PDA固定RGD和bFGF,RGD和bFGF之间有协同协应。

Polydopamine-mediated immobilization of multiple bioactive molecules for the development of functional vascular graft materialsYu Bin Lee a ,Young Min Shin a ,Ji-hye Lee a ,Indong Jun a ,Jae Kyeong Kang b ,Jong-Chul Park b ,Heungsoo Shin a ,c ,d ,*aDepartment of Bioengineering,College of Engineering,Hanyang University,17Haengdang-dong,Seongdong-gu,Seoul 133-791,Republic of KoreabDepartment of Medical Engineering,BK 21Project for Medical Science,Yonsei University College of Medicine,134Shinchon-dong,Seodaemun-ku,Seoul 120-752,Republic of Korea cInstitute for Bioengineering and Biopharmaceutical Research,Hanyang University,17Haengdang-dong,Seongdong-gu,Seoul 133-791,Republic of Korea dInstitute of Aging Society,Hanyang University,17Haengdang-dong,Seongdong-gu,Seoul 133-791,Republic of Koreaa r t i c l e i n f oArticle history:Received 29May 2012Accepted 5August 2012Available online 20August 2012Keywords:Human umbilical vein endothelial cell Polydopamine coating Surface modi fication EndothelializationDual factor immobilizationa b s t r a c tIn this study,we introduced a simple method for polydopamine-mediated immobilization of dual bioactive factors for the preparation of functionalized vascular graft materials.Polydopamine was deposited on elastic and biodegradable poly(lactic acid-co-3-caprolactone)(PLCL)films,and a cell adhesive RGD-containing peptide and basic fibroblast growth factor were subsequently immobilized by simple dipping.We used an enzyme-linked immunosorbent assay and fluorescamine assay to con firm that we had stably immobilized bioactive molecules on the polydopamine-coated PLCL film in a reaction time-dependent manner.When human umbilical vein endothelial cells (HUVEC)were cultured on the prepared substrates,the number of adherent cells and proliferation of HUVEC for up to 14days were greatest on the film immobilized with dual factors.On the other hand,the film immobilized with RGD peptide exhibited the highest migration speed compared to the other groups.The expression of cluster of differentiation 31and von Willebrand factor,which indicates maturation of endothelial cells,was highly stimulated in the dual factor-immobilized group,and passively adsorbed factors showed a negligible effect.The immobilization of bioactive molecules inspired by polydopamine was successful,and adhe-sion,migration,proliferation and differentiation of HUVEC were synergistically accelerated by the presence of multiple signaling factors.Collectively,our results have demonstrated that a simple coating with polydopamine enables the immobilization of multiple bioactive molecules for preparation of polymeric functionalized vascular graft materials.Ó2012Elsevier Ltd.All rights reserved.1.IntroductionCardiovascular diseases that usually result from malfunction in coronary arteries remain the leading cause of mortality worldwide.Severe arterial occlusion is treated by bypass surgery in which saphenous veins and mammary arteries are used as the preferred graft materials;however,this technique suffers from limited availability of suitable autologous tissue and donor site morbidity [1].Synthetic grafts including poly(tetra fluoroethylene),polylac-tide,polycaprolactone,and polyurethane are also routinely used for surgery [2].These polymeric grafts offer compliable mechanicalproperties,but often fail to provide long-term patency as a replacement for small-caliber blood vessels (<6mm in diameter).The reduced success rate is mainly due to the occurrence of the thrombogenic response and tissue thickening by intimal hyper-plasia (IH),which is prevented by the endothelium in normal blood vessels [3].Endothelialization on vascular grafts is a complex process that involves adhesion,migration,proliferation and differentiation of endothelial cells (EC),which are regulated by a myriad of signals.Extracellular matrix (ECM)proteins serve as non-soluble cues to modulate cell fate through cell signaling cascades [4].Collagen type I and fibronectin stimulate