 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 compounds 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 surface layer. The Cr 2p3/2 peaks are curve-fitted with four different peaks

Fig. 3 shows SEM micrographs of neat and chemically treated specimens. As observed in the SEM micrographs, different surface topographies 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 contaminations 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 orientations 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 original 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 effectively 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 no.

Element

Deconvoluted peaks (binding energy)

Neat

C 1s

Neat

O 1s

1

O 1s

1

Cr 2p3/2

1

Fe 2p3/2

7

O 1s

7

Cr 2p3/2

7

Fe 2p3/2

7

F 1s

C\(C,H) (284.98) O\(Fe, Cr) (530.5) O\(Fe, Cr) (530.50) Cr(0): (574.30) Fe3O4 (708.2) O\(Fe, Cr) (530.5) Cr(0) 574.30 Fe3O4 (708.33) Fe\F, Cr\F (685)

FWHM (eV) C\O (286.58) Hydroxides (532.1) Hydroxides (531.90) Cr2O3 (576.80) Fe2O3 (710.87) Hydroxides (532) Cr2O3 576.90 Fe2O3 (711) C\F (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 cardiovascular 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 appearance 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 characteristics of electropolished surfaces [15]. Smoother surfaces with disappearance 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 surface 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, respectively. 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

−

Cr→CrðVIÞ þ 6e :

ð4Þ ð5Þ ð6Þ

The mass of material removed from the metal surfaces during electropolishing process is expressed by Faraday's law of electrolysis [30]: W loss ¼

.

ItM

nF

ð7Þ

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.

C\OO (288.7) \CO3, \COH (533.1) \CO3, \COH (533.20) Cr(OH)3 (577.30) FeOOH (711.8) \CO3,\COH (533.1) Cr(OH)3: 577.30 FeOOH (711.8)

1.83 2.29 2.27 CrO3 (578.30)

3.35 3.96 1.76

CrO3 578.34

2.92 4.05 2.30

According to the Sa and Srms results (Table 5), it can be concluded that the specimens treated by electropolishing plus acid dipping possess 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 durations 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 samples roughness obtained from AFM results, imply that by longer duration 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 biomaterials in acid etching solutions. In contrast, insufficient contamination removal 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 (p b 0.05). Water contact angle of neat stainless steel was 83.6° but decreased to 56.5° after electropolishing (Fig. 5). Since the surface energy increases with decreases in water and diiodomethane contact angles therefore, the electropolished surfaces, have as expected shown higher surface energy (Fig. 5). In electropolished samples, the observed decrease in contact angle of water and diiodomethane indicates significant (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 specimens. Increase in the surface energy can be explained according to the reduced surface roughness and complete removal of surface contaminations 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 Oxygen concentration on the neat and chemical treated specimens. Surface treatment

Oxygen conc. (at.%)

Neat Sample Sample Sample Sample Sample Sample Sample

21.35 22.79 18.05 18.05 13.30 8.12 8.86 9.59

1 2 3 4 5 6 7

smoother surfaces is generally observed which in turn causes lower thrombogenicity [9]. Significant increases in water contact angle (p b 0.05) and lowered surface energy of all chemically-treated specimens (Fig. 5) were observed after extensive acid dipping e.g., for 1200 s. After acid dipping for 1200 s and 1800 s, the water contact angle reached its highest observed value around 98° and surface energy reached its lowest value at around 21 to 25 mN/m. The significant reduction in surface energy can be explained by the presence of fluorine which is already shown by XPS analysis. Fluorine, as an electronegative element with high reactivity, reacts extensively with positive ions like Fe, Cr and Ni ions after acid dipping in hydrofluoric acid ending to 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 contact angle and decrease in the surface energy at higher acid dipping durations can be attributed to the presence of these complexes on the surface.

of 316L stainless steel. The anodic potentiodynamic polarization curve of the neat specimens shows a typical corrosion behavior of a passive material subjected to pitting corrosion (Epit) when the potential is

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 surface treatment. Surface treatment

Sa (nm) ± SD

Srms (nm) ± SD

Neat Sample Sample Sample Sample Sample Sample Sample

161.34 ± 57.15a,b,c,d,e 5.05 ± 0.28a,b,c,d,e 1.54 ± 0.22a 1.40 ± 0.24b 1.43 ± 0.25c 1.23 ± 0.24d 1.28 ± 0.63e 0.96 ± 0.29a

206.58 ± 70.06a,b,c,d,e 8.43 ± 0.40a,b,c,d,e 2.89 ± 0.86a 2.73 ± 0.16b 2.19 ± 0.38c 1.76 ± 0.45b 2.14 ± 1.35d 1.71 ± 0.78e

1 2 3 4 5 6 7

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 passive 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 during the electropolishing process. These changes include formation of different 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 responsible for corrosion resistance of the material due to its low diffusion constant [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 concentration ratio of Cr/Fe was calculated at 1.2. This result indicates Cr-enrichment in the surface due to capability of electropolishing for selective dissolution of elements leading to passive layer formation. Increasing 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. Furthermore, the corrosion current density (icorr) calculated by Tafel extrapolation 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 result 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

solutions at different ratios yield different Cr/Fe ratios on the acid dipped surfaces and lead to surface passivity. XPS results showed significant increase 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 specimens 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 specimens 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 quantitatively 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 chemicallytreated surfaces in comparison to the control group. Significant differences in the number of viable fibroblasts on the surface of neat specimens on day 1 are the direct consequence of surface roughness on cell growth. After 3 days incubation in the cell culture medium, no statistically 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

