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Electrochimica Acta 56 (2011) 7303– 7311

Contents lists available at ScienceDirect

Electrochimica Acta

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olyurethane films modified by antithrombin–heparin complex to enhancendothelialization: An original impedimetric analysis

. Haddada,b, N. Zaninaa,b, A. Othmanea, L. Morab,∗

Biophysic Laboratory, Faculty of Medicine of Monastir, 5019 Monastir, TunisiaINSERM U 698 Laboratoire de Bio-Ingenierie de Polymères Cardiovasculaires, Université Paris 13,99, av JB Clement, Institut Galilée, 93430 Villetaneuse, France

r t i c l e i n f o

rticle history:eceived 22 April 2011eceived in revised form 14 June 2011ccepted 15 June 2011

a b s t r a c t

In this paper, polyurethane (PU) was deposited as a thin layer onto the surface of ITO (indium tin oxide)and was then modified with an antithrombin–heparin complex (ATH). The resulting films were charac-terized by ATR spectroscopy, contact angle measurements and electrochemical impedance spectroscopy(EIS). Physicochemical characterization confirmed the surface modifications.

vailable online 22 June 2011

eywords:olyurethaneeparinntithrombin

The obtained films were used as substrates for endothelial cell attachment and growth. These processeswere characterized using electrochemical impedance spectroscopy (EIS). We observed that the additionof a small amount of heparin and AT additives onto the polymer surface resulted in a considerable changein the surface characteristics, and we found that PU films that were modified by the ATH complex wereable to greatly enhance adhesion and proliferation of endothelial cells (ECs).

ndothelial cells

. Introduction

Polyurethanes (PUs) are commonly used in biomedical appli-ations due to their useful mechanical and physical propertiess well as their excellent blood and tissue compatibility. Theseaterials have thus played an important role in the develop-ent of many medical devices, ranging from catheters to artificial

earts [1]. It has also been shown that use of PUs in the pro-uction of vascular stents or in the coatings of these stents has

ncreased their biological acceptance [2,3]. Further modifications ofolyurethanes with an antithrombin–heparin (ATH) complex [4] orith a zwitterionic sulfobetaine monomer [5] have increased their

iological acceptance in stent applications. Surface modificationssing antithrombogenic substances, such as heparin, urokinase androstacyclin, have also been shown to enhance the blood com-atibility of these materials [6,7]. In addition, in vivo studies inabbit models have shown that ATH-modified catheters are stablend remain effective longer than commercial heparinized catheters4,8].

One frequently employed strategy in tissue engineering involvesmploying synthetic or biological materials in conjunction withells. This approach involves creating biological substitutes to

estore the functions of impaired tissues [9]. For vascular tissuengineering, an embryonic source of endothelial cells (ECs) woulde preferable [10–12]. Endothelialization of the coated surface of

∗ Corresponding author. Tel.: +33 01 49 40 36 63; fax: +33 01 49 40 30 08.E-mail address: Laurence.mora@univ-paris13.fr (L. Mora).

013-4686/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.oi:10.1016/j.electacta.2011.06.048

© 2011 Elsevier Ltd. All rights reserved.

the material under mimetic conditions can be used to evaluate theefficacy of blood compatibility.

ECs are seeded and allowed to generate a monolayer on thesurface of the vascular devices. The formation of such an EC mono-layer is necessary to limit deposition of proteins, such as fibrinogen,leading to thrombus formation.

In this study, the surfaces of PU films were modified by an ATHcomplex. Changes in polymer surface properties were evaluated byinfrared spectral analysis, atomic force microscopy, contact anglemeasurements and electrochemical impedance spectroscopy.

To the ability of the films to support adhesion and growth of ECsin vitro, both surface-modified and unmodified membranes weretested for cell adhesion behavior.

2. Materials and methods

2.1. Materials

Both heparin (Hep) and antithrombin (AT) were pur-chased from Aldrich. Indium tin oxide (ITO)-coated glass plates(10 mm × 20 mm, Merck, Germany) were used in the measurementof electrical impedance spectroscopy.

