Plasma nitriding under low temperature improves the endothelial cell biocompatibility of 316L stainless steel

To evaluate the effects of the surface modification of 316L stainless steel (SS) by low-temperature plasma nitriding on endothelial cells for stent applications. 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. Low temperature plasma nitriding improved the biocompatible of stainless steel for use in 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 (Chichareon 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 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 resistance to corrosion to ensure greater biocompatibility efficiency.Plasma nitriding improves functionalization, 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 properties 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 ˘dil 2006;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 temperatures 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.

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 (36N 2 and 24H 2 ) 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 roughness 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) technique, at a flat and fixed 2h incidence angle sweep detection in the diffractometer.

Cellular morphology
Endothelial cells (5 9 10 4 ) 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-Plus Ò 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/perimeter 2 ) 9 4p (Shah et al. 1999).

MTT assay
The RAEC (2 9 10 3 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-diphenyltetrazoliumbromide (MTT, Invitrogen, Life Technologies, 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 quantified 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 10 3 cells/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 cytometer (BD Facscanto II), atemission and fluorescence wavelengths of 530 nm and 570 nm.The obtained data were analyzed using the FlowJo Analysis software 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, followed 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.

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 (Table 1).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 10 4 cells vs. 4.9 9 10 3 ; p = 0.022) (Fig. 5).

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, CrN 2 ).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 temperature (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 stainless 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 influence 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 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 endothelial 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 endothelial cells to surfaces requires high clustered cell density, which ensures cellular spreading and proliferation 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).However, some authors observed endothelial cell proliferation on 316L stainless steel only 72 h after using different plasma nitriding conditions at low temperatures (400 °C for 5 h) (Martinesi et al. 2013).Thus, the nitriding condition used in our study reduced cytotoxicity in the first hours of adhesion and favored greater proliferation of viable cells.
The plasma nitriding carried out under the conditions 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 viability 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.
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

Fig. 5
Fig. 5 Cell viability by MTT after 24 hours of culture in contact with polystyrene, polished and nitrided surfaces.(a-b) p \ 0.05