Acta Biomaterialia 6 (2010) 3506–3513

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Acta Biomaterialia

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Films of dextran-graft-polybutylmethacrylate to enhance endothelialization ofmaterials

Sidi Mohamed Derkaoui a,b, Amélie Labbé a,b, Agung Purnama c, Virginie Gueguen b, Christel Barbaud a,b,Thierry Avramoglou a,b, Didier Letourneur a,b,*

a INSERM, U698, Bio-ingénierie de Polymères Cardiovasculaires, CHU X. Bichat, Université Paris Cité/Paris Diderot, 75018 Paris, Franceb Institut Galilée, Université Paris Cité/Paris Nord, 99 Av. J.B. Clément, 93430 Villetaneuse, Francec Laboratory for Biomaterials and Bioengineering, Laval University, Metallurgical and Materials Engineering, Que., Canada

a r t i c l e i n f o a b s t r a c t

Article history:Received 20 November 2009Received in revised form 14 March 2010Accepted 31 March 2010Available online 4 April 2010

Keywords:PolybutylmethacrylateDextranCopolymer filmsCell proliferationEndothelial cell migration

1742-7061/$ - see front matter � 2010 Acta Materialdoi:10.1016/j.actbio.2010.03.043

* Corresponding author at: Inserm U698, Institut G99 Av. J.B. Clément, 93430 Villetaneuse, France. Tel./f

E-mail address: didier.letourneur@inserm.fr (D. Le

We have synthesized new structures obtained from amphiphilic copolymers of dextran andpolybutylmethacrylate with the aim of endothelialization of biomaterials. Grafting of butylmethacrylateonto dextran has been carried out using ceric ammonium nitrate as initiator. Three copolymers wereobtained (11, 30 and 37 wt.% dextran) and homogeneous thin films were successfully prepared. In con-trast to dextran, the resulting films were stable in water, and copolymers characterized by Fourier trans-form infrared spectroscopy, differential scanning calorimetry and dynamic mechanical analysis showedevidence of hybrid properties between the parent homopolymers. Surfaces of films were smooth whenanalyzed by atomic force microscopy (roughness 2 ± 1 nm) but greatly differed in their hydrophilicityby increasing the dextran content (water contact angle from 99� to 57�). In contrast to polybutylmethac-rylate, where the proliferation of vascular smooth muscle cells (VSMCs) was excellent but that of endo-thelial cells was very low, the copolymer containing 11% of dextran was excellent for endothelial cells butvery limited for VSMCs. An in vitro wound assay demonstrated that copolymer with 11% dextran is evenmore favorable for endothelial cell migration than tissue-culture polystyrene. Increasing the dextran con-tent in the copolymers decreased the proliferation for both vascular cell types. Altogether, these resultsshow that transparent and water-insoluble films made from copolymers of dextran and butylmethacry-late copolymers with an appropriate composition could enhance endothelial cell proliferation and migra-tion. Therefore, a potential benefit of this approach is the availability of surfaces with tunable propertiesfor the endothelialization of materials.

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1. Introduction

The development of amphiphilic copolymers based on copoly-merization of hydrophilic and hydrophobic parts is of great impor-tance due to their versatility and potential applications inregenerative medicine [1–5]. For instance, methacrylic polymershave been widely studied as a biocompatible matrix for use in tis-sue engineering [6–11]. Other synthetic polymers have shownphysicochemical and mechanical properties comparable to thoseof biological tissues, but have not been sufficiently biocompatible.In this context, natural biological polymers such as polysaccharidespossess good biocompatibility [12–15], but their mechanical prop-erties are often inadequate. Improvements in the characteristics ofsynthetic polymers could be attained by the addition of biologicalcomponents in the structures. The resulting materials could com-

ia Inc. Published by Elsevier Ltd. A

alilée, Université Paris Nord,ax: +33 1 4940 3008.tourneur).