2.1.1. ITO substrate preparation before film depositionThe ITO-coated glass plates were ultrasonically cleaned for

20 min with pure acetone and then for 20 min with methanol,washed thoroughly with Millipore-Q water and dried under a flowof nitrogen. Finally, the ITO slides were sterilized using an autoclaveat 120 ◦C for 20 min.

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304 S. Haddad et al. / Electroch

.1.2. Preparation of PU filmsTo fabricate the PU films, PU (81367, FLUKA) was dissolved in

hloroform and precipitated in methanol. The precipitates wereacuum-dried at 60 ◦C for 1 day to remove the residual solvent. Theurified PU was dissolved in tetrahydrofuran (THF); THF was cho-en after performing solubility tests in different solvents (Table 1).

Ultra-thin PU films were prepared from a 1% solution of PU inHF. The solution was then cast on a clean ITO electrode to form ahin film. Finally, the PU films were dried at 60 ◦C, producing filmsith homogeneous hydrophobic properties on both sides of theembrane.

.1.3. Surface modification of PU filmsPU films were immersed in a solution of distilled 1,6-

exanediamine/propanol at a concentration of 0.06 g/mL for 3 mint 37 ◦C. The films were then rinsed with water for 24 h at roomemperature to remove any free 1,6-hexanediamine (Hex) beforeeing dried under reduced pressure at 30 ◦C to obtain aminolyzedU membranes [13].

A 100 mg sample of non-fractionated heparin (sodium salt) wasissolved in a sodium citrate buffer solution (80 mL, pH 4.7), mixedith EDC (40 mg) and stored at 4 ◦C for 5 h to activate the carboxyl

roups. Then, the aminolyzed Hex/PU membrane was immersedn a sodium citrate buffer solution (80 mL, pH 4.7) that containedeparin that had been previously activated with EDC. The mem-rane was stored at 4 ◦C for 24 h to immobilize the heparin on theembrane surface [14]. Membranes were then washed with phos-

hate buffered saline (PBS, pH 7.2) and finally immersed in a sodiumuffer solution containing AT. Films were kept at 4 ◦C for 4 h beforetilization.

.2. Characterization

.2.1. Infrared spectral analysisThe infrared spectra of PU, hexane-modified PU (Hex/PU) and

eparin modified Hex/PU (Hep/Hex/PU) films were obtained using Perkin-Elmer 1600 spectrometer. Omnic software was used forata acquisition and analysis.

.2.2. Atomic force microscopy (AFM)Surface topography of the films was performed using atomic

orce microscopy (AFM) under ambient conditions with aanoscope III that was equipped with a scanner 1553 D (Digital

nstruments, Santa Barbara, CA, USA).The AFM experiments were performed in the same manner on

he cells that were attached to the different PU films. Cells werelated at density of 5 × 104 cells/mL on unmodified ITO films and

ncubated for 4 h prior to experimentation. After plating the cells,ll samples were fixed and viewed by AFM. All AFM images wereaken using the tapping mode in air at room temperature.

.2.3. Water contact angle measurementTo examine the effects of 1,6-hexanediamine (Hex), heparin

Hep) and ATH treatments on the hydrophilicity of PU films overhe treatment time, the film surface was characterized by staticater contact angle measurements using the sessile drop method.

or this, a water droplet of approximately 0.2 �L was placed onhe dry surface of each composite. The contact angle of water ontohe composite was detected at room temperature using a GBX Sci-ntific Instrument (Romans, France). The contact angle analyzer isquipped with a special optical system and a charge-coupled deviceamera.

Distilled water, formamide (Sigma Chemical CO) andiiodomethane (Sigma Chemical CO, St. Louis, MO, USA) weresed as probe liquids. The surface free energy (SFE) (�S) of theifferent samples was calculated using the Van Oss model [15],

Acta 56 (2011) 7303– 7311

which shows the dispersive (�LW) and the polar acid–base (�AB)components, with the latter component divided into 2 parts, acidic(�A) and basic (�B).

2.3. Cell experiments

2.3.1. Cell cultureThe endothelial cell (EC) line, EAhy926, was grown in Medium

199 that was supplemented with 10% fetal bovine serum (FBS),2 mmol/L glutamine, 100 U/mL penicillin and 100 �g/ml strep-tomycin [16]. Cultures were incubated at 37 ◦C in a humidifiedatmosphere containing 5% CO2. Replicated cultures were obtainedby trypsinization and were used for <5 passages [17]. The endothe-lial cell identification was confirmed by examining their polygonalmorphology.

