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PLLA films: Surface properties, chemical structure and cell affinity
Surface & Coatings Technology 207 (2012) 508–516
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Surface & Coatings Technology journal homepage: www.elsevier.com/locate/surfcoat
DC discharge plasma modification of chitosan/gelatin/PLLA films: Surface properties, chemical structure and cell affinity☆ Tatiana Demina a,⁎, Daria Zaytseva-Zotova c, Michail Yablokov b, Alla Gilman b, Tatiana Akopova a, Elena Markvicheva c, Alexander Zelenetskii a a b c
Laboratory of Solid-State Chemical Reactions, N.S. Enikolopov Institute of Synthetic Polymer Materials, Russian Academy of Sciences, Profsouznaya str., 70, Moscow, 117393, Russia Laboratory of Thermostable Plastics, N.S. Enikolopov Institute of Synthetic Polymer Materials, Russian Academy of Sciences, Profsouznaya str., 70, Moscow, 117393, Russia Polymers for Biology Laboratory, Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry of Russian Academy of Sciences, Miklukho-Maklaya str., 16/10, Moscow, 117997, Russia
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Article history: Received 22 March 2012 Accepted in revised form 20 July 2012 Available online 31 July 2012 Keywords: Air plasma modification DC discharge Chitosan Poly(L,L-lactide) Gelatin Solid-state reactive blending
a b s t r a c t The work was aimed to study an effect of direct current discharge on chemical structure, surface properties and cell response to the plasma modified composite films. The film samples were prepared by solvent casting from colloidal solution of the ternary blend of chitosan, gelatin and poly(L,L-lactide), PLLA, obtained by solid-state reactive blending (SSRB), in CH2Cl2 and treated with air plasma at pressure of 10–20 Pa and a discharge current of 50 mA for 60 s. The model film samples casted from the initial components were treated and studied as well. Contact angle of wettability measurements of the films showed that plasma modification led to increase of hydrophilicity and surface energy. Contact angle changes after plasma treatment of chitosan/gelatin/PLLA (CGP) film were more similar to those for the poly(L,L-lactide) film. X-ray photoelectron spectroscopy (XPS) data confirmed that a surface layer of the blend films was enriched with a polyester component. However, study of mouse fibroblast (L929) attachment and growth on the films showed that the CGP film provided an enhanced cell growth compared to this on the poly(L,L-lactide) film. Plasma modification of the polymer films resulted in a substantial increase in fibroblast viability on the plasma treated poly(L,L-lactide) films and to a rather strong decrease of cell growth on the plasma treated CGP and chitosan ones. Thus, plasma surface modification could be proposed as a good tool to control cell response on the material surface. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Presently, optimization and functionalization of surface properties are of great importance in material science, in order to design materials which could be used in various fields, such as chemistry, physics, electronics, biology and medicine. In biological tissues primary interactions between an extracellular matrix and biomaterial surface include several phenomena, namely hydration, protein adsorption, adhesion, spreading, growth and proliferation of cells [1]. In this regard, scaffold design in tissue engineering is rather difficult challenge which includes not only study of bulk physicochemical properties of the material used (which dictate its mechanical and physical properties), but also evaluation of interfaces with well defined surface chemistry and topography capable to promote cell growth and proliferation.
☆ This work was partially supported by the Russian Foundation for Basic Research (project no. 10-03-01022-а). The authors are grateful to Prof. Ch. Grandfils (Research Center of Biomaterials, University of Liege) for the help in 1HNMR analysis and fruitful discussions. ⁎ Corresponding author. Tel.: +7 495 3325873. E-mail address: [email protected] (T. Demina). 0257-8972/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.surfcoat.2012.07.059
Poly(lactide)s and lactide copolymers are attractive polymers for biomedical applications (pins, plates, screws, intra-bone and soft-tissue implants, and as vectors for sustained release of bioactive compounds) due to its good mechanical properties, processibility, biocompatibility and biodegradability [2]. They are fabricated from renewable sources which are nontoxic both for humans and environment. However, a lack of functional groups needed to promote cell attachment limits their use in some biomedical fields, in particular for tissue engineering [3]. To design the scaffold surface for better cell attachment and spreading, polylactides are modified with polymers of natural origin, such as collagen, chitosan, gelatin, etc. Previously, polylactides were modified by either coating or blending with these molecules [4–7]. One of the most available approaches to polylactide surface modification is a plasma treatment. Various treatment methods and plasma sources are used for surface modification [8–10]. The common plasma sources are the gaseous (corona discharge, dielectric barrier discharge, radio frequency discharge, microwave discharge, low-frequency glow discharge, direct current (DC) discharge) and laser-based ones. Different techniques as well as various working gases are used for different materials and to provide the special requirements. However, even in the case of application of the inert gases, for instance, argon, the interaction between plasma and polymer consists
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in two competitive processes, namely modification and degradation. The surface modification could proceed due to the breaking of bonds at the surface and subsequent formation the new ones or further interaction with the oxygen in the air after sample exposition. Another approach to the surface modification relies on the implantation of elements using a variety of plasmas containing oxygen (carbon mono- and dioxide, nitrogen dioxide and oxide) or other atoms (CF2C, CCl4, SF6, CF4). Another important area of plasma application is a plasma deposition that allows one to synthesize a layer with the properties distinctly different from those of the bulk material [11]. This new surface layer could be created by plasma polymerization of monomers or subsequent placement of them onto the preliminary treated surface enriched by radicals. All this methods are widely used for surface modification of biomaterials to control their hydrophilicity, biocompatibility and other properties. Khorasani et al. showed that plasma treatment with carbon dioxide results in enhanced nervous cell (B65) response on the plasma treated films compared to non-treated whereas L929 fibroblast cells adhesion wasn't influenced significantly [12]. The different response of various cell lines on the plasma modified surface could be used as a promising approach to preparation of scaffolds for tissue engineering which possess selective cell affinity. Thus, suppression of desirable cell types (e.g. fibroblast) could prevent the formation of tissue scars. Various functional groups have been grafted onto the plasma treated polylactide surfaces to improve biocompatibility or to allow a subsequent covalent immobilization of various bioactive molecules [13–15]. In order to control cell affinity to polylactide surface, we suggested to combine both approaches, namely polylactide blending with other biomaterials and plasma surface modification. In this research, poly(L,L-lactide) (PLLA) was modified by naturalorigin polymers, namely chitosan and gelatin, by simple one-step SSRB technique [16,17]. This approach combined mechanical activation of the substrates and their intensive mixing with a reactant under the action of pressure and shear strains which allows one to obtain new polymer materials. The technique has numerous advantages, since the entire modification proceeds in a solid state and does not require any component melting or an employment of any solvents as a reaction medium. Chitosan is a linear polysaccharide consisting of β(1 → 4) linked D-glucosamine residues with a variable number of randomly located N-acetyl-glucosamine groups. Chitosan and its derivatives show excellent biocompatibility, biodegradability and low immunogenicity [18]. The use of chitosan as a component of cartilage tissue scaffolds seems to be a promising approach to enhance chondrogenesis [19]. Recently, chitosan has been rendered as a promising biomaterial for vascular surgery, tissue culture and tissue repair as well as a hemostatic agent [20,21]. Silva et al. showed that radio frequency plasma treatment of chitosan films by argon and nitrogen leads to improvement of the L929 cell adhesion and proliferation [22]. Gelatin was selected as a natural polymer commonly used for pharmaceutical and medical applications because of its biodegradability and biocompatibility [23]. Due to its properties, namely biocompatibility, safety and mainly due to the ability of polyion complexation, gelatin is widely employed for drug delivery and tissue engineering [24,25]. The combination of the properties of synthetic and natural polymers allows one to prepare materials possessed sufficient mechanical characteristics and bioreactivity. Processability of the chitosan/gelatin/PLLA (CGP) blends, including their ability to form colloidal solutions in an organic medium makes it possible to fabricate different types of materials for tissue regeneration (i.e. films, micro- and nanofibers, microbeads) without any additional coating. Due to the strong molecular interactions between the CGP blend components, these materials possess homogenous bulk structure which allows one to keep their properties, including enhanced cell affinity during the process of degradation. The current study is aimed at evaluation of plasma treatment effect on the surface properties and chemical structure of the films made of composite CGP blend obtained by SSRB technique and its ability to support fibroblast cells growth.
