In Vitro Biocompatibility Evaluation of Novel Urethane–Siloxane Co-Polymers Based on Poly(ϵ-Caprolactone)-block-Poly(Dimethylsiloxane)-block-Poly(ϵ-Caprolactone)

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In Vitro BiocompatibilityEvaluation of NovelUrethane–Siloxane Co-PolymersBased on Poly(ϵ-Caprolactone)-block-Poly(Dimethylsiloxane)-block-Poly(ϵ-Caprolactone)Marija V. Pergal a , Vesna V. Antic b , Gordana Tovilovicc , Jelena Nestorov c , Dana Vasiljevic-Radovic a & JasnaDjonlagic da Institute of Chemistry, Technology and Metallurgy,University of Belgrade , Studentski trg 12-16, Belgrade ,Serbiab Faculty of Agriculture , Nemanjina 6, Belgrade , Serbiac Department of Biochemistry , Institute for BiologicalResearch , Bul. Despota Stefana 142, Belgrade , Serbiad Faculty of Technology and Metallurgy , Karnegijeva 4,Belgrade , SerbiaPublished online: 08 May 2012.

To cite this article: Marija V. Pergal , Vesna V. Antic , Gordana Tovilovic , JelenaNestorov , Dana Vasiljevic-Radovic & Jasna Djonlagic (2012) In Vitro BiocompatibilityEvaluation of Novel Urethane–Siloxane Co-Polymers Based on Poly(ϵ-Caprolactone)-block-Poly(Dimethylsiloxane)-block-Poly(ϵ-Caprolactone), Journal of Biomaterials Science,Polymer Edition, 23:13, 1629-1657

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In Vitro Biocompatibility Evaluation of NovelUrethane–Siloxane Co-Polymers Based on

Poly(ε-Caprolactone)-block-Poly(Dimethylsiloxane)-block-Poly(ε-Caprolactone)

Marija V. Pergal a, Vesna V. Antic b, Gordana Tovilovic c, Jelena Nestorov c,

Dana Vasiljevic-Radovic a and Jasna Djonlagic d,∗

a Institute of Chemistry, Technology and Metallurgy, University of Belgrade, Studentski trg 12-16,Belgrade, Serbia

b Faculty of Agriculture, Nemanjina 6, Belgrade, Serbiac Department of Biochemistry, Institute for Biological Research, Bul. Despota Stefana 142,

Belgrade, Serbiad Faculty of Technology and Metallurgy, Karnegijeva 4, Belgrade, Serbia

Received 23 February 2011; accepted 15 July 2011

AbstractNovel polyurethane co-polymers (TPUs), based on poly(ε-caprolactone)-block-poly(dimethylsiloxane)-block-poly(ε-caprolactone) (PCL-PDMS-PCL) as soft segment and 4,4′-methylenediphenyl diisocyanate(MDI) and 1,4-butanediol (BD) as hard segment, were synthesized and evaluated for biomedical applica-tions. The content of hard segments (HS) in the polymer chains was varied from 9 to 63 wt%. The influenceof the content and length of the HS on the thermal, surface, mechanical properties and biocompatibilitywas investigated. The structure, composition and HS length were examined using 1H- and quantitative 13C-NMR spectroscopy. DSC results implied that the synthesized TPUs were semicrystalline polymers in whichboth the hard MDI/BD and soft PCL-PDMS-PCL segments participated. It was found that an increase in theaverage HS length (from 1.2 to 14.4 MDI/BD units) was accompanied by an increase in the crystallinity ofthe hard segments, storage moduli, hydrophilicity and degree of microphase separation of the co-polymers.Depending on the HS content, a gradual variation in surface properties of co-polymers was revealed by FT-IR, AFM and static water contact angle measurements. The in vitro biocompatibility of co-polymers wasevaluated using the endothelial EA.hy926 cell line and protein adsorption on the polyurethane films. All syn-thesized TPUs adsorbed more albumin than fibrinogen from multicomponent protein mixture, which mayindicate biocompatibility. The polyurethane films with high HS content and/or high roughness coefficientexhibit good surface properties and biocompatible behavior, which was confirmed by non-toxic effects tocells and good cell adhesion. Therefore, the non-cytotoxic chemistry of the co-polymers makes them goodcandidates for further development as biomedical implants.© Koninklijke Brill NV, Leiden, 2011

* To whom correspondence should be addressed. E-mail: [email protected]

© Koninklijke Brill NV, Leiden, 2011 DOI:10.1163/092050611X589338

Journal of Biomaterials Science 23 (2012) 1629–1657

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2 M. V. Pergal et al. /

KeywordsSegmented polyurethanes, biocompatibility, surface properties, α,ω-dihydroxy-(PCL-PDMS-PCL), en-dothelial cells

1. Introduction

Segmented thermoplastic polyurethane elastomers (TPUs) are widely used as bio-materials because of their excellent mechanical properties and good biocompat-ibility. They are employed in a wide range of medical applications, includingimplantable medical devices, drug-controlled release systems and biodegradablescaffolds for tissue engineering [1, 2]. TPUs are multi-block co-polymers composedof short, rigid polyurethane sequences and hard segments (HS) connected via longand flexible chains of soft segments (SS). The thermodynamic incompatibility ofthe hard and soft segments at low temperatures results in phase separation and,consequently, in the formation of a domain structure. The hard domains play therole of physical cross-links and act as high-modulus fillers, whereas the soft phaseprovides elasticity. Their two-phase microstructure imparts excellent mechanicalproperties. The properties of TPUs depend on many variables, such as the chemi-cal structure of each segment, the molecular weight of the soft segments, the ratioof the hard/soft segment content, the preparation conditions and, in some cases,crystallization of the soft segments and the ability to form discrete crystalline andrubbery or viscous microdomains [1, 3].

Chain regularity and block length as well as thermal history during and afterpolymerization all play important roles in determining the degree of phase separa-tion. Better phase separation is favored by non-polar soft segments and longer hardsegments. Co-polymers with a low HS content exhibit a morphology in which thehard segments are dispersed in a matrix of SS, while a high HS content can resultin an interconnected and continuous morphology [4, 5]. It is also well known thatthe properties of polyurethanes are greatly affected by the type, content and molec-ular weight of the soft segments [6]. Pre-polymers such as polyester, polyether,polycarbonate and poly(dimethylsiloxane) (PDMS) macrodiols have been utilizedin TPUs intended for biomedical applications. The relationship between the struc-ture and properties of TPUs was extensively investigated and brought in relationto their biocompatibility. Biocompatibility of TPUs is strongly influenced by thechemical composition [7–9], surface hydrophilicity [7], free energy [10], degreeof crystallinity [11] and polymer surface topography [12, 13]. Bil et al. [7] foundthat the microphase-separated morphology of TPUs based on different molecularweights of poly(ε-caprolactone) macrodiol influenced cell responses. Huang et al.[13] found that a TPU surface with suitable microphase separation or surface com-position possessed the lowest fibrinogen/albumin adsorption ratio.

In particular, PDMS-based polyurethanes, which provide a combination of excel-lent biostability, biocompatibility and surface properties, are receiving increasingattention. The introduction of PDMS into the polymer chain has the advantage

1630 Journal of Biomaterials Science 23 (2012) 1629–1657

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of imparting some of the attractive properties of PDMS to polyurethanes such ashigh flexibility, excellent thermal, oxidative and hydrolytic stability and low sur-face energy [14–16]. Initially, PDMS-based TPUs were synthesized by the use ofhydroxypropyl- and hydroxybutyl-terminated pre-polymers as the only SS com-ponent and such TPUs exhibited poor mechanical properties, attributed to the largedifferences between the solubility parameters of the PDMS pre-polymer and the co-monomers, which lead to separation of the reactants during the polyaddition reac-tion. In numerous subsequent papers, the preparation of thermoplastic polyurethaneand polyurea co-polymers based on end-functionalized PDMS pre-polymers, suchas hydroxyhexyl, aminopropyl, methylaminopropyl PDMS, with high molecularweight and good mechanical properties were reported [15, 17, 18]. A mixed orspecial type of polyols as the SS polyurethane were used in order to increase thecompatibility of the reaction mixture and, thus, to achieve better mechanical prop-erties [4, 19–21].