2.3.2. MTT assayThe viability and proliferation of EC cultures on specific days

were determined using an MTT assay. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma, USA) wasprepared as a 5 mg/ml stock solution in PBS. At the specified time(1, 2 or 3 days), 2 mL of fresh culture medium was added to eachwell that contained different PU films. Then, 0.5 mL of MTT solu-tion was added to each well, and the plates were incubated at37 ◦C in a 5% CO2 atmosphere for 4 h. The upper medium (withcolors ranging from yellow to purple) was then carefully removed,and the intracellular formazan was dissolved by adding 2 mL ofdimethyl sulfoxide (DMSO) into each well. The optical density (OD),or absorbance, of the formazan solution was measured at 570 nmwith an ELISA reader. All experiments were repeated three times.The significance of differences between two sets of data was cal-culated using the Student’s t-test. Differences were consideredsignificant when a p-value of 0.05 or less was obtained (correspond-ing to a 95% confidence level).

2.3.3. ITO electrode and cell inoculationFor electrochemical impedance spectroscopy (EIS) measure-

ments, the sterile ITO slides were placed in Petri dishes, and then50 �L of the cell suspension, with a density of 5.0 × 104 cells/mL,was dropped on each slide. Next, the undisturbed cells wereallowed to attach to the substrates in a CO2 incubator (37 ◦C,5% CO2) for approximately 4 h. Then, a desired amount of cul-ture medium was supplied to each Petri dish. EAhy926 cells wereattached to the working electrode and allowed to grow for 2 daysbefore the EIS of the cells was measured.

2.3.4. Immunofluorescence staining for actin in vitroEAhy926 cells were plated onto different PU surfaces at a density

of 5 × 104 cells/mL. After an incubation period of 24 h, all sampleswere washed with PBS. They were then fixed with 3.7% formalde-hyde for 20 min, washed in PBS, permeabilized with 0.5% (v/v)triton X-100 in PBS for 10 min, washed three times in PBS to elim-inate detergent and then incubated for 1 h at room temperaturewith a Phalloidine-FITC. Finally, cells were washed three times (3×2 min) with PBS, viewed and photographed using immunofluores-cence microscopy (Zeiss, SIP42930).

2.3.5. Statistical analysisStatistical analysis of the contact angle was performed using

the Student’s t-test for comparison between two groups (MicrosoftExcel 2007 software). For probabilities of correlation using thePearson’s coefficient (p), corrected p values of below 0.05 wereconsidered as statistically significant.

S. Haddad et al. / Electrochimica Acta 56 (2011) 7303– 7311 7305

Table 1Solubility tests of polyurethane.

Solvents Acetone Methanol Ethanol THF Dichloromethane Chloroform DMF Toluene

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PU I I I SS

: insoluble; S: soluble.

.4. Electrochemical measurements

A conventional three-electrode cell was used for all electro-hemical measurements. A saturated calomel electrode (SCE) wassed as a reference electrode, and platinum foil (0.54 cm2) was useds the counter electrode. The modified ITO electrode (0.21 cm2) wassed as a working electrode. Impedance analysis was performedith a Voltalab PGZ301 analyzer. Measurements were performed

n the presence of a 5 mM [Fe (CN)6]4−/3− (1:1) mixture as the redoxrobe in PBS.

Impedance spectroscopy (EIS) was performed in the frequencyange of 10 mHz to 100 kHz. During measurements, the potentialas maintained at −650 mV. The alternative potential applied to

he working electrode was 10 mV s−1. All electrochemical measure-ents were performed at room temperature in a Faraday cage. The

view modeling program (Scribrer and associates, Charlottesville,A) was used to analyze the impedance data.

All experiments were conducted at ambient temperature20–25 ◦C) and performed in triplicate.

. Results and discussion

.1. Fourier transforms infrared spectroscopy studies

Fig. 1 shows the ATR–FTIR spectra of PU, Hex/PU andep/Hex/PU. The unmodified PU was characterized by a peak atpproximately 1722 cm−1, which corresponds to a hydrogen-bond.ew other differences could be distinguished by visual comparisonf the spectra of PU and PU-Hex due to the strong absorption in theigh-frequency regions.

