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Plasma nitriding under low temperature improves the endothelial cell
biocompatibility of 316L stainless steel
Article in Biotechnology Letters · February 2019
DOI: 10.1007/s10529-019-02657-7 CITATIONS 5 READS 212 10 authors, including:
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O R I G I N A L R E S E A R C H P A P E R
Plasma nitriding under low temperature improves
the endothelial cell biocompatibility of 316L stainless steel
Janine K. F. S. Braz.Gabriel M. Martins.Vladimir Sabino .Jussier O. Vitoriano.
Carlos Augusto G. Barboza.Ana Katarina M. C. Soares.Hugo A. O. Rocha.
Moacir. F. Oliveira.Clodomiro Alves Ju´nior .Carlos Eduardo B. Moura
Received: 29 November 2018 / Accepted: 22 February 2019 Ó Springer Nature B.V. 2019
Abstract
Objectives To evaluate the effects of the surface
modification of 316L stainless steel (SS) by low-temperature plasma nitriding on endothelial cells for stent applications.
Results X-ray diffraction (XRD) confirmed the
incorporation of nitrogen into the treated steel. The surface treatment significantly increased SS roughness and hydrophilic characteristics. After 4 h the cells adhered to the nitride surfaces and formed clusters. During the 24 h incubation period, cell viability on the nitrided surface was higher compared to the polished
surface. Nitriding reduced late apoptosis of rabbit aorta endothelial cell (RAEC) on the SS surface.
Conclusion Low temperature plasma nitriding
improved the biocompatible of stainless steel for use in stents.
Keywords Biomaterial Intravascular devices
Metal surfaces Nitrited Stents
Introduction
316L stainless steel is one of the most frequently applied metals in the manufacturing of cardiovascular stents, because of its mechanical strength, low amount of impurities and low magnetic permeability
(Chi-chareon et al.2019). However, in recent years stainless
steel usage has been reduced due to the dissolution of steel in body fluids, which can lead to the activations of the coagulation cascade and consequent risk of thrombosis, which complicates the tissue integration
process (Butruk-Raszeja et al. 2016). The release of
these metal ions may also be due to wear, but is most
frequently caused by corrosion (Morais et al.2007).
Corrosion and alterations of stainless steel (316L) cardiovascular stents properties make it difficult to
adapt the material to the tissues (Fox et al. 2019). In
addition, blood can induce corrosion by passive oxidation of the stent surface, increasing the risk of
J. K. F. S. Braz G. M. Martins M. F. Oliveira C. E. B. Moura (&)
Departamento de Cieˆncias Animais, Universidade Federal Rural do Semi-A´ rido, UFERSA, Av. Francisco Mota, 572 –Bairro Costa e Silva, Mossoro´, RN CEP: 59.625-900, Brazil
e-mail: carlos.moura@ufersa.edu.br
V. Sabino C. A. G. Barboza
Departamento de Morfologia, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil
J. O. Vitoriano C. Alves Ju´nior
Laborato´rio de Processamento a Plasma, LABPLASMA, Universidade Federal Rural do Semi-A´ rido, UFERSA, Mossoro´, RN, Brazil
A. K. M. C. Soares H. A. O. Rocha
Departamento de Bioquı´mica, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil
ions being released into the bloodstream (toxic and
carcinogenic) and forming thrombi (Kathuria 2006;
Talha et al.2019). Stents should display flexibility and
elasticity, and promote biocompatible biological responses by recruiting growth and chemotactic
factors (Schwartz et al. 2008). These aspects can be
evaluated by in vitro studies using the endothelial cell
model (Arslan et al.2008).
However, it is possible to increase metallic resis-tance to corrosion to ensure greater biocompatibility efficiency. Plasma nitriding improves functionaliza-tion, chemical restructuring, surface compatibilization and the activation of organic and inorganic surfaces of the treated material, such as austenitic stainless steel
(Alves et al.2006; Samanta et al.2017). With this, it is
possible to improve the physical and chemical prop-erties of the material by the formation of a film by ionic bombardment, for example, of nitrogen ions (Lu
et al. 2009). The increase in stainless steel nitrogen
concentrations reduces the toxicity of this metal for
application to cardiovascular devices (Su et al.2018).
The plasma nitriding method is one of the most applied method for modifying stainless steel mechanical and
chemical properties (Trabzon and I˙g˘dil2006; Samanta
et al. 2017). Plasma nitriding significantly improves
the tribological properties of stainless steel (friction, wear and lubrication) and maintains its passive nature
at low temperatures (Zhao et al.2016; Lin et al.2016),
due to increases in hardness and corrosion resistance
to body fluids (Arslan et al.2008).
Some authors state that a decrease in stainless steel corrosion rates after plasma nitriding at low
temper-atures is detected (Zhao et al.2016; Kao et al.2017).