The main objective of this work was to synthesize and investigate novelTPUs based on a biocompatible α,ω-dihydroxy-(PCL-PDMS-PCL) pre-polymerand MDI/BD for biomedical applications. The tri-block pre-polymer containedterminal crystallizable poly(ε-caprolactone) blocks and a central PDMS block.Poly(ε-caprolactone) (PCL) is particularly interesting due to its properties suchas excellent water resistance, slow hydrolytic and enzymatic degradation, goodbiocompatibility and very high flexibility. The combination of the properties ofPCL and PDMS makes these block co-polymers excellent candidates for sur-face modifying additives, drug encapsulation and biomaterial applications [22–24].A series of eight samples of segmented TPUs with different content of hard seg-ments (9–63 wt%) was prepared. The effect of the structure and composition of theco-polymers on the thermal, surface and mechanical properties as well as the invitro biological properties are discussed in this paper.

2. Materials and Methods

α,ω-Dihydroxy-(PCL-PDMS-PCL) (ABCR) was dried at room temperature undervacuum for 2 h. The number-average molecular weight (Mn) of the α,ω-dihydroxy-(PCL-PDMS-PCL), determined by 1H-NMR spectroscopy, was 6100 g/mol. Themolecular weights of the central PDMS-block and the terminal PCL sequenceswere 2000 and 2050 g/mol, respectively. The analysis of GPC chromatograms inTHF showed that Mn of the PCL-PDMS-PCL was 7900 g/mol, Mw = 11 400 g/moland the PDI index (PDI) 1.4. 4,4′-Methylenediphenyl diisocyanate (MDI, Aldrich)with an isocyanate content of 33.6 wt%, was used as received. 1,4-Butanediol (BD,Aldrich) was purified by vacuum distillation. N,N-Dimethylacetamide (DMAc,Acros) was dried over calcium hydride and then distilled under vacuum. Tetrahy-drofuran (THF, J. T. Baker) was dried over lithium aluminum hydride and distilledbefore use. The catalyst was stannous octanoate (Sn(Oct)2), supplied by Aldrich.

1631M. V. Pergal et al. / Journal of Biomaterials Science 23 (2012) 1629–1657

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2.1. Synthesis of the TPUs

The thermoplastic polyurethanes were synthesized by a two-step polymerization insolution, starting from MDI, BD and α,ω-dihydroxy-(PCL-PDMS-PCL). All theco-polymers were synthesized in the same manner under the optimal polymeriza-tion conditions [25]: the molar ratio of NCO/OH groups was 1.05:1, the amount ofthe catalyst Sn(Oct)2 was 0.15 mol% calculated on the amount of α,ω-dihydroxy-(PCL-PDMS-PCL) in a solvent mixture of DMAc/THF (1:1, v/v). Reactions werecarried in a four-neck round-bottom flask equipped with a mechanical stirrer, anargon inlet, a reflux condenser and an addition funnel. Calculated amounts ofα,ω-dihydroxy-(PCL-PDMS-PCL) and MDI were weighed into the reaction flaskat room temperature, dissolved in mixture of DMAc/THF and then heated up to80◦C under an argon atmosphere. The reaction was started by the introduction ofSn(Oct)2. The reaction mixture was stirred for 40 min at 80◦C to prepare the NCO-terminated pre-polymer, when the theoretical NCO content was attained (1.27 wt%)[26]. For chain extension, dilute solutions of BD in DMAc/THF and second portionof MDI also in DMAc/THF were added dropwise to the pre-polymer and reac-tion was continued at 80◦C for 24 h. The polymer solution was precipitated intomethanol/water (1:1, v/v). Finally, the polymer was filtered and dried to constantweight in a vacuum oven. The yields of synthesized polyurethanes after precipita-tion in methanol/water were in the range of 85–93%. The relatively lower yield ofthe co-polymers with high soft segment content was attributed to the low-molecular-weight fraction loss during precipitation (Table 1).

Table 1.Composition of the reaction mixture and the co-polymers, the average length of the hard segmentsand average molecular weights of the TPUs

Polymer Molar Fraction of MDI/BD Ln Mn Mw PDI Yieldratioa segments (wt%) (HS)d (kg/mol)e (kg/mol)e (%)f

In feedb (NMR)c

TPU-9 1:2:1 8.8 8.5 1.2 34.300 53.300 1.6 87TPU-13 1:3:2 13.2 15.3 1.7 32.200 49.800 1.6 85TPU-17 1:4:3 17.2 17.2 1.9 34.400 50.300 1.6 88TPU-21 1:5:4 20.9 19.9 3.8 36.500 53.800 1.5 90TPU-30 1:8:7 30.1 29.4 4.0 37.600 92.800 2.5 91TPU-40 1:12:11 39.5 38.4 4.7 37.500 103.500 2.8 91TPU-50 1:18:17 49.7 48.4 10.2 39.700 69.200 1.7 93TPU-60 1:27:26 59.8 62.6 14.4 53.700 89.300 1.7 92

a PCL-PDMS-PCL:MDI:BD; NCO:OH = 1.05:1.b Predetermined from the composition of the reaction mixtures.c Determined by 1H-NMR spectroscopy.d Determined by quantitative 13C-NMR spectroscopy.e Determined by GPC.f Calculated after precipitation of the co-polymers.

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2.2. Characterization

TPU films were prepared from a 10 wt% solution in DMAc and cast onto a roundTeflon® dish (5 cm diameter). The solvent was evaporated for 48 h at 40◦C in aforce-draft oven and then the films were dried under vacuum at 40◦C for 24 h. Thefilms were about 0.4–0.5 mm thick. Both mold-side and air-side of the films weretested in specific biological assays, and gave no difference in their properties. Thespecimens for characterization were cut from the cast TPU films.

The 1H- and 13C-NMR spectra (Bruker Avance 500 spectrometer, at 500.13 and125.75 MHz, respectively) were measured at 25◦C using DMSO-d6 as solvent.Quantitative 13C-NMR spectra were obtained using the gated-decoupling method.

FT-IR spectra were recorded on an ATR-IR Nicolet 380 instrument. The scan-ning range was from 400 to 4000 cm−1 at a resolution of 4 cm−1 and 64 scans werecollected for each sample.

The GPC chromatograms were obtained on a Waters 600E instrument equippedwith a refractive index detector and three Supelco Pl-Gel columns at 40◦C. DMAcwas used as mobile phase at flow rate of 1.5 ml/min and polystyrene as calibrationstandards.

Differential scanning calorimetry (DSC) was performed using a DSC Q1000V9.0Build 275 thermal analyzer. The samples were analyzed under nitrogen atmospherefrom −90 to 230◦C, at a heating and cooling rate of 10 and 5◦C/min, respectively.

The hardness measurements were performed using a Shore D apparatus (ZwickZ7-2A C.KG.) on molded polymer discs.

Dynamic mechanical analysis was performed on DMA 2980 from TA Instru-ments at 1 Hz and a heating rate of 5◦C/min. The dynamic mechanical analysis wasperformed in tensile mode on polymer bars (13.8 × 5.2 × 1.9 mm) in the temper-ature range from −130 to +150◦C. The TPU specimens for dynamic mechanicalanalysis were prepared by press molding, using a steel mold coated with Teflon®

strips. The specimens were prepared from melt at 230 and 150◦C for samples withhigh HS and low HS content, respectively, under a pressure of 3 MPa, and cooledrapidly. Polymer bars were cut from the specimens using hot blade.

The surface topography of the TPUs was observed by atomic force microscopy(AFM). The AFM characterizations were performed with an AutoProbe CP-Research SPM (TM Microscopes-Veeco) instrument. RMS surface roughness, Rq,of samples was obtained using Region Analysis tool and software Image Processingand Data Analysis Version 2.1.15.

Water contact angle measurements of the co-polymer films were measured in aKrüss DSA100 using the sessile drop method. Single drops of distilled water with avolume of 20 µl were deposited on the polymer surface and the contact angles weremeasured after 30 s, at a temperature of 26◦C. The data presented are the averageof five measurements.

Water absorption of the TPU films was determined by immersing the films inphosphate-buffered saline (PBS, pH 7.4) for 24 h at 37◦C. The wet weight withdifferent immersion times was determined by wiping off the surface water with


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filter paper. The water absorption of the films was calculated as follows: (w−wo)×100/wo, where w is the maximum wet weight measured and wo is the weight ofthe dry sample.

