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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

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ORIGINAL RESEARCH PAPER

Plasma nitriding under low temperature improvesthe 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 Junior . Carlos Eduardo B. Moura

Received: 29 November 2018 / Accepted: 22 February 2019

� 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 Ciencias Animais, Universidade Federal

Rural do Semi-Arido, 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. BarbozaDepartamento de Morfologia, Universidade Federal do

Rio Grande do Norte, Natal, RN, Brazil

J. O. Vitoriano � C. Alves JuniorLaboratorio de Processamento a Plasma, LABPLASMA,

Universidade Federal Rural do Semi-Arido, UFERSA,

Mossoro, RN, Brazil

A. K. M. C. Soares � H. A. O. RochaDepartamento de Bioquımica, Universidade Federal do

Rio Grande do Norte, Natal, RN, Brazil

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Biotechnol Lett

https://doi.org/10.1007/s10529-019-02657-7(0123456789().,-volV)( 0123456789().,-volV)

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 nitridingmethod is one of the most applied

method for modifying stainless steel mechanical and

chemical properties (Trabzon and Igdil 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 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%CO2 chamber, 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

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Biotechnol Lett

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�

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 9 103 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 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 (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 cellswere 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 9 104 cells

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

displaying recent apoptosis are represented in

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Biotechnol Lett

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)

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Biotechnol Lett

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 contactangles (closest to the

surface); 38 Theta; and 78Theta (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)

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Biotechnol Lett

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

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Biotechnol Lett

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

Coordenacao de Aperfeicoamento de Pessoal de Nıvel

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, Sao 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.

References

Alves C, Guerra Neto CLB, Morais GHS et al (2006) Nitriding

of titanium disks and industrial dental implants using hol-

low cathode discharge. Surf Coat Technol 200:3657–3663.

https://doi.org/10.1016/J.SURFCOAT.2005.08.005

Arslan E, Igdil MC, Yazici H et al (2008) Mechanical properties

and biocompatibility of plasma-nitrided laser-cut 316L

cardiovascular stents. J Mater Sci 19:2079–2086. https://

doi.org/10.1007/s10856-007-3302-4

Borgioli F, Galvanetto E, Bacci T (2016) Low temperature

nitriding of AISI 300 and 200 series austenitic stainless

steels. Vacuum 127:51–60. https://doi.org/10.1016/j.

vacuum.2016.02.009

Braceras I, Ibanez I, Dominguez-Meister S et al (2018) Plasma

nitriding of the inner surface of stainless steel tubes. Surf

Coat Technol 1:4. https://doi.org/10.1016/j.surfcoat.2018.

04.057

Butruk-Raszeja B, Dresler M, Kuzminska A, Ciach T (2016)

Endothelialization of polyurethanes: surface silanization

and immobilization of REDV peptide. Elsevier,

Amsterdam

Chichareon P, Katagiri Y, Asano T et al (2019) Mechanical

properties and performances of contemporary drug-eluting

stent: focus on the metallic backbone. Expert Rev Med

Devices 17434440(2019):1573142. https://doi.org/10.

1080/17434440.2019.1573142

De Morais LS, Guimaraes GS, Elias CN (2007) Liberacao de

ıons por biomateriais metalicos. Maringa 12:48–53. https://

doi.org/10.1590/S1415-54192007000600006

Ferraz EP, Sa JC, de Oliveira PT et al (2014) The effect of

plasma-nitrided titanium surfaces on osteoblastic cell

adhesion, proliferation, and differentiation. J Biomed

Mater Res Part A 102:991–998. https://doi.org/10.1002/

jbm.a.34761

123

Biotechnol Lett

Fox KE, Tran NL, Nguyen TA et al (2019) Surface modification

of medical devices at nanoscale—recent development and

translational perspectives. Biomater Transl Med. https://

doi.org/10.1016/b978-0-12-813477-1.00008-6

Jayalakshmi M, Bhat BR, Bhat KU (2018) Enhanced cell

adhesion on severe peened-plasma nitrided 316L stainless

steel. AIP Publishing, New York, p 020086

Kahraman F, Gencer GM, Kahraman AD et al (2018) Low-

temperature nitriding behavior of compressive deformed

AISI 316Ti austenitic stainless steels. Surf Rev Lett.

https://doi.org/10.1142/s0218625x18501883

Kao W-H, Su Y-L, Horng J-H, Hsieh Y-T (2017) Improved

tribological properties, electrochemical resistance and

biocompatibility of AISI 316L stainless steel through

duplex plasma nitriding and TiN coating treatment. J Bio-

mater Appl 32:12–27. https://doi.org/10.1177/

0885328217712109

Kathuria YP (2006) The potential of biocompatible metallic

stents and preventing restenosis. Mater Sci Eng A

417:40–48. https://doi.org/10.1016/J.MSEA.2005.11.007

Li Y, Zhang S, He Y et al (2014) Characteristics of the nitrided

layer formed on AISI 304 austenitic stainless steel by high

temperature nitriding assisted hollow cathode discharge.

Mater Des 64:527–534. https://doi.org/10.1016/J.

MATDES.2014.08.023

Lin N, Liu Q, Zou J et al (2016) Surface texturing-plasma

nitriding duplex treatment for improving tribological per-

formance of AISI 316 stainless steel. Materials (Basel)

9:875. https://doi.org/10.3390/ma9110875

Lin K, Li X, Dong H et al (2018) Nitrogen mass transfer and

surface layer formation during the active screen plasma

nitriding of austenitic stainless steels. Vacuum

148:224–229. https://doi.org/10.1016/J.VACUUM.2017.