In the spectrum of Hex/PU, the peak at 3338 cm−1 correspondso an N–H stretching vibration in the presence of hydrogen bond-ng. A small peak just below 1711 cm−1 was due to H-bonded C Oroups. Two stretching bands at 2938 and 2857 cm−1 correspond tosymmetric and symmetric C–H stretching, respectively, of –CH3roups. The CHN group vibration (C–N stretching) is observed atpproximately 1537 cm−1 for the control polyurethane membranes18].

As shown in the subtraction spectrum of Hep/Hex/PU, newands provide information about the reaction between Hepnd Hex/PU. Heparin-adsorbed samples demonstrated shouldersround 1000–1100 cm−1, which correspond to the symmetrictretching of S O and C–O–C in the saccharide group of hep-rin [19]. A noticeable difference in the absorbances at 3338 and220 cm−1, corresponding to N–H and C–N vibrations, respectively,re observed in the ATR–FTIR spectra of heparin-immobilizedolyurethane samples. These differences were due to a decrease initrogen content on the surfaces after acrylic acid activation, whichas followed by heparin immobilization [19].

.2. Morphology of film coating on ITO by AFM

Films could be analyzed by atomic force microscopy (AFM) andharacterized by the roughness rate root mean square (RMS). The

ariation in the roughness rate suggests that 1,6-hexanediaminend heparin were successfully adsorbed on the PU films (Ra = 1.86,5.82 and 79.33 nm for PU, Hex/PU and Hep/Hex/PU, respectively)Fig. 2).

I S I S

3.3. Contact angle and membrane surface free energy

Contact angles were measured before and after modificationsusing the three probe liquids to evaluate the changes in thehydrophilicity of PU membrane surfaces due to grafting. The resultsare listed in Table 2. Generally, the contact angle decreases whendifferent modifications are performed after PU immobilization. Thewater contact angle decreases significantly after modification withthe ATH complex. The change in the contact angle of the sam-ples functionalized by Hex and by Hep/Hex implied that the Hexand Hep were successfully immobilized on the PU surface, caus-ing the surface to become more hydrophilic. The hydrophilicity ofthe polyurethane thus increased after heparin immobilization. Thisresult can be ascribed to the greater number of hydroxyl groupsin heparin relative to the other compounds. The presence of polarfunctional groups, such as OH groups or NH2 groups, increaseshydrogen-bonding interactions [20] and therefore decreases thecontact angle. This effect can also be observed in the resultsobtained from the surface energy (Table 2), which were determinedaccording to a Van Oss model. All PU membranes have higher sur-face energies after hexanediamine modifications when comparedwith controls. The highest value was observed in the Hep/Hex-modified PU membrane.

The increase of surface energy can be explained by the introduc-tion of polar functional groups, in particular after AT deposition,with the basic component of surface free energy. Variations in con-tact angle can be due to differences not only in surface chemicalcomposition but also in surface structures [21].

3.4. Cell adhesion and proliferation

Cell adhesion is known to be affected by the hydropho-bic/hydrophilic balance, electrostatic interactions, chemical func-tionality and biological cues of the biomaterial surfaces [22]. Mostmaterials that are used for cardiovascular prosthesis, such asPLLA [23], PLGA [24], polyethylene terephthalate (PET) [25] andpolyurethane (PU) [26], are not designed to promote cellular inter-action; hence, an appropriate protein coating is required to supportcell growth.

Cell morphology on unmodified and modified PU films illus-trates that EAhy926 cells adhered on PU films both before and afterfunctionalization.

As shown in Fig. 3, cells had spread out in all directions on themodified surfaces 24 h after seeding and were fully attached to theATH/PU films. However, on untreated PU films, the cells were moreelongated 4 h after seeding and failed to flatten out at their edge(Fig. 4).