2.3. Cell Line for Cell Viability, Cytotoxicity and Adhesion Tests

EA.hy926 cells (kind gift from Dr. Cora Jean Edgell, University of North Carolina,Durham, NC, USA) were cultured in Dulbecco’s Modified Eagle Media supple-mented with 10% fetal bovine serum, 2 mM L-glutamine, 100 µg/ml streptomycin,100 U/ml penicillin and HAT media supplement. The culture was maintained ina humidified atmosphere containing 5% CO2 at 37◦C. Prior to cell seeding, TPUfilms were sterilized by ultraviolet irradiation for 30 min and placed into 96-wellpolystyrene microplates (Sarstedt). The cells were seeded at density of 30 000 cellsper well for all experiments. All tests were performed in triplicate. At 24, 48 and96 h post-seeding, lactate dehydrogenase (LDH) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assays were performed to evaluate LDHrelease and cell viability.

LDH activity in culture medium without phenol red was determined by mix-ing equal volumes of medium and LDH substrate (54 mM lactic acid, 1.3 mMNAD, 0.28 mM N-methylphenazonium methyl sulphate, 0.66 mM INT in 0.2 MTris buffer solution, pH 8.2). After incubation for 10 min at room temperature, ab-sorbance was measured spectrophotometrically at 492 nm using a microplate reader(Multiskan spectrum, Thermo). The percent of cytotoxicity was calculated and ex-pressed relative to control sample (cells grown without sample film), taken as 100%.

Cell viability was evaluated using the MTT assay. Only cells with active mito-chondria can metabolize MTT. The cells were incubated for 1 h at 37◦C with MTTsolution (0.5 mg/ml) and formed crystals were dissolved in 200 µl DMSO. Ab-sorbance was measured spectrophotometrically at 570 nm. The cell viability wasexpressed relative to control sample (cells grown without sample film), taken as100%.

To further examine possible cytotoxic effect of TPU-21, TPU-30 and TPU-40films, films were incubated at 37± 1◦C for 24–26 h [27] in order to extract possibleleachable cytotoxic contents. One day after seeding, cells were exposed to TPU filmextracts for 24, 48 and 96 h. Influence of extracts on cell viability and LDH releasewas assessed at these time points.

Cell adhesion onto the TPU films was examined and photographed by light mi-croscopy using a computer based Carl Zeiss Axiovision microscope, 96 h afterseeding. Cells were fixated with 3% glutaraldehyde solution. The samples wererinsed with PBS in order to remove detached cells. TPU films were placed on micro-scopic slides, stained with 0.4% nigrosin solution and photographed. The attachedcells were counted manually at six different randomly selected positions for eachrepresentative sample and density was expressed as number of cells/mm2.

To assess possible influence of surface preconditioning with most abundantplasma proteins on cell adhesion, the samples were pre-incubated for 2 h at 37◦C


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in a mixture of bovine serum albumin (BSA, 40 mg/ml), bovine γ -globulins (BGG,10 mg/ml) and bovine fibrinogen (FBG, 3 mg/ml) prior to cell seeding. After in-cubation, the TPU films were rinsed two times with PBS and allowed to dry. Thecells were seeded in specified density and TPU films were photographed 96 h afteras indicated above.

Further, to test if preconditioning with chosen proteins corresponds to actual sit-uation that appears in vivo, samples were pre-incubated with diluted plasma and thecell adhesion onto samples prepared in such way was assessed. Peripheral bloodwas obtained from healthy lab members by venipuncture into heparin anticoag-ulant. The blood was diluted with an equal volume of PBS (1.5 mM KH2PO4,6.5 mM Na2HPO4, 2.7 mM KCl, 0.14 M NaCl, pH 7.4), and layered carefully overFicoll-Paque PLUS in a centrifuge tube. Plasma was obtained after density gradientcentrifugation (1900 rpm, 30 min, 20◦C). The TPU samples were incubated withdiluted plasma for 2 h at 37◦C, rinsed two times in PBS, dried and cells were seededonto them. The light microscopy was conducted 96 h after seeding. All experimentswere performed in triplicate.

2.4. Protein Adsorption

Protein adsorption to TPU films was calculated after 2 h incubation with BSA,BGG or FBG. A sample film was incubated with protein solution (40 mg/ml BSA,10 mg/ml BGG or 3 mg/ml FBG) at 37◦C for 2 h. The protein content was measuredcolorimetrically by the method of Markwell [28]. The results were expressed as µgprotein/cm2 film.

In order to assess influence of protein–protein interactions in multi-componentsystems on single protein binding to examined samples, competitive protein ad-sorption experiments were also conducted. TPU films were incubated with proteinmixture (40 mg/ml BSA, 10 mg/ml BGG or 3 mg/ml FBG) for 2 h at 37◦C. Af-ter incubation, TPU films were washed, and the adsorbed proteins were elutedfrom the surfaces with 1× SDS-sample buffer, and boiled for 5 min. Desorbedproteins were resolved on 5% SDS-polyacrylamide gels under non-reducing condi-tions. BSA, BGG and FBG were simultaneously run as standards. Prestained Pro-tein Ladder (Fermentas) was used as molecular mass reference. Gels were stainedwith Coomassie Briliant Blue R-250. Proteins were visualized on a STORM scan-ner (Amersham Biosciences). Quantitative analysis of protein bands was done byImageQuant software. The unknown concentrations for each protein were derivedfrom standard curves and results were expressed as µg protein/cm2 film.

2.5. Statistical Analysis

Data were evaluated statistically by one-way ANOVA, followed by post-comparisonBonferroni’s test. ∗ indicates P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.


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3. Results and Discussion

A series of novel segmented TPUs was synthesized using PCL-PDMS-PCL as theSS and MDI and BD as components of the HS. The GPC analysis of the com-mercial product, i.e., α,ω-dihydroxy-(PCL-PDMS-PCL), confirmed the PDI of thepre-polymer. The TPUs were synthesized by a two-step polyaddition reaction in so-lution under optimized conditions. In the synthesis of TPUs, the PCL blocks servedas compatibilizers for the non-polar PDMS blocks and the polar co-monomers,MDI and BD. The first step of reaction was conducted with the molar ratio MDI/tri-block pre-polymer of 2:1 in order to prepare the NCO-terminated pre-polymer; thisstep was stopped when the theoretical NCO content was attained (1.27 wt%). Inorder to check the molecular weight and PDI of NCO-terminated pre-polymer afterthe first phase, the model compound was prepared with an excess of n-butanol. TheGPC analysis of the n-butanol-terminated pre-polymer (Mn, 8255 g/mol and thePDI index 2.4) confirmed that the chain extension in the first phase had taken placein some extent.

The PDI of the tri-block PCL-PDMS-PCL pre-polymer in molar mass resultedin a fluctuation in the composition of prepared TPUs. It was confirmed by 13C-NMR analysis of the precipitated TPU samples, based on the ratio of the signalsfrom PDMS and PCL, that the fluctuation from the average PCL-PDMS-PCL seg-ment composition was in the range from 1 to 8 wt%. The PDI of the tri-blockPCL-PDMS-PCL pre-polymer, as well as the chain extension reaction after the firstphase together with the precipitation procedure have a significant influence on thestructure and composition of the final co-polymers.

The chemical structure of the TPUs is shown in Scheme 1. The HS content ofTPUs was in the range of 9 to 60 wt%. TPU structures were modified by changingthe molar ratio of α,ω-dihydroxy-(PCL-PDMS-PCL), MDI and BD from 1:2:1 to1:27:26 (Table 1).

3.1. The Structure and Composition of the TPUs

The molecular structure of the polyurethanes was investigated by 1H- and 13C-NMR spectroscopy. The composition of the polyurethanes, i.e., the content of hardand soft segments, was calculated from the 1H-NMR spectra as the relative intensi-ties of the methyl proton signals arising from the –SiCH3 groups and the aromaticproton signals from the MDI moieties [25]. The theoretical and experimental wt%content of the HS of the synthesized co-polymers are presented in Table 1. Theseresults show that the weight fractions of the HS were in the range from 8.5 to

Scheme 1. The chemical structure of the synthesized polyurethanes based on PCL-PDMS-PCL andMDI/BD segments.