However, cellular biocompatibility evaluations were not performed. In addition, stainless steel is exposed to plasma for an extended period ranging from 4 to 168 h, thus leading to high production costs (Braceras
et al.2018). In this context, this study aimed to assess
the effect of low temperature plasma nitriding of 316L stainless steel on endothelial cell viability.
Materials and methods Stainless steel discs
A total of 30 stainless steel discs at 19 mm diameter and 3 mm thickness were used. Their surfaces were gradually sanded with silicon carbide (SiC) 220, 440,
600, 1500 and 2000 MESH granulometries and polished using an aluminum oxide solution for 30 min. Subsequently, the disks were immersed in 0.5% of enzymatic detergent (DEIV) solution in double-distilled water and ultrasound treated for 10 min. The samples were then washed in ethanol and double-distilled water and submitted to ultrasound treatment for another 10 min. Then, the surfaces to be
treated were subjected to a nitriding atmosphere (36N2
and 24H2) in a 200 9 300 mm hermetic cylindrical
chamber (diameter and height) under a pressure of
1 mbar at 450°C for 1 h.
Surface characterization
Surface nanotopography analysis was based on rough-ness parameters (Ra, Rp, Rz and Rp/Rz), obtained using an atomic force microscope (AFM, SPM 9700, Shimadzu). Wettability was evaluated through the
sessile drop method (Silva et al.2015), which consists
in measuring the angle formed by a drop of 20 lL of deionized water pipetted onto the samples (polished and nitrided). Then images were captured by the goniometer video camera and the Suftens program was used to obtain the contact angles. The stainless steel surfaces were chemically evaluated by the Grazing Incidence X-ray Diffraction (GIXRD) tech-nique, at a flat and fixed 2h incidence angle sweep detection in the diffractometer.
Rabbit aorta endothelial cell culture (RAEC)
Rabbit aortic endothelial cells (RAEC) were cultured in HAM-F12 medium, supplemented with fetal bovine serum (10%), and penicillin/streptomycin antibiotics (100 mU/mL; 100 lg/mL, respectively), and
subse-quently incubated at 37°C in a 5% CO2chamber, with
exchanges of culture medium every 72 h. Cellular morphology
Endothelial cells (5 9 104) were cultured on the
stainless steel discs (polished and nitrided) for 4 h to describe cellular morphology. The disks were then washed with a phosphate buffer solution (PBS), fixed with 2.5% glutaraldehyde in PBS, pH 7.0, and postfixed with osmium tetroxide. The samples were then serially dehydrated in increasing concentrations, and plated with gold (Q Plus Series, Quorum
Technologies Ltd., Laughton, England). Images were captured by Scanning Electron Microscope (SEM) (SEM-SSX 550 Superscan, Shimadzu Corporation,
Tokyo, Japan)and analyzed using theImage Pro-PlusÒ
software (Version 4.5.0.29). Cell morphology was evaluated by capturing 30 cells per surface to obtain the Form Factor (FF), which consists of the product of the division between area and cellular perimeter:
FF = (area/perimeter2) 9 4p (Shah et al.1999).
MTT assay
The RAEC (2 9 103 cells/disk) was grown on the
stainless steel surfaces for 24 h, followed by dilution of 1 mL of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazoliumbromide (MTT, Invitrogen, Life Technolo-gies, Carlsbad, CA, USA) in the culture medium (1 mg/mL). After 3 h of incubation, the formazan crystals produced by MTT reduction were dissolved after adding 1 mL of ethanol to each well for 15 min under constant stirring. Then, 100 lL from each well were transferred to 96-well culture plates and quan-tified by absorbance spectrophotometry at 570 nm using a microplate reader (Quant MKX200, BioTek Instruments, Winooski, VT, USA).
Apoptosis assay
A FITC/Annexin V Dead Cell Apoptosis Kit with FITC Annexin and PI (Invitrogen, USA) was used for
apoptosis detection. The RAEC (2 9 103cells/disc)
was cultured on the two different surfaces. After 24 h, adherent cells were released by viokase, washed twice in ice-cold PBS and then incubated with 5 lL of annexin V-FITC and 1 lL of propidium iodide (PI) at 100 lg/mL PBS at room temperature for 20 min, protected from light. The apoptosis percentage was determined every 10,000 events using a flow cytome-ter (BD Facscanto II), atemission and fluorescence wavelengths of 530 nm and 570 nm. The obtained data were analyzed using the FlowJo Analysis soft-ware version 9.3.2 (Tree Star Incorporation, OR, USA).
Statistical analyses
The experiments were performed in duplicate for each surface. Student’s t test was applied to the RAEC Form Factor, roughness and surface wettability parameters.