migration and proliferation of EC [5,6].In addition,vitronectin reduces the apoptosis rate through binding to a v integrin in the EC membrane [7].Numerous soluble growth factors are also implicated in new blood vessel formation and EC activities either by direct binding to cellular transmembrane receptors or to ECM proteins.In particular,vascular endothelial*Corresponding author.Department of Bioengineering,College of Engineering,Hanyang University,17Haengdang-dong,Seongdong-gu,Seoul 133-791,Republic of Korea.Tel.:þ82222202346;fax:þ82222982346.E-mail address:hshin@hanyang.ac.kr (H.Shin).Contents lists available at SciVerse ScienceDirectBiomaterialsjournal homepage:w ww.elsevi/locate/biomaterials0142-9612/$e see front matter Ó2012Elsevier Ltd.All rights reserved./10.1016/j.biomaterials.2012.08.011Biomaterials 33(2012)8343e 8352growth factor (VEGF)and basic fibroblast growth factor (bFGF)induce overexpression of their respective receptors within the EC membrane and regulate adhesion,migration,proliferation,and physical organization [8].To recapitulate these multi-step events,one effective engi-neering approach is to immobilize signaling bioactive molecules affecting EC function on synthetic vascular grafts.ECM proteins such as collagen,bovine serum albumin (BSA),fibronectin,and laminin have been immobilized on the surface of grafts,demon-strating improvement of adhesion,spreading,and proliferation of the EC [9].On the other hand,selected domains of ECM proteins have been used to functionalize the graft materials,which affect cell behaviors in the same way as ECM proteins [10].There have been reports that RGD (Arg-Gly-Asp)peptide recognized by integrin can enhance adhesion and migration of EC [11].None-theless,whereas immobilization of these molecules presents some success in enhanced endothelization,no ideal system has beenproposed.In particular,most studies have focused on one aspect of EC function,but few studies have included the multiple signals required for precise mimicry of the real endothelialization condi-tion (i.e.,combination of ECM proteins and growth factors).Although mainly delivered in the soluble form,growth factors such as VEGF,bFGF,and platelet-derived growth factor-BB have been covalently immobilized on the material surface.This has been shown to accelerate proliferation of HUVEC,implying that conju-gation of some growth factors preserves functional activity and stability in a long-term manner [12,13].The aim of this study was,therefore,to test our hypothesis that immobilization of dual factors (ECM components and growth factor)can synergistically modulate EC function.Various techniques are available for immobilization of biomol-ecules on polymeric biomaterials.Most vascular grafts are produced from inert hydrophobic polymers lacking functional moieties for chemical conjugation;therefore,selection of an appropriate functionalization method is critical.Plasma treatment and gamma ray irradiation use high energy sources to introduce reactive groups for subsequent immobilization of target biomole-cules such as fibronectin,collagen,gelatin,and RGD [14e 19].However,these methods have limitations in terms of penetration depth and possible cleavage in polymeric chains negatively affecting the mechanical property and long-term stability of the vascular graft [20].Recently,a facile surface modi fication method that can be applied to versatile solid materials from metal to synthetic polymers by simple dip-coating with dopamine solutionTable 1Sample conditions.Sample code ConditionDP Polydopamine-coated onto PLCL film DPr RGD-immobilized onto DP DPbf bFGF immobilized onto DPFig.1.Schematic diagram of polydopamine-mediated immobilization of bioactive molecules onto PLCL films that can be utilized as a functionalized vascular graft material.Y.B.Lee et al./Biomaterials 33(2012)8343e 83528344was reported [21].Under slightly basic conditions,dopamine can undergo oxidative polymerization,which creates a stable layer that is adherent to the surface of