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Towards cell selective surfaces: cell adhesion and proliferation on breath figures with antifouling surface chemistry Enrique Martinez-Campos, Tamara Elzein, Alice Bejjani, Maria Jesus Garcia-Granda, Ana Santos-Coquillat, Viviana Ramos, Alexandra Munoz-Bonilla, and Juan Rodriguez-Hernandez ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b12832 • Publication Date (Web): 24 Feb 2016 Downloaded from http://pubs.acs.org on February 25, 2016

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ACS Applied Materials & Interfaces

Towards Cell Selective Surfaces: Cell Adhesion and Proliferation on Breath Figures with Antifouling Surface Chemistry

Enrique Martínez-Campos,1 Tamara Elzein,2 Alice Bejjani,2 Maria Jesús GarcíaGranda,1 Ana Santos-Coquillat,1 Viviana Ramos,1 Alexandra Muñoz-Bonilla3 and Juan Rodríguez-Hernández4*

1. Tissue Engineering Group; Instituto de Estudios Biofuncionales, Universidad Complutense de Madrid. Paseo Juan XXIII, nº 1 28040 Madrid, Spain. 2. Lebanese Atomic Energy Commission-National Council for Scientific Research CNRS-L. P.O. Box: 11-8281, Riad El Solh, 1107 2260, Beirut, Lebanon 3. Departamento de Química Física Aplicada, Facultad de Ciencias, Universidad Autónoma de Madrid, C/Francisco Tomás y Valiente 7, Cantoblanco, 28049 Madrid, Spain. 4. Instituto de Ciencia y Tecnología de Polímeros (ICTP), Consejo Superior de Investigaciones Científicas (CSIC), C/Juan de la Cierva 3, 28006 Madrid, Spain. Email: [email protected] . 1 ACS Paragon Plus Environment


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Abstract We report the preparation of microporous functional polymer surfaces that have been proved to be selective surfaces towards eukaryotic cells while maintaining antifouling properties against bacteria. The fabrication of functional porous films has been carried out by the breath figures approach that allowed us to create porous interfaces with either poly(ethylene glycol) methyl ether methacrylate (PEGMA) or 2,3,4,5,6-pentafluorostyrene (5FS). For this purpose, blends of block copolymers in a polystyrene homopolymer matrix have been employed. In contrast to the case of single functional polymer, using blends allowed to vary the chemical distribution of the functional groups inside and outside the formed pores. In particular, fluorinated groups were positioned at the edges while the hydrophilic PEGMA groups were selectively located inside the pores, as demonstrated by TOF-SIMS. More interestingly, studies of cell adhesion, growth and proliferation on these surfaces confirmed that PEGMA functionalized interfaces are excellent candidates to selectively allow cell growth and proliferation while maintaining antifouling properties.

Keywords Antifouling surfaces, selective cell adhesive, porous films, breath figures, functional polymer surfaces, microstructures

2 ACS Paragon Plus Environment

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Introduction

A crucial issue in materials applications for biorelated purposes concerns the contamination by microorganisms and in particular by bacteria. This problem affects many different applications of these materials such as medical devices, healthcare products, water purification systems, hospitals or dental office equipment, food packaging, food storage, household sanitation, just to menta ion few of them.1-2 Bacterial contamination is a common and still unresolved problem that affects most of the commonly used biomaterials. Although this is a general problem independently of the biomaterial considered, it is particularly serious in long-term implants. For instance, long-term catheters can be affected by implant-associated infections.3-5 As a result, the development of novel interfaces with controlled adhesion properties appears to be crucial. Different strategies have been developed to achieve this goal, including the incorporation of antibiotics within the material for further release or the immobilization of antimicrobial coatings.6 In addition, surface structuration has been highlighted to play a key role on bacterial adhesion. Surface roughness as well as the shape of micro and nanopatterns have been recently explored as a mean to reduce bacterial adhesion7. For instance, several studies have evidenced the direct relation between the size of surface moieties and the amount of microorganisms immobilized. Nevertheless, antimicrobial properties need to be accompanied by biocompatibility, including eventually cell adhesion and proliferation studies, especially in the evaluation of a particular material for implants. Most of the studies reported so far have been focused on studying either one these issues separately. Herein, we propose model surfaces that can serve as basis for the development of potential antimicrobial and simultaneously biocompatible implants. In this design, we have taken into consideration both the chemistry required at the surface level and the microstructuration, in order to produce polymeric materials that favor the growth and proliferation of cells at the surface while preventing bacterial adhesion. For that purpose, we