In addition, it is clear in Fig. 3 that endothelial cells adhered to allPU films within 24 h. Cells presented a good contrast under the fluo-rescence microscope. Actin cytoskeletal proteins are clearly visible,revealing close cell arrangements. Cells spread out in all directionson the modified surfaces and were fully attached to the ATH/PUfilms. The cells were homogeneously distributed and well spread;their morphology changed to flat polygonal or spindle-shaped cells,

and tight junctions had formed between cells. However, on thePU and 1,6-hexanediamine-modified PU films, the cells were morespherical or spindle-shaped and failed to flatten out at the edge.Cells on the untreated PU films appeared to be round and not as

7306 S. Haddad et al. / Electrochimica Acta 56 (2011) 7303– 7311

Fig. 1. Fourier transforms infrared spectra of polyurethane (PU); 1,6-hexanediamine modified PU (Hex/PU) and Heparin modified Hex/PU films.

Fig. 2. AFM topography images: (a) PU, (b) Hex/PU and (c) Hep/Hex/PU.

S. Haddad et al. / Electrochimica Acta 56 (2011) 7303– 7311 7307

Table 2Contact angles and surface energy components of modified and unmodified PU films measured by sessile drop technique.

Surface Contact angle Composants of surface energy

Water Formamide Diiodomethane �LW �AB �+ �− �Total

ITO bare 58.0 ± 2◦ 62.8 ± 2◦ 51.5 ± 2◦ 33.4 6 0.2 37.5 39.4PU/ITO 88.4 ± 2◦ 79.95 ± 2◦ 53.7 ± 2◦ 32.2 6.1 0.9 9.8 38.2Hex/PU/ITO 79.6 ± 2◦ 44.8 ± 2◦ 0◦ 50.8 1.3 0.2 2.1 52.1Hep/Hex/PU/ITO 63.7 ± 2◦ 17.8 ± 2◦ 12.8 ± 2◦ 49.6 7.0 2.0 6.1 56.6

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ATH/Hex/PU/ITO 16.2 ± 2◦ 0◦ 38.1◦

ell spread as those observed on the other samples (Hep/Hex/PUnd AT/Hep/Hex/PU (or ATH/PU)).

Growth kinetics further demonstrated that the EAhy926 cellsaintained significantly higher proliferation rates on the ATH/PU

s. the unmodified PU during the 3 days of culturing (Fig. 5).pecifically, the ATH/PU cells were proliferated approximatelywice as fast as those cultured on unmodified PU. The statis-ical evaluation of all samples examined from the two sampleeries is shown in Fig. 5. These proliferation results confirm themportance of the nature of the film coating on endothelial cellehavior.

.5. EIS analysis

.5.1. EIS analysis of ITO coatingsFig. 6 shows Nyquist plots of polyurethane before and after mod-

fication. After functionalization of the PU films, we show only one

Fig. 3. Actin filament immunofluorescent staining of endothelial cells adhered to

40.6 17.0 1.5 49.5 57.6

semi-circle in the Nyquist plots. The polyurethane coating systeminvestigated in this study follows the classical behavior that hasbeen described in the literature [27,28].

The Nyquist semi-circle of PU-modified electrodes is not per-fect. We can see a small semicircle in the Nyquist diagrams at thelower frequencies (Fig. 6), suggesting that there is a slow reactionoccurring at the film–solution interface at these higher poten-tials.

As shown in Fig. 6, the diameters of the semicircles increasedsignificantly after the adsorption of both 1,6-hexanediamine andheparin on the PU film, which indicates a continuous growthof the surface film. In contrast, in the case of the ATH com-plex, we observed a decrease in the diameter of the semicircle,

which indicates a decrease in Rct and in the capacitance of thefilm.

The impedance spectra were fitted with electrical equivalent cir-cuit models. Electrical equivalent circuits are powerful tools if each

different PU films. (a) PU, (b) Hex/PU, (c) Hep/Hex/PU and (d) ATH/Hex/PU.

7308 S. Haddad et al. / Electrochimica Acta 56 (2011) 7303– 7311

Fig. 4. AFM photos of adhered endothelial cells to different PU films. (a) PU, (b) Hex/PU, (c) Hep/Hex/PU and (d) ATH/Hex/PU.

Fig. 5. Proliferation test of endothelial cells to different PU films for a time periods of 24, 48 and 72 h. The error bars represent standard deviations on cells counting. *Meanssignificantly higher proliferating rate.