materials.Aside from the simplicity and cleanliness of the reaction environment,unlike with other chemical immobilization methods,post-modi fication of the poly-dopamine layer allows for the functionalization of bioactive molecules containing thiols or primary amines via imine formation and/or Micheal addition.Previous work has utilized the reactivity of polydopamine-coated substrate for immobilization of thiolated methoxy-poly(ethylene glycol)(PEG),animated methoxy-PEG,trypsin,BSA,concanavalin A-R Nase B,and antibody [22].In addi-tion,it was shown that growth factors such as VEGF and bone morphogenetic protein 2(BMP-2)immobilized on a titanium-based biomaterial surface following polydopamine coating enhanced proliferation of EC and differentiation of rat mesen-chymal stem cells,respectively [22].In this study,we developed a polydopamine-mediated immobi-lization platform together with a cell adhesive peptide,RGD,and an angiogenic growth factor,bFGF,on elastic biodegradable poly(lactide-co-caprolactone)(PLCL)substrates.We then investi-gated the effect of dual factors on regulation of HUVEC activity.Speci fically,we measured the immobilized amounts of RGD and bFGF on the polydopamine-coated PLCL film in a time-dependent manner and examined the synergistic or independent effects of each factor on adhesion,migration,proliferation and differentiation of HUVEC.2.Experimental 2.1.Materials3,4-Dihydroxyphenylamine was purchased from Sigma (St.Louis,MO,USA).Chloroform was purchased from Junsei (Tokyo,Japan).Hexa fluoroisopropanol (HFIP)was purchased from Wako (Osaka,Japan).Endothelial Cell Growth Medium-2(EGM-2)was obtained from Lonza (Basel,Switzerland).Tris e HCl was purchased from Shelton Scienti fic,Inc.(Peosta,IA,USA).BSA was purchased from Bioshop (Ontario,Canada).The fluorescent probes Hoechst 33,258,rhodamine-phalloidin,and Alexa Fluor 488rabbit anti-mouse IgG were purchased from Molecular Probes (Eugene,OR,USA).Other unspeci fied chemicals were purchased from Sigma.Distilled water (DW)was obtained using Milli-Q Plus System (Millipore,Billerrica,MA,USA).2.2.Preparation of PLCL films immobilized with bioactive molecule immobilization We synthesized poly(L -lactide-co-ε-caprolactone)(PLCL)by ring open poly-merization as previously described [23,24].To prepare PLCL film,PLCL was dissolved in chloroform (10wt%)and poured on a glass plate and dried at room temperature for 3days.For polydopamine coating,the PLCL film was submerged in 3,4-dihydroxyphenylamine (2mg/ml)dissolved in Tris e HCl buffer (10m M ,pH 8.5)and shaken on a rocker for 1h.After this process,the film was washed three times in DW.Immobilization of bioactive molecules was performed similarly to the poly-dopamine coating process.Brie fly,we prepared separate solutions of 1m M RGD peptide (GGGRGDS,Anygen,Gwangju,Korea)and 500ng/ml bFGF (Peprotech,Rocky Hill,NJ,USA)(dissolved in 10m M Tris e HCl buffer,pH 8.5).Polydopamine-coated PLCL film was cut into a circular shape (area :1.99cm 2),which was then incubated with 400m l of the prepared peptide or growth factor solution at 37 C for various times as indicated in Table 1.2.3.Quanti fication and stability of immobilized bioactive moleculesImmobilized RGD peptide on the surface was quanti fied by fluorescamine assay.After the immobilization process,the supernatant was obtained and reacted with fluorescamine solution (100m g/ml in acetone)in a 96-well plate (RGD sol-ution ¼75m l,fluorescamine solution ¼25m l).The reaction was performed for 1min at RT.The fluorescent intensity of each sample was observed (480nm of excitation,520nm of emission)using a plate reader (Spectramax M2,Molecular Device,CA,USA).Known concentrations of RGD peptide (20m g/ml e 625m g/ml)solution were also reacted with fluorescamine solution to obtain a standard curve.Similarly,reacted bFGF solution was collected and quanti fied through the bFGF enzyme-linked immunosorbent assay (ELISA)development kit (Peprotech,Rocky Hill,NJ,USA),following the manufacturer ’s guidelines.To calculate