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Biomaterials 28 (2007) 806–813

www.elsevier.com/locate/biomaterials

Response of human endothelial cells to oxidative stress onTi6Al4V alloy

Roman Tsaryka, Marie Kalbacovab, Ute Hempelb, Dieter Scharnweberc, Ronald E. Ungera,Peter Dieterb, C.James Kirkpatricka, Kirsten Petersa,�

aInstitute of Pathology, Johannes Gutenberg-University Mainz, Langenbeckstr. 1, 55101 Mainz, GermanybInstitute of Physiological Chemistry, Technische Universitat Dresden, Fiedlerstr. 42, 01307 Dresden, Germany

cMax Bergmann Center of Biomaterials, Technische Universitat Dresden, Budapester Str. 27, 01069 Dresden, Germany

Received 18 July 2006; accepted 22 September 2006

Available online 16 October 2006

Abstract

Titanium and its alloys are amongst the most frequently used materials in bone and dental implantology. The good biocompatibility of

titanium(-alloys) is attributed to the formation of a titanium oxide layer on the implant surface. However, implant failures do occur and

this appears to be due to titanium corrosion. Thus, cells participating in the wound healing processes around an implanted material,

among them endothelial cells, might be subjected to reactive oxygen species (ROS) formed by electrochemical processes during titanium

corrosion. Therefore, we studied the response of endothelial cells grown on Ti6Al4V alloy to H2O2 and compared this with the response

of endothelial cells grown on cell culture polystyrene (PS). We could show that although the cell number was the same on both surfaces,

metabolic activity of endothelial cells grown on Ti6Al4V alloy was reduced compared to the cells on PS and further decreased following

prototypic oxidative stress (H2O2-treatment). The analysis of H2O2-induced oxidative stress showed a higher ROS formation in

endothelial cells on Ti6Al4V than on PS. This correlated with the depletion of reduced glutathione (GSH) in endothelial cells grown on

Ti6Al4V surfaces and indicated permanent oxidative stress. Thus, endothelial cells in direct contact with Ti6Al4V showed signs of

oxidative stress and higher impairment of cell vitality after an additional oxidative stress. However, the exact nature of the agent of

oxidative stress generated from Ti6Al4V remains unclear and requires further investigation.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: Titanium alloy; Endothelial cells; Free radicals; Corrosion; In vitro; Oxidative stress

1. Introduction

Successful use of titanium-based implants for numerousapplications is attributed to the excellent mechanicalproperties, corrosion resistance and biocompatibility ofcommercially pure titanium and titanium alloys. However,some cases of aseptic loosening of titanium alloy implantstake place and tissue-affecting processes occurring at thetissue–implant interface may be responsible. The surface oftitanium(-alloy) implant is covered with a titanium oxidefilm which reduces the corrosion potential of the metal [1].In the body, however, mechanical friction and chemicalinfluences might lead to rupture or weakening of the

e front matter r 2006 Elsevier Ltd. All rights reserved.

omaterials.2006.09.033

ing author. Tel.: +496131 174522; fax: +49 6131 173448.

ess: peters@pathologie.klinik.uni-mainz.de (K. Peters).

TiO2-layer, leading to a corrosion processes and theformation of wear debris in such regions. Increased releaseof titanium and an elevated level of titanium in the serumwas observed in some patients after implantation [2]. Metalions are the products of the anodic part of the corrosionreaction. The cathodic reaction that always occurssimultaneously with the anodic one leads to the reductionof oxygen at physiological pH values. This reductionproceeds via a number of partial reactions, with radicalsand hydrogen peroxide being formed as intermediateproducts [3]. Formation of reactive oxygen species (ROS)on the titanium(-alloy) surface might influence the viabilityand activity of the surrounding cells, but this process hasnot yet been studied in any detail.Immediately after implantation the material is exposed to

biological fluids and cells. Inflammatory cells, particularly

ARTICLE IN PRESSR. Tsaryk et al. / Biomaterials 28 (2007) 806–813 807

macrophages and neutrophils, are among the first cells tocontact the implant surface. Activation of macrophagesleads to the production of high amounts of ROS and H2O2,which are important for the wound healing process. It isassumed that the formation of ROS by activated cells couldin turn modify the TiO2-layer, since H2O2 in a phosphate-buffered solution leads to pronounced thickening of theTiO2-layer [4].

