Materials Science and Engineering C 33 (2013) 362–369

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Materials Science and Engineering C

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The surface modification of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)copolymers to improve the attachment of urothelial cells

José M. García-García a, Isabel Quijada-Garrido a,⁎, Laura López b, Rodrigo París a,María Teresa Núñez-López b, Enrique de la Peña Zarzuelo c, Leoncio Garrido a

a Departamento de Química-Física de Polímeros, Instituto de Ciencia y Tecnología de Polímeros, Consejo Superior de Investigaciones Científicas (ICTP-CSIC), Juan de la Cierva, 3,E-28006, Madrid, Spainb Hospital Universitario Fundación de Alcorcón C/Budapest, 1, E-28922, Alcorcón, Madrid, Spainc Servicio de Urología del Hospital Universitario Fundación de Alcorcón, Avda. Budapest 1, E-28922, Alcorcón, Spain

⁎ Corresponding author. Tel.: +34 91 258 74 30, +91 564 48 53.

E-mail address: iquijada@ictp.csic.es (I. Quijada-Gar

0928-4931/$ – see front matter © 2012 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.msec.2012.08.052

a b s t r a c t

a r t i c l e i n f o

Article history:Received 20 February 2012Received in revised form 3 August 2012Accepted 29 August 2012Available online 6 September 2012

Keywords:Poly(hydroxybutyrate-co-hydroxyhexanoate)Mechanical propertiesUrotheliumYIGSRCell attachment

Biocompatible and biodegradable poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(HB-co-HHx)] sub-strates were modified to improve the attachment of porcine urothelial cell culture. The pristine copolymerexhibits excellent mechanical properties to replace the bladder tissue, but its surface lacks chemical function-alities to interact with cells. Thus, wet chemical treatments based on NaOH and ethylenediamine in aqueous[ED(aq)] and isopropanol [ED(isoOH)] media to functionalize the P(HB-co-HHx) films surfaces were com-pared. Among these treatments, short ED(aq) treatment was able to decrease the hydrophobicity, renderinga surface with amino groups and without a significant alteration of the mechanical properties. Furthermore,to enhance the interaction with urothelial cells, laminin derived YIGSR sequence was covalently bound tothese amino functionalized substrates. The focal attachment was clearly improved with this last treatment,comparing with those results found with the unmodified and first-step functionalized P(HB-co-HHx).

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Poly(hydroxyalkanoate)s [P(HA)s], a family of natural biodegradableand semicrystalline polymers, produced by many types of micro-organisms, have been widely employed for biomedical applications[1–3]. Among them, in the last few years, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(HB-co-HHx)] (Fig. 1), a random copolymer ofHB with a small amount of HHx, has revealed as a biomaterial withpromising properties [1,4,5]. The propyl side groups of HHx reduce themelt temperature and crystallinity of the copolymers and, therefore,the flexibility is significantly improved comparedwith that of P(HB) ho-mopolymer [6]. In fact, the secondary crystallization, which reduces theamorphous phase and produces a notorious embrittlement of P(HB)[7–10], is almost suppressed in copolymers with HHx fraction higherthan 10% mol [6]. Furthermore, P(HB-co-HHx) exhibits some other at-tractive properties, such as anaerobic and aerobic biodegradability, hotalkaline digestibility, hydrolytic stability, good odor and oxygen barrierand good biocompatibility for many types of cells [11–17]. For all thesereasons, P(HB-co-HHx) is a potentially ideal biomaterial to be used intissue engineering applications.

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rido).

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Often bladder dysfunction and diseases lead to therapeutic inter-ventions that require partial or complete replacement of damaged tis-sue. For this reason, the development of biomaterials to repair thebladder by promoting the adhesion and growth of urothelial cells isof interest. Relatively few studies have investigated the interactionsof human urinary tract-derived cells with biodegradable polyesters[18–22]. Recently, the use of PHAs as substrate for the attachmentof bladder cells has been reported [23,24]. Thus, Renard et al.[23] studied the cell adhesion and proliferation of human bladdercarcinoma RT112 cells on unfunctionalized and functionalizedpolyalkanoates. On other hand, our research group [24] demonstrat-ed that poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(HB-co-HV)]functionalized with a laminin fragment clearly improved the adhesionof urothelial cells. However, considering the flexibility of bladder tissue,the deterioration of the mechanical properties of P(HB-co-HV) sub-strates due to the modification treatments [24], together with the em-brittlement due to the secondary crystallization could compromise thepolymer use for this in vivo application. Since, as indicated above,P(HB-co-HHx) exhibits higher flexibility and does not experience a de-cline of mechanical properties with time (secondary crystallization), itwas selected in thiswork as substrate to study in vitro bladder cell adhe-sion. Thus, the main objective of this work will be to optimize the sur-face properties of P(HB-co-HHx) films for improving attachment ofurothelial cells. As it occurs with other polyesters, the hydrophobicproperties of P(HB-co-HHx) surfaces could be a disadvantage for cell

Fig. 1. Chemical structure of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)[P(HB-co-HHx)].