the detached amount of bioactive molecules,samples were sonicated in tris e HCl buffer (400m l)for 30min,and quanti fication was performed with the same ELISA development kit.The detached amount was calculated as the percentage compared to the immobilized amount for each incubation time.2.4.X-ray photoelectron spectrometer (XPS)The atomic composition of the bioactive surface was analyzed using an XPS.Analysis was performed with an ESCA LAB 200I (Thermo VG Scienti fic,MA,USA)spectrometer using a magnesium anode source.The changes in atomic composition by surface modi fication were investigated with peak fitting software.2.5.Culture of human umbilical vein endothelial cells (HUVEC)HUVEC were purchased from Lonza Group Ltd.(Basel,Switzerland).Sub-culture of HUVEC was performed in endothelial growth medium (EGM-2bulletkit,LonzaFig.2.Quanti fication of the immobilized bioactive molecules on polydopamine-coated PLCL film.(a)The amount of immobilized RGD peptide on the polydopamine-coated surface was measured by fluorescamine assay.(b)The amount of immobilized bFGF on the polydopamine-coated surface was measured by ELISA.(c)The percentages of detached bioactive molecules after application of sonication were calculated by normalization to the immobilized amount of bioactive molecules at each time point.Y.B.Lee et al./Biomaterials 33(2012)8343e 83528345Group Ltd.,Basel,Switzerland),and other conditions were the same as standard cell culture conditions(37 C,5%CO2,95%humidity).Every cell test was carried out using HUVEC passage number7.Other specific culture conditions(medium treat-ment,culture day)for each analysis are described later.The medium was changed every2days.2.6.Cell adhesion and immunofluorescent stainingFor cell adhesion study,circular-shapedfilms(1.99cm2)were placed inside the 24well polystyrene culture dish and HUVEC were seeded at the density of 1Â104cells/cm2with endothelial basal medium(EBM-2,Lonza Group Ltd.,Basel, Switzerland)supplemented with FBS(5%).For the positive control group,HUVEC were seeded at the same density on the tissue culture plate under the same media condition,but we also added bFGF(10ng/ml).In order to preventfilmsfloating from the surface,samples were overlaid with stainless rings(24well size)and HUEVEC were cultured for24h.For immunostaining of cluster of differentiation31 (CD31)expression,HUVEC were seeded at the higher density of4Â104cells/cm2 with EBM-2.After24h,the media was replenished with EBM-2supplemented with FBS(5%),and additionally cultured for3days(n¼5).The cells(for adhesion test and CD31staining)were thenfixed with4%paraformaldehyde for20min, gently washed with PBS,and treated with cytoskeletal(CSK)buffer(pH6.8,50m M NaCl,150m M sucrose,3m M MgCl2,50m M trizma-base,0.5%Triton X-100)for cell permeabilization(20min,4 C).Then,the samples were treated with blocking buffer(5%BSA in PBS)for1h at37 C,followed by treatment with anti-paxillin solution(1:200,BD science,NJ,USA)for1h at37 C.For CD31staining,CD31 antibody(Cell Signaling Technology Inc.,Danvers,MA,USA)solution(1:200)was used instead of anti-paxillin solution.The samples were incubated with Alexaflour 488rabbit anti-mouse IgG(1:100),rhodamine-phalloidin(1:200)and Hoechst 33,258(1:10,000)-containing solution to visualize focal contact or CD31expression and cell skeletal structure(1h,37 C).After incubation,cells were gently washed with PBS and mounted with Vectashield mounting medium(Vector Laboratory, UK).Immunofluorescence images were taken using a confocal microscope(Nikon, Tokyo,Japan).2.7.Cell migration assayThe migratory behavior of HUVEC was observed using a wound healing model. Briefly,a square silicon-insert with two rectangular wells(Ibidi,Munich,Germany) was laid on the PLCLfilms to create a cell-free gap(500m m).In each rectangular well,HUVEC(2Â105cells/cm2)were seeded with EBM-2supplemented with FBS (5%)and incubated for24h to achieve confluent cell adhesion.After this process, samples were placed on an inverted microscope(Olympus Optical