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2. Materials and methods 2.1. Reagents Commercially available poly(L,L-lactide) (Sigma-Aldrich, Germany) with an average Mw of 160 kDa and gelatin (Chimmed, Russia) was used in this study as received. Chitosan (Mw = 60 kDa; acetylation degree of 0.10) was prepared by the solid-state synthesis as reported earlier [26]. All solvents were purchased from Acros Organics as analytical grade and were used without further purification. Dulbecco's modified Eagle's medium (DMEM), gentamicin, Trypsin/EDTA solution and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenil-2 H-tetrazolium bromide (MTT) were from PanEco (Russia). Fetal bovine serum (FBS) was purchased from HyClone (USA). 2.2. Sample preparation CGP blend stabilized by graft-copolymer fraction was prepared by SSRB in a semi-industrial co-rotating twin-screw extruder (ZE 40A × 40D UTS, Berstorff, Germany) specifically designed for powerful dispersion of solids. Before final processing, a chitosan–gelatin mixture was first pre-mixed by passing previously weighed amounts of the components once through the extruder at 50 °C. The final CGP (chitosan:gelatin:PLLA, 52:13:35 wt.%) blend was synthesized at 100 °C. The treatment of the initial components in the extruder allowed us to carry out their modification in order to give them possibility to form a stable dispersion in organic solvents. The physical mixture of the parent materials dissolved in the chlorinated solvents will separate into two phases: PLLA would dissolve instantaneously whereas natural polymers (chitosan and gelatin) would never dissociate in CH2Cl2 at all. The stable colloidal dispersion of the prepared blend was further used for film fabrication. The film samples were prepared by casting stable colloidal CH2Cl2 solution of the CGP blend (concentration 5 wt.%) on a glass substrate. The model film samples casted from the initial components were prepared from 5 wt.% solutions: chitosan in 4% acetic acid, PLLA in CH2Cl2; gelatin in deionized water. All films were dried in a dust-free chamber at RT. 2.3. Dynamic light scattering Solubility characteristics of the CGP blend were assessed by dynamic light scattering (DLS) using a Zetatrac particle size analyzer (Microtrac, Inc., USA) with the aid of Microtrac application software program (V.10.5.3). The CGP blend solution in CH2Cl2 (2 mg/mL) was prepared under magnetic stirrer agitation for 2 h at RT. The solutions kept idle within 48 h. The stable colloidal fraction was carefully selected by thin autopippete tip and studied by DLS. 2.4.
1H
NMR spectroscopy
The weighted amount of PLLA or stable CGP fraction (the film made of this fraction was used as a source) was inserted into NMR tube; required amount of deuterated chloroform was added (10 mg of sample per 600 μL of solvent). Samples were dissolved at RT for 2 h. Bruker 250 spectrometer was used to carry out 1HNMR spectroscopy. The obtained NMR data were analysed using MestReNova V. 5-3-0-4536 (Mestrelab Research S.L.). 2.5. Plasma treatment Plasma modification of the prepared film samples was carried out using a DC discharge set-up (Fig. 1). The polymer samples were placed inside a vacuum reaction chamber (1) on one of two symmetrical parallel plate aluminum electrodes (cathode (2) or anode (3); 18 cm in diameter; 5 сm distance between electrodes) and treated by DC discharge (800 V) induced from DC power supply (5). The reactor was preliminary
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2.7. X-ray photoelectron spectroscopy The surface chemical structure of the initial and the plasma modified films was analyzed by XPS. The XPS measurements were carried out using LAS‐3000 (“Riber”, France) equipped with a hemispherical analyzer with retarding field of OPX‐150. The radiation of Al Kα (hν=1486.6 eV) with power of 12 kW and emission current of 20 mA was used. Binding energies were referenced to the hydrocarbon (C\C and/or C\H) С1S peak set at 284.8 eV [28,29]. Atomic concentrations of elements were calculated according to peak areas and coefficient of element sensitivity [30]. 2.8. CHN elemental analysis CHN elemental composition of the film sample was obtained using a FLASH-2000 Organic Elemental Analyzer (Thermo, UK). The percentage of carbon, hydrogen and nitrogen was estimated. 2.9. Scanning electron microscopy The initial and the plasma treated film samples were coated with gold and examined by Jeol JSM-5300LV at a voltage of 15 kV. Fig. 1. A scheme of a DC discharge set-up: (1) a vacuum reaction chamber, (2) a cathode, (3) an anode, (4) a film sample, (5) a DC power supply, (6) a milliammeter, (7) an avacuation system, (8) a pressure measure system.
evacuated (7) to ~2 Pa (8), and then a filtered air was used as a working gas. The samples were treated in the continuous-flow mode at an air pressure of 10–20 Pa and a discharge current of 50 mA for 60 s.
2.6. Contact angle measurements Contact angles of wettability were measured in an air using an Easy Drop DSA100 (KRUSS, Germany), and images were acquired using a Drop Shape Analysis V.1.90.0.14 software. All measurements were carried out at RT with 1 μL drops of deionized water and glycerin as testing liquids, in order to evaluate contact angles and to calculate adhesion work, total surface energy, and its polar and dispersive components according to the Young equation [27].
2.10. Surface charge measurements Measurements of surface charge value of the film samples were carried out by dynamic capacitance technique [31–33]. The system for surface potential measurements consisted of two parallel electrodes – the holder and vibrating one – having the same diameter equal to 20 mm with the distance between them ≈0.5 mm. The film sample was placed on the holder. The movement of the vibrating electrode was induced by an electro-magnetic actuator (60 Hz) connected to the holder. The appearance of an electric current in the circuit was induced by an electret charge on the polymer film. The surface potential value (U) of the film sample was measured by applying a compensatory DC voltage U to the holder and zero current in the circuit. A surface potential of the films was measured with averaging on the film area of 300 mm2 while a surface charge density (μC/m2) was calculated as follow: σ ¼ ε0 εU=L;
Fig. 2. Effect of plasma treatment on surface properties of the films casted from (a) CGP blend and from initial components: (b) PLLA, (c) chitosan and (d) gelatin. Θwater contact angle air/water, %; γ surface energy, mJ m−2; γp polar surface energy, mJ m−2.
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Fig. 3.
1H
511
MNR spectra of (a) PLLA and (b) stable fraction of CGP.
where ε0 is permittivity of free space (ε0 = 8.85 × 10 −12 F/m); ε is dielectric conductivity (accepted as 2) and L is thickness of the film sample.
2.11. In vitro cell growth on the films In our study mouse fibroblast cell line (L929) was cultured in DMEM medium supplemented with 10 vol.% FBS and 1 vol.% gentamicin in a 5%
CO2 humidified atmosphere at 37 °C (CO2-incubator HERAEUS B5060 EK/CO2) and was reseeded every 2–3 days. Before cell culture on the prepared films, film samples (5×5 mm) were placed on a bottom of 96-well polystyrene culture plates (SPL Lifesciences, Korea) and sterilized with a 70 vol.% ethanol solution for 1 h. Then 60 μL of cell suspension was seeded into each film sample (3×103 cell/cm2) and after cell attachment to the films, 200 μL of culture medium was added into each well. The cells were cultivated on the films in a 5% CO2 humidified atmosphere at 37 °C for 1 week. The medium was
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replaced every 2–3 days. Blank wells seeded with the cells were used as controls. Cell viability was assessed by the MTT colorimetric assay. Briefly, 100 μL of a 1.2 mM MTT solution was added into each well and incubated for 3 h at 37 °C. After incubation, formazan crystals were solubilized by adding DMSO solution (50 μL) into each well. Absorbance was measured at 540/690 nm using a Multiscan plate reader (Flow Laboratories, USA). The relative viability of the cells related to the control wells was calculated as 100×[test viability]/[control viability]. Results were expressed as mean±standard deviation for three replicates. Cell overall morphology was characterized using optical microscopy (Reichert Microstar 1820E, Germany). 3. Results and discussion 3.1. Characterization of the films The plasma treatment of all samples in general caused a reduction in the contact angle of wettability and an increase in the surface energy, as shown in Fig. 2. This increase in the surface energy reflected an increase of surface hydrophilicity. The obtained results are in good agreement with the literature data in terms of the effect of plasma treatment of PLLA and chitosan [8,34–36]. According to the data of the contact angle measurements (Fig. 2), the initial CGP films were characterized by the contact angle of wettability similar to ones of the initial PLLA. These results led us to the conclusion that the surface of the CGP film in general consisted of polyester chains. This surface structure can be explained by the method of film casting. The obtained CGP blend has demonstrated amphiphilic properties forming micelle-like stable ultra fine dispersions with a mean particle size of stable fraction of 150–400 nm (data are not presented). In order to verify the structure of aggregates in organic medium the 1HNMR spectroscopy of non-modified PLLA and stable CGP fraction in CDCl3 (Fig. 3) was carried out. The main proton peaks in the spectra of CGP blend corresponded mainly to the signals coming from the polyester sequences: chemical shifts at 1.57 ppm for \CH3 and at 5.16 ppm for \CH. Although, they were not resolved so well as in the spectra observed for non-modified PLLA (cf. Fig. 3 a and b). The NMR data showed that the polysaccharide moieties are mainly internalized within a core of the micelle-like aggregates generated within organic solvent, and therefore they are not visible in this nuclear spectrometry mode. Thus, the CGP films casted from organic solution had a surface layer enriched by polyester component while chitosan and gelatin were incorporated into a polyester matrix. As can be seen from Fig. 2, plasma modification of the CGP films both on the cathode and the anode resulted in a significant reduction of contact angles of wettability up to full drop spreading, while decrease of glycerin contact angles varied in a range of 9–50% relative to an initial value [37]. The plasma treated films possessed increased 3–4.7-fold total surface energy through its polar part (Fig. 2). According to other reports, interaction of low-temperature air plasma with polymer films caused changes in surface polarity, which can be explained as a result of polar group formation (e.g. C_O, \OH, and COOH at the oxidation) [38,39]. Another factor which could cause the contact angle change after plasma treatment is the change of surface charge. According to the data presented in Fig. 4, surface charge density changes were similar for the PLLA and CGP films. Both initial films possessed a negative surface charge, namely −6 μC/m2 and −11 μC/m2 for the PLLA and CGP samples, respectively. Plasma treatment on the anode in both cases led to not so pronounced changes (−5 μC/m2 and −23 μC/m2, respectively), whereas treatment on the cathode resulted in a significant charge increase together with sign reversal (+37 μC/m2 and +43 μC/m2). These results are in agreement with the data of contact angle measurements. The increase of surface charge should affect the polar component of surface energy more strongly than dispersive one. As it can be seen from Fig. 2 in case of plasma treatment on the cathode both PLLA and CGP films the increase of polar components is higher than in case of the samples treated on the
Fig. 4. Surface potential of the initial and plasma treated (a) PLLA and (b) CGP films.