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Figure 1. Quantitative 13C-NMR spectrum of the TPU co-polymer with 40 wt% PCL-PDMS-PCL.

62.6 wt%. It could be concluded that the compositions of the co-polymers werein good agreement with those expected from the composition of the feed.

The length of the hard segments in co-polymer chains is defined as the numberof repeating units (MDI-BD)n (where n = 1,2,3,4) and is designated as Ln(HS).The Ln(HS) was calculated from quantitative 13C-NMR spectroscopy and is shownin Table 1. A typical 13C-NMR spectrum of TPU-60 is shown in Fig. 1. FromFig. 1, it could be seen that the peaks of the original symmetrical aromatic carbonscontained in MDI split and different signals (r, r′ at 134.9–135.5 ppm and t, t′ at137.1–137.7 ppm) result from the existence of MDI-BD and MDI-PCL linkages[29]. In addition, two signals, one at 152.5 ppm and the other at 153.4 ppm, thatare related to –NHCOO– were found, which could be attributed to the –NHCOO–formed by reaction of MDI with BD and with ε-caprolactone, respectively. TheLn(HS) was calculated from the ratio of the integral of aromatic carbon signal fromthe MDI connected to BD (r, Fig. 1) and that of the aromatic carbon signal fromthe MDI connected to ε-caprolactone (r′, Fig. 1). The TPUs showed that the av-erage HS lengths increased from 1.2 to 14.4 MDI/BD units with increasing HScontent. The main reason that the target values for the hard segment length werenot reached could be due to the PDI of the PCL-PDMS-PCL pre-polymer and for-


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mation of polydisperse NCO-pre-polymer in the first stage of the reaction as well asthe fractionation of sample during their precipitation. The yields of TPUs after pre-cipitation were in the range of 85–93%, indicating that the low-molecular-weightfractions are rich in MDI residues. The PDI of both commercial pre-polymer andNCO-pre-polymer after the first-stage of reaction has the main influence on the in-crease of randomness in polyurethanes chains and therefore on the increase in PDIof the hard segments length.

The results of GPC analysis of the polyurethanes are presented in Table 1. TheMn of the TPUs was in the range from 32.2 to 53.7 kg/mol with a PDI between 1.5and 2.8 (Table 1).

3.2. Hydrogen Bonding in the TPUs Determined by FT-IR Spectroscopy

In order to study the influence of the HS content on the formation of hydro-gen bonds, the carbonyl and NH regions of the spectra were examined in moredetail. The carbonyl (from 1650 to 1780 cm−1) and NH regions (from 3150 to3500 cm−1) for all the synthesized co-polymers are shown in Fig. 2a and b, respec-tively. The synthesized TPUs exhibited four absorbance peaks in the CO region:hydrogen-bonded carbonyl groups in the ordered (crystalline) hard domains at1702 cm−1, free (non-bonded) carbonyl groups at 1733 cm−1, hydrogen-bondedcarbonyl groups in the disordered (amorphous) domains at 1714 cm−1 and car-bonyl groups from PCL segments at 1722 cm−1. Similarly, in the –NH stretchingregion, the two distinct bands at 3320 cm−1 and 3435 cm−1 are connected to H-bonded and free –NH groups, respectively [3]. The CO and NH regions were fittedby a Gaussian deconvolution technique, resulting in the locations and areas of allof these individual peaks. The fractions of hydrogen-bonded carbonyl and N–Hgroups of the TPUs in dependence on the weight fraction of the HS are presented inFig. 3. The IR spectra of co-polymers showed that the fraction of hydrogen-bonded–NH groups increased from 88.6 to 100% with increasing HS content, which means

Figure 2. FT-IR spectra of the TPUs (a) the C=O stretching region from 1650 to 1780 cm−1 and(b) the N–H stretching region from 3150 to 3500 cm−1. This figure is published in colour in theonline edition of this journal, which can be accessed via http://www.brill.nl/jbs


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Figure 3. Fraction of hydrogen-bonded urethane carbonyl and NH groups as a function of the contentof hard segments, obtained from curve fitting of the IR spectra.

that the urethane –NH groups were almost completely hydrogen bonded. Simulta-neously, a small shoulder at 3435 cm−1, assigned to non-H-bonded urethane NHgroups, decreased with increasing content of hard segments.

With increasing HS content, the fraction of H-bonded urethane carbonyl groupsincreased and their IR stretching frequency shifted to lower wavenumbers while thefraction of free urethane carbonyl groups decreased, indicating a decrease in seg-ment mixing. Simultaneously, the fraction of free PCL carbonyl groups decreased.The band at 1714 cm−1, associated with disordered hard domains, showed the high-est value for TPUs with a low HS content, which could be related to major phasemixing of the hard and soft segments. This led to the conclusion that substantialhydrogen bonding must occur between the urethane –NH and ester linkages in thesoft segments. Incomplete domain separation resulting in dispersion of the hardsegments in the soft segment matrix and in hydrogen bonding at the hard–soft inter-face was the suggested explanation. In the case of polyurethanes with a HS content(>30 wt%), the band at 1702 cm−1 increased and, as consequence, provoked a de-cline in the values of the areas related to the bands at 1733 and 1714 cm−1. Thisindicates that the hard segments composed of more MDI/BD units decreased theinteractions between the hard and soft segments and instigate a phase-separatedmicrostructure [21, 30]. For this reason, TPUs with a higher content of HS exhib-ited a highly ordered structure due to the incorporated long hard segments. Thesefindings are consistent with the found changes in properties, as shown below for thethermal and mechanical properties of the TPUs.

3.3. Thermal Properties of the TPUs

The TPUs were semicrystalline polymers; hence, melting and crystallization tem-peratures were observed by DSC analysis. The DSC thermograms of the TPUsrecorded during second heating and cooling are presented in Fig. 4a and b, respec-tively. The results obtained from DSC analysis, the melting temperature (Tm), theenthalpies of melting (�Hm), the crystallization temperatures (Tc), the enthalpies


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Figure 4. DSC thermograms of some synthesized polyurethanes obtained during (a) the second heat-ing run and (b) the cooling run.

Table 2.DSC analysis of the TPUs and α,ω-dihydroxy-(PCL-PDMS-PCL) pre-polymer

Polymer MDI/BD segment PCL soft segment

Tm1 �Hm1 Tc1 �Hc1 Xc1 Tg2 Tm2 �Hm2 Tc2 �Hc2 Xc2(◦C) (J/g) (◦C) (J/g) (%) (◦C) (◦C) (J/g) (◦C) (J/g) (%)

TPU-9 – – – – – −64 49.3 38.1 23.9 37.3 41.8TPU-13 – – – – – −58 46.5 32.3 21.2 31.4 38.3TPU-17 – – – – – −51 45.5 28.8 12.2 27.6 35.0TPU-21 209.3 0.5 186.5 1.1 2.8 −56 45.2 25.0 11.5 24.2 31.4TPU-30 209.2 3.4 186.5 5.2 12.7 −60 45.6 22.2 13.5 20.8 31.6TPU-40 208.0 4.4 178.9 5.0 12.6 −53 43.8 18.3 3.1 15.9 29.8TPU-50 208.1 6.4 182.4 8.4 14.5 −58 43.9 14.1 0.2 10.2 27.4TPU-60 213.4 8.0 188.5 16.7 14.0 −49 36.4 5.3 −19.2 1.6 14.3α,ω-dihydroxy- – – – – – −69 50.0 53.1 34.2 51.8 53.3(PCL-PDMS-PCL)

of crystallization (�Hc) of the polyurethanes and, for sake of comparison, of theα,ω-dihydroxy-(PCL-PDMS-PCL) pre-polymer are given in Table 2.

The second heating shows one or two clear endotherm peaks, attributed to theTm of the SS only or the Tm of the SS and HS depending on composition. The DSCcurves of TPUs with a HS content above 20 wt% showed different high-temperaturetransitions corresponding to the melting and crystallization temperatures of the hardMDI/BD segments (Fig. 4a and b). The Tm of the MDI/BD crystallites of the TPUsoccurred in the temperature region of 208–213◦C, while the Tc occurred in the re-gion of 179–189◦C. As the HS content increased, the Tm was slightly shifted tohigher temperatures, which indicates better ordered hard domains. In the thermo-grams of the TPUs with a HS content below 20 wt%, the absence of a melting peak


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indicates that the HS could not form a crystalline structure, probably because oftheir short chain length and the lack of organization.