After implantation the formation of new blood vessels inthe region of wound healing takes place. Blood vesselformation is driven by endothelial cells and is considered tobe one of the main processes that determines the success ofthe integration of the biomaterial [5]. This inducedneovascularization in the area of wound healing providesa supply of oxygen and nutrition to the cells surroundingthe implant. Besides their role in angiogenesis, endothelialcells are important in inflammation by secreting cytokinesand expressing surface adhesion molecules for attractionand attachment of leucocytes. At the site of acuteinflammation, endothelial cells are subjected to ROSproduced by macrophages and neutrophils. Chronicoxidative stress is one cause of endothelial dysfunction invascular diseases such as atherosclerosis [6].

The cells possess mechanisms to defend themselvesagainst ROS. These include antioxidant enzymes, such ascatalase, superoxide dismutase (SOD) and glutathioneperoxidase, which determine the response of cells tooxidative stress [7]. Physiological concentrations of ROS,however, lead to the activation of endothelial cells thuscontributing to inflammation and wound healing [6].These inflammatory events at the early stages of implantintegration are necessary for efficient wound healing.However, a dysregulation of inflammatory processes dueto the constant formation of ROS at the surface oftitanium(-alloy) might contribute to aseptic loosening ofthe implant.

While the response of endothelial cells to oxidativestress has been well studied, the effects of ROS produced asthe result of cathodic corrosion of titanium alloy onendothelial cells have not been addressed as yet. Tosimulate inflammation- and corrosion-induced oxidativestress H2O2 was applied to endothelial cells grown onTi6Al4V alloy and on cell culture polystyrene (PS) as acontrol. It was shown that human endothelial cells grownon Ti6Al4V displayed signs of persistent oxidativestress. Furthermore, the extent of H2O2-induced effectswas significantly higher in endothelial cells in contactwith Ti6Al4V compared to cells on PS. Thus, there areindications that endothelial cells in vitro in contact with atitanium alloy are subjected to higher ROS-levels in whichthe exact source for the increased ROS-development is asyet unknown.

2. Materials and methods

All chemicals were obtained from Sigma unless otherwise indicated.

2.1. Ti6Al4V preparation

Titanium alloy Ti6Al4V (ASTM 136) disks with 16mm diameter and

2mm thickness were used in this study. The titanium surfaces were

prepared by grinding and polishing to roughness values below 25nm

(RMS) on a 100mm length scale by Fa.Hegedus. Before use, ultrasonic

cleaning was done in 1% Triton X-100, acetone, and ethanol for 20min

each. Titanium samples were sterilized with ethylene oxide.

2.2. Cell culture

Human dermal microvascular endothelial cells (HDMEC) were

isolated from juvenile foreskin as described before [8] and cultivated in

endothelial cell basal medium MV (PromoCell) supplemented with 15%

fetal bovine serum (Invitrogen), penicillin/streptomycin (40 units peni-

cillin/ml, 40 mg streptomycin sulphate/ml, Invitrogen), sodium heparin

(10mg/ml) and basic fibroblast growth factor (bFGF, 2.5 ng/ml) in a

humidified atmosphere at 37 1C (5% CO2). Endothelial cells in passage

four were seeded on polystyrene (TPP) pre-coated with fibronectin

(5mg/ml solution) or on Ti6Al4V alloy disks (110,000 cells/cm2). After 2

days H2O2 solution (0.25/0.5mM) in endothelial cell basal medium MV

with lowered ascorbic acid content (0.5mg/l) and without phenol red was

applied to the cells for 24 h.