S. Haddad et al. / Electrochimica Acta 56 (2011) 7303– 7311 7309

0 1x 105 2x105 3x105 4x105 5x105 6x105 7x105 8x105 9x1050.0

5.0x1 041.0x1 051.5x1 052.0x1 052.5x1 053.0x1 053.5x1 054.0x1 054.5x1 05

AT/Hep/Hex/I TOHep/hex/PU/I TOHex/PU/I TO

Zr (Ω . cm2)

- Z Im

(Ω.c

m2 )

PU/I TO ITO bare

Fig. 6. Nyquists diagrams for the impedance measurements corresponding to ITOemv

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Table 3Fitted parameters of all films modified bare ITO electrode.

Rf (�) Cf (F) Rct (�) CPE (F) �2

ITO bare – – 8.63E4 5.57E−6 1.1E−3

PU/ITO 1431 1.99E−7 3.51E5 3.32E−6 1.6E−2

Hex/PU/ITO 1783 1.45E−7 4.76E5 5.69E−6 2.1E−2

lectrode, PU/ITO, Hex/PU/ITO, Hep/Hex/PU/ITO and ATH/Hex/PU/ITO. All measure-ents were performed in culture medium pH 7.8. Amplitude of the alternating

oltage is 10 mV. Symbols show the experimental data in culture medium.

lement used corresponds to a physical process. Considering thatrocesses taking place on an intact sample, an artificially damagedample or on a heated sample are all different, different circuits cane proposed [29].

Fig. 7a and b shows the equivalent electric circuits used fornterpreting the spectra obtained from untreated ITO and from ITOoated with different PU films. The equivalent circuit presented inig. 7b has been used for coating analysis throughout this paper30,31].

The equivalent circuits include the ohmic resistance of thelectrolyte solution (Rs); the film ohmic resistance (Rf); the filmapacitance (Cf); the interfacial charge-transfer resistance (Rct) andhe constant phase element (CPE).

The coating resistance (Rf) reflects the ability of the circuit toesist electrolyte penetration and may be used to evaluate therotective performance of the coatings [32,33]. The coating capac-

tance (Cf) is related to the behavior of electrolyte penetration inoatings. A CPE was used to provide a better fit of the model to theata, which was most likely necessary due to the inhomogeneity ofhe coating [34–37].

The calculated coating capacitance and resistance are presentedn Table 3 as the average of three replications. In addition to other

ell-known approaches for the interpretation of EIS results, such asoating resistance, coating capacitance, charge transfer resistance

ig. 7. Equivalent circuit models used in this study: (a) represent the circuit for theare ITO electrode, (b) equivalent circuit often used to simulate EIS data obtainedith coated systems (films coatings) and (c) for the cells functionalization.

Hep/Hex/PU/ITO 1985 1.11E−8 8.55E5 4.65E−7 2.6E−2

ATH/Hex/PU/ITO 2194 5.47E−9 7.09E5 5.67E−7 1.4E−2

and double layer capacitance [38–42], the impedance magnitudeat low frequencies [43–45] can also provide general informationabout the performance of the coatings, avoiding errors in calcula-tions. Calculation errors are hidden in chi-square when measureddata points are fitted.

The Rct values for the untreated ITO, PU/ITO, Hex/PU/ITO,Hep/Hex/PU/ITO and ATH/Hex/PU/ITO were 8.63E4, 3.51E5, 4.76E5,8.55E5 and 7.09E5 �, respectively. These results indicated that thecharge transfer resistivity was significantly increased when the PUfilm was modified by heparin and the ATH complex. The charge-transfer resistance is a measure of the electron exchange betweenthe electrode and the redox coupling of K3[Fe(CN)6]/K4[Fe(CN)6]in solution, which depends on the interfacial structure of the elec-trode/electrolyte solution and on the electrostatic forces betweenthe interface and the redox probe [46].

For PU coated ITOs, the Rct was found to be greater than that ofthe untreated ITO, which can be explained by the PU/ITO surfaceobstructing the electron transfer from the electrochemical probe.The buildup of the polymer layer on the electrode introduces abarrier to electron transfer; thus, the interfacial charge-transferresistance is expected to rise as the layer thickness increases [46].