bine the appropriate mechanical properties of the synthetic poly-mer with the biocompatibility of the natural polymers. The mainchallenge in achieving this combination relates to the thermody-namic incompatibility of natural and synthetic polymers that isthe result of enthalpy and entropy barriers caused by the differ-ences in hydrophilicity/hydrophobicity. This incompatibility is aphenomenon that has been frequently observed for many polymermixtures and results in phase separation [16,17]. In order to over-come this problem, we and others investigated graft copolymeriza-tion of a synthetic polymer, polymethylmethacrylate, onto thenatural dextran macromolecule using ceric ammonium nitrate asa redox initiator [18–22]. This strategy consisted of creating free-radical grafting sites on a dextran backbone by reaction with cer-ium IV, the radicals so formed initiating copolymerization withmethylmethacrylate monomers. We describe here new com-pounds obtained from graft polymerization of dextran with butyl-methacrylate. The materials were prepared as a series of films,differing in their wettability by varying the concentration of dex-tran, and we studied their behaviors with vascular cells [23,24].

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We found that films with a particular composition were able togreatly enhance the migration and proliferation of endothelial cellsbut not of vascular smooth muscle cells.

2. Materials and methods

2.1. Materials

Dextran (70,000 g mol�1), obtained from Sigma, was dried un-der vacuum at 60 �C for 24 h. Butylmethacrylate monomers wereobtained from Acros, and were purified by washing with 5% NaOH,20% NaCl, and three times with distilled water. Ce(IV) ammoniumnitrate and nitric acid were obtained from Acros.

2.2. Graft copolymerization

Three types of copolymers were synthesized by varying theamount of dextran. The general experimental procedure was as fol-lows: the reactions were carried out in a 1 l five-neck flaskequipped with a stirrer and a condenser, then the flask was im-mersed into a thermostated oil bath at 40 �C and purged withnitrogen gas. Three different mass ratios of dextran to butylmeth-acrylate (0.125/2, 0.5/2 and 0.75/2 w/w) were used. Dextran wasdissolved in 500 ml of 0.2 M nitric acid for 10 min. Five millilitersof solution of Ce(IV) dissolved in 0.2 M HNO3 and 2 g of monomerswere simultaneously added. The system was continuously stirredfor 50 min and the solution was neutralized to pH 8 by the additionof a 10 M NaOH aqueous solution and concentrated with an evap-orator. Methanol was used as a non-solvent to precipitate the poly-meric material, which was washed with 50 ml of 10�2 M EDTA toremove the cerium ions. The three purified copolymers, namedDB1, DB2 and DB3, were frozen at �20 �C and lyophilized for24 h. To obtain pure graft copolymers, the resulting products wereextracted with acetone in a Soxhlet extractor for 24 h. The purecopolymers were dried under vacuum and weighted.

2.3. Preparation of films

Preparation of copolymers films was carried out by dissolving6% (w/v) of copolymers in a 90/10 (v/v) tetrahydrofuran (THF)/H2O mixture. A 500 ll aliquot of the transparent solution obtainedwas molded into Petri dishes (15 mm in diameter) for 24 h at roomtemperature in a saturated atmosphere of THF and in presence ofCaCl2 to absorb water. After solvent evaporation, films were re-leased from the mold upon addition of distilled water, while inthe case of polybutylmethacrylate (PBMA) (very fragile) the filmswere soaked in distilled water for 2 h and then carefully removedfrom the Petri dishes. The samples were air dried for 24 h to re-move the solvent and obtained films are 35 lm in thickness. Thefilms were rinsed several times with phosphate-buffered saline(PBS). Films of copolymers were cut for mechanical tests in theshape of 15 mm � 5 mm, but PBMA film was not prepared becauseit was very brittle.

2.4. Infra-red spectral analysis

The infrared spectra of copolymers films (DB1, DB2 and DB3)were obtained using a Perkin-Elmer 1600 spectrometer. Each filmwas mounted on the sample holder. The atmosphere spectrum wassubtracted as the background for each scan. Each spectrum was ob-tained at a resolution of 1 cm�1 using 32 scans. The studied sam-ples did not gaining weight by moisture uptake during theacquisition, similar data being obtained after 1 h. Omnic softwarewas used for data analysis. Fourier transform infrared spectroscopy(FTIR) was also used to semi-quantitatively estimate the percent-

age of dextran in the copolymers using physical mixtures of dex-tran and PBMA at different ratios. A 15 mg quantity of fine KBrpowder was mixed with 1.5 mg of a physical blend of dextran/PBMA weight ratios (5/95, 10/90, 15/85, 20/80, 25/75, 30/70, 35/65 and 40/60). We used the integrated area of the (O–H) band at3400 cm�1 with different concentrations of dextran, PBMA andeach synthesized copolymer.