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adhesion. But, on the other hand and due to the flexibility of the bladdertissue, it is necessary to design a reliable method to modify the surfaceof the P(HB-co-HHx) substratewithout a significant alteration of itsme-chanical properties. The surface modification of P(HB-co-HHx) filmswith NaOH wet methods has been explored [25–28] but, to the best ofour knowledge, aminolysis treatments have not yet been investigated.Thus, in this work wet chemical methods based on alkaline hydrolysiswith NaOH and aminolysis with ethylenediamine (ED)were compared.On other hand, it is also known that the attachment of active peptidicfragments into the polymer surface is a successful procedure toincrease cell attachment and proliferation [29–33]. Furthermoreand taking into account the desired application, these functionalgroups introduced at the surface were used to covalently linkYIGSR, a laminin fragment which has been found to be a function-ally active region for cell attachment, spreading and migration, inextracellular matrices and, in particular, in the urothelial basementmembrane [34].

2. Materials and methods

2.1. Materials

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(HB-co-HHx)]with a 12% by mol of 3-hydroxyhexanoate units was kindly providedby Professor Chen GQ, Tsinghua University. Ethylenediamine (ED)employed for surface modification was supplied by Fluka (Steinheim,Germany) while isopropanol (isoOH) was supplied by Panreac(Barcelona, Spain) and chloroform (CHCl3) to dissolve P(HB-co-HHx) forcasting by Carlo Erba Réactifs-SdS (Val de Reuil, France). Water for allreactions and solutions was Milli‐Q from water purification facility(Millipore Milli-U10). Ninhydrin to evaluate amino group concentrationwas provided by Sigma-Aldrich (Steinheim, Germany). N-Ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide sodium salt (sulfo-NH), 2-(N-morpholino)ethane sulfonic acid (MES) and laminin fragment YIGSR (Tyr-Ile-Gly-Ser-Arg) was provided by Sigma-Aldrich. All other items were pur-chased from Sigma-Aldrich Co. and used as received, if not otherwisestated.

2.2. Preparation of P(HB-co-HHx) films

P(HB-co-HHx) films were prepared by solvent casting from poly-mer solutions in chloroform at a concentration of 50 mg mL−1 intoa glass mold, and maintained at room temperature 48 h to allow theevaporation of the solvent and, after that, vacuum dried during 8 h.After this time elapsed, sample weight remained constant. The filmsobtained with a thickness of 70±1 μm were stored at −18 °C.

2.3. Surface functionalization of P(HB-co-HHx) films by ethylenediamineaminolysis

To introduce amino functional groups onto P(HB-co-HHx) filmssurface, two different reaction solutions were employed. For treat-ments with aqueous solution, P(HB-co-HHx) films of 4×2 cm wereimmersed in ED aqueous solutions at 20 and 40% (v/v) and 50 °C,with continuous magnetic stirring for 20, 40, 60, 80, 120, 200, 280and 360 min. When isopropanol solutions were used, the films wereimmersed in solutions at 5 and 20% (v/v) and 30 °C, during 20, 30,40, 60, 120 and 200 min, under continuous magnetic stirring. Afterthese treatments, samples were subjected to extensive washingwith water Milli-Q during 24 h refreshing the water bath, at least,three times during this period. Then, samples were dried 4 h in avacuum desiccator and stored at −18 °C.

2.4. Surface functionalization of P(HB-co-HHx) films by NaOH hydrolysis

The generation of carboxyl and hydroxyl functional groups ontoP(HB-co-HHx) films (4×2 cm) was accomplished with immersionin 0.25, 0.5 and 1 M NaOH solutions at 30 °C with continuous stirringfor 5, 10, 20, 30, 40, 50 and 60 min. After treatment, samples weresubjected to extensive washing with water Milli-Q during 24 h, re-freshing the bath, at least, three times during this period. Washed sam-ples were dried 4 h in a vacuum desiccator and stored at −18 °C untilfurther use.