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employed the Breath Figures approach that permits the elaboration of porous surfaces with controlled chemistry and pore sizes. The Breath Figures (BF) methodology has been largely employed to prepare porous films based on the evaporation of polymeric solution under moist atmosphere. As a result of the solvent evaporation, the temperature of the solvent surface dramatically decreases, favoring water vapor condensation. Thus, water droplets grow at the solution-air interface. Finally, solvent and water droplets are evaporated leaving porous films. The Breath Figures approach has been extensively employed to produce porous interfaces with variable pore sizes ranging from few tens of nanometers up to ~20 µm. The simplicity of the procedure has attracted the attention of different research groups and pioneer studies focused on the understanding of the physics behind this phenomenon. Nowadays, many studies are focused on the elaboration of porous films having variable pore shapes, chemistry and even exhibiting hierarchical organization by using block copolymers. 8-10 The Breath Figures approach has been already studied in cell adhesion processes for the development of cell culture scaffolds with a great potential in different areas including tissue engineering.11-12 Besides the requirements of biocompatibility or mechanical properties, these scaffolds should present high porosity. Since the outermost surface of the scaffolds is in contact with the cell, the properties of the surface will direct the interactions and, consequently, cell adhesion. In this sense, BF technique has been proposed as a very simple and versatile method to fabricate micropatterned cell culture substrates 12-24 and also as adhesion barriers or to reduce postoperative adhesion using fibrinolytic agents or anticoagulants, among others. 25-26 However, most of the previous reports over the cell adhesion onto micropatterned honeycomb films focused on the initial stages of protein adhesion 27, the role of the pore size 20 or the influence of the porous topography in comparison to planar films.28 A key step towards the understanding of cells and bacterial adhesion requires more sophisticated interfaces in which not only the chemical composition or the pores size but also their distribution could be varied. The systems explored up to date involve chemically different


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polymeric materials such as commercially available polymers, e.g. polycaprolactone

27

or

amphiphilic copolymers.29 In those systems, the analysis of the chemical contribution to the cell adhesion was just limited to those particular polymers and no systematic analyses varying gradually and spatially the chemical composition were carried out. As will be described below, blending is an interesting alternative to concentrate particular functional groups on precise positions: inside, outside or at the edge of the pores. Moreover, there are no previous investigations that combine studies with microorganisms and cells using porous films prepared by the breath figures approach. Both aspects have been studied separately and only few studies have evaluated the antimicrobial/antifouling properties of the honeycomb patterned surfaces.26 Within this context, recent reports of our group 13, 30-31 and other teams 26 have been focused on understanding the role of pore size and surface chemistry on the bacterial adhesion, or on the development of honeycomb-structured films with antimicrobial/antifouling properties in order to reduce the bacterial adhesion, which is essential for biomaterials used in vivo. Our recent findings demonstrate that both pore size and chemistry clearly direct bacterial adhesion. In this study we explored the formation of micrometer sized porous films having similar pore dimensions but varying the surface chemistry and the distribution. In particular, we will describe the preparation of porous interfaces based on the Breath Figures methodology and the evaluation of cell adhesion/growth on these surfaces. The surfaces evaluated contain polystyrene (PS: employed as reference) and variable amounts of either poly(2,3,4,5,6pentafluorostyrene) (P5FS) or poly(poly(ethylene glycol) methyl ether methacrylate) (PPEGMA). The rationale behind the selection of materials is based on the antifouling properties against microorganisms exhibited by both P5FS and PPEGMA, which imply two different principles. On the one hand, P5FS increases the hydrophobicity of the material surfaces preventing bacterial adhesion by reducing the contact between the environmental solution and the material surface. On the other hand, PPEGMA has been extensively explored for its capability in the prevention of protein adhesion, which generates antiadhesive bacterial properties.



