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Vacuum 117 (2015) 73e80

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Corrosion resistance of praseodymium-ion-implanted TiN coatings inblood and cytocompatibility with vascular endothelial cells

Ming Zhang a, b, Shengli Ma a, Kewei Xu a, Paul K. Chu b, *

a State Key Laboratory for Mechanical Behavior of Materials, Xi'an Jiaotong University, Xi'an 710049, Chinab Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, China

a r t i c l e i n f o

Article history:Received 2 March 2015Received in revised form5 April 2015Accepted 6 April 2015Available online 14 April 2015

Keywords:TiN coatingsPraseodymium ion implantationCorrosion resistanceCytocompatibility

* Corresponding author.E-mail address: paul.chu@cityu.edu.hk (P.K. Chu).

http://dx.doi.org/10.1016/j.vacuum.2015.04.0140042-207X/© 2015 Elsevier Ltd. All rights reserved.

a b s t r a c t

Praseodymium (Pr) is implanted into TiN coatings to improve the corrosion resistance and cyto-compatibility in blood plasma. The corrosion resistance of the Pr-doped TiN coatings in blood plasma isimproved based on electrochemical measurements. Pr ion implantation dramatically decreases the he-molysis rate of the TiN coatings suggesting their suitability in cardiovascular applications. The viability ofvascular endothelial cells seeded on the untreated TiN coatings and two Pr implanted TiN coatingsimplanted for different time is assessed. The vascular endothelial cell attach and grow to confluence onthe Pr implanted TiN coatings and a network composed of vascular tissues is observed from the 0.5 h Primplanted coatings. The results suggest that Pr ion implantation can effectively improve the corrosionresistance as well as cytocompatibility of TiN coatings in blood plasma.

© 2015 Elsevier Ltd. All rights reserved.

1. Introduction

The essential properties of biomaterials are good corrosionresistance, suitable mechanical properties, and good cytocompati-bility [1,2] and the surface properties of biomaterials play animportant role in the interactions with tissues and cells. Thephysical and chemical properties as well as surface morphology areimportant [3] and biomaterials are often surface modified to meetbiomedical and clinical needs [4,5]. Although titanium and tita-nium alloys are used in biomedical implants, surfacemodification isfrequently needed to improve the corrosion resistance and bloodcompatibility in the physiological environment [6e12]. In fact,surface modification is an effective way to enhance the surfacecytocompatibility without affecting the favorable bulk propertiessuch as materials strength and sturdiness. Transition-metal-basedbinary or ternary nitride coatings have been fabricated on im-plants by PVD (physical vapor deposition), CVD (chemical vapordeposition), and ion implantation to improve the cytocompatibility,mechanical properties, and corrosion resistance [13e17].

Titanium nitride (TiN) coatings which can reduce wear damageand improve corrosion resistance of titanium alloys [18e22] aregood biomedical materials [23e26], especially as coatings on

blood-contacting biomedical implants [27]. An anticoagulantceramic heart valve comprising a single crystal alumina disk andTiN valve ring has been developed and the heart valve surface doesnot exhibit platelet aggregation or fibrin deposition [28]. TiN-coated surfaces have been observed to reduce the presence ofbacteria significantly [29]. Compared to bare nickeletitanium al-loys, TiN coatings improve the physiological activity associatedwith gene expression of cardiovascular endothelial cells promotinganti-inflammatory and cell adhesion gene expression [30e33].Generally, TiN coatings have good mechanical properties andchemical inertness, but there are often some inherent small poresand defects such as pinholes in these coatings thereby degradingthe corrosion resistance [34]. Ion implantation has been applied toreduce corrosion and wear of many types of biomaterials. Inaddition to modifying the surface structure and composition, ionimplantation can introduce a suitable amount of ions into the nearsurface of the materials without forming a discrete interface like inthe case of a deposited coating, and so the risk of delamination canbe minimized. Ion implantation of rare earth elements has beenshown to improve the corrosion resistance of materials in aqueousmedia [35]. For instance, ion implantation of praseodymium (Pr)has been shown to improve the corrosion resistance in aggressivemedia such as NaCl solutions [36].