The MTT data and apoptosis assay were submitted to an analysis of variance (ANOVA) assessment, fol-lowed by a post hoc student’s t-test. The analyses were performed with the Graph Pad Instat software, version 3.5, assuming p \ 0.05.
Results
Surface characterization
The roughness profiles are displayed in Fig.1A–D.
Plasma nitriding generated peaks on the treated surface compared to the polished surface. The Ra,
Rp and Rz roughness parameters (Table 1) were
obtained based on these profiles. Plasma nitriding significantly increased all analyzed stainless steel roughness parameters (Ra, Rp and Rz) compared to
the polished surface (Table1). In addition, the shapes
of the surface peaks were evaluated by the Rp/Rz ratio, which indicated no significant difference between samples. However, the contact angle of the nitrided surface was significantly lower when compared to the
polished surface (71.81° ± 2.10 versus
100.13° ± 2.49, p \ 0.05) (Fig.2). Thus, plasma
nitriding increased surface hydrophilicity.
Next, GIXRD confirmed nitrogen incorporation to the treated steel, with the formation of small chrome nitrite
(CrN) peaks (Fig.3). Adherent cells were detected on the
samples after 4 h. Cell morphology on the nitrided
stainless steel was elliptical with projections (Fig.4A,
B). Despite the morphological similarity on the different surfaces, confirmed by the results of the form factor (0.37 ± 0.1 vs. 0.40 ± 0.1; p [ 0.05 for nitrided and polished, respectively) cell clusters were observed on the
nitrided surface (Fig.4C).
Cellular viability
Cell viability on the nitrided surface detected via the MTT assay was significantly higher after 24 h in
comparison to the polished surface (1.73 9 104cells
vs. 4.9 9 103; p = 0.022) (Fig.5).
Cell death
Cellular apoptosis was quantified by flow cytometry. Living cells are reported in quadrant 3 (Q3). Cells
Fig. 1 Nanotipography of stainless steel by AFM. A–B Surface of polished stainless steel. C–D Surface of plasma nitrided stainless steel. Area = 10 9 10 lm
Table 1 Roughness parameters (nm) of the polished and nitrided plasma stainless steel
Surface Ra Rp Rz Rp/Rz
Polished 0.9 ± 0.05a 3.9 ± 1.67a 5.7 ± 0.61a 0.7 ± 0.2 Nitrided 2.4 ± 0.6b 10.1 ± 3.3b 15.9 ± 5.8b 0.6 ± 0.1 Data are expressed as average ± standard deviation. Averages with different pairs of lower case letters on the same line (a–b) (p \ 0.001)
quadrant 4 (Q4), while those presenting late apoptosis are shown in quadrant 2 (Q2). No significant differ-ence (p [ 0.05) in initial cellular apoptosis on the nitrided surface (1.34 ± 0.09%) was detected when compared to the control group (1.35 ± 0.2%) and the
polished surface (1.36 ± 0.5%) (Fig.6A–C). Late
cellular apoptosis was significantly reduced
(p \ 0.05) in the plasma nitrided group
(0.42 ± 0.1%) when compared to the control
(0.67 ± 0.2%) and the polished surface
(0.54 ± 0.2%) group.
Fig. 2 Contact angle by sessile drop for polished and nitrided stainless steel surface. ***p \ 0.001
Fig. 3 Diffractogram of the stainless steel surface nitrided at 0.58 theta contact angles (closest to the surface); 38 Theta; and 78 Theta (deeper)
Fig. 4 Scanning electron microscopy of RAEC cells cultured on plasma-nitrated and polished stainless steel discs. A RAECs grown on the polished surface with elongated morphology and low emission of cytoplasmic extensions. B RAECs grown on
plasma nitrided stainless steel surface with elongated morphol-ogy and formation of clusters. C Endothelial cell clusters on nitride surface (black line)
Discussion
The vascular biocompatibility of plasma-nitrided stainless steel has not yet been well established in scientific literature. According to Braceras et al. (2018), nitriding treatments at high temperatures
(above 500°C) can decrease corrosion stainless steel
due resistance due to high nitrogen incorporation in
the form of chromium nitrides (CrN, CrN2). However,
the use of the planar cathode plasma nitriding method on 316L stainless steel samples at a temperature of
450 °C during 1 h in our study did not lead to the
formation of iron nitrides. A similar result was
observed at 400°C for a longer exposure time of 8 h
in another study (Samanta et al.2017). Therefore, this
new surface was evaluated by in vitro tests using endothelial cells applied to consolidated morphology, viability and cellular apoptosis assays.