Table 6 Corrosion behavior of neat and chemical treated specimens in ringer's solution. Surface treatment

icorr (μA/cm2)

Ecorr (mV)

Neat Sample Sample Sample Sample Sample Sample Sample

0.921 0.61 0.0170 0.0070 0.0185 0.0043 0.0061 0.0066

−343 −292 −227 −52 −25 −30 25 −16

1 2 3 4 5 6 7

9

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 glutaraldehyde 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

11

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 examinations that can be considered as an evidence for nontoxic effects. Furthermore, microscopic examinations revealed different cellular population 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 obvious 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 roughness, topography and corrosion resistance of 316L stainless steel samples. According to the results, electropolishing process has effectively improved the surface quality. The experimental conditions of electropolishing process applied in this study (combination of phosphoric 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

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 significantly 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. Corrosion 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. Additionally, it was found that acid dipping at higher duration i.e., 1200 s provides lower corrosion current density and higher corrosion potential. 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 specimens. 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

References [1] C. Liu, G. Lin, D. Yang, M. Qi, Surf. Coat. Technol. 200 (2006) 4011. [2] K.Y. Chiu, F.T. Cheng, H.C. Man, Surf. Coat. Technol. 200 (2006) 6054. [3] V. Muthukumaran, V. Selladurai, S. Nandhakumar, et al., Mater. Des. 31 (2010) 2813. [4] A. Tiwari, J. Zhu, L.H. Hihara, Surf. Coat. Technol. 202 (2008) 4620. [5] W. Wu, X. Liu, H. Han, et al., J. Mater. Sci. Technol. 24 (2008) 926. [6] Y. Okazaki, E. Gotoh, Corros. Sci. 50 (2008) 3429. [7] I. Gurappa, Mater. Charact. 49 (2002) 73. [8] A. Samide, I. Bibicu, B. Opera, et al., J. Optoelectron. Adv. Mater. 10 (2008) 1431. [9] H. Zhao, J.V. Humbeeck, J. Mater. Sci. Mater. Med. 13 (2002) 911. [10] M. Haïdopoulos, S. Turgeon, G. Laroche, et al., Surf. Coat. Technol. 197 (2005) 278. [11] J.M. Seeger, M.D. Ingegno, E. Bigatan, et al., J. Vasc. Surg. 22 (1995) 327. [12] J.J. Ramsden, D.M. Allen, D.J. Stephenson, et al., CIRP Ann. Manuf. Technol. 56 (2007) 687. [13] M. Haïdopoulos, S. Turgeon, C. Sarra-Bournet, et al., J. Mater. Sci. Mater. Med. 17 (2006) 647. [14] A. Baron, W. Simka, G. Nawrat, et al., J. Achiev. Mater. Manuf. Eng. 18 (2006) 55. [15] C.C. Irving, ASTM Special Tech. Publ. 859 (1985) 136. [16] T. Hryniewicz, K. Rokosz, M. Filippi, Materials 2 (2009) 129. [17] S.J. Lee, J.J. Lai, J. Mater. Process. Technol. 140 (2003) 206. [18] T. Hryniewicz, K. Rokosz, R. Rokicki, Corros. Sci. 50 (2008) 2676.

[19] C.C. Shih, C.M. Shih, Y.Y. Su, et al., Corros. Sci. 462 (2004) 427. [20] Standard Practice for Cleaning, Descaling, and Passivation of Stainless Steel Parts, Equipment, and Systems, American Society for Testing and Materials (ASTM), 2005, p. A380. [21] M. da Cunha Belo, B. Rondot, C. Compere, et al., Corros. Sci. 40 (1998) 481. [22] J. Okado, K. Okada, A. Ishiyama, et al., Surf. Coat. Technol. 202 (2008) 5595. [23] M. Seo, N. Sato, Trans. Jpn. Inst. Met. 21 (1980) 805. [24] D.K. Owens, R.C. Wendt, J. Appl. Polym. Sci. 13 (1969) 1741. [25] F. Zhang, E.T. Kang, et al., Biomaterials 22 (2001) 1541. [26] N. Naseri, S. Yousefzadeh, E. Daryaei, et al., Int. J. Hydrogen Energy 36 (2011) 13462. [27] Z. Feng, X. Cheng, C. Dong, et al., Corros. Sci. 52 (2010) 3646. [28] Y. Shinonaga, K. Arita, Dent. Mater. 28 (2009) 735. [29] http://henke.lbl.gov/optical_constants/atten2.html. [30] Y. Li, Microelectronic Applications of Chemical Mechanical Planarization, First ed., John Wiley, New York, 2007. [31] C.J. Lee, S.K. Lee, D.C. KO, et al., J. Mater. Process. Technol. 32 (2009) 4769. [32] L. Dahlgren, Treatment of Spent Pickling Acid from Stainless Steel Production, Royal Institute of Technology, 2010. [33] A. Vesel, M. Mozetic, A. Drenik, et al., Appl. Surf. Sci. 32 (2008) 1759. [34] Y.B. Wang, et al., Mater. Sci. Eng. C 32 (2012) 599. [35] F. Unger, U. Westedt, P. Hanefeld, et al., J. Control. Release 117 (2007) 312.