anode. It's quite likely that the increase of hydrophilicity after plasma treatment was caused in general by an increase of surface charge density. Study of chemical structure of the CGP films by XPS showed that the surface of the initial CGP film consisted of polyester chains (Table 1). These data are in good agreement with contact angle measurements and a theory of core–shell structure of the CGP aggregates in an organic medium. The О/С ratio of the initial CGP films (0.85) was similar to the ratio calculated from chemical structure of the initial PLLA (0.89). Fig. 5 showed binding energies and related functional groups obtained by fitting C1S and N1S peaks of the initial and plasma treated samples. The C1S peaks had three components: main one at 284–285 eV was due to C\C and/or C\H, a component at about 286–288 eV corresponded to C\O, a component at about 288–289 eV related to COO. However, plasma treatment on both cathode and anode electrodes resulted in a decrease of oxygen-containing groups in the surface layer of the films (Fig. 5, b, d). According to the literature, immobilization of chitosan or gelatin moieties on PLLA surfaces leads to decrease of C_O and C\O groups as well [13,14]. The local destruction of polyester chains on the CGP film surface, which can be caused by plasma treatment, probably led to the similar effect. The N1S peak observed for the plasma treated CGP films was placed at 400.1 eV, corresponding to N\C, N\O and N\H. An additional peak at 407.0 eV (\NO2 groups) was observed for the cathode treated film (Fig. 5, e). The appearance of nitrogen-containing groups at the surface of the plasma treated films (Fig. 5, c, e) could be caused by the interactions of surface molecules with nitrogen from the working gas (air) as well as by local destructions of the polylactide surface layer leading to the Table 1 Surface chemical structure of the film samples. Samples
Initial CGP CGP treated on the anode CGP treated on the cathode PLLA treated on the cathode
Atomic concentration [%] С
О
N
54.1 74.8 60.8 57
45.9 21.9 34.4 39.1
– 3.3 4.8 3.9
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Fig. 5. XPS C1S and N1S core-level spectra of: (a) the initial CGP, (b, c) the anode treated CGP and (d, e) the cathode treated CGP film.
uncovering of nitrogen groups contained in the bulk of the CGP films. In order to evaluate the contribution of each process, the appearance of nitrogen in the PLLA films after plasma modification at the same conditions was determined (Table 1). Although nitrogen was found on the surface of PLLA samples treated by plasma, its atomic percentage was lower than that in the case of CGP plasma treated films (3.9 and 4.8, respectively). That difference in nitrogen content was in good agreement with elemental analysis data according to which the initial CGP film contained 0.75± 0.06% of nitrogen. At the same time, another reason which could cause the difference of nitrogen content in plasma modified PLLA and CGP films is various effectiveness of the plasma treatment on these samples. The amount of nitrogen entrapped from the air into CGP treated films could be higher than that in the case of PLLA because of the lower crystallinity of the composite films. According to Riccardi et al. [40], the plasma treatment affects the amorphous regions preferentially than crystalline ones, leaving them practically unaltered. However, the destruction of the CGP surface layer could also make a contribution to the nitrogen appearance on the film surface as mentioned above and thus should be taken into consideration since significant changes in film surface morphology were observed (see next paragraph).
Study of the surface topography by SEM (Fig. 6) showed a significant difference in film sample topography. While the initial PLLA sample possessed heterogeneous structure consisted of spherulites with diameter in a range of 50–100 μm (Fig. 6, a), the initial CGP film had a homogenous structure (Fig. 6, b). The plasma treatment of the CGP films led to a local destruction which was more pronounced for the anode treated film compared to the cathode treated one (cf. b and c of Fig. 6). At the same time, it's worth to note that the amount of nitrogen in surface layer after plasma treatment on the cathode was higher than that in case of on the anode treated one: 4.8 and 3.3, respectively (Table 1). According to the literature data, different conditions of plasma treatment lead, in some cases, to appearance of local defects or to homogenous destruction of surface layer or overlapping of both processes [8,22]. As we suppose, in case of treatment on the cathode, both processes took a place, which explain less heterogeneity of the surface and higher amount of nitrogen. 3.2. Cell growth on the films The ability of the prepared films to support animal cells growth was tested using mouse fibroblasts (L929) as a model cell line. It should be
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Fig. 6. SEM microphotographs of (a) the initial PLLA, (b) initial CGP film and treated (c) on the anode and (d) the cathode. Scale bar is 50 μm.
taken into account that cells do not directly interact with the film surface but respond on the conformation and composition of layer of biomolecules adsorbed onto the film surface from environmental media (DMEM supplemented with 10% FBS). The results of MTT-test after cell culture on the film surface for 1 week are shown in Fig. 7. We have found that the number of alive fibroblasts on the initial PLLA films was rather small, in particular less than 30%, compared to that grown on polystyrene culture plates (100%). These data are in good agreement with the results obtained earlier by Y. Wang and Y. Lin [3,41]. Although the surface of the CGP films was enriched with polyester moieties, the fibroblast growth was significantly enhanced on the initial CGP film compared to the initial PLLA one. Moreover, the initial CGP films provided cell viability similar to that on the chitosan films. Probably, the presence of natural polymers, namely chitosan and gelatin, in the bulk of the films gave a knock-on effect on the film bioactivity. Plasma modification of the prepared films affected cell viability and cell morphology in different ways. Plasma treatment of the PLLA films significantly enhanced cell growth on PLLA films treated both on the anode and cathode. It is very likely that the observed effect is caused mainly by the changes in wettability, in particular a reduction in contact angles. However, as seen from Fig. 7, the number of alive cells on the treated CGP and chitosan films was smaller than that on the initial films in both cases (on the cathode and anode). Plasma treatment of the chitosan films slightly affected cell viability, namely the decrease of cell viability was less than 25%. This difference in cell viability on the plasma treated films led us to a conclusion that cell affinity to the samples was not exclusively governed by wettability of the surfaces in the case of the PLLA film. Thus, we assume that the other physicochemical surface properties may play a significant role, e.g. surface morphology and charge.
Along with wettability and surface morphology a surface charge change after plasma treatment can affect cell-to-film affinity through altered protein adsorption to the film surface [42,43]. As it has been previously mentioned, plasma treatment of the PLLA and CGP films resulted in similar change of surface charge which, in this case, can't be explanation of different bioreactivities of the materials. Another possible reasons for the cell viability decrease on the plasma treated CGP films are changes in surface morphology after plasma treatment. Fig. 8 demonstrates an overall morphology of mouse fibroblasts cultured on the polystyrene culture plate, as well as on the PLLA, CGP
Fig. 7. Relative viability of L929 mouse fibroblasts cultured on the PLLA and CGP films for 1 week. Results of MTT-test expressed as mean± standard deviation for three replicates. Viability of the cells cultivated on polystyrene plates was used as a control (100%).