Co.Ltd.,Tokyo,Japan)with a micro culture system(37 C,5%CO2,95%humidity).Then,the insert was removed,and cell migration to the gap was observed with a change-coupled device camera(Electric Biomedical Co.Ltd.,Osaka,Japan),and images were ob-tained every5min for up to12h.From these images,the migratory speed was calculated and statistically analyzed.2.8.HUVEC proliferationTo observe the effect of bioactive molecules on proliferation of HUVEC,we performed cell counting for up to14days.HUVEC were seeded at1Â104cells/cm2 on the circular-shapedfilms with EBM-2and cultured for24h.Then,we added5% FBS to the EBM-2media and the cells were cultured for14days with the media change in every2day.Sample images were taken at1,3,7,10,and14days,andfive spots(center,right,left,top and bottom)for each sample group(n¼5)were captured for cell counting(magnification,40x).2.9.Reverse transcription-polymerase chain reaction(RT-PCR)We used RT-PCR to analyze the expressions of von Willebrand factor1(vWF-1), CD31,and GAPDH as a housekeeping gene.The circular-shapedfilms were placed inside the24well tissue culture dish and HUVEC were seeded at4Â104cells/cm2 with EBM-2.After24h,the media was changed with EBM-2supplemented with FBS (5%)and cultured for3days.In the positive control group,bFGF(10ng/ml)was additionally supplemented.RNA was obtained from these samples and reverse-transcribed to cDNA.200ng of cDNA was used for each PCR reaction.The cDNA was denatured at95 C for5min and then95 C for30s,and annealing was per-formed for45s at56 C for CD31,54 C for vWF-1,and55 C for GAPDH(GAPDH:fw. 50-ACCACAGTCCATGCCATCAC-30rev.50-TCCACCACCCTGTTGCTGTA-30,CD31:fw.50-GAGTCCTGCTGACCCTTCTG-30rev.50-CACTCCTCCCACCAACACCT-30,vWF-1:fw.50-ATGATTCCTGFFAGATTTGC-30rev.50-AGACTCTTTGGTCCCCCTGT-30).Elongation was performed at72 C for60s,and the reaction was repeated for35cycles.Thefinal elongation was performed for5min at72 C.The RT-PCR product was loaded in a1.5%agarose gel with buffer(7m l)and separated by gel electrophoresis.Separated bands were stained with ethidium bromide,and images were obtained using the Gel Logic100Imaging System(Kodak,Rochester,NY,USA)UV illumination tool.The band intensity was measured with image software(Image J),and statistical quan-titative analysis was performed.2.10.Western blottingSample preparation for Western blotting was performed in the same way as for RT-PCR.After3days of culture,cells were lysed with400m l of RIPA buffer,andtotal Fig.3.Chemical composition analysis.(a)XPS spectra of the substrates,and(b)high resolution spectra of the carbon peak(C1s).Y.B.Lee et al./Biomaterials33(2012)8343e83528346protein was measured using the BCA protein assay kit (Pierce,Rockford,IL,USA).Cell lysates were loaded on SDS-PAGE gels to observe the b -actin and CD31bands separated through electrophoresis.Then,the proteins were transferred onto a nitrocellulose membrane (0.45m m pore size,Invitrogen,Carlsbad,Ca,USA).The membrane was treated with blocking buffer for 1h at 37 C and then with diluted primary antibody solution (1:100)overnight at 4 C for b -actin (Applied Biological Materials Inc.,Richmond,BC,USA)and CD31detection.After reaction with primary antibodies,the membrane was washed with PBS-T (0.01%Tween-20in PBS)for 15min and then incubated with anti-mouse secondary antibody (Applied Biological Materials Inc.,Richmond,BC,USA)for 2h at 25 C.We used the ECL plus Western blotting detection system (Amersham Bioscience,Buckinghamshire,MO,USA)to visualize the protein bands.The bands intensity was measured with Image J,and statistical quantitative analysis was performed.2.11.Statistical analysisAll quantitative data were obtained from triplicate samples,and the results are reported as mean Æstandard deviation (SD).Statistical signi ficance was distin-guished through one-way ANOVA,Tukey ’s HSD test,and Student ’s t -test (p <0.05).3.Results and discussionLimited endothelialization on synthetic vascular graft mate-rials often causes undesirable complications such as in flamma-tory reactions,thrombogenic