2.5. Differential scanning calorimetry (DSC)

The thermal behavior of homopolymers (dextran and PBMA)and all copolymers (DB1, DB2 and DB3) was analyzed with a Seta-ram DSC131 thermal analyzer, operating in combination with a li-quid nitrogen cooling accessory. A mixture of dextran + PBMA (50/50 w/w) was also tested. The furnaces were purged with nitrogenand samples were passed in the temperature range from �50 to+200 �C at a heating rate of 10 �C min�1. In each case, two consec-utive scans were carried out on each sample. The glass transitiontemperature (Tg) of the products was taken as the mid-point inthe shift of heat flow baseline.

2.6. Dynamic mechanical analysis (DMA) on copolymer films

DMA was used to analyze loss modulus, storage modulus andtan d of all films of copolymers (DB1, DB2 and DB3). PBMA wasnot tested because it was rigid and very fragile at room tempera-ture. Films of 15 � 5 � 0.035 mm were analyzed on a DMA Q800dynamic mechanical analyzer (TA Instruments). The equipmentwas operated in a single cantilever bending mode at a frequencyof 1 Hz, an amplitude of 5 lm and a heating rate of 3 �C min�1 from�100 to +200 �C.

2.7. Creep test

To determine the deformation of DB copolymers, a creep test atambient temperature was carried out by applying a constant loadto DB1, DB2 and DB3 and observing the deformation with time.Several loads (100, 125, 150, 175 and 200 g) were suspended tothe extremity of films of 15 mm length, 5 mm width and0.035 mm thickness. The expansion of films was measured frompictures taken every minute until rupture. The results were ob-tained as creep curves according to the equation:

e ¼ a½1� expð�btÞ� þ ct

where e is the distortion and t is the time. Curves were drawn forthe percentage of expansion vs. time, and the initial deformationvelocity, obtained from

V0 ¼ ðde=dtÞt ! 0 ¼ abþ c

was then deduced for all samples.

2.8. Contact angle measurements

The water contact angles of films were measured at room tem-perature using a contact angle system (Krüss, model DSA 10-Mk2).A droplet of deionized water (2 ll) was put on the air-side surfaceof the films and the contact angle was measured after 10 s. Theprocess was monitored by a video contact angle system. Ten mea-surements were carried out for each sample (PBMA, DB1, DB2 andDB3; dextran gave any film) and the values obtained wereaveraged.

2.9. Topography and roughness

Atomic force microscopy (AFM) was performed to determinethe surface roughness and the uniformity of coating. All films were

Fig. 1. Variation of pH (N) and optical density (j) during copolymerization.Dextran-graft-polybutylmethacrylate copolymers were synthesized via redox ini-tiation. The decrease in pH (left) and the increase in optical density at 650 nm(right) were plotted as functions of time. No change was observed in the absence ofdextran or butylmethacrylate monomer.

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imaged with a Nanoscope III Digital Instruments dimension 3100atomic force microscope (Veeco Metrology Group) at a resonancefrequency range of 50–100 Hz. Three areas per sample were im-aged under tapping contact mode with a silicon nitride probe(5 lm � 3.5 lm) and analyzed with WSxM 3.0 beta 10.0 to obtainroughness (Ra) and peak heights of each film. The mean with 256independent measurements of surface roughness was calculatedon a 100 lm � 100 lm surface.

2.10. Protein adhesion study

The protein adsorption study was performed using a MicroBCA™ Protein Assay Reagent Kit purchased from Sigma–Aldrich.In order to analyze protein adsorption on the surfaces (15 mm indiameter), tissue culture-treated polystyrene surface (TCPS), PBMAand DB1 copolymer were exposed to 2 mg ml�1 bovine serumalbumin (BSA). Following overnight incubation at 37 �C, each sur-face was gently rinsed with PBS and any adsorbed proteins weredesorbed by incubation with 1 M NaCl for 1 h at 37 �C. The concen-tration of BSA was then determined according to the kit’s instruc-tions. The protein concentration was calibrated with the standardBSA solutions included in the test kit. Samples were incubatedfor 1 h at 60 �C and the absorbance was then read at 540 nm usinga Multiskan EX Primary EIA V.2.1-0 microplate reader. At leastthree replicates were read for each sample.