Recently there has been a growing interest in science andapplication of rare earth materials. Pr has been shown to providelevels of corrosion protection to the underlyingmetal substrate and

Table 2Praseodymium ion implantation conditions.

Substrate used Acceleratedvoltage

Deceleratedvoltage

Implantationtime

TiN coatings 15 kV 1.3 kV 0.5 h/1 h

M. Zhang et al. / Vacuum 117 (2015) 73e8074

their protective properties have been reported. The corrosion in-hibition mechanism of Pr is often attributed to the deposition of anoxide/hydroxide thin film at cathodic domains, facilitated by thealkaline pHwhich arises from the reduction of water and/or oxygen[37]. However, under physiological blood condition, may be thethin oxide/hydroxide film is not sufficient for corrosion resistance.For TiN coating, the structural defects such as pores are responsiblefor the corrosion initiation. Implantation is used to change thesurface microstructure of materials without changing the bulkproperties in various fields such as biomaterials. Corrosion resis-tance should be advanced in the TiN coating with the help of Pr ionimplantation.

In previous studies of the biological role of rare earth elementssuch as praseodymium, scandium, and lanthanum, soluble salts(citrate or chloride) were introduced into animals by injection oringestion [38,39]. Although the direct biological role of praseo-dymiumcompounds is still controversial, ion implantation of Pr intoartificial biomedical implants is potentially useful. Past in-vestigations indicate that water soluble compounds of rare earthelements are mildly toxic, but insoluble rare earth compounds arenontoxic [40]. Generally, some physiological disorder has beenobserved.When the elements enter the cell nuclei, a stable complexis formed with the biological macromolecules affecting geneexpression and chromosome replication. However, in the case ofpraseodymium-doped ceramic coatings prepared by ion implanta-tion, whether or not soluble compounds are produced in the humanenvironment requires more research. Although rare earth elementsare typically not considered essential to the cellular life cycle,beneficial effects have been observed fromcrops. Hence, rare-earth-element-enriched fertilizers have been used in China since the1980s and so far, there have been no reported incidents about toxiceffects in humans as a result of uptake through the food chain [41].In addition, Pr possesses good antibacterial characteristics, does notcause inflammation of human umbilical cord perivascular cells(HUCPV), and is considered nontoxic at the proper concentrations[42,43]. The objective of this study is to study the effects of Pr ionimplantation on the corrosion resistance and cytocompatibility ofTiN coatings in blood plasma. Real blood is chosen in this studyinstead of commonly used artificial media such as SBF (simulatedbody fluid) and PBS (phosphate buffered saline) to better simulatethe physiological conditions for cardiovascular devices.

2. Experimental details

2.1. Sample preparation

The TiN coatings were deposited on medical titanium alloy(Ti6Al4V) by DC magnetron sputtering. The substrate was cleanedultrasonically in acetone and methanol and dried by nitrogen. Thesputtering targets were composed of 99.99% pure Ti (5.08 cm indiameter and 5 mm thick) oriented at 45� from each other. Thesubstrate was rotated continuously to face the twoTi targets from adistance of 70 mm. The sputtering parameters used in the depo-sition of TiN coatings are listed in Table 1. The TiN coatings were ionimplanted with praseodymium on the Plasma Technology Ltd.HEMII-80 cathodic arc plasma ion implanter in City University ofHong Kong. The base pressure in the vacuum chamber was 10�4 Paand the important ion implantation parameters are listed in Table 2.

Table 1Sputtering parameters for the PVD TiN coatings.

Target Base pressure Gas used Sputtering power

Ti 6.8 � 10�4 Pa N2:Ar2 (8:11) 75w

The sample holder was water cooled during ion implantation tokeep the temperature below 30 �C.

2.2. Characterization

The structure of the TiN coatings was characterized by X-raydiffraction (XRD, XRD-7000, SHIMADZU LIMITED) using Cu Ka ra-diation and the elemental depth profiles and chemical states weredetermined by X-ray photoelectron spectroscopy (XPS, AXIS ULTRA,KRATOS ANALYTICAL Ltd.). Atomic force microscopy (AFM,SPI3800-SPA-400, Seiko Instruments Inc.) was conducted toexamine the surface morphology of the samples before and after Prion implantation.