responses,and intimal hyperplasia.Surface modi fication of vascular grafts may be a promising approach;however,an ideal material that canprecisely emulate complex endothelialization processes has never been proposed.Our ultimate goal is to develop vascular grafts that can provide multiple signals to facilitate endotheli-alization in a stable and long-term manner.Given that,in this study,we investigated how EC function is affected by the coordinated effects of a cell adhesive peptide and an angiogenic growth factor-immobilized onto polydopamine-coated PLCL films.Fig.1shows the scheme of our strategy,and sample codes are presented in Table 1.Fig.4.Adhesion morphology and spreading of HUVEC on the substrates.Immuno fluorescent images of adherent HUVEC on substrates,which were stained for f-actin (red),paxillin (green),and nuclei (blue),and the number of adherent cells following in vitro culture for 1day (scale bar ¼100m m).“*”indicates signi ficance as compared to the DPbf group,and “x ”indicates signi ficance as compared to the DPrbf group (p <0.05).(For interpretation of the references to colour in this figure legend,the reader is referred to the web version of this article.)Y.B.Lee et al./Biomaterials 33(2012)8343e 835283473.1.Quanti fication and stability of immobilized bioactive moleculesWe coated polydopamine on PLCL films,which were subse-quently immobilized with RGD-containing peptide and bFGF by simple dipping.As shown in Fig.2a and b,the amount of immo-bilized peptide was recorded as 66.60Æ1.97mg/cm 2after 24h of incubation.Since the concentration of bFGF was much less than that of peptide,the complete immobilization of bFGF was more rapidly achieved.The amount of immobilized bFGF was 8.32Æ0.27ng/cm 2for 10min of incubation,which did not change despite prolonged incubation.The calculated values of immobilized factors suggest that the polydopamine layer coated on PLCL films enables effective immobilization of peptides and growth factors.Previously,peptide or growth factors have been immobilized on the surfaces of biomaterial substrates via physical adsorption or chemical reaction,demonstrating bioactivity of the immobilized molecules [25e 28].However,the previous immobilization processes left signi ficant room for improvement since they oftenrequired a long process time,extensive labor,complicated expertise and uncontrolled immobilization ef ficiency.Our results suggest that we could more easily immobilize bioactive molecules with relatively high ef ficiency within a few hours.Considering that bFGF at concentrations greater than 10ng/ml can modulate proliferative properties of HUVEC,the immobilized amount of bioactive mole-cules is expected to be suf ficient to induce speci fic cell behaviors [29].We then tested the stability of immobilized bioactive mole-cules by applying sonication.As shown in Fig.2c,the detached amount following employment of sonication was less than 2%and 0.2%for RGD peptide and bFGF immobilized substrates,respec-tively,suggesting that the immobilization onto polydopamine-coated PLCL films supported stable binding of bioactive molecules.3.2.X-ray photoelectron spectrometry (XPS)Surface chemical composition of functionalized substrates was investigated with XPS.As shown in Fig.3a,three separate peaksFig.5.Migration of HUVEC on the substrates.Images of migrated cells at 12h.The top left image shows the initial condition (0h)with a 500-m m defect in the middle of the cell monolayer.The lower graph presents the migration speed of HUVEC for 12h “*”indicates signi ficance as compared to the DP group (p <0.05).Y.B.Lee et al./Biomaterials 33(2012)8343e 83528348corresponding to C1s (288eV),N1s (399eV),and O1s (533eV)were observed as the main elements.Notably,the intensity of the N1s peak increased in samples immobilized with peptide and/or growth factor compared to the DP.All groups had C e H (284.6eV),C e O (286.6eV),C ]O (288.6eV),C e N (286.0eV)peaks from high resolution spectra of C1s,as shown in Fig.3b,and the DP group had the