2.3. Cell number determination

Relative cell number was determined by means of nuclei quantification.

Nuclei were stained with Hoechst 33342 solution (1 mg/ml, Sigma) and

nine digital microscopic images were randomly taken for each sample with

a Leica DC 300F camera. The number of nuclei on the images was

quantified with Image Tool software (freeware of the University of

Texas, Health Science Center San Antonio: http://ddsdx.uthscsa.edu/dig/

download.html).

2.4. Cytotoxicity assays

The CytoTox 96s Non-Radioactive Cytotoxicity Assay was performed

following the manufacturer’s protocol (Promega). This assay measures

lactate dehydrogenase (LDH) activity in the cell culture supernatants.

LDH release was calculated as the percentage of total LDH activity of the

samples obtained after lysis of cells in a maximum release control sample.

The metabolic cell activity (an indirect measure of cytotoxicity)

was measured by the conversion of MTS (3-(4,5-dimethylthiazol-2-yl)-

5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, Promega)

which was quantified by photometrical analysis at 492 nm of cell culture

medium 1.5 h after addition of MTS to the cells.

2.5. H2O2 concentration measurement

H2O2 concentration was measured using the Bioxytechs H2O2-560 kit

(Oxis) according to the manufacturer’s instructions.

2.6. Determination of GSH amount

The amount of reduced glutathione (GSH) was determined using the

fluorescent dye monochlorobimane (Fluka). Briefly, cells were lysed with

20mM Tris-HCl buffer pH 7.4 containing 150mM NaCl, 1mM ethylene-

diamine-N,N,N0,N0-tetraacetic acid (EDTA), 1mM ethylene glycol-bis(2-

aminoethyl ether)-N,N,N0,N0-tetraacetic acid (EGTA) 1% Triton X-100

and 1mM phenylmethansulfonyl fluoride (PMSF), the lysate was stained

with monochlorobimane solution (final concentration 0.1mM) containing

equine liver glutathione-S-transferase (1U/ml, Sigma) for 30min at 37 1C

in the darkness and fluorescence was measured using excitation filter for

395 nm and emission filter for 480 nm. GSH amount was normalized to the

protein concentration of the lysates measured using the Bradford assay [9].

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Fig. 1. Quantification of HDMEC number grown on PS and Ti6Al4V

24h after treatment with H2O2 (means7SDs; untreated control on PS set

as 100%).

R. Tsaryk et al. / Biomaterials 28 (2007) 806–813808

2.7. DCF-assay

Oxidative stress was assayed utilising the fluorescent probe 20,70-

dichlorodihydrofluoresceine-diacetate (DCDHF-DA, Alexis). DCDHF-

DA enters the cells where it is converted to 20,70-dichlorodihydrofluor-

esceine (DCDHF) by cellular esterase activity which is further oxidized by

ROS with the formation of a fluorescent product (DCF). The cells were

incubated with DCDHF-DA solution (50 mM) in the cell culture medium

for 30min, then the cells were washed with medium and H2O2-containing

medium was added. Fluorescence was measured 1 h after incubation with

H2O2 using a 485 nm excitation filter and a 535 nm emission filter.

2.8. Catalase and SOD activity assays

After a particular treatment (indicated in the text) the cells were lysed

with 10mM Tris-HCl buffer pH7.4 containing 150mM NaCl, 0.1mM

EDTA, 0.5% Triton X-100 and 1mM PMSF for 10min on ice. The

enzyme activities were measured with catalase and SOD activity assay

(Calbiochem) following the manufacturer’s instructions. The assay used

measures total SOD activity. Enzyme activity was normalized to the

protein content of the sample. Protein concentration was determined using

the BCA Protein Assay Kit (Pierce).

2.9. Statistical analysis

All experiments were repeated at least three times and the results are

presented as means7standard deviations (SDs). Statistical analysis was

carried out with Microsoft Excel one-way ANOVA test for independent

samples. Normal distribution was tested by the Shapiro–Wilk test.