Figure 4b shows the crystallization of different polyurethane samples. The TPUswith higher HS contents show well-defined peaks of the crystallization of the HS athigh temperatures below 200◦C. An increase in the MDI/BD units of the HS leadsto the formation of large-sized hard domains and, therefore, to higher degrees ofphase separation.

The pre-polymer, α,ω-dihydroxy-(PCL-PDMS-PCL), exhibited sharp meltingand crystallization peaks at 50 and 34◦C, respectively. These data are in agreementwith those previously reported [31]. The Tm of PCL segments in co-polymers werein the range from 36 to 49◦C, while the Tc were in the range from −19 to 24◦C.The Tm and Tc of PCL segments in co-polymers were lower than that of the PCLsegments in pre-polymer and decreased with increasing HS content. This can beexplained if the introduction of a covalent bond between the PCL segments and theurethane blocks restricts both the phase separation and crystallization of the PCLsegments [32].

The enthalpies of melting and crystallization of MDI/BD (�Hm1 and �Hc1,respectively) and the PCL segments (�Hm2 and �Hc2, respectively) in the TPUs(Table 2).

The degrees of crystallinity of MDI/BD and PCL segments (Xc1 and Xc2) of theTPUs were calculated by means of the following equation:

Xc(1,2) = �Hm(1,2)/(w(1,2)�Hθ

m(1,2)

),

where w(1,2) is the weight fraction of hard and PCL segments determined by 1H-NMR spectroscopy (w1 and w2, respectively) and �Hθ

m1 and �Hθm2 are, respec-

tively, the theoretical values of the enthalpy of the melting of perfectly crystallineMDI/BD homo-polymer and PCL-homo-polymer, calculated based on the groupcontribution method (�Hθ

m1 = 91.2 J/g and �Hθm2 = 148.2 J/g) [33].

The degrees of crystallinity of hard MDI/BD segments in the TPUs, Xc1, in thesecond heating run, were in the range from 3–15% (Table 2). The value of Xc1 of thehard segments tended to increase with increasing content of hard segments, i.e., HSlength. In addition, the degrees of crystallinity of PCL segments in the TPUs, Xc2,were in the range from 14–42%, as compared to 53% for the pre-polymer (Table 2).The value of Xc2 of the PCL segments tended to decrease with increasing contentof hard segments, from which it can be concluded that the presence of the hardsegments probably disturbed the crystal growth of the PCL segments.

TPUs that consist of crystallizable HS and SS segments belong to double crys-talline co-polymers. They could exhibit various morphological structures in thenanoscale as a result of competition between crystallization and phase segregation.It is known that block co-polymers that consist of chemically different segmentscan self-assemble into various periodic nanostructures in bulk, depending on thesegregation strength χN , where χ and N represent the Flory–Huggins interaction


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parameter and the degree of polymerization, respectively, as well as the volumeratio of the constituent components [34].

The Flory–Huggins interaction parameter was calculated using the equationχ = Vu(δ1 − δ2)

2/RT , where the arbitrary reference volume Vu was convenientlyselected as 100 ml/mol, R is the universal gas constant, T is the absolute tem-perature and δ is the solubility parameter of the polymer. Solubility parameters,calculated using the group contribution method [35] are: 11.9 (cal/cm3)1/2 for thehard (composed of one BD and two MDI units), and 8.55 and 7.3 (cal/cm3)1/2 forthe PCL and PDMS units of soft segments, respectively.

The estimation of χN of the PCL-PDMS-PCL tri-block co-polymer rangedfrom 9.3 to 7.9 at room temperature and at 10◦C above the Tm of the PCL-homo-polymer, respectively, suggesting that the sample exhibited a homogeneous mixedphase (χN < 10.5) according to the mean-field theory. The results are in agree-ment with those of Childs et al. [22] who reported that PCL-PDMS-PCL tri-blockco-polymers are either mixed in the melt or weakly segregated.

Since synthesized TPUs are multi-block co-polymers, we also calculated the χN

parameters for covalently bonded HS (MDI/BD) and the PCL segment from PCL-PDMS-PCL, both being crystallizable. It was roughly estimated that χN for theHS-PCL segments in the TPUs varied from 1.4 to 16.5 (at temperatures between180 and 223◦C) and the average degree of polymerization of HS (from 1.2 to 14.4);thus, they either form a homogeneous melt or are weakly segregated. It could beconcluded that crystallization of TPUs occurred from a homogeneous or weaklysegregated melt, depending on HS content.

The glass transition temperatures of the PCL segments were determined fromDSC thermograms (second heating run) in the low temperature range from −80 to0◦C. Although not so pronounced, the change in heat capacity of flexible segmentswas in the range of 0.048 to 0.144 J/g per ◦C. The Tg values of the TPUs are givenin Table 2. DSC thermograms are given only for a few representative samples inorder to better present the change in heat capacity of the samples (Fig. 4). The glasstransitions of the PCL segments were not pronounced in the cooling run. The glasstransition temperature of the prepared TPUs could be determined more accuratelyfrom DMA measurements.

3.4. Hardness and Dynamic Mechanical Analysis of the TPUs

The results of hardness measurements show that the hardness of the TPUs increasedwith increasing HS content, from 25 ShoreD for TPU-13 to 41 ShoreD for TPU-60 (Table 3). An exception from these results was TPU-9 with a hardness valueof 29 due to the high degree of PCL crystallinity. The TPUs with higher HS con-tents showed higher hardness values because of the stiffness that the ordered harddomains induced in the co-polymers.

DMA experiments were performed in order to obtain further information on themicrophase morphologies of the TPUs. Velankar and Cooper [36, 37] observed thatthe rheological properties of polyurethanes are determined by block length, block


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Table 3.Dynamic mechanical analysis and hardness of the TPUs

Polymer Tg (PDMS),tan δpeak (◦C)

Tg (PCL) (◦C) Tm (PCL) (◦C) Hardness(ShoreD)

E′′peak Infl. E′ tan δpeak Infl. E′ tan δpeak

TPU-9 −102 −50 −52 −44 59 70 29TPU-13 −112 −53; −6 −44 −40; −12 61 70 25TPU-17 −116 −55 −48 −44; 44 63 68 27TPU-21 −111 −53 −53 −46 62 68 28TPU-30 −116 −47; 27 −44 −48; 30 58 70 29TPU-40 −98 −61; 15 −37 −28 59 73 31TPU-50 −95 −52 −49 −49 68 78 34TPU-60 −116 −49 −46 −46 70 78 41

Figure 5. Storage modulus and tan δ of the TPUs vs. temperature at 1 Hz and a heating rate of5◦C/min.

incompatibility and composition of the polyurethane. The storage modulus, E′ andtan δ values of the TPUs as a function of temperature at a fixed frequency of 1 Hzare displayed in Fig. 5.

The storage modulus, E′, vs. temperature plots show a slight decrease at lowtemperatures associated with the glass transition temperature of the PCL segmentsof the SS. At these temperatures, tan δ showed a peak located in the temperaturerange from −49 to −28◦C associated with the α transition, which shifted to highertemperatures as the hard segment content increased (Table 3). In addition, a slightincrease in Tg of the PCL segments in the TPUs indicates the mixing of some pre-sumably short HS into the soft phase, thus meaning there was a decrease in theoverall degree of phase separation. These results are in agreement with FT-IR re-sults that confirmed segment mixing which decreased with increasing HS content.The DSC results showed that the Tg of the PCL segments in the TPUs were be-tween −64 and −49◦C. It was reported that a pure PCL-PDMS-PCL tri-blockco-polymer formed a two-phase morphology and exhibited two glass transitions,


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one of the siloxane segment at −128.3◦C and the other at −72◦C of the amorphousPCL segments, and a melting point of PCL at +54.6◦C [38]. The results from theDSC measurements indicated an increase in the Tg of the PCL segments in the co-polymers in comparison with the Tg of the PCL blocks in the pre-polymer, due to thedecrease in the chain flexibility imparted by the hard segments. In addition, theseresults indicated a relatively small amount of HS mixing within the soft domain.For the TPU-13, TPU-17 and TPU-30 samples, tan δ showed an additional peak,attributed to various degrees of mixing between the ester and urethane blocks. TheTg in these samples would be related to the glass transition of this amorphous re-gion formed by the entanglement of urethane and ester blocks. As shown in Fig. 5,the segmental relaxation peak, tan δ, broadens and shifts to higher temperatures withincreasing HS content, suggesting that a greater fraction of the hard segments is dis-solved in the soft phase. It was also confirmed that the magnitude of the tan δ peakdecreases with increasing the hard segment length and content as a consequence ofa decrease in the soft segment content. In addition, for the TPUs, a relaxation peakcorresponding to the Tg of the PDMS-segments appeared in temperature range from−95 to −116◦C on the tan δ curve (Table 3).