2.11. Cell proliferation on copolymer films

Endothelial cells (ECs; human umbilical vein endothelial cells(HUVECs) from ATCC) and smooth muscle cells (a rabbit aortic vas-cular smooth muscle cell line Rb1 (VSMCs) [23,24]) were used in thisstudy. The films (15 mm in diameter) were placed at the bottom of24-well plates and the two surfaces were submitted to ultravioletlight at 254 nm for 15 min. The ECs and VSMCs cells were seededon each film at 5 � 103 cell cm�2. Cells were cultured in Dulbecco’smodified Eagle’s medium (DMEM, Gibco) with 4.5 g l�1 of glucoseand 2% L-glutamine supplemented with 10% calf serum and 1% pen-icillin and streptomycin. The cells were incubated for 5 days at 37 �Cin air containing 5% CO2 in a humidified incubator. The transparencyof all films allowed the cell morphology to be seen with a classical in-verted phase contrast light microscope (Zeiss, Axiovert 100). Thekinetics of proliferation was determined daily for 5 days after start-ing cell culture. The cell layer adhering to the each disk was washedtwice with PBS to remove any remaining material and loose cells,and the cells were detached with trypsin/EDTA 0.25/0.02 (w/v),resuspended and counted with a ZM Coulter Counter. The mean cellnumber from two wells per tested material was counted and the re-sults from two separate experiments were averaged. Cell prolifera-tion was also observed by fluorescence microscopy, with cellscultured on films of copolymers for 5 days. Cells were fixed with10% paraformaldehyde, permeabilized with 0.1% Triton X-100 andlabeled with intracellular F-actin (phalloïdin FluoProbes547) forthe cytoskeleton and with 40,6-diamidino-2-phenylindole (DAPI)for the nuclei.

2.12. Endothelial cell migration

In vitro migration assay of ECs was performed as described [23].Briefly, ECs at 5 � 103 cell cm�2 were seeded into 24-well plates,where DB1 film had been placed in the bottom of the wells. Equiv-alent numbers of cells were also seeded directly into 24-well platesof TCPS as a reference surface. After 5 days, EC monolayers werescraped (denuded) using a 10 ll plastic micropipette tip andwashed with sterile PBS (0.01 M, pH 7.4), after which the PBSwas replaced with DMEM. The area of migration was observed

by optical microscopy and measured at 8, 24, 32 and 48 h after tak-ing pictures with a digital camera (Pentax istD).

3. Results and discussion

Dextran-graft-polybutylmethacrylate copolymers were synthe-sized in this work. A redox initiation was performed with an acidifiedaqueous solution (pH 0.32). The decrease in pH of the reaction mix-ture and the increase in optical density (caused by the appearance oftiny white particles shortly after the addition of the cerium andmonomer) indicated the initiation of copolymerization (Fig. 1). Incontrast, this reaction was not obtained in the absence of either dex-tran or butylmethacrylate monomer, indicating that the reactionwas not the formation of homopolymers. The resulting copolymerswere then purified by extensive extraction with acetone in a Soxhletextractor to eliminate butylmethacrylate monomer. Three copoly-mers (DB1, DB2 and DB3) were successfully synthesized by increas-ing the dextran content in the starting solution. Interestingly, theresulting copolymers were not soluble in any pure solvents of theparent homopolymers (water, DMSO, dichloromethane, acetone,chloroform, dioxane and THF), but only in the 90/10 mixture ofTHF/water.

The absence of a common solvent between the copolymers andthe parent homopolymers prevented a nuclear magnetic resonancestudy from being performed. However, for semi-quantitative anal-ysis, comparison of the FTIR spectra of PBMA and dextran for thethree copolymers showed the presence of a typical absorptionband of PBMA at 1730 cm�1, corresponding to C@O stretchingvibrations, and a characteristic band for dextran, correspondingto OH stretching absorption at 3400 cm�1 (Fig. 2). The intensityof the OAH band increased with increasing proportion of dextranin the copolymers. Semi-quantitative analysis of the integratedarea of the 3400 cm�1 peak as previously described [25] gave a cal-ibration curve using physical mixtures of known proportions ofdextran and PBMA powders, and for the copolymers with dextrancontents of 11, 30 and 37 wt.% (DB1, DB2 and DB3, respectively).