3. Results

3.1. Cytotoxic effects of H2O2 on endothelial cells grown on

Ti6Al4V

To study oxidative stress in cells grown on titanium alloysurfaces we compared the response to H2O2 elicited inHDMEC grown on Ti6Al4V with the reaction of HDMECgrown on cell culture PS. The cell numbers between bothgrowth substrates tested did not differ significantly in theuntreated state (Fig. 1). On both surfaces H2O2-treatmentled to the reduction of cell number after 24 h withoutsignificant differences (0.25mM H2O2 led to a reduction of15% on PS and to 10% reduction on Ti6Al4V, 0.5mM

H2O2 induced a reduction of 430% on PS and 420% onTi6Al4V).

The reduction of the number of HDMEC points to thecytotoxicity of H2O2 at the concentrations used in thisstudy (0.25 and 0.5mM). To determine the cytotoxicity ofH2O2 on HDMEC grown on titanium alloy and PS wemeasured LDH-activity in the cell culture supernatant 24 hafter the addition of H2O2. An increased release of LDHfrom HDMEC was observed after H2O2 treatment, withthe higher H2O2-induced LDH release being observed incells grown on Ti6Al4V alloy (Fig. 2A, significant increasein LDH-release of ca. 10% on PS and 20% on Ti6Al4V by0.5mM H2O2 in comparison to the untreated control,po0.05).

In the MTS-conversion assay (a measurement of cellularmetabolic activity), it became apparent that untreated

HDMEC grown on Ti6Al4V alloy surfaces displayed asignificantly (po0.01) lower MTS conversion rate thanthe cells cultured on PS, thus indicating reduced metabolicactivity of cells grown on Ti6Al4V. Furthermore, theH2O2 treatment of HDMEC grown on PS did notsignificantly change metabolic activity of the cells,whereas 0.5mM H2O2 led to a significant reduction ofabout 20% of the MTS conversion in comparison tothe untreated cells on Ti6Al4V (Fig. 2B). Thus, thebasic metabolic activity of cells grown on Ti6Al4V wasreduced and the highest tested H2O2-concentration(0.5mM) exerted a unequivocal cytotoxic effect (shown byLDH release and MTS conversion) only in cells in contactwith Ti6Al4V.H2O2 is unstable and may be rapidly decomposed in the

cell culture medium by e.g., catalase present in the serum orreleased by cells. To determine whether the differences inthe sensitivity of HDMEC grown on titanium alloy and PSmight be induced by differences in H2O2 decay rates in cellculture medium depending on the adhesion substrates theH2O2-concentration in the medium was analysed (Fig. 3).One hour after the addition of 0.5mM to the medium H2O2

was nearly completely decomposed (i.e., ca. 0.02mM H2O2

for PS and 0.015mM for Ti6Al4V). This indicated that onlyabout 3% of the added amount of H2O2 remained incontact with PS and Ti6Al4V. When a 50% higher H2O2-concentration (0.75mM) was added, the decompositionwas not as pronounced. After 1 h a H2O2-concentrationof approximately 0.1mM was detectable, which wasapproximately 14% of the initial amount of H2O2 added.In the presence of HDMEC, the H2O2-decompositionoccurred even more rapidly. There were no statisticallysignificant differences in H2O2-decomposition betweenTi6Al4V and PS.

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Fig. 2. H2O2 cytotoxicity in HDMEC. (A) LDH release from EC grown on PS and Ti6Al4V 24 h after treatment H2O2. (B). MTS conversion by EC

grown on PS and Ti6Al4V 24h after H2O2 application (untreated control on PS set as 100%; means7SDs; significant difference: *po0.05, **po0.01).

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Fig. 3. Measurement of H2O2 concentration in cell culture medium in

contact with PS and Ti6Al4V with and without growing cells 1 h after

addition of H2O2 (means7SDs).

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Fig. 4. Determination of oxidative stress in EC grown on PS and Ti6Al4V

1h after addition of H2O2 (DCF fluorescence in untreated cells on each

surface set as 1; means7SDs; significant difference: *po0.05).