2.5. Covalent attachment of YIGSR sequence onto pretreatedP(HB-co-HHx) films

YIGSR was covalently attached to the P(HB-co-HHx) surface byamine functionalities previously introduced with the ED treatment.Solutions of the peptide, EDC and sulfo-NH were prepared in a0.1 M solution of MES buffer (pH 6.0). The peptide and the EDC andsulfo-NH solutions were combined for peptide activation, in a 1000:1:1(peptide:EDC:sulfo-NH) molar ratio. Then, the pretreated films were in-troduced in the new solution and were shaken at 120 rpm during 24 hat room temperature. Afterwards, the surface-activated films were im-mersed in a PBS buffer solution (pH 7.4) for one hour, under stirring(120 rpm). Then, the samples were removed from solution, washedwith deionized water several times and dried. The films were stored inhydrophilic conditions, at−18 °C, until their use. A schematic represen-tation of the P(HB-co-HHx) surfacemodification employed in the presentwork is shown in Fig. 2.

2.6. Contact angle measurement

Contact angles with Milli‐Q water on nontreated and treatedP(HB-co-HHx) films were measured at room temperature and air at-mosphere using the sessile drop method with a goniometer KSVTheta (KSV Instruments Ltd, Finland). Contact angles were measuredat five different sites.

2.7. Ninhydrin assay

The presence of amine groups onto the P(HB-co-HHx) surface wasassessed by a ninhydrin assay. It is well known that ninhydrin bindsto amine groups present on the substrate surface producing a purplepigment [35], with a maximum absorption at a wavelength of 560 nmin the solvent mixture chloroform/isopropanol (1:1), that can bedetected with UV-Visible spectrophotometer (Cary 3 BIO-Varian).Thus, a solution 1 M of ninhydrin in ethanol is prepared and sixP(HB-co-HHx) disks (ϕ=4 mm) of each sample were immersed insolution aliquots for 1 min. After reaction, each disk was transferredinto a glass vial and heated for 15 min at 70 °C in a water bath. Thetreated disks were then dissolved in 4 mL of chloroform/isopropanol

Fig. 2. (a) Amino surface functionalization by aminolysis treatment in aqueous medium, (b) activation of the carboxylic groups of YIGSR and (c) amide formation and attachment ofthe YIGSR peptide sequence onto the P(HB-co-HHx) surface.

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and the absorbance measured at 560 nm. The amine functionalizationwas estimated from a calibration curve (molar extinction coefficientε560 nm=701.2±58.5 L mol−1 cm−1), previously determined usingdifferent solutions of known ethylenediamine concentrations inchloroform/isopropanol (1:1).

2.8. Tensile test

Tensile properties were measured in an Instron 3866. Bone-shaped cut films (width: 2.2 mm, length: 20 mm and thickness~70 μm) were assayed for each sample studied, with a preload of0.3 N at 5 mm min−1 and at room temperature. The results representthe average of three measurements.

2.9. Porcine urothelial cells procurement and culture

Urothelial cells of porcine origin were used to determine the opti-mal treatment of P(HB-co-HHx) to facilitate the interaction withthese cells. Animal procedures were performed in accordance withthe European Communities Council Directive 86/609/EEC and approvedby the institutional ethics and animal research committees. Bladder bi-opsies were provided by the Laparoscopy Department of “Jesús Usón”at the Minimally Invasive Surgery Centre (Cáceres, Spain). Cell isolationfrom tissue and culture was carried out as follows. Briefly, cell sampleswere immediately obtained from biopsies and processed in asepticconditions. Mechanical dissection was performed to separate theurothelium from the subjacentmuscle and subsequent enzymatic diges-tion on the urothelial sample with 3 mg mL−1 Dispase (Gibco) at 4 °CO/N and constant agitation. The cells obtained were directly seeded on6-cm culture plates and expanded until adequate quantities wereachieved for the experiments with polymer substrates. For cell expan-sion, complete medium for growth of keratinocytes with compositionof 3DMEM:1F12 supplemented with growth and hormone factors:adenine (0.18 mM), choleric toxin (0.1 nM), thyroid hormone(2 nM), bovine insulin (5 μg mL−1), OH-cortisone (0.4 μg mL−1),epidermal growth factor (10 ng mL−1), and HEPES buffer (20 mM)was used.