The plasma nitriding treatment significantly
increased steel roughness, making it more irregular
and rough when compared to the untreated surface. The average roughness elevation (Ra = 2.4 ± 0.6 nm) observed here in was similar to that described for the 316L stainless steel plasma nitride surface
obtained at 430°C for 5 h (Ra = 2.5 ± 0.1 nm), as
roughness tends to increase with increasing
tempera-ture (Borgioli et al. 2016). Moreover, the XRD
analysis confirmed that condensation of the ejected atoms occurred, or deposition of surface iron nitrides from the mass transfer of cathodic sputtering nitriding
(Ribeiro et al.2008; Lin et al.2018). The formation of
these nitrites enhances plastic deformation and stain-less steel resistance, providing a vital advantage in the
production of cardiovascular stents (Arslan et al.2008;
Li et al.2014; Kahraman et al.2018).
Roughness and wettability properties may influ-ence cell viability differently, depending on the surface composition that affects protein adsorption
(Vilardell et al. 2018). The Rp/Rz ratio provides a
roughness profile of the surface by its shape and, when this value is greater than 0.5, it is indicative of pointed peaks, while a value lower than 0.5 refers to more
rounded peaks (Whitehead et al. 1995). Previous
reports indicate that osteoblasts display a higher affinity for surfaces presenting rounded peaks (Rp/
Rz = 0.45 nm) (Silva et al. 2015). However, this
behavior had not yet been described for endothelial cells. In addition, this variable can aid in estimating
surface wettability (Nunes Filho et al.2018). Although
no significant difference was found for the Rp/Rz ratio for the evaluated surfaces, a significant difference in wettability was noted. Thus, the lower the Rp/Rz ratio value, the lower the contact angle and, consequently, the higher its hydrophilicity. This can trigger increased
Fig. 5 Cell viability by MTT after 24 hours of culture in contact with polystyrene, polished and nitrided surfaces. (a–b) p \ 0.05
Fig. 6 Apoptosis of RAEC cells on stainless steel surfaces. A RAEC control cultured on polystyrene of the culture plate. B RAEC on the polished surface. C RAEC on the nitrided surface, all double-incubated with annexin-V and propidium iodide
proliferation and cell differentiation (Vilardell et al. 2018).
Both focal adhesion and the cell spreading area are important parameters used to assess cell-biomaterial
interactions (Turner et al.2004). Here in, the
endothe-lial cells showed adhesion and spreading in the first 4 h after incubation on the nitrided surface. Therefore, it is probable that the chemical and physical changes due to plasma nitriding stimulated protein adsorption on the surface, being important for the activation of
cell adhesion proteins (Ferraz et al.2014; Moura et al.
2016; Talha et al.2019). Both roughness and nitriding
conditions play an important role in promoting
adhesion (Martinesi et al. 2013; Jayalakshmi et al.
2018). According to van Wachem et al. (1985) the initial adhesion of human umbilical cord vein endothe-lial cells to surfaces requires high clustered cell density, which ensures cellular spreading and prolif-eration on the polymer surface. This implies that the applied metal nitriding stimulated the colonization of endothelial cells, an important feature to increase vascularization and re-endothelialization, which aid in functionalizing stainless steel implants (Offner et al. 2017).
Surface cell adhesion does not necessarily imply that the cells maintain their viability (Popat et al. 2007). However, a significant increase in cell viability on the treated surface was observed 24 h after adhesion, indicating that plasma nitriding indirectly
improves biocompatibility (Arslan et al.2008).
How-ever, some authors observed endothelial cell prolifer-ation on 316L stainless steel only 72 h after using different plasma nitriding conditions at low
tempera-tures (400°C for 5 h) (Martinesi et al.2013). Thus, the
nitriding condition used in our study reduced cytotox-icity in the first hours of adhesion and favored greater proliferation of viable cells.
The plasma nitriding carried out under the condi-tions applied in the present study reduced the late apoptosis of endothelial cells. It is possible that the treatment reduced the release of nickel ions, which raises the cytotoxic effect of the surface, since it is then necessary to add high nitrogen concentrations to stainless steel to produce a nickel-free metal (Lo
et al.2009). In the present study, the application of low
temperature plasma nitriding on stainless steel, besides promoting better adhesion and greater viabil-ity of endothelial cells, also reduced the cytotoxic effect of the stainless steel in the first 24 h. Nitrogen
incorporation, carried out at 450°C for only 1 h, was
able to increase stainless steel corrosion resistance. Therefore, this treatment is a possible candidate for use in cardiovascular stainless steel devices.
Acknowledgements This study was financed in part by the Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior—Brasil (CAPES)—Finance Code 001. The authors wish to acknowledge the professional efforts of team of the Laboratory of Structural Characterization of Materials at UFRN and Dr. Helena B. Nader of UNIFESP, Sa˜o Paulo, Brazil for contributing with the endothelial rabbit aorta cell line.
Compliance with ethical standards
Conflict of interest All authors declares that they have no conflict of interest.
Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.
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