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Experimental Section Materials. High molecular weight polystyrene (PS2400) (Aldrich, Mw = 2.50•105g/mol) was used as polymeric matrix while tetrahydrofuran (THF), chloroform (CHCl3) and carbon disulfide (CS2) were purchased from Scharlau and employed as solvents. Round glass coverslips of 12 mm diameter were supplied from Ted Pella Inc. Polymer synthesis. The

hydrophobic

poly(2,3,4,5,6-pentafluorostyrene)-b-polystyrene

(P5FS21-b-PS31)

and

amphiphilic block copolymers polystyrene-b-poly[poly(ethylene glycol) methyl ether methacrylate] i.e. PS45-b-PPEGMA34 were synthesized via atom transfer radical polymerization (ATRP) as previously reported.32-33. The monomer units of each block are indicated by the number depicted below and is the result of 1H-NMR analysis. Equally, GPC analysis indicated a monomodal distribution of the block copolymers with narrow polydispersities below 1.2 for both cases. For clarity purposes we will referee to those block copolymers without the numbers. Measurements. Scanning electron microscopy (SEM) micrographs were taken using a Philips XL30 with an acceleration voltage of 25 kV. The samples were coated with gold-palladium (80/20) prior to scanning. Time Of Flight-Secondary Ion Mass Spectrometry (TOF-SIMS) measurements were carried out at the Lebanese Atomic energy commission using a TOF-SIMS 5-100 (IONTOF GmbH, Münster, Germany) equipped with two sources; Bi liquid-metal ion source for imaging and for spectroscopy analysis and an dual Argon ion source for sputtering and analysis, both with an incident angle of 45 degrees to the surface of the sample. The images of the negative ions, presented in this work, were collected using the Bi3+ on different area sizes with a primary dose of more than 8 x102 ions/cm2 and at least a pixel size of 0.2 x 0.2 µm2. The secondary ions were extracted with 2 keV energy passing throw a single stage reflector before hitting a single micro channel plate detector. Low energy flood gun is used for the surface charge compensation.


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All the samples were first cleaned by a 3 nA cluster Ar1500 at an energy of 5 keV, for 50 seconds to remove all the organic contaminants from the surface of interest. The internal mass calibration of the acquired spectra is done using C-, C2-, C3- in the negative mode, which permits a mass accuracy of few ppm. The data were acquired and processed with SurfaceLab 6.5 software from IONTOF GmbH. Preparation of the honeycomb films with variable surface chemical composition. Different blends were prepared by mixing high molecular weight polystyrene matrix (75, 50 and 25 wt. %) with the appropriate block copolymer (25, 50, 75 wt.%), maintaining constant the total concentration of polymer in the solution (30 mg/mL). Films were obtained from these solutions by casting onto glass wafers at room temperature under controlled humidity inside of a closed chamber. The samples using blends of the block copolymer P5FS-b-PS and PS were obtained using CS2 as solvent and a saturated vapor humidity (> 99% relative humidity (RH)) whereas the films from the blends of PS-b-PPEGMA and PS were prepared using THF as solvent and 70 % RH. In addition, for comparative purposes, a porous surface was prepared using chloroform as solvent and cast under saturated vapor atmosphere.

Bacterial adhesion evaluation. Bacteria Immobilization. Staphylococcus aureus strain RN4220 carrying the plasmid pCN57 for GFP expression was grown overnight at 37 °C in Luria−Bertani (LB) media with erythromycin (10µg/mL). The cells were centrifuged and washed three times in PBS buffer (150 mM NaCl, 50 mM sodium phosphate pH 7.4). The solution was adjusted to a fixed cell concentration that corresponds to an optical density (OD) at 600 nm of 1.0. The patterned polymeric surfaces were incubated during 1 h with a bacterial suspension at OD = 1.0 in PBS buffer with 0.05% v/v Tween 20. After incubation the surfaces were thoroughly washed with PBS. Fluorescence microscopy for bacteria imaging was performed using a Leica DMI-3000-B fluorescence microscope. Images were acquired using different magnifications (10×, 20×, 40×, and 60×) and the corresponding set of filters for imaging green fluorescence and bright field.