The inflexion point observed in the storage modulus of the TPUs in range from58 to 70◦C can be assigned to the melting of the crystalline PCL segments. The PCLsegment crystallinity enhances the low-temperature stiffness of the TPUs. The crys-talline regions of the soft domain serve as reinforcing fillers and contribute to themechanical integrity of the polyurethanes. Upon melting of the PCL crystallites, thehard domains support the microphase separated morphology and the TPU samplesexhibited elastomeric behavior. Figure 5 shows that the rubbery plateau modulusof the TPUs increased with increasing HS content and its length, which is believedto be due to the increased formation of a more continuous hard phase morphology.These results indicate microphase separated morphologies. The storage modulus at100◦C was in the range from 0.1 to 110 MPa. In addition, the mechanical proper-ties of these TPUs could be adjusted depending on requirements by variation theHS content. With a change of the HS content from 9 to 63 wt%, the properties ofthe TPUs changed from that of a soft to a tougher polymeric material.

3.5. Topographical Investigation of the TPUs by AFM

The effect of the content of the PCL-PDMS-PCL soft segments was explored inorder to understand its influence on the formation of the microtopology of the sur-face of the co-polymers. AFM images of the surface topology obtained in a contactmode were used to characterize the TPUs with different contents of PCL-PDMS-PCL segments and the results are presented in Fig. 6.

The distribution of hard and soft phases of the polymer surface was analyzedby 3D- and 2D-topographic images (the results of 2D-contact mode are not pre-sented). Based on prior studies, it is known that the bright regions represent thehard phase (hard ordered domains or crystalline regions in a polyurethane), whilethe darker regions represent the soft phase. The three-dimensional large-scale reso-


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Figure 6. 3D AFM images of the TPUs with different contents of PCL-PDMS-PCL segment (scanarea 10 µm × 10 µm).


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lution images clearly showed that the surface structure changes and the roughnesscoefficient varies with increasing content of soft segments containing PDMS (Ta-ble 4). The obtained results are also in agreement with those previously reportedbased on surface composition analysis, which showed that PDMS predominateson the surface of block and graft co-polymers containing PDMS segments due toits immiscibility with the other polymers and its low surface energy [22, 39]. Thesamples with higher Rq values showed a rougher co-polymer surface. The AFM im-ages clearly show that the formation of a spherulitic superstructure was increasedon with increasing HS content. These images represent clear visual evidence for theappearance of microphase separated structures in the synthesized TPUs.

3.6. Water Contact Angle of the TPUs

The surface of TPUs was characterized by water static contact angle measurementswith special attention paid to the wettability and hydrophobicity of the samples.A water contact angle of 90◦ or more indicates a non-wetting surface. The resultsof measurements of the water contact angles (WCA) for the polyurethanes are re-ported in Table 4. The value of the water contact angle for the TPUs increasedfrom 79 to 100◦ with increasing content of PDMS in the co-polymers. This behav-ior may be ascribed to a tendency of the PDMS segments to migrate to the surfacecaused by very low surface energy of PDMS, which results in a reduction of surfacetension and which covers most of the surface of the TPUs [22]. As expected, thewettability of the co-polymers decreased, i.e., the hydrophobicity increased, withincreasing weight fraction of PDMS. The large contact angles obtained for TPUswith high contents of PDMS segments indicate the presence of a large hydropho-bic surface at the air interface with a tendency to reach the contact angle value ofthe pre-polymer PCL-PDMS-PCL (99 ± 1◦). This clearly indicates that the TPUsurface wettability was controlled by the chemical composition. TPU-60 is the syn-thesized polyurethane with the most hydrophilic surface. The water contact anglesfor the TPU-40, TPU-50 and TPU-60 samples were comparable with the contact an-gles of TPUs used in commercial implantable devices (75–80◦) and culture gradepolystyrene (80–85◦) [40].

3.7. Water Absorption of the TPUs

Water uptake was measured to determine the bulk hydrophobicity of the TPUs.The chemical composition was the main factor controlling the amount of absorbedwater. Water absorption increased slightly with increasing immersion times anddoes not change after 24 h. The water uptake for the TPU samples in PBS at 37◦Cafter 24 h is shown in Table 4. It can be seen that the water uptake of TPUs af-ter 24 h was in the range from 1.42 to 2.90% while for pre-polymer was 1.50%.The value of the water uptake for the TPUs increased with decreasing contentof PDMS in the co-polymers. The lowest water uptake was observed for TPU-9sample with the highest PCL-PDMS-PCL content, which showed high degree ofcrystallinity (Table 2). These results showed that the hydrophobicity increased with


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19

Tabl

e4.

RM

Ssu

rfac

ero

ughn

ess

(Rq),

wat

erco

ntac

tan

gle

(WC

A),

wat

erab

sorp

tion,

fibri

noge

n/al

bum

inad

sorp

tion

ratio

(FB

G/B

SA)

from

com

petit

ive

prot

ein

adso

rptio

nex

peri

men

tof

the

TPU

san

dce

llde

nsity

Poly

mer

Rq

(nm

)aW

CA

(◦)

Wat

erab

sorp

tion

FBG

/BSA

Cel

lden

sity

Cel

lden

sity

Cel

lden

sity

afte

r24

h(%

)ra

tio(c

ells

/mm

2)b

(cel

ls/m

m2)c

(cel

ls/m

m2)d

TPU

-984

99.8

±0.

91.

420.

147

600

±10

011

00±

210

1060

±15

0T

PU-1

342

96.4

±0.

71.

730.

098

240

±11

058

0±

130

460

±90

TPU

-17

2294

.7±

1.0

1.74

0.12

120

0±

4016

0±

8012

0±

80T

PU-2

138

92.1

±1.

02.

300.

086

260

±70

440

±13

050

0±

290

TPU

-30

4888

.6±

0.2

2.30

0.10

348

0±

160

370

±16

042

0±

190

TPU

-40

5384

.0±

0.5

2.46

0.10

322

0±

6078

0±

230

960

±19

0T

PU-5

059

81.5

±0.

92.

830.

094

1230

±31

010

50±

310

1100

±31

0T

PU-6

062

78.9

±0.

92.

900.

091

1430

±18

010

80±

8012

60±

210

α,ω

-dih

ydro

xy-

–98

.9±

1.1

1.50

0.10

363

0±

6048

0±

150

540

±26

0(P

CL

-PD

MS-

PCL

)

aO

btai

ned

from

AFM

imag

es(s

can

area

10µm

×10

µm).

bD

eter

min

edfo

run

trea

ted

TPU

film

s.c

Det

erm

ined

for

prot

ein

pre-

adso

rbed

TPU

film

s.d

Det

erm

ined

for

plas

ma

pre-

adso

rbed

TPU

film

s.


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increasing weight fraction of PDMS. The synthesized TPUs are considered ratherhydrophobic in comparison with TPUs based on more hydrophilic polyols reportedby other authors [41], due to the hydrophobic nature of PDMS and PCL. Thus,PDMS-containing polyurethane with its water-resistant properties demonstrated agreat promise for use as implantable material. Polyurethane membranes containingPDMS have been used as interfaces between implantable devices and biologicaltissues to operate as a protective barrier from water exchange and to enhance bio-compatibility [42].