11.022

Lo KH, Shek CH, Lai JKL (2009) Recent developments in

stainless steels. Mater Sci Eng R 65:39–104. https://doi.

org/10.1016/j.mser.2009.03.001

Lu B, Malcuit C, Wang S et al (2009) Long-term safety and

function of RPE from human embryonic stem cells in

preclinical models of macular degeneration. Stem Cells

27:2126–2135. https://doi.org/10.1002/stem.149

Martinesi M, Stio M, Treves C, Borgioli F (2013) Biocompat-

ibility studies of low temperature nitrided and collagen-I

coated AISI 316L austenitic stainless steel. J Mater Sci

24:1501–1513. https://doi.org/10.1007/s10856-013-4902-

9

Moura CEB, Silva NB, Sa JC et al (2016) MC3T3-E1 cells

behavior on surfaces bombarded by argon ions in planar

cathode discharge. Artif Organs 40:497–504. https://doi.

org/10.1111/aor.12597

Nunes Filho A, de Aires M, Braz DC et al (2018) Titanium

surface chemical composition interferes in the Pseu-

domonas aeruginosa biofilm formation. Artif Organs

42:193–199. https://doi.org/10.1111/aor.12983

Offner D, Wagner Q, Idoux-Gillet Y et al (2017) Hybrid col-

lagen sponge and stem cells as a new combined scaffold

able to induce the re-organization of endothelial cells into

clustered networks. Biomed Mater Eng 28:S185–S192.

https://doi.org/10.3233/BME-171640

Popat KC, Leoni L, Grimes CA, Desai TA (2007) Influence of

engineered titania nanotubular surfaces on bone cells.

Biomaterials 28:3188–3197. https://doi.org/10.1016/J.

BIOMATERIALS.2007.03.020

Ribeiro KJB, de Sousa RRM, de Araujo FO et al (2008)

Industrial application of AISI 4340 steels treated in

cathodic cage plasma nitriding technique. Mater Sci Eng A

479:142–147. https://doi.org/10.1016/j.msea.2007.06.033

Samanta A, Chakraborty H, Bhattacharya M et al (2017) Nan-

otribological response of a plasma nitrided bio-steel.

J Mech Behav Biomed Mater 65:584–599. https://doi.org/

10.1016/j.jmbbm.2016.09.017

Schwartz RS, Edelman E, Virmani R et al (2008) Drug-eluting

stents in preclinical studies: updated consensus recom-

mendations for preclinical evaluation. Circ Cardiovasc

Interv 1:143–153. https://doi.org/10.1161/CIRCINTER

VENTIONS.108.789974

Shah A, Sinha R, Hickok N, Tuan R (1999) High-resolution

morphometric analysis of human osteoblastic cell adhesion

on clinically relevant orthopedic alloys. Bone 24:499–506.

https://doi.org/10.1016/S8756-3282(99)00077-0

Silva MAM, Valentim RAM, Guerra PVA et al (2015) Influ-

encia da topografia na molhabilidade em superfıcies de

titanio tratadas por plasma. Rev Bras Inov Technol Saude.

https://doi.org/10.18816/r-bits.v5i2.7247Su Y, Luo C, Zhang Z et al (2018) Bioinspired surface functional-

ization of metallic biomaterials. J Mech Behav BiomedMater

77:90–105. https://doi.org/10.1016/j.jmbbm.2017.08.035

Talha M, Ma Y, Kumar P et al (2019) Role of protein adsorption

in the bio corrosion of metallic implants—a review. Col-

loids Surf B 176:494–506. https://doi.org/10.1016/j.

colsurfb.2019.01.038

Trabzon L, Igdil MC (2006) On the materials properties of thin

film plasma-nitrided austenitic stainless steel. Surf Coat

Technol 200:4195–4200. https://doi.org/10.1016/J.

SURFCOAT.2004.12.012

Turner N, Armitage M, Butler R, Ireland G (2004) An in vitro

model to evaluate cell adhesion to metals used in implan-

tation shows significant differences between palladium and

gold or platinum. Cell Biol Int 28:541–547. https://doi.org/

10.1016/j.cellbi.2004.04.009

van Wachem PB, Beugeling T, Feijen J et al (1985) Interaction

of cultured human endothelial cells with polymeric sur-

faces of different wettabilities. Biomaterials 6:403–408.

https://doi.org/10.1016/0142-9612(85)90101-2

Vilardell AM, Cinca N, Garcia-Giralt N et al (2018)

Osteoblastic cell response on high-rough titanium coatings

by cold spray. J Mater Sci 29:1–10. https://doi.org/10.

1007/s10856-018-6026-8

Whitehead SA, Shearer AC, Watts DC, Wilson NHF (1995)

Comparison of methods for measuring surface roughness

of ceramic. J Oral Rehabil 22:421–427. https://doi.org/10.

1111/j.1365-2842.1995.tb00795.x

Zhao G-H, Aune RE, Espallargas N (2016) Tribocorrosion

studies of metallic biomaterials: the effect of plasma

nitriding and DLC surface modifications. J Mech Behav

Biomed Mater 63:100–114. https://doi.org/10.1016/J.

JMBBM.2016.06.014

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