2.3. Corrosion behavior

The corrosion behavior of the control and implanted sampleswas evaluated by potentiodynamic polarization tests conducted ona Zahner Zennium electro-chemical workstation at 37 �C. A three-electrode cell with the sample as the working electrode, saturatedcalomel electrode (SCE) as the reference electrode, and platinumelectrode as the counter electrode was adopted. The medium wasgoat blood plasma (RUITE BIOTECH Ltd., Guang Zhou, China) afterseparating the blood plasma from whole blood by gradient centri-fugation. The sample with a surface area of 75 mm2 was exposed tothe blood plasma. In the potentiodynamic polarization test, thescanning rate was 1 mV s�1. The corrosion potential (Ecorr) wasderived from the curves and the corrosion current density (Icorr) wasdetermined from the polarization curves by Tafel extrapolation.

2.4. Hemolysis ratios

In this hemolysis ratio determination, fresh blood samples wereused. Four ml of blood was diluted with 5 ml of 0.9% (w/v) NaCl and10ml of 0.9% (w/v) NaCl were added. Additionally, 10 ml of the 0.9%(w/v) NaCl (negative controls, n ¼ 3) and 10 ml of doubly distilledwater (positive control, n ¼ 3) were prepared for antitheses. All thesamples were kept at 37 �C for 30 min and incubated in 0.2 ml ofwhole blood at the same temperature. After 60 min, the sampleswere centrifuged for 5 min and the supernatant was analyzed at540 nm to determine the absorbance of cells undergoing hemolysison a microplate reader (Powerwave XS MQX200R). The hemolysisratios were calculated by the following relationship: R ¼ (A � C1)/(C2 � C1) � 100% where R was the hemolysis ratio (%), A was theabsorbance (%), C1 was the absorbance of the negative controls (%),and C2 was the absorbance of the positive control (%) [44].

2.5. Vascular endothelial cell culture

The endothelial cell line (EAhy926) was provided by Shanghaicell bank (catalog number GNHu39) of The Chinese Academy of

Deposition time Sputtering pressure Substrate temperature

90 min 0.8 Pa 400 �C

Fig. 1. XRD patterns of the PVD TiN coatings.

M. Zhang et al. / Vacuum 117 (2015) 73e80 75

Sciences. They served differentiated endothelial cell functions,namely angiogenesis, homeostasis, thrombosis, blood pressure, andinflammation. Furthermore, they could be cultured to high pas-sages without appreciable changes in the growth rate and pheno-type, thus avoiding the diversity of primary isolated endothelialcells from different individuals, limitation of replication potential,and senescent tendency in cultures. The cells were cultured in high-glucose DMEM supplemented with 10% fetal bovine serum (FBS)and 1% (v/v) penicillin/streptomycin on culture dishes and incu-bated in a humidified atmosphere of 5% CO2 at 37 �C [45].

2.6. Cell proliferation

After sterilization with 75% alcohol, the samples were placed ona 12-well culture plate and 1 ml of the DMEM medium was intro-duced. The concentration of endothelial cells was 5 � 104 cells/ml.Endothelial cell proliferation was investigated by the CCK-8 kit(Biotime, China) after incubation for 1 and 6 days. After removingthe medium, the samples were washed twice with PBS. The freshmedium containing the CCK-8 reagent was added to each sampleand incubated at 37 �C for 3 h under standard culturing conditions.Afterwards,100 ml of the blue solutionwere transferred to a 96-wellplate. The absorbance was measured at 450 nm on a microplatereader and all the proliferation experiments were performed intriplicates [46].

2.7. Cell morphology and spreading

5 � 104 Endothelial cells were seeded on the surface of thesamples and cultured for 1 and 6 days in an incubator to study cellspreading. The CFDA SE kit and FITC kit (Beyotime China) was usedfor endothelial cells staining and the staining method was based onthemanufacturer's instructions. After staining was completed, eachsample was put in 1 ml of fresh medium and cultured for 24 h toensure that all cells were stained. A fluorescence microscope wasused to examine the samples afterwards.