R. Tsaryk et al. / Biomaterials 28 (2007) 806–813 809

3.2. Oxidative stress in endothelial cells exposed to Ti6Al4V

surface

H2O2 is known to induce the formation of other radicalsin the cell. To study intracellular radical formation afterH2O2 treatment we utilized the DCF-assay. This assaydemonstrated that H2O2 induced a dose-dependent forma-tion of ROS in HDMEC as early as 1 h after treatment(Fig. 4). One hour after applying 0.5mM H2O2, DCFfluorescence in the cells grown on Ti6Al4V alloy wassignificantly higher (po0.05) then the corresponding valuesin cells grown on PS (i.e., 45-fold higher on Ti6Al4V vs.43-fold higher on PS compared to the untreated control).

These data suggest that H2O2 provokes a strongeroxidative stress in HDMEC in contact with Ti6Al4V alloycompared to HDMEC grown on PS.GSH is a component of the cellular antioxidant defence

system and is oxidized during detoxification of H2O2 andH2O2-induced organic peroxides. Therefore, to estimatethe intracellular oxidation status induced by exposure toH2O2 we analysed the amount of GSH in HDMECcultured on Ti6Al4V and PS 24 h after treatment. Inter-estingly, the level of GSH in the cells exposed to theTi6Al4V alloy surface without the addition of H2O2

was significantly lower (po0.01) and exhibited only 40%of the GSH amount in the cells grown on PS (Fig. 5).

ARTICLE IN PRESSR. Tsaryk et al. / Biomaterials 28 (2007) 806–813810

Furthermore, while the concentration of GSH increased inthe cells cultured on PS following H2O2 treatment (around20% increase for treatment with 0.5mM H2O2, po0.01),the GSH-concentration was slightly reduced in the cellsgrown on titanium alloy after 24 h (Fig. 5). Theseobservations indicate a permanent oxidative stress stateof HDMEC in contact with Ti6Al4V surfaces.

To further investigate the reactions of HDMEC onTi6Al4V and PS to H2O2 we studied the activity of twoantioxidant enzymes, catalase and SOD, 24 h after treat-ment. Catalase activity in untreated HDMEC grown onTi6Al4V was slightly lower than in cells grown on PS,although the difference was not statistically significant

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Fig. 5. Evaluation of GSH amount in HDMEC grown on PS and

Ti6Al4V 24 h after H2O2 treatment (untreated control on PS set as 100%;

means7SDs; significant difference: **po0.01).

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Fig. 6. Measurement of antioxidant enzymes activity in HDMEC grown on P

activity (untreated HDMEC on PS are set as 100%; means7SDs; significant

(Fig. 6A). However, the changes in catalase activity ofHDMEC in response to H2O2 were divergent on thedifferent adhesion substrates. On PS, catalase activity wasincreased 24 h after treatment with 0.25mM H2O2 but wasat the control level after exposure to 0.5mM (Fig. 6A). Incontrast, catalase activity in HDMEC grown on Ti6Al4Vdecreased upon addition of 0.25mM H2O2 (30% reduction)but was around the control levels after exposure to 0.5mM

H2O2.The activity of SOD was significantly lower in untreated

HDMEC grown on Ti6Al4V than in cells grown on PS(Fig. 6B, ca. 60% less SOD-activity on Ti6Al4V than onPS). The H2O2 treatment resulted in more pronounceddifferences in the overall SOD activity on the twomaterials. Although low H2O2-concentrations (0.25mM)did not induce significant changes compared to therespective untreated control, higher H2O2-amounts(0.5mM) increased SOD activity in HDMEC grown onPS (around 50% increase after treatment with 0.5mM

H2O2, po0.01) and decreased SOD-activity in HDMEC onTi6Al4V following H2O2 treatment (around 40% reduc-tion). Altogether, the data point to the occurrence ofpermanent oxidative stress in HDMEC on Ti6Al4Vcompared to HDMEC on PS.