3.8. In Vitro Cell Compatibility of the TPUs

To assess biocompatibility of novel polyurethane co-polymers, a preliminary eval-uation of TPU film cytotoxicity and influence on cell viability was carried out usingthe EA.hy926 cell line. Biological compatibility of the TPUs was evaluated via di-rect contact with endothelial cells, as well as after surface preconditioning with amulti-component system consisting of three most abundant blood proteins or di-luted plasma. Endothelial cells have frequently been used in cytotoxicity testing ofpolymers, blood-contacting implants, as well as for investigating seeding technolo-gies for vascular prostheses [43].

Any material preparation and manufacturing procedure may induce some tox-icity. Although specifically prepared for medical use, polymeric materials maycontain in addition to the relatively inert high-molecular-weight polymer, othercomponents such residual monomers, catalysts and processing aids. Besides that,additional chemical species may be generated during the polymer preparation pro-cedure, for example, during reaction carried out for long time at high temperatureand manufacturing processes, such as heat sealing, spraying or sterilization of thedevices. All these chemical species can migrate from the device into the humanbody and should be the subject of risk assessments [44].

Quantitative assessment of the cytotoxicity by LDH assay after cells contact withthe TPU co-polymers was presented in Fig. 7a. Statistically significant increase ofthe LDH activity by 91 and 227% was observed in culture medium of cells incu-bated with TPU-40 film for 48 h and 96 h, respectively. Two-fold elevation of theLDH activity was registered in culture medium of cells incubated for 96 h withfilms TPU-21 and TPU-30.

Influence of incubation with co-polymers on cell viability was evaluated by MTTassay and the results are shown in Fig. 7b. Consistent patterns of changes in cellviability were observed at all three time points. Namely, 24 h after cells seeding ontothe TPU films with HS content of 15–38 wt% a statistically significant reduction incell viability (34–42%) as compared to the control was found. Similarly, a 45–60%decrease in cell viability was observed 48 h after growing in the presence of theabove-mentioned TPU films, while a decrease in cell viability by 48, 41, 52, 55and 71% was observed 96 h post-seeding onto TPU-13, TPU-17, TPU-21, TPU-30and TPU-40 films, respectively. The reduction in cell viability can indicate a lower


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Figure 7. Influence of novel synthesized polyurethane films and PCL-PDMS-PCL pre-polymer filmon LDH release (a) and mitochondrial activity (b) in EA.hy926 cells after 24, 48 and 96 h.

number of cells on the surface of investigated co-polymers in relation to the controlsurface (polystyrene), probably as a consequence of low EA.hy926 cells adherence.

In order to check whether the increased activity of LDH in the medium after 48and 96 h was a result of release of residual monomers, oligomers, catalysts andprocessing aids from the films TPU-21, TPU-30 and TPU-40 during incubationwith cells, a test with co-polymer extracts was also performed. Results of LDHand MTT assays showed that TPU-21, TPU-30 and TPU-40 film extracts were nottoxic to EA.hy926 cells after 24, 48 and 96 h (Fig. 8). It can be concluded that theincreased LDH activity (Fig. 7a) is probably a result of cell non-adherence to thesurface of TPU films. That was indirectly confirmed in experiment with EA.hy926cells grown in plate for suspension cells. LDH activity in medium of cells seeded on


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Figure 8. Influence of TPU-21, TPU-30 and TPU-40 films extracts on LDH release and mitochondrialactivity of EA.hy926 cells after 24, 48 and 96 h.

Figure 9. Photographs of (a) PCL-PDMS-PCL pre-polymer and TPU films with EA.hy926 cells ad-hered on their surface, (b) PCL-PDMS-PCL pre-polymer and TPU films pre-adsorbed with proteinmixture and adhered with EA.hy926 cells.

plates with non-adhering surfaces after 24, 48 and 96 h was significantly increased(data not shown). This result shows that inability of adhering to an appropriatesurface represents stress for these cells, leading to LDH release into the medium.

Cell adhesion to the TPU films was examined by light microscopy 96 h post-seeding. The representative photographs of EA.hy926 cells on the surface of TPUfilms are shown in Fig. 9. The cell adhesion and growth appeared to depend on themicrophase separation, surface roughness as well as surface hydrophilicity. The ap-pearance and density of EA.hy926 cells attached at PCL-PDMS-PCL pre-polymer


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and TPU-9 films are comparable (Fig. 9). However, exposure to samples with HScontent from 15 to 38 wt% clearly results in cell detachment from the TPU filmsurfaces. Finally, after seeding onto the TPU-50 and TPU-60 films, an increase incell density as compared to the PCL-PDMS-PCL film was observed (Fig. 9). Thenumber of attached EA.hy926 cells on the polymer surface 96 h post-seeding wasevidently higher on TPU-9, TPU-50 and TPU-60 than other samples, which is con-sistent with the MTT test data in Fig. 7b. In addition, cell morphology was preservedon the surface of TPU-9, TPU-50 and TPU-60 samples. In the case of TPUs withHS content from 15 to 38 wt%, most of the cells have spherical shape and were notuniformly distributed on the polymer film surfaces, presenting small cell clusters,whereas on TPU-9, TPU-50 and TPU-60 films, they appeared to be flat, polygonalin shape, and were better spread out than those adhering to other synthesized TPUs.The density of attached endothelial cells was in the range from 600 (TPU-9) to 1400(TPU-60) cells/mm2 while for pre-polymer was 630 cells/mm2 (Table 4).

The influence of preconditioning of TPU film surfaces with BSA, BGG and FBGmixture or diluted plasma on cell adhesion was also examined. Similar results wereobtained with protein mixture (Fig. 9) and diluted plasma (data not shown). Rep-resentative micrographs of EA.hy926 cells adhesion on protein pre-adsorbed TPUsare shown in Fig. 9 and cell densities are reported in Table 4. These results showconsistency with previous experiment with untreated samples in endothelial cell ap-pearance and attachment to the pre-polymer, TPU-50 and TPU-60 films. However,the cell densities for TPU-9, TPU-13 and TPU-40 pre-incubated with proteins wereincreased by 180, 240 and 350%, respectively, in comparison to untreated sam-ples. These cell densities are comparable with those for TPU films and pre-polymerpre-incubated with diluted plasma (Table 4). In comparison to cells adhered on non-treated TPU-13 and TPU-40 films, cells attached to preconditioned samples appearmorphologically preserved and show tendency to form typical network (Fig. 9).Results of cell adhesion onto the other samples pretreated with protein mixture anddiluted plasma were similar as for untreated samples. The obtained results showthat cell density is affected by surface roughness and FBG/BSA ratio (Table 4). Thecell density increased with increasing roughness coefficient for untreated samples.However, untreated samples TPU-50 and TPU-60 have higher cell densities thenother samples probably due to the higher hydrophilicity of these TPUs.

The results obtained for untreated samples imply that the surface of TPU-9, TPU-50 and TPU-60 films might be more favorable for the cell spreading and growththan the surface of other synthesized TPUs, probably because of higher surfaceroughness and better microphase separation structure. In addition, more hydrophilicsurfaces are thought to better promote cell attachment, spreading and proliferation[45]. The highest viability/cell density was observed for more hydrophilic sam-ples with 48 and 63 wt% hard segments. De et al. [46] have already reported thatthe atmospheric helium plasma treatment of the polyurethane film resulted in bet-ter human coronary artery endothelial cell attachment compared to the untreatedpolyurethane film due to increase in roughness and reduction in hydrophobicity.


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According to Yaseen et al. [47] the most important influence on further cell ad-hesion is governed by the presence of FBG. Interactions of cells with fibrinogenare strongly determined by the presence of adhesion binding sites for vascularcell receptors in fibrinogen molecule. These binding sites are two Arg-Gly-Asp(RGD) motifs within the Aα chain, and a non-RGD dodecapeptide sequence inthe γ chain [48, 49]. In addition, Lin et al. [50] have shown that RGD-containingpeptide-grafted polyurethane co-polymers supported endothelial cells adhesion andspreading. Exposure of these sequences to the cells depends on the quantity, as wellas conformation and orientation of adsorbed fibrinogen, which is in turn affectedby the surface topography and the roughness coefficient. Other authors previouslyobserved that fibrinogen preferentially adsorbs into the low valley regions of silicananocages-polyurethane co-polymers, where it takes on conformation which pro-motes cell adhesion [47]. In our study, this could particularly be the case for thesample TPU-9, with the lowest HS content. This sample has a very sharp roughnesspeak and sharp crevice topography, and consequently the highest FBG/BSA ratio(Fig. 6, Table 4). Therefore, it can be concluded that FBG adsorbed in tight cervicescan increase the cell attachment. Similar presumption can be made for TPU-13 andTPU-40 samples, which displayed better cell adhesion after pretreatment with pro-teins and plasma. In contrast, pre-treatment did not influence the cell adhesion ontothe TPU-50 and TPU-60 samples, which had the lowest FBG/BSA ratios (Table 4).