Adsorption of 1,6-hexanediamine involves an increase in Rct anda decrease in Cf. This can be explained by an increase in film thick-ness following the adsorption of 1,6-hexanediamine on the surfaceof the PU film.

The charge-transfer resistance increase after assembly of hep-arin on the 1,6-hexanediamine-modified PU films can be explainedby the electrostatic repulsion between the negatively chargedheparin and the negatively charged redox label Fe(CN)6

3−/4−. Gen-erally, the deposition of the heparin layer on the electrode yields alower peak current due to the electrostatic repulsion between theFe (CN)6

3−/4− and the heparin-modified electrode, resulting in slowinterfacial kinetics. Association of AT eliminates the electrostaticrepulsion, resulting in a lessening of charge-transfer resistance.The Rct decrease is caused by AT binding to heparin, and subse-quently, the negative charge of heparin is decreased.Antithrombinhas a heparin-binding site (HBS) located near the N-terminus thatcontains several lysine residues (high positive charge) and has astrong affinity for heparin (negative charge).

Results show that the film resistance (Rf) increases and the filmcapacitance (Cf) decreases after each modification (Table 3). Theseresults coincide with an increase in the film thickness. The Rf isdirectly proportional to the thickness of the film. If the coating dete-riorates and absorbs large amounts of water, then the Cf shouldincrease [47,48].

3.5.2. EIS and Cell adhesion: impedance spectra of endothelial cellmonolayers grown on different PU films

A linear relationship between the cell adhesion rate and thespecific resistance of the cell layer to negatively charged proteinswas shown using EIS [12]. Endothelial cell adhesion was greater onpreconditioned protein surfaces of lower specific resistance. We

conclude that EIS could be a quantitative tool for estimating theadhesion rate of EC cells onto material surfaces.

Biological cells are very poor conductors at low frequencies(below 10 kHz), and therefore, they force electrical currents to

7310 S. Haddad et al. / Electrochimica Acta 56 (2011) 7303– 7311

Table 4Fitted parameters of endothelial cells functionalized all PU films.

Ccell (F) Rcell (�) Cf (F) Rf (�) CPE (F) �2

Cell/PU/ITO 1.26E−6 567 2.42E−9 1332 5.32E−6 5.4E−3

−6 −6 −6 −2

bmdtpt(

iofe

rtlfip

Fp(e

Cell/Hex/PU/ITO 1.65E 490Cell/Hep/Hex/PU/ITO 1.77E−6 455.7

Cell/AT/Hep/PU/ITO 6.65E−6 511.1

ypass them. The influence of cells on the impedance measure-ents is plotted in Figs. 8 and 9, which present the Nyquist

iagrams obtained on a confluent endothelial cell monolayer onhe PU before and after functionalization. The attachment androliferation of cells on different PU films is expected to retardhe interfacial electron transfer kinetics and increase the Rct

Figs. 8 and 9; Table 4).Biological cells often act as insulating particles when attach-

ng and spreading on substrates; therefore, the existence of cellsn electrode surfaces will undoubtedly inhibit the electron trans-er between the redox probe in the electrolyte solution and thelectrode [49].

The impedance of the cell layer was set as parallel-connectedesistance (Rcell) and capacitance [50]. The parameter Rcell reflectshe morphological and physiological changes in the extracellu-

ar space (e.g., cell/cell or cell/substrate gap). By non-linear curvetting with the equivalent circuit model (Fig. 7c; Table 4), thearameters in the model were adjusted to minimize the sum

a

b

0 1x105 2x10 5 3x10 5 4x10 5 5x10 5 6x10 50.0

5.0x104

1.0x105

1.5x105

2.0x105

2.5x105

3.0x105

3.5x105

4.0x105

Z r (Ω . cm2 )

- Z Im

(Ω. c

m2 )

Hex/PU/ITO Cells/Hex/PU/ITO

0 1x105 2x105 3x10 5 4x10 5 5x10 50.0

5.0x104

1.0x105

1.5x105

2.0x105

- Z Im

(Ω. c

m2 )

Z r (Ω . cm2 )

PU/ITO Cells/PU/ITO

ig. 8. Nyquists diagrams for the impedance measurements corresponding to (a)olyurethane coated ITO before and after functionalization by endothelial cells andb) corresponding to Hex/PU modified ITO before and after functionalization byndothelial cells.