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1
2
3
4a
4b
4c
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Fig. 8. Microphotographs of the L929 cells on polystyrene plate (1) and on the initial PLLA (2), the initial CGP (4a), the CGP films treated by plasma on the cathode (4b) and anode (4c); the initial chitosan (3). Cultivation for 7 days.
and chitosan films for 1 week. As well known, mouse fibroblast L929 can grow after being attached and spread on the surface. From Fig. 8 it is obviously that not all cells were spread on the film surface. Noteworthy, the lowest number of round cells (not spread cells on the film surface) was observed on the initial CGP and initial chitosan films as well as on the polystyrene culture plates. According to SEM images, these surfaces listed above were the most smooth and homogeneous unlike to initial PLLA on which the cells spread respectively worse. Comparing L929 spreading on plasma modified CGP films, it can be seen that cells flatten better on the cathode treated CGP films in comparison with the anode treated samples (cf. Fig. 8 4b and 4c). These data are in good agreement with the SEM images of CGP films treated by plasma. The higher amount of local defects on the anode treated sample prevented the cell spreading whereas more homogenous surface destruction during plasma treatment on the cathode gave to cells the space for attachment. However, in spite of better cell attachment onto cathode treated CGP films the cell viability in this case was lower than that onto anode treated sample. It's possible to assume that the different cell viability on the samples can be explained by different composition of the layer of the absorbed biomolecules (from culture medium) that depended on the amount of nitrogen containing groups on the surface layer after plasma treatment (Table 1). Additionally, cell growth on the low-temperature plasma treated CGP films which were kept for 5 months in the polyethylene bags at RT was evaluated. The MTT assay revealed that viability of the cells cultivated on the initial CGP films maintained at the same level, namely 63%, compared to that in the case of the films after being stored for a few days. At the same time, cell viability on the plasma treated samples changed after 5 months of storage. In particular, we have found that a percentage of viable cells increased from 22% to 52% and from 9% to 29% for the films treated on the anode and cathode, respectively. These data confirmed our suggestion that the surface properties affected by plasma treatment could partly be restored at storage. According to the literature, these changes
could be explained by surface rearrangement, e.g. thermally activated macromolecular motions to minimize the interfacial energy [44,45]. 4. Conclusion For the first time, we fabricated and studied the films from chitosan/ gelatin/PLLA blend obtained by SSRB technique. The obtained biocompatible and biodegradable films combine the useful properties of natural and synthetic polymers which makes them a promising material for tissue engineering purposes. The XPS and contact angle measurements revealed that the surface layer of the film casted after dissolution of the blend in organic solvent was enriched with the polyester component. The plasma treatment of the composite films as well as the films prepared from initial components (chitosan, gelatin and PLLA) led to the surface hydrophilicity increase. However, plasma treatment resulted in a decrease of oxygen-containing groups and in the appearance of nitrogen-containing ones. Study of the surface topography showed that the untreated film possessed homogenous structure, whereas plasma treatment led to local destruction which was more pronounced on the anode treated film than on the cathode one. The MTT-test and microscopic observations confirmed that mouse fibroblast adherence and growth on the initial CGP films were higher compared to that on the PLLA films, while plasma treatment led to a reduction of fibroblast cell attachment and viability. Depending on the application of the materials, the surface can be treated with the low temperature plasma with the aim to suppress growth of the desired cell lines on the materials. Thus, plasma treatment of the CGP films could be proposed as a good tool to control cell/interface interactions. References [1] P. Roach, P.D. Eglin, K. Rohde, C.C. Perry, J. Mater. Sci. Mater. 18 (2007) 1263. [2] P. Simamora, W. Chen, J. Drugs Dermatol. 5 (2006) 436.
516 [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25]
T. Demina et al. / Surface & Coatings Technology 207 (2012) 508–516 Y.Q. Wang, J.Y. Cai, Curr. Appl. Phys. 7 (2007) 108. N. Nafee, S. Taetz, M. Schneider, U.F. Schaefer, C.-M. Lehr, Nanomedicine 3 (2007) 173. L. Lao, H. Tan, Y. Wang, C. Gao, Colloids Surf. B Biointerfaces 66 (2008) 218. S.A. Martel-Estrada, C.A. Martínez-Pérez, J.G. Chacón-Nava, P.E. García-Casillas, I. Olivas-Armendariz, Carbohydr. Polym. 81 (2010) 775. A.R.C. Duarte, J.F. Manoa, R.L. Reis, J. Supercrit. Fluids 54 (2010) 282. B.M.P. Ferreira, L.M.P. Pinheiro, P.A.P. Nascente, M.J. Ferreira, E.A.R. Duek, Mater. Sci. Eng. C 29 (2009) 806. P.K. Chu, J.Y. Chen, L.P. Wang, N. Huang, Mater. Sci. Eng. R. 36 (2002) 143. F. Intanuovo, R. Sardella, R. Cristina, M. Nardulli, L. White, D. Howard, K.M. Shakesheff, M.R. Alexander, P. Favia, Surf. Coat. Technol. 205 (2011) S542. G. Da Ponte, E. Sardella, F. Fanelli, A. Van Hoeck, R. d'Agostino, S. Paulussen, P. Favia, Surf. Coat. Technol. 205 (2011) S525. M.T. Khorasani, H. Mirzadeh, S. Irani, J. Appl. Polym. Sci. 112 (2009) 3429. Z. Ding, J. Chen, G. Sao, J. Chang, J. Zhanga, E.T. Kang, Biomaterials 25 (2004) 1059. J.P. Chen, C.H. Su, Acta Biomater. 7 (2011) 234. J. Yang, J.Z. Bei, S.G. Wang, Biomaterials 23 (2002) 2607. T.A. Akopova, A.N. Zelenetskii, A.N. Ozerin, A.N. Ferguson, A.G. O'Neill, Ch. 8, in: Focus on Chitosan Research, Nova Science Publishers, N.Y., USA, 2011, p. 223. A. N. Ozerin, A. N. Zelenetskii, T. A. Akopova, et al., RF Patent 2,292,354, 2007. R.A.A. Muzzarelli, C. Muzzarelli, Adv. Polym. Sci. 186 (2005) 151. R. Jin, L.S. Moreira Teixeira, P.J. Dijkstra, M. Karperien, C.A. van Blitterswijk, Z.Y. Zhong, J. Biomater. 30 (2009) 2544. C. Shi, Y. Zhu, X. Ran, M. Wang, Y. Su, T. Cheng, J. Surg. Res. 133 (2006) 185. J.K.F. Suh, H.W.T. Matthew, Biomaterials 2 (2000) 2589. S. Silva, S. Luna, M. Gomes, J. Benesch, I. Pashkuleva, J. Mano, R. Reis, Macromol. Biosci. 8 (2008) 568. P.B. Malafaya, G.A. Silva, R.L. Reis, Adv. Drug Delivery Rev. 59 (2007) 207. H.A. Awad, M.Q. Wickham, H.A. Leddy, J.M. Gimble, F. Guilak, Biomaterials 25 (2004) 3211. M.S. Ponticiello, R.M. Schinagl, S. Kadiyala, F.P. Barry, J. Biomed. Mater. Res. 52 (2000) 246.
[26] S.Z. Rogovina, T.A. Akopova, G.A. Vikhoreva, J. Appl. Polym. Sci. 70 (1998) 927. [27] S. Wu, Polymer Interfaces and Adhesion, Marcel Dekker Inc., N.Y., USA, 1982. [28] J.F. Rabek, Experimental Methods in Polymer Chemistry, Wiley Inc., N.Y., USA, 1980. [29] http://srdata.nist.gov. [30] D. Briggs, M.P. Seah, Practical Surface Analysis by Auger and X-Ray Photoelectron Spectroscopy, Wiley Inc., N.Y., USA, 1983. [31] G.M. Sessler, Electets, third ed. Laplacian press, CA, USA, 1999. [32] N. Behrendt, C. Greiner, F. Fischer, T. Frese, V. Altstadt, H.-W. Schmidt, R. Giesa, J. Hillenbrand, G.M. Sessler, Appl. Phys. A: Mater. Sci. Process. 85 (2006) 87. [33] T. Frese, D. Lovera, D.J.K.W. Sandler, G.-T. Lim, V. Altstadt, R. Giesa, H.-W. Schmidt, Macromol. Mater. Eng. 292 (2007) 582. [34] Y. Wan, J. Yang, J. Yang, J. Bei, S. Wang, Biomaterials 24 (2003) 3757. [35] L.J. Matienzo, S.K. Winnacker, Macromol. Mater. Eng. 287 (2002) 871. [36] M.T. Khorasani, H. Mirzadeh, S. Irani, Radiat. Phys. Chem. 77 (2008) 280. [37] T. Demina, T. Demina, D. Zaytseva-Zotova, M. Yablokov, A. Gilman, T. Akopova, E. Markvicheva, A. Zelenetskii, High Energy Chem. 46 (2012) 60. [38] N. Inagaki, Plasma Surface Modification and Plasma Polymerization, Technomic. Publ. Co., Lancaster, PA, 1996. [39] A. Kaminska, H. Kaczmarek, J. Kowalonek, Eur. Polym. J. 38 (2002) 1915. [40] C. Riccardi, R. Barni, E. Selli, G. Mazzone, M.N.R. Massafra, B. Marcandalli, G. Poletti, Appl. Surf. Sci. 211 (2003) 386. [41] Y. Lin, L. Wang, P. Zhang, X. Wang, X. Chen, X. Jing, Z. Su, Acta Biomater. 2 (2006) 155. [42] P.B. van Wachem, A.H. Hogt, T. Beugeling, J. Feijen, A. Bantjes, J.P. Detmers, W.G. van Aken, Biomaterials 8 (1987) 323. [43] H. Chen, J. Huang, J. Yu, S. Liu, P. Gu, Int. J. Biol. Macromol. 48 (2011) 13. [44] L. Safinia, N. Datan, M. Höhse, A. Mantalaris, A. Bismarck, Biomaterials 26 (2005) 7537. [45] X. Ximing, T.R. Gengenbach, H.J. Griesser, J. Adhes. Sci. Technol. 6 (1992) 1411.