Our results suggest that HS content, surface roughness and microphase separa-tion of TPUs could have an important influence on surface properties necessary forendothelial cells attachment and growth. This observation was further supported bythe fact that samples with the lowest roughness coefficient (TPU-17 and TPU-21,Table 4) and higher segment mixing (TPU-17 and TPU-30, Fig. 5) attached mini-mal number of cells. In addition to surface roughness and microphase separation,preconditioning of some TPUs with plasma proteins may contribute to better cellattachment and growth.

3.9. Protein Adsorption on the TPU Films

Adsorption of proteins onto synthetic material surface is the first event that takesplace when it is exposed to blood, which is then followed by platelet adhesionand activation. Albumins, fibrinogen and γ -globulins are most common proteinsin blood; therefore, the investigation of adsorption of these proteins on polymersurface can contribute to better understanding biocompatibility. To that end, the ad-sorption of BSA, FBG and BGG on TPU films were studied separately, as wellas from three-component mixture, at physiological conditions and results are pre-sented in Figs 10 and 11.

Albumin may tend to interact with domains of similar size and, thus, passivateand protect the biomaterial surface from thrombosis, whereas fibrinogen promotesplatelet adhesion on the polymer surface. Polymers that show improved blood com-patibility and less platelet adhesion have a smaller ratio of FBG/BSA [13]. Whenexamined separately (Fig. 10), the adsorbed amounts of BSA on TPU films with


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Figure 10. The adsorption of albumin (BSA), globulin (BGG) and fibrinogen (FBG) on PCL-PDM-S-PCL pre-polymer and TPU films, after 2 h incubation at 37◦C.

low HS content (<30 wt%) were significantly reduced by 35–39%, as compared toPCL-PDMS-PCL pre-polymer film. Also, a decrease (37–54%) in BGG adsorptionon TPU films with low HS content (<30 wt%), in comparison to pre-polymer film,was found. Slight increase in fibrinogen adsorption was observed for all samples,in comparison to pre-polymer film.

In order to assess the influence of protein–protein interactions in multi-compo-nent systems on single protein binding to examined polyurethane films, competitiveprotein adsorption experiments were conducted. The adsorbed proteins were ana-lyzed by electrophoresis (Fig. 11a) and the relative amounts of adsorbed proteinswere determined (Fig. 11b). The adsorption of BSA was approx. 9-fold higher in re-lation to adsorbed FBG, at all TPU films. Also, the level of BSA was approx. 3-foldhigher than the amount of adsorbed BGG. The competitive protein adsorption ex-periments showed that BSA adsorption was higher than BGG and FBG. The resultsof single and competitive experiments show that TPU films with high HS content(>30 wt%) adsorb higher amounts of examined proteins in comparison with TPUfilms with low HS content (<30 wt%) (Figs 10 and 11).

The influence of surface topography of polyurethanes on protein adsorption dis-tribution has been reported in numerous studies. Hergenrother et al. [15] reportedthat polyurethane-containing PDMS possessed the advantageous qualities of re-duced fibrinogen adsorption and platelet adhesion due to migration of PDMS on thesurface of co-polymers. On the other side, Ma et al. [51] reported that polyurethanesbased on poly(propylene oxide) or PDMS segments can adsorb proteins such asfibrinogen, albumin and lysozyme due to the hydrophobic interaction between the


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Figure 11. The adsorption of albumin (BSA), globulin (BGG) and fibrinogen (FBG) from the three–component mixture on PCL-PDMS-PCL pre-polymer and TPU films. (a) Electrophoresis of adsorbedproteins and (b) competitive adsorption of proteins.

polyurethane surface and proteins. Fibrinogen adsorption on polyurethanes withdifferent surface-modifying end-groups (SMEs) has been studied by Chen et al.[52]. It was found that fibrinogen binds weakly on the hydrophilic backbone ofa PDMS-modified polyurethane surface but leaves the hydrophobic PDMS partuntouched. On sulfonate end-group-modified polyurethane surfaces, fibrinogen isadsorbed well. However, on poly(ethylene oxide) (PEO)-modified surfaces, it is ad-sorbed poorly. Another study has shown that albumin predominantly adsorbed onthe soft, hydrophobic PTMO segments of poly(urethane-urea)s using AFM witha nanogold-labeled BSA [53]. Further, it is well known that polyurethanes may


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undergo significant rearrangement and reorientation upon hydration, leading tochanges in both microphase separation and surface morphology. Xu et al. [53, 54]reported that the surface of the polyurethanes with different soft segments (PTMO,PDMS) enriched in hard domains during hydration, which led to decrease of fib-rinogen activity and, therefore, to decrease in platelet adhesion on TPU surfaces.

Our results show that dominant protein adsorption was observed on TPU sampleswith high HS content as a consequence of microphase separated structure. There-fore, it may be presumed that microphase separation and HS content overcomethe effect of TPUs hydrophilicity on protein adsorption. This presumption is inthe agreement with previously reported study that the protein adsorption enhancesproportionally with urethane and urea concentrations within the hard segments ofpolyurethane-urea based on MDI and PTMO [55].

The ratios of fibrinogen and albumin adsorption (FBG/BSA ratio) onto the sur-faces of the TPU films from competitive adsorption experiments are presented inTable 4. The protein adsorption ratio of FBG/BSA for the TPUs, was in the rangefrom 0.086 to 0.147. All TPU samples preferred to adsorb BSA, which was sup-posed to be beneficial for biocompatibility, according to findings of other authors[56, 57].

4. Conclusions

A series of novel segmented TPUs based on a soft PCL-PDMS-PCL segment withdifferent HS content (9–63 wt%) was synthesized by a two-step solution polymer-ization. The average length of the HS varied from 1.2 to 14.4 MDI/BD units. Theproperties of TPU co-polymers crucial for endothelial cell viability and adhesionare influenced by the HS content, surface roughness and microphase separation.FT-IR spectroscopy confirmed that the fractions of strongly hydrogen-bonded car-bonyl and NH groups, which are correlated with the formation of the hard domains,increased with increasing length of the HS in the co-polymers. Moreover, with in-creasing HS content, the modulus, hydrophilicity and microphase separation of thepolyurethanes increased. The highest viability/cell density was observed for sam-ples with high HS content. Nevertheless, the obtained result suggested that thesurface of the sample with the lowest HS content was also favorable for the cellspreading and growth probably due to a high roughness coefficient. Keeping inmind high the RMS roughness observed for TPU-9, as well as for TPU-50 and TPU-60, it can be concluded that the surface roughness is one of the factors contributingto cell adhesion. Therefore, good microphase separation and higher surface rough-ness improved adhesion of endothelial cells on the surface of the co-polymers.Pretreatment with protein mixture and diluted plasma improved cell adhesion onsome TPU samples, probably due to the presence of cell-binding sites in fibrino-gen molecules. The synthesized TPUs do not exhibit any cytotoxic effects to cells.All TPU samples adsorbed more albumin than fibrinogen in competitive proteinadsorption, which can be regarded as beneficial for biocompatibility. Thus, their


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non-cytotoxic chemistry makes TPUs with rougher surface and good microphaseseparation promising biomaterials for use in implantable medical devices. Furtherresearch should also examine the possible use of non-adhering TPU films as poten-tial biomedical devices for short-term uses.

Acknowledgements

This work was financially supported by the Ministry of Education and Science ofthe Republic of Serbia (Project No. 172062). The authors thank Prof. Dr. ZoranPetrovic from (Kansas Polymer Research Center, Pittsburg State University, Pitts-burg, KS, USA) for the DMA.

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