4. Discussion

In this study, the development of ROS in a model ofcathodic corrosion of Ti6Al4V by the addition of H2O2

and their effects on human endothelial cell viability andfunction were examined. The endothelial cells were grownon Ti6Al4V surfaces and studied for signs of oxidativestress. The surface of Ti6Al4V is covered with a TiO2-layer,which is thought to be extremely protective and considered

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S and Ti6Al4V 24h after H2O2 treatment. (A) Catalase activity. (B) SOD

difference: **po0.01).

ARTICLE IN PRESSR. Tsaryk et al. / Biomaterials 28 (2007) 806–813 811

to be the main reason for the high biocompatibility oftitanium(-alloy) implants [1]. However, several facts pointto the reactivity of titanium surfaces. Recently Bikondoaet al. [10] showed that defects such as oxygen vacancies in amodel oxide surface, rutile TiO2 (1 1 0), mediate thedissociation of water. Another indication for titaniumalloy reactivity was an elevated concentration of titaniumin the serum of patients after implantation. Hallab et al. [2]demonstrated the increase in titanium concentration fromundetectable amounts to 5.95 mM in the serum of patientswithin 1 month of revision surgery after total knee replace-ment. Titanium release may be a result of the anodiccorrosion process, which takes place at the sites of defectsin TiO2-layer. The cathodic part of the corrosion processresults in the reduction of oxygen at physiological pH withthe formation of ROS and H2O2 as intermediate products[3]. Thickening of the TiO2-layer in biological solutionssubstantiate the fact that corrosion processes permanentlyoccur at titanium implant surfaces [4,11]. Wear particlesformed at the interface bone/implant or implant/cement(used for the fixation of the implant) can disrupt theprotective TiO2 film, thus further inducing corrosion,leading to the synergistic fretting-wear process. Besidesthe possible formation of ROS by the titanium(-alloy) itselfas the result of cathodic corrosion, titanium may besubjected to the ROS produced by inflammatory cellscoming into contact with titanium(-alloy) surfaces directlyafter implantation. One of the mediators is H2O2 releasedby monocytes, macrophages and granulocytes. The TiO2-layer may interact with H2O2 leading to formation ofhydroxyl radicals [12]. Altogether, these facts prompted usto hypothesize that endothelial cells that take part in thewound healing early after implantation, might permanentlybe subjected to ROS formed at the titanium implant surface,which can be referred to as oxidative stress. To confirm thishypothesis, we studied the reaction of endothelial cells to theTi6Al4V alloy in the presence of the oxidative stress inducerH2O2 in comparison to the reaction elicited in endothelialcells grown on cell culture PS.

The quantification of HDMEC growing on Ti6Al4V andPS revealed the dose-dependent reduction of cell number tothe same extent on PS and Ti6Al4V alloy 24 h afterH2O2 treatment compared to the untreated control. Thereduced cell number after H2O2-treatment cannot exclu-sively be explained by inhibition of cell proliferation, sincesome cytotoxicity was also observed. It has been shownin fibroblasts that H2O2 concentrations between 0.12and 0.4mM led to a growth arrest, whereas higher H2O2-concentrations induced cell death via apoptosis(0.5–1.0mM) or necrosis (5.0–10.0mM) (reviewed in [13]).

The LDH-release cytotoxicity assay revealed a concen-tration-dependent increase of LDH release in endothelialcells after H2O2-treatment. The LDH release indicates abreakdown of the cell membrane characteristic of necroticcell death. The LDH release was higher in cells grown onTi6Al4V alloy compared to those in contact with PS.Another indication for cytotoxicity, the reduction of