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Surface & Coatings Technology journal homepage: www.elsevier.com/locate/surfcoat
DC discharge plasma modification of chitosan/gelatin/PLLA films: Surface properties, chemical structure and cell affinity☆ Tatiana Demina a,⁎, Daria Zaytseva-Zotova c, Michail Yablokov b, Alla Gilman b, Tatiana Akopova a, Elena Markvicheva c, Alexander Zelenetskii a a b c
Laboratory of Solid-State Chemical Reactions, N.S. Enikolopov Institute of Synthetic Polymer Materials, Russian Academy of Sciences, Profsouznaya str., 70, Moscow, 117393, Russia Laboratory of Thermostable Plastics, N.S. Enikolopov Institute of Synthetic Polymer Materials, Russian Academy of Sciences, Profsouznaya str., 70, Moscow, 117393, Russia Polymers for Biology Laboratory, Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry of Russian Academy of Sciences, Miklukho-Maklaya str., 16/10, Moscow, 117997, Russia
a r t i c l e
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Article history: Received 22 March 2012 Accepted in revised form 20 July 2012 Available online 31 July 2012 Keywords: Air plasma modification DC discharge Chitosan Poly(L,L-lactide) Gelatin Solid-state reactive blending
a b s t r a c t The work was aimed to study an effect of direct current discharge on chemical structure, surface properties and cell response to the plasma modified composite films. The film samples were prepared by solvent casting from colloidal solution of the ternary blend of chitosan, gelatin and poly(L,L-lactide), PLLA, obtained by solid-state reactive blending (SSRB), in CH2Cl2 and treated with air plasma at pressure of 10–20 Pa and a discharge current of 50 mA for 60 s. The model film samples casted from the initial components were treated and studied as well. Contact angle of wettability measurements of the films showed that plasma modification led to increase of hydrophilicity and surface energy. Contact angle changes after plasma treatment of chitosan/gelatin/PLLA (CGP) film were more similar to those for the poly(L,L-lactide) film. X-ray photoelectron spectroscopy (XPS) data confirmed that a surface layer of the blend films was enriched with a polyester component. However, study of mouse fibroblast (L929) attachment and growth on the films showed that the CGP film provided an enhanced cell growth compared to this on the poly(L,L-lactide) film. Plasma modification of the polymer films resulted in a substantial increase in fibroblast viability on the plasma treated poly(L,L-lactide) films and to a rather strong decrease of cell growth on the plasma treated CGP and chitosan ones. Thus, plasma surface modification could be proposed as a good tool to control cell response on the material surface. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Presently, optimization and functionalization of surface properties are of great importance in material science, in order to design materials which could be used in various fields, such as chemistry, physics, electronics, biology and medicine. In biological tissues primary interactions between an extracellular matrix and biomaterial surface include several phenomena, namely hydration, protein adsorption, adhesion, spreading, growth and proliferation of cells [1]. In this regard, scaffold design in tissue engineering is rather difficult challenge which includes not only study of bulk physicochemical properties of the material used (which dictate its mechanical and physical properties), but also evaluation of interfaces with well defined surface chemistry and topography capable to promote cell growth and proliferation.
☆ This work was partially supported by the Russian Foundation for Basic Research (project no. 10-03-01022-а). The authors are grateful to Prof. Ch. Grandfils (Research Center of Biomaterials, University of Liege) for the help in 1HNMR analysis and fruitful discussions. ⁎ Corresponding author. Tel.: +7 495 3325873. E-mail address: [email protected] (T. Demina). 0257-8972/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.surfcoat.2012.07.059
Poly(lactide)s and lactide copolymers are attractive polymers for biomedical applications (pins, plates, screws, intra-bone and soft-tissue implants, and as vectors for sustained release of bioactive compounds) due to its good mechanical properties, processibility, biocompatibility and biodegradability [2]. They are fabricated from renewable sources which are nontoxic both for humans and environment. However, a lack of functional groups needed to promote cell attachment limits their use in some biomedical fields, in particular for tissue engineering [3]. To design the scaffold surface for better cell attachment and spreading, polylactides are modified with polymers of natural origin, such as collagen, chitosan, gelatin, etc. Previously, polylactides were modified by either coating or blending with these molecules [4–7]. One of the most available approaches to polylactide surface modification is a plasma treatment. Various treatment methods and plasma sources are used for surface modification [8–10]. The common plasma sources are the gaseous (corona discharge, dielectric barrier discharge, radio frequency discharge, microwave discharge, low-frequency glow discharge, direct current (DC) discharge) and laser-based ones. Different techniques as well as various working gases are used for different materials and to provide the special requirements. However, even in the case of application of the inert gases, for instance, argon, the interaction between plasma and polymer consists
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in two competitive processes, namely modification and degradation. The surface modification could proceed due to the breaking of bonds at the surface and subsequent formation the new ones or further interaction with the oxygen in the air after sample exposition. Another approach to the surface modification relies on the implantation of elements using a variety of plasmas containing oxygen (carbon mono- and dioxide, nitrogen dioxide and oxide) or other atoms (CF2C, CCl4, SF6, CF4). Another important area of plasma application is a plasma deposition that allows one to synthesize a layer with the properties distinctly different from those of the bulk material [11]. This new surface layer could be created by plasma polymerization of monomers or subsequent placement of them onto the preliminary treated surface enriched by radicals. All this methods are widely used for surface modification of biomaterials to control their hydrophilicity, biocompatibility and other properties. Khorasani et al. showed that plasma treatment with carbon dioxide results in enhanced nervous cell (B65) response on the plasma treated films compared to non-treated whereas L929 fibroblast cells adhesion wasn't influenced significantly [12]. The different response of various cell lines on the plasma modified surface could be used as a promising approach to preparation of scaffolds for tissue engineering which possess selective cell affinity. Thus, suppression of desirable cell types (e.g. fibroblast) could prevent the formation of tissue scars. Various functional groups have been grafted onto the plasma treated polylactide surfaces to improve biocompatibility or to allow a subsequent covalent immobilization of various bioactive molecules [13–15]. In order to control cell affinity to polylactide surface, we suggested to combine both approaches, namely polylactide blending with other biomaterials and plasma surface modification. In this research, poly(L,L-lactide) (PLLA) was modified by naturalorigin polymers, namely chitosan and gelatin, by simple one-step SSRB technique [16,17]. This approach combined mechanical activation of the substrates and their intensive mixing with a reactant under the action of pressure and shear strains which allows one to obtain new polymer materials. The technique has numerous advantages, since the entire modification proceeds in a solid state and does not require any component melting or an employment of any solvents as a reaction medium. Chitosan is a linear polysaccharide consisting of β(1 → 4) linked D-glucosamine residues with a variable number of randomly located N-acetyl-glucosamine groups. Chitosan and its derivatives show excellent biocompatibility, biodegradability and low immunogenicity [18]. The use of chitosan as a component of cartilage tissue scaffolds seems to be a promising approach to enhance chondrogenesis [19]. Recently, chitosan has been rendered as a promising biomaterial for vascular surgery, tissue culture and tissue repair as well as a hemostatic agent [20,21]. Silva et al. showed that radio frequency plasma treatment of chitosan films by argon and nitrogen leads to improvement of the L929 cell adhesion and proliferation [22]. Gelatin was selected as a natural polymer commonly used for pharmaceutical and medical applications because of its biodegradability and biocompatibility [23]. Due to its properties, namely biocompatibility, safety and mainly due to the ability of polyion complexation, gelatin is widely employed for drug delivery and tissue engineering [24,25]. The combination of the properties of synthetic and natural polymers allows one to prepare materials possessed sufficient mechanical characteristics and bioreactivity. Processability of the chitosan/gelatin/PLLA (CGP) blends, including their ability to form colloidal solutions in an organic medium makes it possible to fabricate different types of materials for tissue regeneration (i.e. films, micro- and nanofibers, microbeads) without any additional coating. Due to the strong molecular interactions between the CGP blend components, these materials possess homogenous bulk structure which allows one to keep their properties, including enhanced cell affinity during the process of degradation. The current study is aimed at evaluation of plasma treatment effect on the surface properties and chemical structure of the films made of composite CGP blend obtained by SSRB technique and its ability to support fibroblast cells growth.