3. Results and discussion

3.1. Coatings characteristics

Fig. 1 presents the XRD results of the TiN coatings with Primplanted at different duration. The primary phases of the Primplanted TiN coatings were approximately identical to those ofthe un-implanted TiN coating and the (1 1 1) plane was thepreferred orientation. However, after Pr implanted, the intensity of(2 2 0) peak increased slightly, and (2 0 0) peak decreased. For TiNcoatings, (2 2 0) peak with a higher surface energy adsorbs moreplasma protein [47]. With increasing the implantation duration, therelative diffraction intensities ratio of I(1 1 1)/I(2 0 0) increased. It wassuggested a change of the partial microstructural after ion im-plantation, although it could not change the preferred orientation.

After Pr ions were implanted into the TiN coating, some PrNpeaks appear in the XRD spectrum. Some literatures have shownthat the TiN and PrN have the same cubic structure [48,49]. Fromthe XRD results, chemical state of Pr is dominated present in thenitride state. Some studies report, in the TiN coatings, the newexogenous nitride phases were formed by ion implantation treat-ment [50,51]. There may be complex compounds such as TiePreNin the Pr ion-implanted TiN coatings althoughmore work is neededto elucidate the phenomenon clearly. In addition, after the ionimplantation, [2 0 0] peak decreased, may be due to the effects ofion bombardment.

To investigate the surface properties of the TiN coatingsimplanted with praseodymium for different time and determine

the amount of Pr in the coatings, XPS is conducted on two samples,one implanted for 0.5 h and the other for 1 h. The XPS depth profilesand high-resolution Ti2p and Pr3d spectra obtained from thepraseodymium-implanted TiN coatings are depicted in Fig. 2. Thesputtering rate during the XPS analysis was 4 nm/min. The pra-seodymium concentration reaches about 50 at% in the 1 h sampleand about 25 at% in the 0.5 h sample. Fig. 2b and e shows the Pr 3d3/

2 and Pr 3d5/2 peaks in the two samples allowing unambiguousidentification even without exact knowledge of the peak positions.The intensity of the peaks increases with implantation time and thechemical state changes as well. Fig. 2b reveals characteristicshoulders on the low-energy side of both Pr 3d peaks and Prmay bepresent in the Pr3þ in accordance with the Pr 3d5/2 binding energyrange [52]. Due to this peak position different with Pr oxide bindingenergy range, indicate some other Pr compound formed not onlyoxide. In spite of there were a small amount of Pr oxide on thesurface of the coatings as a result of air oxidation, but few small, sonot the main reason for the performance impact. Pr in PrN com-pound is in the Pr3þ state. This inference is reliable and in agree-ment with the XRD result. The details not yet clear, more work needto do in the future.

Fig. 2e shows that the peak position of Pr 3d changes with ionimplantation duration. The peak shift may arise from the differentpraseodymium chemical states such as metallic ones (binding en-ergy between 931 eV and 932 eV) and other species. Due to PrNlattice parameter larger than TiN, may easily integrate the lattice ofPrN, which can subsequently alter the electron field of Pr, and hencedecrease the binding energy of Pr3þ. In the view of the analysisabove, TiN should be the main components in both un-implantedand Pr implanted TiN coatings and the PrN could be found afterPr ion implantation.

The AFM images in Fig. 3 show the surface morphology of theuntreated TiN coatings and Pr-implanted coatings. The morphologyis obviously altered by ion implantation. The surface of the un-treated TiN coatings shows large and uniformly distributed cones(Fig. 3a) with a root-mean-square (RMS) roughness of 3.25 nm. Incomparison, the surface after ion implantation is smooth (Fig. 3band c) with root-mean-square (RMS) roughness values of 0.21 nm[Pr(0.5 h)] and 0.17 nm [Pr(1 h)]. This smoothing effect may be dueto ion beam sputtering during ion implantation. However, there isno significant difference in the surface roughness between the twoPr implanted TiN coatings.

Fig. 2. XPS depth profile and high-resolution XPS Pr 3d and Ti 2p spectra acquired from the Pr-implanted TiN coatings at different sputtering time, with the numbers in the figuresdenoting the sputtering time. (a) Depth profile in 0.5 h, the ion implanted layer depth is about 68 nm, (b) Pr 3d(0.5 h), (c) Ti 2p(0.5 h), (d) depth profile in 1 h, the ion implanted layerdepth is about 80 nm, (e) Pr 3d(1 h), (f) Ti 2p(1 h).