The Tgs of the copolymers were measured by DSC, taking themid-point in the heat flow vs. temperature transition as the Tg va-lue. The DSC curves of the single homopolymers showed single Tg

values, of 153 �C for dextran (Fig. 3a) and 28 �C for PBMA (Fig. 3b).The DSC curves of the physical mixtures dextran + PBMA showedtwo separate Tg values, corresponding to the Tgs of the singlehomopolymers (Fig. 3c). Each DB copolymer showed a single glasstransition, with a value intermediate between the parent

Fig. 2. FTIR spectra of (a) PBMA, (b) dextran, (c) DB1, (d) DB2 and (e) DB3. Note thepresence of a typical absorption band of PBMA at 1730 cm�1 corresponding to C@Ostretching vibrations, and a characteristic band for dextran corresponding to OHstretching absorption at 3400 cm�1.

S.M. Derkaoui et al. / Acta Biomaterialia 6 (2010) 3506–3513 3509

homopolymers (Fig. 3d–f). The Tg values of DB copolymers in-creased with increasing dextran content (31, 38 and 42 �C forDB1, DB2 and DB3, respectively). Thus, the existence of a singleglass transition in the copolymers confirmed the grafting of BMAto dextran in DB copolymers. These values also greatly differedfrom the copolymers when methylmethacrylate was used insteadof butylmethacrylate (Tg = 126 �C) [22].

Thin copolymer films were then obtained by dissolving copoly-mers in 90/10 (v/v) THF/H2O followed by controlled solvent evap-oration. The films were homogeneous, transparent and insoluble inwater (in contrast to dextran). The DMA gave three parameters: (i)the storage modulus, E0; (ii) the loss modulus, E0 0; and (iii) tan d,which is the ratio E0 0/E0, for determining the occurrence of molecu-lar mobility transition. DMA is a technique whereby a small defor-mation is applied to a sample in a cyclic manner. This allows thepolymer response to stress, temperature and frequency to be stud-ied. The data revealed a storage modulus E0 at 37 �C that increasedwith increasing dextran, with values of 480 MPa for DB1, 680 MPa

Fig. 3. DSC curves showing the Tg of polymers. Curves for (a) dextran, (b) PBMA, (c) mixtemperature range from �50 to +200 �C at a heating rate of 10 �C min�1. In each case, twpoint in the shift of heat flow baseline.

for DB2 and 1010 MPa for DB3. In all cases, E0 decreased withincreasing temperature. The elastic modulus, i.e. the stiffness, de-creased with increasing amount of dextran in the copolymers. Onthe other hand, the temperature dependence of tan d showed max-imum peaks for DB1 (57 �C), DB2 (60 �C) and DB3 (64 �C). Thesevalues were much lower than that for pure polymethylmethacry-late (109 �C) or dextran-graft-polymethylmethacrylate (141 �C)[22]. DMA on films and DSC from polymer powders gave respectivetransition temperatures obtained in different conditions. Never-theless, the results obtained were all consistent for DB copolymers.They both showed the appearance of new structures (not a mix ofpolymers) with properties quite different from the homopolymers.Similar to the DSC results, each film exhibited one glass transitionrelaxation, confirming the graft copolymerization, and the detec-tion of a single peak in tan d is a sufficient criterion to assumethe miscibility. Differences in temperature transition values withDSC are attributed to the presence of water tightly associated tocopolymers, which induced changes in the mobility of themacromolecules.

Creep test was also carried out in order to study differences be-tween DB1, DB2 and DB3 copolymers. Elongation of copolymersunder load is high (Fig. 4, left part), with values reaching 380%.As shown in Fig. 3, the deformation velocity increases with strain.The elasticity of the films decreases with increasing dextran con-tent in the DB copolymer (Fig. 4, right part).