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2. Materials and methods 2.1. Reagents Commercially available poly(L,L-lactide) (Sigma-Aldrich, Germany) with an average Mw of 160 kDa and gelatin (Chimmed, Russia) was used in this study as received. Chitosan (Mw = 60 kDa; acetylation degree of 0.10) was prepared by the solid-state synthesis as reported earlier [26]. All solvents were purchased from Acros Organics as analytical grade and were used without further purification. Dulbecco's modified Eagle's medium (DMEM), gentamicin, Trypsin/EDTA solution and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenil-2 H-tetrazolium bromide (MTT) were from PanEco (Russia). Fetal bovine serum (FBS) was purchased from HyClone (USA). 2.2. Sample preparation CGP blend stabilized by graft-copolymer fraction was prepared by SSRB in a semi-industrial co-rotating twin-screw extruder (ZE 40A × 40D UTS, Berstorff, Germany) specifically designed for powerful dispersion of solids. Before final processing, a chitosan–gelatin mixture was first pre-mixed by passing previously weighed amounts of the components once through the extruder at 50 °C. The final CGP (chitosan:gelatin:PLLA, 52:13:35 wt.%) blend was synthesized at 100 °C. The treatment of the initial components in the extruder allowed us to carry out their modification in order to give them possibility to form a stable dispersion in organic solvents. The physical mixture of the parent materials dissolved in the chlorinated solvents will separate into two phases: PLLA would dissolve instantaneously whereas natural polymers (chitosan and gelatin) would never dissociate in CH2Cl2 at all. The stable colloidal dispersion of the prepared blend was further used for film fabrication. The film samples were prepared by casting stable colloidal CH2Cl2 solution of the CGP blend (concentration 5 wt.%) on a glass substrate. The model film samples casted from the initial components were prepared from 5 wt.% solutions: chitosan in 4% acetic acid, PLLA in CH2Cl2; gelatin in deionized water. All films were dried in a dust-free chamber at RT. 2.3. Dynamic light scattering Solubility characteristics of the CGP blend were assessed by dynamic light scattering (DLS) using a Zetatrac particle size analyzer (Microtrac, Inc., USA) with the aid of Microtrac application software program (V.10.5.3). The CGP blend solution in CH2Cl2 (2 mg/mL) was prepared under magnetic stirrer agitation for 2 h at RT. The solutions kept idle within 48 h. The stable colloidal fraction was carefully selected by thin autopippete tip and studied by DLS. 2.4.
1H
NMR spectroscopy
The weighted amount of PLLA or stable CGP fraction (the film made of this fraction was used as a source) was inserted into NMR tube; required amount of deuterated chloroform was added (10 mg of sample per 600 μL of solvent). Samples were dissolved at RT for 2 h. Bruker 250 spectrometer was used to carry out 1HNMR spectroscopy. The obtained NMR data were analysed using MestReNova V. 5-3-0-4536 (Mestrelab Research S.L.). 2.5. Plasma treatment Plasma modification of the prepared film samples was carried out using a DC discharge set-up (Fig. 1). The polymer samples were placed inside a vacuum reaction chamber (1) on one of two symmetrical parallel plate aluminum electrodes (cathode (2) or anode (3); 18 cm in diameter; 5 сm distance between electrodes) and treated by DC discharge (800 V) induced from DC power supply (5). The reactor was preliminary
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2.7. X-ray photoelectron spectroscopy The surface chemical structure of the initial and the plasma modified films was analyzed by XPS. The XPS measurements were carried out using LAS‐3000 (“Riber”, France) equipped with a hemispherical analyzer with retarding field of OPX‐150. The radiation of Al Kα (hν=1486.6 eV) with power of 12 kW and emission current of 20 mA was used. Binding energies were referenced to the hydrocarbon (C\C and/or C\H) С1S peak set at 284.8 eV [28,29]. Atomic concentrations of elements were calculated according to peak areas and coefficient of element sensitivity [30]. 2.8. CHN elemental analysis CHN elemental composition of the film sample was obtained using a FLASH-2000 Organic Elemental Analyzer (Thermo, UK). The percentage of carbon, hydrogen and nitrogen was estimated. 2.9. Scanning electron microscopy The initial and the plasma treated film samples were coated with gold and examined by Jeol JSM-5300LV at a voltage of 15 kV. Fig. 1. A scheme of a DC discharge set-up: (1) a vacuum reaction chamber, (2) a cathode, (3) an anode, (4) a film sample, (5) a DC power supply, (6) a milliammeter, (7) an avacuation system, (8) a pressure measure system.
evacuated (7) to ~2 Pa (8), and then a filtered air was used as a working gas. The samples were treated in the continuous-flow mode at an air pressure of 10–20 Pa and a discharge current of 50 mA for 60 s.
2.6. Contact angle measurements Contact angles of wettability were measured in an air using an Easy Drop DSA100 (KRUSS, Germany), and images were acquired using a Drop Shape Analysis V.1.90.0.14 software. All measurements were carried out at RT with 1 μL drops of deionized water and glycerin as testing liquids, in order to evaluate contact angles and to calculate adhesion work, total surface energy, and its polar and dispersive components according to the Young equation [27].
2.10. Surface charge measurements Measurements of surface charge value of the film samples were carried out by dynamic capacitance technique [31–33]. The system for surface potential measurements consisted of two parallel electrodes – the holder and vibrating one – having the same diameter equal to 20 mm with the distance between them ≈0.5 mm. The film sample was placed on the holder. The movement of the vibrating electrode was induced by an electro-magnetic actuator (60 Hz) connected to the holder. The appearance of an electric current in the circuit was induced by an electret charge on the polymer film. The surface potential value (U) of the film sample was measured by applying a compensatory DC voltage U to the holder and zero current in the circuit. A surface potential of the films was measured with averaging on the film area of 300 mm2 while a surface charge density (μC/m2) was calculated as follow: σ ¼ ε0 εU=L;
Fig. 2. Effect of plasma treatment on surface properties of the films casted from (a) CGP blend and from initial components: (b) PLLA, (c) chitosan and (d) gelatin. Θwater contact angle air/water, %; γ surface energy, mJ m−2; γp polar surface energy, mJ m−2.
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Fig. 3.
1H
511
MNR spectra of (a) PLLA and (b) stable fraction of CGP.
where ε0 is permittivity of free space (ε0 = 8.85 × 10 −12 F/m); ε is dielectric conductivity (accepted as 2) and L is thickness of the film sample.
2.11. In vitro cell growth on the films In our study mouse fibroblast cell line (L929) was cultured in DMEM medium supplemented with 10 vol.% FBS and 1 vol.% gentamicin in a 5%
CO2 humidified atmosphere at 37 °C (CO2-incubator HERAEUS B5060 EK/CO2) and was reseeded every 2–3 days. Before cell culture on the prepared films, film samples (5×5 mm) were placed on a bottom of 96-well polystyrene culture plates (SPL Lifesciences, Korea) and sterilized with a 70 vol.% ethanol solution for 1 h. Then 60 μL of cell suspension was seeded into each film sample (3×103 cell/cm2) and after cell attachment to the films, 200 μL of culture medium was added into each well. The cells were cultivated on the films in a 5% CO2 humidified atmosphere at 37 °C for 1 week. The medium was
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replaced every 2–3 days. Blank wells seeded with the cells were used as controls. Cell viability was assessed by the MTT colorimetric assay. Briefly, 100 μL of a 1.2 mM MTT solution was added into each well and incubated for 3 h at 37 °C. After incubation, formazan crystals were solubilized by adding DMSO solution (50 μL) into each well. Absorbance was measured at 540/690 nm using a Multiscan plate reader (Flow Laboratories, USA). The relative viability of the cells related to the control wells was calculated as 100×[test viability]/[control viability]. Results were expressed as mean±standard deviation for three replicates. Cell overall morphology was characterized using optical microscopy (Reichert Microstar 1820E, Germany). 3. Results and discussion 3.1. Characterization of the films The plasma treatment of all samples in general caused a reduction in the contact angle of wettability and an increase in the surface energy, as shown in Fig. 2. This increase in the surface energy reflected an increase of surface hydrophilicity. The obtained results are in good agreement with the literature data in terms of the effect of plasma treatment of PLLA and chitosan [8,34–36]. According to the data of the contact angle measurements (Fig. 2), the initial CGP films were characterized by the contact angle of wettability similar to ones of the initial PLLA. These results led us to the conclusion that the surface of the CGP film in general consisted of polyester chains. This surface structure can be explained by the method of film casting. The obtained CGP blend has demonstrated amphiphilic properties forming micelle-like stable ultra fine dispersions with a mean particle size of stable fraction of 150–400 nm (data are not presented). In order to verify the structure of aggregates in organic medium the 1HNMR spectroscopy of non-modified PLLA and stable CGP fraction in CDCl3 (Fig. 3) was carried out. The main proton peaks in the spectra of CGP blend corresponded mainly to the signals coming from the polyester sequences: chemical shifts at 1.57 ppm for \CH3 and at 5.16 ppm for \CH. Although, they were not resolved so well as in the spectra observed for non-modified PLLA (cf. Fig. 3 a and b). The NMR data showed that the polysaccharide moieties are mainly internalized within a core of the micelle-like aggregates generated within organic solvent, and therefore they are not visible in this nuclear spectrometry mode. Thus, the CGP films casted from organic solution had a surface layer enriched by polyester component while chitosan and gelatin were incorporated into a polyester matrix. As can be seen from Fig. 2, plasma modification of the CGP films both on the cathode and the anode resulted in a significant reduction of contact angles of wettability up to full drop spreading, while decrease of glycerin contact angles varied in a range of 9–50% relative to an initial value [37]. The plasma treated films possessed increased 3–4.7-fold total surface energy through its polar part (Fig. 2). According to other reports, interaction of low-temperature air plasma with polymer films caused changes in surface polarity, which can be explained as a result of polar group formation (e.g. C_O, \OH, and COOH at the oxidation) [38,39]. Another factor which could cause the contact angle change after plasma treatment is the change of surface charge. According to the data presented in Fig. 4, surface charge density changes were similar for the PLLA and CGP films. Both initial films possessed a negative surface charge, namely −6 μC/m2 and −11 μC/m2 for the PLLA and CGP samples, respectively. Plasma treatment on the anode in both cases led to not so pronounced changes (−5 μC/m2 and −23 μC/m2, respectively), whereas treatment on the cathode resulted in a significant charge increase together with sign reversal (+37 μC/m2 and +43 μC/m2). These results are in agreement with the data of contact angle measurements. The increase of surface charge should affect the polar component of surface energy more strongly than dispersive one. As it can be seen from Fig. 2 in case of plasma treatment on the cathode both PLLA and CGP films the increase of polar components is higher than in case of the samples treated on the
Fig. 4. Surface potential of the initial and plasma treated (a) PLLA and (b) CGP films.