cellular metabolic activity (determined by the MTSviability assay which mirrors the development of NADH)also occurred after H2O2-treatment. While the H2O2-concentrations tested did not induce major changes in themetabolic activity of endothelial cells grown on PS, theysignificantly decreased MTS conversion in the cells grownon the Ti6Al4V alloy. Interestingly, even the basic MTSconversion rates (i.e., in cells not treated with H2O2) werelower in the cells grown on Ti6Al4V alloy compared to thecells grown on PS. It has been shown that oxidative stressinduced by H2O2-treatment can lead to a rapid depletion ofNADH and ATP due to an increased catabolic rate [14].Thus, the reduction of metabolic activity in cells grown onTi6Al4V could be a result of H2O2-induced oxidativestress. However, this does not explain the absence ofmetabolic reduction of cells in contact with PS.As already mentioned, H2O2 can induce oxidative stress

in endothelial cells by promoting ROS formation. This isachieved by several mechanisms including activation ofROS production by mitochondria, NAD(P)H oxidases,xanthine oxidase and uncoupled eNOS [7]. H2O2 in the cellcan also undergo the Fenton reaction in the presence ofmetal ions, resulting in the formation of highly toxichydroxyl radicals. Using the DCF-assay we observedhigher levels of ROS in endothelial cells grown on Ti6Al4Valloy 1 h after H2O2 addition compared to the cells on PS.DCF is the resulting oxidation product from DCDHF,which is oxidized by peroxyl radical, peroxynitrite and alsoH2O2 [15]. Therefore, increases in DCF-fluorescence shownin this study can be explained through oxidation by H2O2,which enters the cell. However, since the H2O2-concentra-tion remaining in the medium 1h after its addition is nearlysimilar on both materials the higher DCF-fluorescence incells grown on Ti6Al4V alloy might reflect further ROSproduction. One possible explanation for this could be theformation of ROS due to the reactivity of H2O2 with theTiO2-layer. A recent study by Lee et al. [12] showed thathydroxyl radicals were formed during the interaction ofTiO2 and H2O2 in vitro. The amount of hydroxyl radicalsformed was comparable to the level of ROS produced bystimulated inflammatory cells. It is also known that tracesof iron are present in titanium alloys (according to ASTMF 136 Ti6Al4V is allowed to have up to 0.2% Fe). Thepresence of iron in titanium alloy surface could also lead tothe formation of ROS due to the Fenton reaction uponH2O2 exposure.Cells have several mechanisms to protect themselves

against oxidative stress. One of the central defencemechanisms is an enzymatic ROS detoxification carriedout by SOD, catalase and glutathione peroxidase. WhileSOD and catalase directly catalyse the reduction ofsuperoxide anion and H2O2, respectively, glutathioneperoxidase utilizes thiol-containing GSH for detoxifica-tion of H2O2 [7]. GSH can also reduce the products oflipid peroxidation. This reaction can occur non-enzymati-cally but is usually mediated by glutathione-S-transferase.The result of two reactions is the formation of oxidized

ARTICLE IN PRESSR. Tsaryk et al. / Biomaterials 28 (2007) 806–813812

glutathione (GSSG) or glutathione conjugates with lipids.The balance between GSH and GSSG is critical forprotecting cells against oxidants. Excessive formation ofGSSG and glutathione conjugates during prolongedoxidative stress leads to the depletion of the GSH pool[16]. Thus, the reduced GSH-concentrations in endothelialcells grown on Ti6Al4V alloy compared to the cells grownon PS could reflect a state of permanent oxidative stress incells in contact with Ti6Al4V alloy due to ROS formationon the titanium surface. This is supported by the fact thatthe GSH-concentration in endothelial cells on Ti6Al4V isreduced by H2O2-treatment, whereas in cells grown on PSthe H2O2-treatment induced an elevation of GSH-level.The effects of H2O2-treatment on PS are in agreement withreports of adaptive increase in GSH concentration afterexposure to H2O2, a fact which can be explained by theonset of GSH de novo synthesis and H2O2-inducedupregulation of the expression of GSH synthesis enzymes(glutamate cysteine ligase) by this temporary oxidativestressor [17].