The hydrophilicity of materials was evaluated by measuringtheir water contact angle (h), an indication of surface wettability.PBMA exhibited the highest contact angle (h = 99�) and was themost hydrophobic material. For comparison, the average contactangle for a TCPS surface was 60�. The DB1 (h = 80�) and DB2(h = 73�) films had moderate levels of wettability, whereas theDB3 (h = 57�) film showed the lowest contact angle. The contactangles of copolymer films exhibited a progressive decrease withincreasing dextran content, thus indicating increased hydrophilic-ity. All films were further characterized by AFM in tapping modeimaging to obtain information on their surface morphology. DBfilms exhibited a smooth surface, with no notable trends in topo-graphical features upon increasing dextran content (Fig. 5). Thevalues for roughness of films were similar (PBMA Ra = 1.9; DB1Ra = 1.6; DB2 Ra = 2; DB3 Ra = 2.7). The roughnesses observed onthese surfaces were low, and suitable for comparison betweensamples for the cell adhesion assay [26].

The copolymers were tested in vitro by seeding ECs and VSMCson films. After 6 h of culture, analysis of the VSMC morphology on

ture of dextran + PBMA (50% + 50%), (d) DB1, (e) DB2 and (f) DB3 are reported in ao consecutive scans were carried out on each sample. The Tg was taken as the mid-

Fig. 4. (Left) Representative example of elongation of copolymer DB1; (right) comparison of initial deformation velocity of DB1 (�), DB2 (j) and DB3 (N). A constant load(100, 125, 150, 175 and 200 g) was applied to DB1, DB2 and DB3 films (15 mm length, 5 mm width and 0.035 mm thickness). Elongation of films was measured from picturesevery minute until rupture. Curves were obtained for the percentage of expansion vs. time, and initial deformation velocity was then deduced for all copolymers. PBMA wastoo fragile to be characterized, and dextran did not allow obtaining any film.

Fig. 5. AFM of PBMA (top left), copolymers DB1 (top right), DB2 (bottom left) andDB3 (bottom right). All films were imaged under tapping contact mode with aresonance frequency range of 50–100 Hz. Three areas per sample were imaged andanalyzed to obtain the roughness (Ra) of each film.

Fig. 6. Effect of film copolymers on endothelial cell proliferation. ECs were culturedon films of PBMA or copolymers DB1, DB2 and DB3. Top panel: pictures were takenafter 5 days in culture (original magnification 12.5�). Bottom panel: endothelial cellgrowth curve for PBMA and copolymers DB1, DB2 and DB3. Values are means ofduplicate wells in two experiments.

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PBMA film showed clear adhesion with cells, which changed fromspherical to fusiform in shape and spread out via pseudopodia, butno adhesion of ECs on hydrophobic PBMA was observed. After5 days, VSMCs attained confluence on PBMA (never with ECs),and the VSMC proliferation and morphology were comparable tocontrol cultures on TCPS (Fig. 6).

VSMCs did not proliferate on DB1 (Fig. 6). Interestingly, analysisof endothelial cell numbers indicated that ECs proliferated verywell on copolymer film DB1 (dextran 11%), reaching confluenceafter 5 days (Fig. 7). This marked difference for DB1 film (Fig. 8)was unexpected, but is likely to be related to the particular surfaceenergy of the film and its subsequent adhesion of serum proteinsthat could differentially interact with the membrane componentsinvolved in cell adhesion. When the dextran content in the copoly-mers increased to 30% or 37%, the number of spreading cells (ECsand VSMCs) decreased considerably, and proliferation of bothtypes of cells was very limited (Figs. 6–8). This result indicated thatEC proliferation is sensitive to the surface energy of films, thechemical components being the same between the copolymers.In this context, many studies have proved that surface characteris-tics of biomaterials such as wettability greatly affect the cell adhe-

sion and growth on materials [27,28], with the preferentialadsorption of some serum proteins. In the work of Risbud et al.[29], poly(methyl methacrylate) (PMMA) substrates were exposedto radiofrequency-induced argon and nitrogen plasmas to decreasethe water contact angle. They found that the untreated PMMA sur-face did not support endothelial growth and that PMMA treated byplasma was better, but exhibited lower growth rates than TCPS. Ina recently published work [30], we studied cell adhesion and pro-liferation on aluminum surfaces covered by five different chemicalgroups. Neither human VSMCs nor HUVECs could be cultured onaluminum spots presenting alkyl, carboxyl, quaternary aminesand nitrilotriacetic acid chemical functions. Both the wettabilityand chemical nature of the surface appeared to be importantparameters that could explain these differences. Indeed, humanvascular cells were only cultured on silica-coated spots presentinga moderate wettability (h = 67.5� ± 3.5). HUVECs were also de-