anode. It's quite likely that the increase of hydrophilicity after plasma treatment was caused in general by an increase of surface charge density. Study of chemical structure of the CGP films by XPS showed that the surface of the initial CGP film consisted of polyester chains (Table 1). These data are in good agreement with contact angle measurements and a theory of core–shell structure of the CGP aggregates in an organic medium. The О/С ratio of the initial CGP films (0.85) was similar to the ratio calculated from chemical structure of the initial PLLA (0.89). Fig. 5 showed binding energies and related functional groups obtained by fitting C1S and N1S peaks of the initial and plasma treated samples. The C1S peaks had three components: main one at 284–285 eV was due to C\C and/or C\H, a component at about 286–288 eV corresponded to C\O, a component at about 288–289 eV related to COO. However, plasma treatment on both cathode and anode electrodes resulted in a decrease of oxygen-containing groups in the surface layer of the films (Fig. 5, b, d). According to the literature, immobilization of chitosan or gelatin moieties on PLLA surfaces leads to decrease of C_O and C\O groups as well [13,14]. The local destruction of polyester chains on the CGP film surface, which can be caused by plasma treatment, probably led to the similar effect. The N1S peak observed for the plasma treated CGP films was placed at 400.1 eV, corresponding to N\C, N\O and N\H. An additional peak at 407.0 eV (\NO2 groups) was observed for the cathode treated film (Fig. 5, e). The appearance of nitrogen-containing groups at the surface of the plasma treated films (Fig. 5, c, e) could be caused by the interactions of surface molecules with nitrogen from the working gas (air) as well as by local destructions of the polylactide surface layer leading to the Table 1 Surface chemical structure of the film samples. Samples
Initial CGP CGP treated on the anode CGP treated on the cathode PLLA treated on the cathode
Atomic concentration [%] С
О
N
54.1 74.8 60.8 57
45.9 21.9 34.4 39.1
– 3.3 4.8 3.9
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Fig. 5. XPS C1S and N1S core-level spectra of: (a) the initial CGP, (b, c) the anode treated CGP and (d, e) the cathode treated CGP film.
uncovering of nitrogen groups contained in the bulk of the CGP films. In order to evaluate the contribution of each process, the appearance of nitrogen in the PLLA films after plasma modification at the same conditions was determined (Table 1). Although nitrogen was found on the surface of PLLA samples treated by plasma, its atomic percentage was lower than that in the case of CGP plasma treated films (3.9 and 4.8, respectively). That difference in nitrogen content was in good agreement with elemental analysis data according to which the initial CGP film contained 0.75± 0.06% of nitrogen. At the same time, another reason which could cause the difference of nitrogen content in plasma modified PLLA and CGP films is various effectiveness of the plasma treatment on these samples. The amount of nitrogen entrapped from the air into CGP treated films could be higher than that in the case of PLLA because of the lower crystallinity of the composite films. According to Riccardi et al. [40], the plasma treatment affects the amorphous regions preferentially than crystalline ones, leaving them practically unaltered. However, the destruction of the CGP surface layer could also make a contribution to the nitrogen appearance on the film surface as mentioned above and thus should be taken into consideration since significant changes in film surface morphology were observed (see next paragraph).
Study of the surface topography by SEM (Fig. 6) showed a significant difference in film sample topography. While the initial PLLA sample possessed heterogeneous structure consisted of spherulites with diameter in a range of 50–100 μm (Fig. 6, a), the initial CGP film had a homogenous structure (Fig. 6, b). The plasma treatment of the CGP films led to a local destruction which was more pronounced for the anode treated film compared to the cathode treated one (cf. b and c of Fig. 6). At the same time, it's worth to note that the amount of nitrogen in surface layer after plasma treatment on the cathode was higher than that in case of on the anode treated one: 4.8 and 3.3, respectively (Table 1). According to the literature data, different conditions of plasma treatment lead, in some cases, to appearance of local defects or to homogenous destruction of surface layer or overlapping of both processes [8,22]. As we suppose, in case of treatment on the cathode, both processes took a place, which explain less heterogeneity of the surface and higher amount of nitrogen. 3.2. Cell growth on the films The ability of the prepared films to support animal cells growth was tested using mouse fibroblasts (L929) as a model cell line. It should be
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Fig. 6. SEM microphotographs of (a) the initial PLLA, (b) initial CGP film and treated (c) on the anode and (d) the cathode. Scale bar is 50 μm.
taken into account that cells do not directly interact with the film surface but respond on the conformation and composition of layer of biomolecules adsorbed onto the film surface from environmental media (DMEM supplemented with 10% FBS). The results of MTT-test after cell culture on the film surface for 1 week are shown in Fig. 7. We have found that the number of alive fibroblasts on the initial PLLA films was rather small, in particular less than 30%, compared to that grown on polystyrene culture plates (100%). These data are in good agreement with the results obtained earlier by Y. Wang and Y. Lin [3,41]. Although the surface of the CGP films was enriched with polyester moieties, the fibroblast growth was significantly enhanced on the initial CGP film compared to the initial PLLA one. Moreover, the initial CGP films provided cell viability similar to that on the chitosan films. Probably, the presence of natural polymers, namely chitosan and gelatin, in the bulk of the films gave a knock-on effect on the film bioactivity. Plasma modification of the prepared films affected cell viability and cell morphology in different ways. Plasma treatment of the PLLA films significantly enhanced cell growth on PLLA films treated both on the anode and cathode. It is very likely that the observed effect is caused mainly by the changes in wettability, in particular a reduction in contact angles. However, as seen from Fig. 7, the number of alive cells on the treated CGP and chitosan films was smaller than that on the initial films in both cases (on the cathode and anode). Plasma treatment of the chitosan films slightly affected cell viability, namely the decrease of cell viability was less than 25%. This difference in cell viability on the plasma treated films led us to a conclusion that cell affinity to the samples was not exclusively governed by wettability of the surfaces in the case of the PLLA film. Thus, we assume that the other physicochemical surface properties may play a significant role, e.g. surface morphology and charge.
Along with wettability and surface morphology a surface charge change after plasma treatment can affect cell-to-film affinity through altered protein adsorption to the film surface [42,43]. As it has been previously mentioned, plasma treatment of the PLLA and CGP films resulted in similar change of surface charge which, in this case, can't be explanation of different bioreactivities of the materials. Another possible reasons for the cell viability decrease on the plasma treated CGP films are changes in surface morphology after plasma treatment. Fig. 8 demonstrates an overall morphology of mouse fibroblasts cultured on the polystyrene culture plate, as well as on the PLLA, CGP
Fig. 7. Relative viability of L929 mouse fibroblasts cultured on the PLLA and CGP films for 1 week. Results of MTT-test expressed as mean± standard deviation for three replicates. Viability of the cells cultivated on polystyrene plates was used as a control (100%).