2.10. In vitro study of the P(HB-co-HHx)-urothelial cell interactions andmorphological observation by scanning electron microscope (SEM)

Urothelial cells were seeded on modified P(HB-co-HHx) films(disks of 14 mm Ø) and were incubated in vitro. P(HB-co-HHx)

films were sterilized with a 70% ethanol solution, dried and placedat the bottom of 24 well plates. The experiments were performedseeding 1.3 105 cells/cm2 on standard tissue culture plates and onthe substrates, and adding carefully complete keratinocyte growthmedia to avoid flotation of P(HB-co-HHx) disks. After the specifiedtime elapsed, samples were removed from the medium and gentlywashed to remove unattached cells. Then, cell cultures were fixedwith glutaraldehyde (1:100), dehydrated stepwise with mixtures ofethanol/water, progressively rich in alcohol. Samples of fixed cellswere coated with Au/Pd and observed on a Philips XL30 (Philips,Eindhoven, the Netherlands) SEM at ambient temperature using theparameters indicated in each photograph.

3. Results and discussion

Wet chemical methods were used to modify P(HB-co-HHx)substrates because they allow a well control and reproducibility ofthe polymer functionalization [36] These treatments had two aims:(i) to improve the interaction with cells by decreasing the hydropho-bicity of the surface and (ii) to provide the surface with active func-tional groups for a further modification with the YIGSR sequence.The measurement of water contact angles on modified substratesallows the evaluation of changes in hydrophilicity produced in theirsurface properties. Fig. 3 shows the variation of the contact anglesfor P(HB-co-HHx) films treated with the three wet methods used inthis work, ED/water (ED(aq)), ED/isopropanol (ED(isoOH)) andNaOH solutions. The contact angle of pristine PHBHHx was about93°, in agreement with other values reported in the literature [37].This value somewhat higher than the typical angle obtained forP(HB-co-HV) copolymers (about 80°) [12] is attributed to the higherhydrophobic character of HHx compared to that of HV. Apart fromthat, it can be observed that, an increase of the hydrophylicity,reflected in a decrease of the contact angle, was obtained in allcases with increasing the treatment time as it was observed forP(HB-co-HV) [19]. It is important to remark that different treatmenttimes were used with each methodology since, as it will be laterexplained, they also affect the mechanical properties and a require-ment for the final applications is to maintain them in acceptablevalues. Using isopropanol as the aminolysis medium, only a smoothdecrease of the contact angle was observed, in the same way thatwhen samples were incubated in NaOH solutions. These results arein contrast with those obtained for P(HB-co-HV) copolymers, wherea significant reduction of contact angle values was also obtained

Fig. 4. Amine surface density estimated by the ninhydrin assay for P(HB-co-HHx) filmstreated with (a) ED aqueous solutions and (b) with ED isopropanol solutions.

Fig. 3. Contact angle measurements of P(HB-co-HHx) films treated with (a) ED aqueoussolutions, (b) ED isopropanol solutions and (c) NaOH solutions.

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with 1,6-hexanediamine treatment in isopropanol medium [38].However, the reduction of the contact angle is very remarkable forthe ED(aq) treatment, in which a decrease to about 68° was attained,independently of the ED concentration employed.

Ninhydrin assay was used to estimate the number of aminogroups introduced at the surfaces using both aminolysis media.Fig. 4a shows the density of surface amino groups as a function ofthe aminolysis time for the ED(aq) treatment. The number of −NH2

groups increases almost exponentially with the treatment time,being higher for the most concentrated ED solution. Fig. 4b shows

the results for the aminolysis in isopropanol medium. In this case,the degree of functionalization with the ED(isoOH) treatment is sig-nificantly higher than when ED(aq) is employed, about seven timeshigher for a similar ED concentration. For both media, the concentra-tion of amine groups introduced increases with augmenting EDconcentration.

These results are in clear contrast with the wettability resultsobtained by contact angle measurements. However, it is known thatthe contact angle depends not only on the number of functionalgroups, but also on other factors such as the roughness of thesubstrate [36,39]. For this reason, the morphology of the surface ofP(HB-co-HHx) substrates was investigated by SEM. Thus, Fig. 5shows the effect of the three treatments, ED(aq), ED(isoOH) andNaOH on the P(HB-co-HHx) surface.