2.00E 10078 3.8E 3.1E1.55E−6 9986 2.33E−6 2.9E−2

2.63E−6 46406 1.92E−6 6.9E−2

of squared deviations between the model and the measuredimpedance spectra.

The specific resistance of the cell layer (Rcell) was between 455and 567 �. These values are consistent with Rutten et al. [51], whoanalyzed endothelial cells initially isolated as individual clonesfrom bovine brain microvessels. Cells of this type, which weregrown on permeable glutaraldehyde-treated collagen gels, yieldedspecific cell layer resistances that were between 160 and 800 �. Itis clear that the cell type and the culture medium are importantparameters on which cell resistance depends.

Bouafsoun et al. [52] analyzed endothelial cells (EAhy926)for lipopolysaccharide detection. Cells yielded specific cell layerresistances of 1552 �. However, much smaller values for spe-cific endothelial cell layer resistances were reported by Wegneret al., who obtained a value of 30–80 � for porcine brain capillary

endothelial cells [50] and only 4 � for bovine aortic endothelial cells[53]. Finally, for endothelial cells from brain capillaries, very high

a

b

0.0 2.0x105 4.0x 105 6.0x1 05 8.0x 105 1.0x1 06 1.2x1060

1x105

2x105

3x105

4x105

5x105

6x105

He p/Hex/PU /ITO

Z r (Ω . cm2 )

- Z Im

(Ω.c

m2)

Cell s/Hep/Hex/PU/ITO

0.0 2.0x1 05 4.0x1 05 6.0x1 05 8.0x1 05 1.0x1060

1x105

2x105

3x105

4x105

5x105

6x105

7x105

AT/Hep/Hex/PU/I TO

Z r (Ω . cm2 )

- Z Im

(Ω.c

m2)

Cell s/AT /He p/He x/PU/I TO

Fig. 9. Nyquists diagrams for the impedance measurements corresponding to (a)Hep/Hex/PU/ITO before and after functionalization by endothelial cells and (b) cor-responding to ATH/Hex/PU/ITO before and after functionalization by endothelialcells.

imica

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ttp0Iw

tlcd

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ley, 1972.

S. Haddad et al. / Electroch

pecific resistances of up to 2000 � have been reported by Cronet al. [54].

The specific capacitance of cell membranes is generally regardedo be approximately 1 �F cm−2 [55]. Steinem et al. have shownhat the specific capacitance of the endothelial cell layer (inorcine brain microvessels and bovine aorta) showed a value of.5–0.8 �F cm−2 and a value of 1–3 �F cm−2 for epithelial cells [56].n our experiments, the specific endothelial layer capacitance (Ccell)

as found to be between 1.26 and 6.65 �F cm−2 (Table 4).The specific endothelial layer capacitance was found to be equal

o 6.65 �F cm−2 for the ATH/Hex/PU-modified ITO electrode. Aarger specific cell layer capacitance can be explained in terms ofell membrane folding and/or the existence of microvilli and pseu-opods [57] caused by the presence of the ATH complex.

. Conclusion

PU films were elaborated to investigate the effects of theTH complex on cell adhesion. Electrochemical impedance spec-

roscopy (EIS) was used to study the interface between endothelialells and ATH-modified PU. The interaction between endothelialells and various biomaterials is of interest for the development ofardiovascular grafts. This method is also suitable for monitoringhe specific charge transfer resistance and the specific capacitancef the total cell layer.

Cell culture studies showed that ATH was significantly benefi-ial to endothelial cell spreading and adhesion, which subsequentlyromoted cell growth and proliferation. From this result, we expecthat biomedical implants, especially catheters, can be preferablyndothelialized and show improved cell adhesion by the simpledsorption of ATH complexes on their surface.

cknowledgments

This work was financially supported by CMCU No. 09G0835.e thank Eric AIT, from the LSPM, for his help with the AFM and

r. Laure BACHELET for her help with the ATR–FTIR analyses. Weould thank Ms. Mary Osborne-Pelligrin for her help in English

hecking of the paper.

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