Fig. 7. Effect of film copolymers on VSMC proliferation in vitro. VSMCs werecultured on films of PBMA or copolymers DB1, DB2 and DB3. Top panel: pictureswere taken after 5 days in culture (original magnification 12.5�). Bottom panel: cellgrowth curve for PBMA and copolymers DB1, DB2 and DB3. Values are means ofduplicate wells in two experiments.

Fig. 9. BSA adsorption on the surface of TCPS, PBMA and DB1. Dextran did not allowobtaining any film for protein adhesion study. Surfaces (15 mm in diameter) ofTCPS, PBMA and DB1 copolymer were exposed to 2 mg ml�1 BSA overnight at 37 �C.Each surface was rinsed with PBS and the concentration of BSA desorbed byincubation with 1 M NaCl for 1 h at 37 �C was then determined according to theinstructions of the micro-BCA kit with an absorbance reading at 540 nm and acalibration curve. At least three replicates were applied for each sample. Nosignificant difference was obtained between DB1 and TCPS, whereas BSA adsorptionon PBMA was significantly higher (*p < 0.05).

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scribed by others to adhere to self-assembled monolayers of alkan-ethiols having CH3/OH with h = 40�, and HeLa cells on both CH3/OHand CH3/COOH with h = 50� [31]. The results obtained here withvascular cells on DB1 film are in agreement with these observa-tions, with PBMA exhibiting the highest contact angle (h = 99�)and DB1 film (h = 80�) having moderate wettability. To explainthe cell response, it has been shown in the literature that the ser-um proteins necessary for cell adhesion adhere according to thechemical groups and wettability of surfaces. By comparison of me-tal-oxide-based surfaces comprising combinations of titanium,aluminum, vanadium and niobium, both fibronectin and albuminadsorption were seen to be significantly greater on the non-alumi-num regions, suggesting that differences in cellular response corre-late with a modulation of the concentration of serum proteins onthe surface [32]. On low density polyethylene, maximum adsorp-

Fig. 8. Effect of DB1 film on EC (left), or VSMC proliferation in vitro (right). Cells were clabeled with intracellular F-actin (phallo FluoProbes547 in red) for cytoskeleton and DAPare representative of three experiments.

tion of albumin, fibrinogen and human Factor XII was observedon surfaces in the range of h = 60–65� [33]. In this context, we havecarried out a preliminary study with BSA. BSA was used as a modelprotein, and is the most abundant protein in blood. The data dem-onstrated a significantly lower BSA adsorption on DB1 as comparedto PBMA (Fig. 9). Interestingly, the BSA adsorption on DB1 did notdiffer from that on TCPS used as a positive control surface in cellculture experiments (Fig. 9). Further experiments will be per-formed on DB1 with different specific proteins, such as vitronectin,fibronectin and laminin, involved in cell adhesion, migration andproliferation.

Surface roughness is another important parameter in biomedi-cal materials. It may affect cell adhesion [34,35], and could com-promise hemocompatibility in the case of vascular prostheses[36]. According to the AFM results, the roughnesses of the differentfilms were similar. Consequently, the proportion of dextran in thecopolymer, which greatly affected the hydrophilicity, seems to bethe main parameter determining the favorable endothelializationof the surface of these materials.