Interestingly, the micrographs corresponding to samples subjectedto both treatments which lead to the lower reduction of the contactangle, i.e. ED(isoOH) andNaOH, displaymany holes and suggest a strongdegradation of the surface. In fact, similar results were observed by Shenet al. [28] for P(HB-co-HHx) films treatedwith NaOH solutions. In accor-dancewithWenzel'smodel, hydrophobic surfaces (θ>90°)will becomemore hydrophobic with increasing degree of roughness [40]. Therefore,the high values of contact angles measured in samples modified with

Fig. 5. Scanning electron micrographs corresponding to: (a) pristine P(HB-co-HHx) film, (b) P(HB-co-HHx) film treated with 20% ED(aq) for 280 min, (c) film treated with 5%ED(isoOH) for 20 min and (d) film treated with 0.25 M NaOH for 30 min.

Fig. 6. Stress–strain curves for P(HB-co-HHx) films treated with (a) 20% ED aqueoussolution and (b) 40% ED aqueous solution at the times indicated in the figure.

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both ED(isoOH) and NaOH could be attributed to an increase of the sur-face roughness and not to a lower degree of chemical modification.

As indicated earlier, the P(HB-co-HHx) substrates were modifiedto grow autologous bladder tissue in vitro. To find a material for thereplacement of the bladder, in addition to biocompatibility, biode-gradability and proper surface characteristics, it is important thatthis material exhibits mechanical properties that mimic those of thebladder native tissue, since some studies have revealed that cells aresensitive to the mechanical properties of the substrate [41,42]. Fur-thermore, the biomaterial must withstand the surgical requirements,i.e. the suture to surrounding tissues. For these reasons, the mechan-ical properties of the modified substrates were also investigated.Strain–stress curves of pristine P(HB-co-HHx) were compared tothose obtained for substrates subjected to both ED treatments.Fig. 6a illustrates the strain–stress curves for the 20% ED(aq) treat-ment. This mild treatment shows slight influence on the mechanicalproperties. The modulus does not change and the elongation tobreak only decreases after 60 min of treatment. However, in allcases, the results are those typical of a ductile material. As it isshown in Fig. 6b, increasing the ED concentration in aqueous mediumproduces a strong effect on the mechanical properties. Consequently,a treatment of 60 min in 40% ED(aq) leads to high embrittlement ofthe material. In both treatments, ED(isoOH) and NaOH, a large reduc-tion of the elongation at break occurred in all cases, which means aremarkable detriment of the mechanical properties. As an example,the effect of the ED in isopropanol on the mechanical properties ofthe polymer substrates is shown in the Supporting Information. Theincubation of the sample for as short as 20 min in a 5% ED(isoOH) so-lution leads to a reduction of the elongation at break of 500%. Theseresults are in agreement with the deterioration of the surface ob-served in the SEM micrographs of samples treated with ED(isoOH)and NaOH. As shown in our previous work [24], the worsening ofthe mechanical properties is attributed to the breaking of polymerchains by ED and NaOH. This degradation occurs preferentially inamorphous regions leading to more rigid (higher modulus) and, atthe same time, brittle substrates.

Fig. 7. Scanning electron micrographs at two magnifications of porcine urothelial cells 8 h after of seeding in: (a) and (b) pristine P(HB-co-HHx) films, (c) and (d) films treated with20% ED(aq) for 40 min, (e) and (f) films treated with 20% ED(aq) for 120 min, (g) and (h) films treated with 20% ED(aq) for 40 min and YIGSR, and (i) and (j) films treated with 20%ED(aq) for 120 min and YIGSR.

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Taking into account the balance between the degree of modificationand the mechanical properties, it is clear that the treatment with ED inaqueous medium is the more suitable for the desired application, evenconsidering thehigh density of amino groups providedwith the ED alco-holic treatment. Therefore, two P(HB-co-HHx) substrates modifiedwith20% ED(aq) during 40 and 120 minwere selected to bind covalently theYIGSR peptide sequence (Tyr-Ile-Gly-Ser-Arg). This pentapeptidesequence YIGSR, from the B1 chain of laminin, was selected because ithas been found to be a functionally active fragment for cell attachment,spreading and migration [43] and, in particular, in the urothelial base-ment membrane [34]. This sequence has been studied to modified scaf-folds for improving adhesion of several cell lines [30,44]. Our group alsofound a clear improvement in urothelial adhesion on P(HB-co-HV) sub-strates modified with YIGSR laminin sequence [24]. Thus, it was cova-lently attached to the amine-functionalized P(HB-co-HHx) surface by acarbodiimide coupling reaction [45], as illustrated in Fig. 2. The densityof amine groups in films modified with laminin was determined usingthe procedure previously described for the characterization of precursorfilms. Amine densities of 0.9 and 3.7 μmol cm−2 for the samples modi-fied during 40 and 120 min, respectively, were determined. These re-sults represent a balance between the reduction of free amino groupsat the surface, due to the reaction process, and the increasing of aminogroups present in the attached YIGSR.