lowest ratio of C e N bonding (0.028).In contrast,the DPrbf had the C e N peak with the highest ratio (0.155),which is directly related to the presence of a large number of amino acids.In previous research on the immobilization of RGD using diamine-PEG as a spacer on polydopamine-coated poly(methyl methacrylate),the nitrogen composition of the RGD-immobilized surface was 7.4%,which is higher than that of the polydopamine-coated surface (7.2%)[30].Another study that immobilized VEGF on polydopamine-coated substrate presented a higher value for the nitrogen ratio in the VEGF-immobilized group (13.5%)in compar-ison to the polydopamine-coated group (9.1%)[31].Therefore,the change in the C e N peak suggests that RGD and bFGF were successively immobilized on the polydopamine-coated PLCL film.3.3.Cell adhesion study and immuno fluorescent stainingAs shown in Fig.4,HUVEC were adherent and well-dispersed on all samples.Overall,the morphology of EC on the surface appeared to be similar among all groups,and it was clear that co-immobilized factors produced a synergistic effect on cell adhesion by increasing the number of adherent cells;DPrbf presented the highest cell number (465Æ23),and DPbf,P,DPr,DP recorded sequentially lower cell numbers (360Æ9,351Æ24,245Æ17,148Æ18,respectively).Although DPr had a relatively lower value than the other groups,the combination of RGD peptide and bFGF synergis-tically affected the adhesion of HUVEC.Collectively,adhesion and spreading were enhanced by polydopamine coating,which were further promoted features by immobilization of bioactive factors,particularly in the case of dual factors.Adhesion is the initialprocess in endothelialization and is closely implicated in later stages of cell behavior,including migration,proliferation,and differentiation.Therefore,regulating cell adhesion is crucial for constructing a healthy endothelium [32].There are some reports that cell adhesion is enhanced on polydopamine-coated surfaces [33,34].In addition to that,other reports have observed enhanced EC adhesion by introducing RGD or bFGF through plasma treat-ment,secondary conjugation,or chemical immobilization [28,35,36].Considering these previous reports,we suggest that enhanced hydrophilicity of the polydopamine-coated surface and the effects of RGD peptide and bFGF could synergistically in fluence HUVEC adhesion.3.4.Cell migration assayCell migration plays an important role in endothelialization,organization of tightened endothelium surface and angiogenesis [11].Therefore,we investigated the effect of immobilized mole-cules on migratory behavior of HUVEC using a simple wound healing assay model.As shown in Fig.5,the wound was effec-tively generated by removal of a square silicon-insert,leaving a cell-free defect with a pre-determined size (500m m).Time-lapse monitoring for longer than 12h showed that migrating HUVEC partially covered the defects.The migration speed of HUVEC on the PLCL film immobilized with bioactive molecules was signi ficantly greater than that on the film coated with pol-ydopamine;the migration speed was 20.64Æ2.04m m/h on DP,which signi ficantly increased to 31.74Æ 3.43,25.2Æ 2.42,30.06Æ2.68,and 26.4Æ2.43m m/h for DPr,DPbf,DPrbf,and P,respectively.Interestingly,the migration speed of HUVEC on DPbf and P was signi ficantly lower than that on DPr and DPrbf,indi-cating that cell adhesive RGD-containing peptide dominantly in fluenced migration of HUVEC,and no synergistic effects of bFGF were observed.In addition,we concluded that bFGF can be physically adsorbed onto the PLCL film,which affected theFig.6.Proliferation of HUVEC on the substrates.(a)Images of proliferated cells at 3and 14days (scale bar ¼200m m),and (b)the number of proliferating HUVEC on the substrates.Immobilized molecules in fluenced the proliferation of HUVEC for 14days and co-immobilized factors produced a synergistic effect.“*”indicates signi ficance as compared to the DPrbf group at each time point (p <0.05).Y.B.Lee et al./Biomaterials 33(2012)8343e 83528349。