Fig. 3. AFM images: (a) TiN coating, (b) 0.5 h Pr implanted TiN coating, and (c) 1 h Pr implanted TiN coating.

M. Zhang et al. / Vacuum 117 (2015) 73e8076

According to previous reports, ion implantation of Pr and otherrare earth elements can reduce the friction coefficient of the ma-terials, make the surface smooth, and increase the microhardness[53]. In this study, surface morphology improvement is believed tostem from the Pr ion bombardment as well as surface atomic mo-bile due to Pr ion implantation.

Our AFM results are consistent and a smooth surface bodingwell for blood cells and consequently cardiovascular implants.Blood cells are broken down after exposure to the implant in aprocess called hemolysis. Ion implantation is an effective means toreduce damage to blood cells and the hemolysis rate is an objectiveindicator whether the surface roughness affects the blood cells.

After implanted with Pr, the crystal TiN mixed with crystal PrNis better suited for the biological application. The Pr implanted TiNsurface topography is generally island-like and the size of theseislands in the crystal depends on the volume entrapped. Thechanges in the island dimensions may also affect the functions ofthe vascular endothelial cells.

3.2. Corrosion behavior

The potentiodynamic polarization curves acquired from theuntreated and implanted samples in blood plasma at 37 �C aredisplayed in Fig. 4 showing that the corrosion potential of the un-treated TiN coatings is lower. After Pr ion implantation, the corro-sion potential is significantly improved while the corrosion currentdensity decreases. A possible reason is the protection offered by thebarrier provided by the Pr implanted TiN coatings and another onemay be that inter granular corrosion is suppressed by implantationof praseodymium into the TiN coatings [54]. Nitride of Pr existedaround TiN grain boundaries, both them had the same crystalstructure, which might be beneficial to the compact coating for-mation of PrN and TiN. Because the poor corrosion properties of TiNcoating are dependent on the structural defects. The TiN coatingsexhibit the columnar structure with pores between the inter-granular column, forming a direct path for the corrosive mediumto pass through the coating defects. The crystalline particles located

Fig. 4. Polarization curves of Pr-implanted TiN coatings.

Fig. 5. Hemolysis rates of the unimplanted TiN coating and two different Pr implantedTiN coatings.

M. Zhang et al. / Vacuum 117 (2015) 73e80 77

at the boundaries of the pores are weakly bonded. The pores maybe the most possible spots for corrosion initiation [55]. When theTiN coatings were immersed in blood, the corrosion initiates atsome structural pores, which will easily filled in by blood plasma orcorrosive medium.

After implanted with Pr, the coatings corrosion resistance isenhanced. From the XRD result, the reason maybe was that formedcrystalline PrN phase or the ternary TiePreN complex compoundsplug the transport path for the corrosive medium, which couldrestrain the penetration and diffraction of corrosive medium intothe inner of TiN coatings.

Generally, a pore-free coating gives the substrate greaterpassivity than a coating with pores [56]. From the AFM result, thecoating surface morphology is obviously altered by ion implanta-tion. This smoothing effect may be due to Pr ion bombardmentwhich leads to change microstructural, result in pores reduce orvanish.

On the other hand, plasma protein adsorption also influencesthe corrosion process of cardiovascular implants when surfacecoverage by blood components occurs. In case of surface coveragethe layers formed may act as a diffusion barrier leading to reducedcorrosion. After Pr ion implantation, the coatings exhibitedincreased surface energy due to enhanced TiN (2 2 0) plane in-tensity, with a higher surface energy adsorbs more plasma proteins.