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Fig. 8. Microphotographs of the L929 cells on polystyrene plate (1) and on the initial PLLA (2), the initial CGP (4a), the CGP films treated by plasma on the cathode (4b) and anode (4c); the initial chitosan (3). Cultivation for 7 days.
and chitosan films for 1 week. As well known, mouse fibroblast L929 can grow after being attached and spread on the surface. From Fig. 8 it is obviously that not all cells were spread on the film surface. Noteworthy, the lowest number of round cells (not spread cells on the film surface) was observed on the initial CGP and initial chitosan films as well as on the polystyrene culture plates. According to SEM images, these surfaces listed above were the most smooth and homogeneous unlike to initial PLLA on which the cells spread respectively worse. Comparing L929 spreading on plasma modified CGP films, it can be seen that cells flatten better on the cathode treated CGP films in comparison with the anode treated samples (cf. Fig. 8 4b and 4c). These data are in good agreement with the SEM images of CGP films treated by plasma. The higher amount of local defects on the anode treated sample prevented the cell spreading whereas more homogenous surface destruction during plasma treatment on the cathode gave to cells the space for attachment. However, in spite of better cell attachment onto cathode treated CGP films the cell viability in this case was lower than that onto anode treated sample. It's possible to assume that the different cell viability on the samples can be explained by different composition of the layer of the absorbed biomolecules (from culture medium) that depended on the amount of nitrogen containing groups on the surface layer after plasma treatment (Table 1). Additionally, cell growth on the low-temperature plasma treated CGP films which were kept for 5 months in the polyethylene bags at RT was evaluated. The MTT assay revealed that viability of the cells cultivated on the initial CGP films maintained at the same level, namely 63%, compared to that in the case of the films after being stored for a few days. At the same time, cell viability on the plasma treated samples changed after 5 months of storage. In particular, we have found that a percentage of viable cells increased from 22% to 52% and from 9% to 29% for the films treated on the anode and cathode, respectively. These data confirmed our suggestion that the surface properties affected by plasma treatment could partly be restored at storage. According to the literature, these changes
could be explained by surface rearrangement, e.g. thermally activated macromolecular motions to minimize the interfacial energy [44,45]. 4. Conclusion For the first time, we fabricated and studied the films from chitosan/ gelatin/PLLA blend obtained by SSRB technique. The obtained biocompatible and biodegradable films combine the useful properties of natural and synthetic polymers which makes them a promising material for tissue engineering purposes. The XPS and contact angle measurements revealed that the surface layer of the film casted after dissolution of the blend in organic solvent was enriched with the polyester component. The plasma treatment of the composite films as well as the films prepared from initial components (chitosan, gelatin and PLLA) led to the surface hydrophilicity increase. However, plasma treatment resulted in a decrease of oxygen-containing groups and in the appearance of nitrogen-containing ones. Study of the surface topography showed that the untreated film possessed homogenous structure, whereas plasma treatment led to local destruction which was more pronounced on the anode treated film than on the cathode one. The MTT-test and microscopic observations confirmed that mouse fibroblast adherence and growth on the initial CGP films were higher compared to that on the PLLA films, while plasma treatment led to a reduction of fibroblast cell attachment and viability. Depending on the application of the materials, the surface can be treated with the low temperature plasma with the aim to suppress growth of the desired cell lines on the materials. Thus, plasma treatment of the CGP films could be proposed as a good tool to control cell/interface interactions. References [1] P. Roach, P.D. Eglin, K. Rohde, C.C. Perry, J. Mater. Sci. Mater. 18 (2007) 1263. [2] P. Simamora, W. Chen, J. Drugs Dermatol. 5 (2006) 436.
516 [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25]
T. Demina et al. / Surface & Coatings Technology 207 (2012) 508–516 Y.Q. Wang, J.Y. Cai, Curr. Appl. Phys. 7 (2007) 108. N. Nafee, S. Taetz, M. Schneider, U.F. Schaefer, C.-M. Lehr, Nanomedicine 3 (2007) 173. L. Lao, H. Tan, Y. Wang, C. Gao, Colloids Surf. B Biointerfaces 66 (2008) 218. S.A. Martel-Estrada, C.A. Martínez-Pérez, J.G. Chacón-Nava, P.E. García-Casillas, I. Olivas-Armendariz, Carbohydr. Polym. 81 (2010) 775. A.R.C. Duarte, J.F. Manoa, R.L. Reis, J. Supercrit. Fluids 54 (2010) 282. B.M.P. Ferreira, L.M.P. Pinheiro, P.A.P. Nascente, M.J. Ferreira, E.A.R. Duek, Mater. Sci. Eng. C 29 (2009) 806. P.K. Chu, J.Y. Chen, L.P. Wang, N. Huang, Mater. Sci. Eng. R. 36 (2002) 143. F. Intanuovo, R. Sardella, R. Cristina, M. Nardulli, L. White, D. Howard, K.M. Shakesheff, M.R. Alexander, P. Favia, Surf. Coat. Technol. 205 (2011) S542. G. Da Ponte, E. Sardella, F. Fanelli, A. Van Hoeck, R. d'Agostino, S. Paulussen, P. Favia, Surf. Coat. Technol. 205 (2011) S525. M.T. Khorasani, H. Mirzadeh, S. Irani, J. Appl. Polym. Sci. 112 (2009) 3429. Z. Ding, J. Chen, G. Sao, J. Chang, J. Zhanga, E.T. Kang, Biomaterials 25 (2004) 1059. J.P. Chen, C.H. Su, Acta Biomater. 7 (2011) 234. J. Yang, J.Z. Bei, S.G. Wang, Biomaterials 23 (2002) 2607. T.A. Akopova, A.N. Zelenetskii, A.N. Ozerin, A.N. Ferguson, A.G. O'Neill, Ch. 8, in: Focus on Chitosan Research, Nova Science Publishers, N.Y., USA, 2011, p. 223. A. N. Ozerin, A. N. Zelenetskii, T. A. Akopova, et al., RF Patent 2,292,354, 2007. R.A.A. Muzzarelli, C. Muzzarelli, Adv. Polym. Sci. 186 (2005) 151. R. Jin, L.S. Moreira Teixeira, P.J. Dijkstra, M. Karperien, C.A. van Blitterswijk, Z.Y. Zhong, J. Biomater. 30 (2009) 2544. C. Shi, Y. Zhu, X. Ran, M. Wang, Y. Su, T. Cheng, J. Surg. Res. 133 (2006) 185. J.K.F. Suh, H.W.T. Matthew, Biomaterials 2 (2000) 2589. S. Silva, S. Luna, M. Gomes, J. Benesch, I. Pashkuleva, J. Mano, R. Reis, Macromol. Biosci. 8 (2008) 568. P.B. Malafaya, G.A. Silva, R.L. Reis, Adv. Drug Delivery Rev. 59 (2007) 207. H.A. Awad, M.Q. Wickham, H.A. Leddy, J.M. Gimble, F. Guilak, Biomaterials 25 (2004) 3211. M.S. Ponticiello, R.M. Schinagl, S. Kadiyala, F.P. Barry, J. Biomed. Mater. Res. 52 (2000) 246.
[26] S.Z. Rogovina, T.A. Akopova, G.A. Vikhoreva, J. Appl. Polym. Sci. 70 (1998) 927. [27] S. Wu, Polymer Interfaces and Adhesion, Marcel Dekker Inc., N.Y., USA, 1982. [28] J.F. Rabek, Experimental Methods in Polymer Chemistry, Wiley Inc., N.Y., USA, 1980. [29] http://srdata.nist.gov. [30] D. Briggs, M.P. Seah, Practical Surface Analysis by Auger and X-Ray Photoelectron Spectroscopy, Wiley Inc., N.Y., USA, 1983. [31] G.M. Sessler, Electets, third ed. Laplacian press, CA, USA, 1999. [32] N. Behrendt, C. Greiner, F. Fischer, T. Frese, V. Altstadt, H.-W. Schmidt, R. Giesa, J. Hillenbrand, G.M. Sessler, Appl. Phys. A: Mater. Sci. Process. 85 (2006) 87. [33] T. Frese, D. Lovera, D.J.K.W. Sandler, G.-T. Lim, V. Altstadt, R. Giesa, H.-W. Schmidt, Macromol. Mater. Eng. 292 (2007) 582. [34] Y. Wan, J. Yang, J. Yang, J. Bei, S. Wang, Biomaterials 24 (2003) 3757. [35] L.J. Matienzo, S.K. Winnacker, Macromol. Mater. Eng. 287 (2002) 871. [36] M.T. Khorasani, H. Mirzadeh, S. Irani, Radiat. Phys. Chem. 77 (2008) 280. [37] T. Demina, T. Demina, D. Zaytseva-Zotova, M. Yablokov, A. Gilman, T. Akopova, E. Markvicheva, A. Zelenetskii, High Energy Chem. 46 (2012) 60. [38] N. Inagaki, Plasma Surface Modification and Plasma Polymerization, Technomic. Publ. Co., Lancaster, PA, 1996. [39] A. Kaminska, H. Kaczmarek, J. Kowalonek, Eur. Polym. J. 38 (2002) 1915. [40] C. Riccardi, R. Barni, E. Selli, G. Mazzone, M.N.R. Massafra, B. Marcandalli, G. Poletti, Appl. Surf. Sci. 211 (2003) 386. [41] Y. Lin, L. Wang, P. Zhang, X. Wang, X. Chen, X. Jing, Z. Su, Acta Biomater. 2 (2006) 155. [42] P.B. van Wachem, A.H. Hogt, T. Beugeling, J. Feijen, A. Bantjes, J.P. Detmers, W.G. van Aken, Biomaterials 8 (1987) 323. [43] H. Chen, J. Huang, J. Yu, S. Liu, P. Gu, Int. J. Biol. Macromol. 48 (2011) 13. [44] L. Safinia, N. Datan, M. Höhse, A. Mantalaris, A. Bismarck, Biomaterials 26 (2005) 7537. [45] X. Ximing, T.R. Gengenbach, H.J. Griesser, J. Adhes. Sci. Technol. 6 (1992) 1411.