The chemical approach reported here by copolymerization be-tween two different macromolecular structures allowed tunableproperties to be obtained for excellent endothelial cell coverageof DB1. The influence of mechanical properties of polymeric sur-faces on cell adhesion was largely reported in the literature. For in-stance, Yung and Cooper [38] have synthesized polyurethanesusing glycerophosphorylcholine (GPC) as a chain extender. By

ultured on films of copolymer DB1 for 5 days. Cells were fixed, permeabilized andI (in blue) for nucleus. Cells were observed under a fluorescent microscope. Pictures

Fig. 10. EC migration of a denuded EC monolayer on DB1 film (top left panel) or onTCPS (top right panel). The bar in each picture is 100 lm. Bottom panel:quantitative analysis of EC migration vs. time. The area of migration was observedby optical microscopy and measured at 8, 24, 32 and 48 h. Values are representativedata obtained in two independent experiments.

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altering the ratio of GPC to butanediol, a series of polymers was ob-tained. DSC and DMA showed that the polymer with the highestphosphorylcholine content had the lowest Tg and the highest ten-sile strength and Young’s modulus among the polymers studied.Under shear rates from 20 to 120 s�1, neutrophils did not adhereto this surface, though they did adhere to the control polyurethane.Cell spreading was observed on the control polyurethane but noton the modified polyurethane. Hsu and Kao [37] have also studied

the bulk and surface properties of various cast poly(carbonate ure-thane) films. The glass transition temperatures measured by DMAand the surface images obtained by AFM indicated that these sur-faces with various degrees of nanophase separation correlated verywell with endothelial cell attachment and proliferation. We alsorecently studied the influence of the local stiffness of polysaccha-ride scaffolds on stem cell adhesion [39]. Numerous studies in factfocus on the relationship between local mechanical properties andstem cell differentiation [40]. By modulating the mechanical prop-erties of the copolymers obtained here with various butylmethac-rylate and dextran contents, we could expect to influence the celladhesion/proliferation/differentiation of, for instance, progenitorendothelial cells on these surfaces. Further experiments need tobe performed to investigate this question.

Finally, a migration assay using a wound created in a confluentEC monolayer was used to assess the effect of DB1 on EC migrationcompared to TCPS (the other polymer surfaces did not allow a con-fluent EC monolayer). Cell migration was measured as the denudedarea into which ECs were invading. The endothelial cells on DB1copolymer migrated extensively from the edge of the scrape intothe denuded space (Fig. 10, top left panel), reaching confluenceat 32 h on copolymer. In contrast, EC migration on TCPS was slower(Fig. 10, right panel), and migration area remained non-confluentuntil 48 h. This showed that the surface of the DB1 film was suit-able for both EC migration and proliferation.

4. Conclusion

In this study we have synthesized new copolymers having thebiological properties of polysaccharides and the mechanical prop-erties of polyacrylate. Copolymers of dextran and butylmethacry-late were successfully synthesized and characterized. FTIR, DSCand DMA revealed that graft copolymerization has taken place be-tween the two non-miscible polymers with different natures. Incontrast to dextran, water-insoluble, homogeneous and transpar-ent films were prepared from all DB copolymers. The mechanicalproperties were enhanced as compared to our previous report withdextran-graft-polymethylmethacrylate [22]. Interestingly, DB1was able to inhibit VSMC proliferation and at the same time sup-port endothelial cell proliferation and migration. Lastly, cell adhe-sion and proliferation can be controlled by varying the content ofdextran incorporated into the DB copolymer. DB1 copolymerexhibited excellent characteristics for applications requiring theendothelialization of materials. Therefore, a potential benefit ofthis approach is the availability of bioactive surfaces with tunableproperties for biomedical devices. We can thus propose its use forapplications such as the coating of catheters or endovascular pros-theses (stents). Interestingly, some clinical stents are already cov-ered by a layer of PBMA; the reported copolymers could furtherimprove the biocompatibility of these materials.

Acknowledgements

This work was supported by INSERM, University Paris 13 andIFR Paris Nord-Plaine de France. A.P. was supported by programFranco-québécois Samuel de Champlain. The authors wish to ex-press their gratitude to Dr. Cédric Lorthioir (CNRS Thiais, France)for his excellent technical assistance in DMA tests.

Appendix A. Figures with essential colour discrimination

Certain figures in this article, particularly Figures 5–8, are diffi-cult to interpret in black and white. The full colour images can befound in the on-line version, at doi:10.1016/j.actbio.2010.03.043.

S.M. Derkaoui et al. / Acta Biomaterialia 6 (2010) 3506–3513 3513

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