It has been shown that magnetron sputtered TiN coatingsimprove the corrosion resistance of hard coatings such as TieSieNand TieAleN and that ion implantation of Pr and other rare earthelements promotes the wear and corrosion resistance due to theformation of surface barriers [57]. In this work, the TiN coating istreated as the control. The purpose of the experiment is to deter-mine whether the Pr-implanted TiN coatings possess enhancedcorrosion resistance in blood. The corrosion potential of the un-coated Ti6Al4V alloy in artificial saliva is �0.3 V which is smallerthan that of the TiN coating under the same conditions [58]. Thecomposition of blood is more complex than that of artificial salivaand the TiN coating shows better corrosion resistance than theuncoated Ti6Al4V alloy in blood. In biomedical applications, manydifferent types of coatings have been proposed to protect titaniumalloys.With respect to biodegradable implantsmade of polymers ormagnesium alloys, extensive corrosion can create mechanicalproblems prior to tissue healing [59].

After ion implantation, the implanted samples exhibitedincreased surface hardness, and improve corrosion property [35].

The improvement is believed to stem from the implantationinduced smoothing of the surface as well as surface hardening dueto ion implantation. In this study, Pr implanted TiN coatings arebetter than un-implanted TiN coating corrosion resistance.

All in all, our experiments corroborate that Pr ion implantationindeed improves the corrosion resistance of TiN coatings in a realblood environment.

3.3. Hemolysis rate

The hemolysis rate is an important indicator of the effectivenessof biomaterials in the blood environment and a smaller hemolysisrate translates into better blood compatibility. The surface rough-ness is indirectly related to the hemolysis rate. A hemolysis ratebelow the threshold of 5% means little harm to red blood cells [60].Fig. 5 shows the hemolysis rate of the untreated and Pr implantedTiN coatings and the hemolysis rates of two different Pr implantedTiN coatings are significantly smaller than that of the control whichshows a hemolysis rate larger than 5% possibly due to the surfaceroughness. The Pr implanted materials show that a hemolysis rateof less than 3% is more suitable clinically.

From the AFM results, Pr implanted TiN coatings surfaceroughness decreased, may be due to the effects of ion bombard-ment. A smoother surface may induce less damage to blood cells inflowing blood. The blood components are blood cells and plasma.Rapid movement of blood cells in a blood vessel does not ruptureendothelial tissues and a smoother surface can also reduce themechanical friction loss.

3.4. Cell proliferation

Vascular endothelial cells in the inner walls of blood vessels arein direct contact with blood and implants and vascular endothelialcell proliferation is an important index gauging the cytocompati-bility of the implants [61]. The effects of Pr ion implantation onendothelial cell proliferation are investigated by a CCK-8 viabilityassay after incubation for 1 and 6 days, respectively. Fig. 6 showsthe amount and proliferation of endothelial cells on the surface ofthe three samples. The absorbance is proportional to the amount ofcells. That is, the more the cells on the surface, the larger is theabsorbance. After 1 and 6 days, increased proliferation is observedand the two kinds of Pr samples show more prominent

Fig. 6. Endothelial cell proliferation on the unimplanted TiN coating and two Primplanted TiN coatings using the CCK-8 assay.

M. Zhang et al. / Vacuum 117 (2015) 73e8078

proliferation together with the TiN coatings. Pr ion implantationchanges the chemical properties of the coatings and tends to adsorbmore extracellular matrix that is conducive to cell adhesion andproliferation.

The surface characteristics of the materials such as surfacechemistry, roughness, and other topographic features play impor-tant roles in cell proliferation and also the internal environmentinside the organism [62,63]. It has been shown that Pr does notinhibit proliferation of human umbilical cord perivascular cells(HUCPV) [43] and our data further demonstrate that Pr ion im-plantation does not inhibit proliferation of vascular endothelialcells rendering the materials desirable in the blood environment.

After implanted with Pr, TiN coatings [2 2 0] plane intensityincreased, and this crystal plane with a higher surface energy ad-sorbs more extracellular matrix proteins. Extracellular matrix

Fig. 7. Endothelial cell morphology at day 1: (a) unimplanted TiN coating, (b) 0.5 h Pr implanthe samples after 6 days: (d) unimplanted TiN coating, (e) 0.5 h Pr implanted TiN coating

proteins are known to both maintain cellular morphology and actas conduits between extracellular stimuli and cells by regulatingproliferation, migration, differentiation, and survival. The proteinadsorption is the key determinant of cell proliferation on the ma-terial surface.