SEM images allow the study of the urothelial cells morphologyafter seeding on the P(HB-co-HHx) films subjected to different treat-ments, providing information about cytocompatibility of the sub-strate. In Fig. 7, SEM micrographs of urothelial cells after 8 h ofculture, on pristine and several modified P(HB-co-HHx) films areshown. Fig. 7a and b shows the morphology of cells seeded on anunmodified P(HB-co-HHx). Spheroid cells with almost no filopodiaextension are observed, suggesting a poor interaction with the sur-face of the substrates, which is attributed to the high hydrophobicityof the substrate coupled to the lack of chemical functionality. Fig. 7c–fshows SEM images of cells 8 h after seeding on 20% ED(aq) modifiedP(HB-co-HHx) surfaces for 40 and 120 min. After these treatments, aslight improvement in cell-substrate interactions could be appreciated.In fact, compared with unmodified P(HB-co-HHx), an increase in cellpopulation is observed. Cells exhibit a less rounded morphology whencompared with those on pristine polyalkanoate and some of them ex-hibit filopodial extensions, suggesting an improved interaction withthe substrate. This behavior could be associated to the reduction of thesurface contact angle observed in substrates after a long treatmenttime with ED. The cell morphology on ED modified P(HB-co-HHx) sub-strates grafted with the YIGSR laminin sequence is shown in Fig. 7g–j.These figures clearly illustrate how, on these substrates, cells appearedto be capable of reorganizing their basementmembranematrices by cel-lular traction forces, leading to the formation of dense cellular networks.Comparing Fig. 7g and hwith Fig. 7i and j, it can be observed that, in thelatter, there is an increase in the number of cell colonies and their con-nections. This indicates that samples with higher YIGSR content, treatedpreviously with ED(aq) during 120 min, still exhibit better cell interac-tion than samples with lower YIGSR content. This result provides evi-dence that modifying the surface with specific cell-signaling moleculesenhances the cell-substrate interaction more significantly than usingunspecific chemical modifications.

4. Conclusions

The mechanical properties of P(HB-co-HHx) substrates areideal for applications where flexibility is required. Contrary tothe P(HB-co-HV), copolymers based on 3-hydroxyhexanoate showedno signs of aging. Thus, from the mechanical point of view, it seems ap-propriate for the replacement of bladder tissue. However, as it occurswith other polyesters, its surface needs to be modified for a betterinteraction with urothelial cells. Although three different treatmentshave been tested, the treatment of P(HB-co-HHx) substrates with

ethylenediamine in aqueous medium renders the surface with activeamino groups without dramatic loss of their mechanical properties.Aminolysis treatments in alcoholic medium lead to higher density ofamino groups, but also to a considerable decrease of the mechanicalproperties. Thus, short treatment times with ED in aqueous mediumwere used to obtain amino functionalized surfaces and covalently attachYIGSR laminin fragments for improving urothelial cell adhesion. Thismild surfacemodificationwas very effective and substantially enhancedcellular attachment in all experiments performed, without a deteriora-tion of the mechanical properties of the substrate.

Acknowledgements

Financial support provided by the Ministerio de Economía yCompetitividad (MEC) through the Projects MAT2011-22861 andFIS-PI081677 is acknowledged. Authors would like to thank MiguelAngel Sánchez Hurtado from the Laparoscopy Department of “JesúsUsón” Minimally Invasive Surgery Centre for providing the bladder bi-opsies. R. P. thanks the MICINN for a “Juan de la Cierva” contract. Also,J.G. thanks the CSIC for a JAE predoc fellowship.

Appendix A. Supplementary data

Stress–strain curves corresponding to P(HB-co-HHx) films treatedwith ED isopropanol solutions. This material is available free of chargevia the Internet at http://www.sciencedirect.com. Supplementarydata associated with this article can be found, in the online version,at http://dx.doi.org/10.1016/j.msec.2012.08.052.

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