Additional evidence for permanent oxidative stress onTi6Al4V is based on the analysis of enzymatic activities ofcatalase and SOD. Cells grown on Ti6Al4V alloy showedsignificantly lower SOD activities than cells grown on PS.This could be explained by a persistent ROS-formation incells on the Ti6Al4V-surface resulting in exhaustion ofenzyme activity due to protein oxidation by ROS [18]. Incontrast, catalase activity in endothelial cells grown onTi6Al4V was only slightly reduced compared to cellscultured on PS. Moreover H2O2-treatment of endothelialcells grown on PS elevated catalase and SOD activity. It isknown that oxidative stress may increase the activity ofantioxidant enzymes. The mechanism of this induction mayinclude changes in enzymatic activity of existing proteinsand de novo enzyme synthesis [19]. Interestingly, 0.5mM

H2O2 did not change catalase-activity, whereas this H2O2-concentration induced an increase in SOD activity suggest-ing an uncoupled regulation of catalase and SOD activity.In contrast, H2O2-exposure on Ti6Al4V led to a decrease inSOD activity in endothelial cells. We hypothesize that thisreduction in SOD activity could be caused by a cumulativeeffect of the temporary H2O2-induced and the permanentTi6Al4V-induced oxidative stress. Consequently, theabove-demonstrated facts indicated an elevated sensitivityof endothelial cells to oxidative stress when growing incontact with Ti6Al4V and a thus reduced antioxidantdefence potential.

An animal study of Ozmen et al. [20] indicated a similareffect in vivo. It is shown that 1 month after the insertion oftitanium implants in rabbits an increase in lipid peroxida-tion (a possible result of oxidative stress) in the tissuessurrounding the titanium implants occurred, whereas therewas a decrease in the activities of antioxidant enzymes (e.g.,catalase, SOD, glutathione peroxidase). These observationsindicate a state of permanent oxidative stress at the surfaceof titanium implants leading to exhaustion of antioxidantenzymes in the surrounding tissues. In a recent study,

endothelial cells were shown to undergo oxidative stresswhen growing in contact with a NiTi alloy. However, thiswas attributed to the presence of the transition metal Ni inthe TiO2 layer [21]. Vanadium, which is a component of thealloy used in this study, is known to be toxic and to induceformation of ROS [22]. However, on analysis of the surfaceoxide film of the Ti6Al4V alloy using X-ray photoelectronspectroscopy vanadium oxide was not detected [23], but thepresence of traces of vanadium on the surface of titaniumalloy cannot be excluded completely.In conclusion, a permanent state of oxidative stress

appears to exist in endothelial cells grown in direct contactwith Ti6Al4V surfaces. However, the nature of the stressoris as yet unknown and must be further investigated.Therefore, experiments under conditions stimulatingTi6Al4V-corrosion are required to analyse the developingROS and the response of endothelial cells. In addition tothe pathophysiological impact of oxidative stress, lowconcentrations of ROS exert a role as signalling moleculesthat are involved in signal transduction cascades ofnumerous growth factor-, cytokine-, and hormone-mediated pathways (reviewed in [24]). Thus, a deeperinsight into the processes occurring at the cell–implantinterface can be useful in understanding the reactionsoccurring at the interface in implant acceptance andfailure.

5. Conclusions

Endothelial cells grown in vitro on Ti6Al4V alloydisplayed signs of permanent oxidative stress and exhibitedless tolerance to additional oxidative stress (induced byH2O2) with respect to metabolic activity, radical formationand antioxidant defence molecules in comparison to cellscultured on PS. Whether these in vitro results reflect thesituation of endothelial cells at the implant surface in vivoremains unclear. However, increased knowledge of themechanisms of Ti6Al4V alloy-induced oxidative stress mayprove useful for understanding the causes of titaniumimplant failure.

Acknowledgements

This work was supported by the German ResearchFoundation (DFG, KI 601/4-1). We would like to thankDr. Sophie RoXler for the helpful discussions and SusanneBarth and Andrea Kolzow for their excellent technicalassistance.

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