Numerous clinical studies with bare metal implants showedseveral limitations under cardiovascular condition. Especially, in-stent stenosis, late stent thrombosis and problems with revascu-larization are still unsolved major critical issues. Therefore, theideal cardiovascular implants should prevent smooth muscle cellhyperplasia, and support a fast healing of the endothelial tissue[64]. In this study, Pr implanted TiN coating promote endothelialcell proliferation due to coatings micro-structural adjustment by Prion implantation, which consistent with the clinical requirements.Anti-carcinogenic properties of Pr have been shown in number ofstudies [65,66]. This also indicated that, Pr implanted TiN coatingsshould be adjustable in the normal range of cell cycle geneexpression, rather than the malignant proliferation.

3.5. Cell morphology and spreading

The morphology and arrangement of the vascular endothelialcells on the samples are determined by immunofluorescencestaining for the actin expression. Fig. 7aec depicts the cytoskeletonof the vascular endothelial cells after incubation for 1 day. Thevascular endothelial cells on all the samples are similar exhibitingan elliptical or round morphology.

The cell morphology and functions are related. The morphologychanges after cell necrosis, aging, damage, and so on and so the cellmorphology is an important index of cytocompatibility [67]. Ourcell proliferation experiments show that the two Pr implantedsamples do not harm vascular endothelial cells that can adhere andproliferate on the surface. The observed cytoskeleton andmorphology of the vascular endothelial cells are normal indicativeof normal physiological functions. Vascular endothelial cells havemany important physiological functions such as anticoagulation,blood clotting, vasodilatation, etc [68]. Our results indicate the Pr

ted TiN coating, and (c) 1 h Pr implanted TiN coating. The endothelial cell spread on allforming avascular tissue-like network in vitro, and (f) 1 h Pr implanted TiN coatings.

M. Zhang et al. / Vacuum 117 (2015) 73e80 79

implanted TiN coatings maintain the normal functions of the bloodvessel inner wall while the corrosion resistance is improved.

One of the important requirements for cytocompatibility is theformation of a dense layer of cells on the materials surface. Thevascular endothelial cells also show good spreading and prolifera-tion on the TiN coatings and two Pr implanted TiN coatings. Almostconfluent coverage of vascular endothelial cells with ellipticalmorphology is observed after culturing for 6 days. Fig. 7d showsthat after 6 days, the cells spread on the TiN coatings, but there arestill gaps between the endothelial cells. In comparison, the endo-thelial cells are very dense on the 0.5 h Pr sample (Fig. 7e) and avascular tissue-like network is observed in vitro implying goodcytocompatibility. Good cell spreading is also observed from the 1 hPr implanted TiN coating (Fig. 7f).

The surface morphology containing micro/nano scale featuresshould be designed to enhance the cell functions [69]. The tissuestructure is a part of the endothelial cell property that is necessaryfor proper cell morphology, attachment, and secretion. The coatingtopography with a mixture of micro-/nano-scale features is animportant factor for enhancing cellular responses. The Pr implantedTiN coatings with a micro-/nano-scale crystals' surface topographyprovided an excellent substrate for cell adhesion. The cell adhesionmorphology with better tissue structure, The cell adhesionmorphology with better tissue structure, cell spreading, andcellecell interaction on the Pr implanted TiN coatings clearly sug-gests that the Pr implanted TiN coatings had favorable surfacecharacteristics for cell property. The experimental results areconsistent with cell proliferation data demonstrating that Pr ionimplantation enhances the cytocompatibility of TiN coatings.

4. Conclusion

In this study, the Pr ion implantation was used to improve thecorrosion resistance and cytocompatibility of TiN coatings. Fromthe XRD and XPS result, it was found formed Pr nitride in Primplantated TiN coatings. May be the corrosion path is blocked inthe Pr implanted TiN coatings. The implantation treatment en-hances the corrosion resistance of the TiN coating in blood condi-tion. The change of TiN crystal (2 2 0) plane intensity and formationof smooth surface produce a relatively stable and biofriendlyinterface for red blood cell intact and endothelial cells growth,resulting in enhanced cytocompatibility in vitro.

Acknowledgments

The work was financially supported by Hong Kong ResearchGrants Council (RGC) General Research Funds (GRF) No. 112212.

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