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Experimental protocol for the cell-adhesion experiments. Prior to cell studies, all surfaces were sterilized with a 70% ethanol solution rinsing four times during 10 minutes. Then, the honeycomb surfaces were washed with PBS four times, exposed to UV radiation during 20 minutes, washed two times with incomplete culture medium (DMEM), and finally washed twice with complete culture medium (FBS and antibiotics). The cell studies were carried out using C2C12-GFP, a mouse pre-myoblast cell line (CRL 1772™, obtained from ATCC®, USA). Green Fluorescent Protein (GFP) was expressed due to previous lentivirus infection of this cell line. Routine passaging of the cell line was performed with DMEM high in glucose (GIBCO, UK), supplemented with 10% fetal bovine serum (FBS, 10500-064, Gibco, UK) plus antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin sulfate) (Gibco, UK). The medium was refreshed every two or three days. These cells were chosen as in vitro model because they are able to differentiate toward osteoblastic or myoblastic phenotype, depending on the surrounding microenvironment. Another interesting characteristic of these cells concerns their self-fluorescence. Due to this property, they can be analyzed through some non-opaque/translucent surfaces, such as the polymer films employed here.

In order to test the versatility of the platforms, a second cell line, i.e. 3T3-Swiss albino, was employed in the experiments. This is a mouse embryo fibroblast cell line (CCL-92™, ATCC®, USA). The routine passaging and culture conditions were the same as for C2C12-GFP cells. In particular, these cells were chosen as model in vitro phenotype of fibroblast. For culturing cells over the porous films, the three cell types were seeded singly on the samples at different densities in supplemented DMEM, and the polymers were plated in a 24-well plate in maintenance medium, incubated at 37 °C with 5% CO2 in a humidified incubator. For experiments on PS-copolymers, cells were seeded at a density of 5x103/polymers.


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Metabolic activity study: Alamar Blue assay. Metabolic activity of cells was measured by Alamar Blue assay; this was performed following the manufacturer’s instructions (Biosource, CA, USA). Assays were carried out in triplicate on each sample type. This method is non-toxic, scalable and uses the natural reducing power of living cells, generating a quantitative measure of cell viability and cytotoxicity. Briefly, Alamar Blue dye (10 % of the culture volume) was added to each well, containing living cells seeded over films, and incubated for 90 minutes. The fluorescence (λex/λem 535/590 nm) of each well was measured using a plate-reader (Synergy HT, Brotek).

Actin and Hoechst staining. Actin labelling was performed in order to evaluate qualitatively F-actin microfilaments of cytoskeleton (involved in mobility and contraction of muscle cells). Additionally, Hoechst staining was carried out to determinate the number of viable versus apoptotic cells. Cells on the films were fixed with 4% paraformaldehyde (PFA) solution for 10 min. Once the PFA was removed, cells were rinsed with PBS twice and permeabilized with 0.1% (v/v) Triton X-100. Then, the cells were washed with PBS and stained with Texas Red®-X phalloidin (Life Technologies, Grand Island, NY), a high-affinity F-actin probe conjugated to red fluorochrome, for 20 minutes at room temperature and in darkness, followed by Hoechst staining (Invitrogen, Molecular Probes®). Finally, fluorescent-labelled cells were observed using an inverted fluorescence microscope (Olympus IX51) with a TRICT filter (λex/λem=550/600 nm) for Actin and DAPI filter for Hoechst (λex/λem = 380/455nm) using CellD analysis software (Olympus).

Immunocytochemistry Focal adhesions (FAs) of seeded cells were studied using immunocytochemistry. The staining protocol was performed as standard procedures: PFA 4% fixation, Blocking Solution (5% Normal Donkey Serum) and 0.3% Triton X-100 in 1x PBS. Later, cells were incubated with mouse monoclonal anti-vinculin antibody (1/150, Sigma-Aldrich) at 4 °C overnight and rinsed four times with PBS and finally one additional time with blocking solution for 30 min at room 9 ACS Paragon Plus Environment


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temperature. Then, the FAs were labeled by anti-mouse IgG(y1)-Alexa 488 antibody (1/200, Life technologies) at 37 °C for 2 h in darkness. Finally, cells were rinsed 4 times with PBS and results were visualized and recorded under the microscope, camera and program described above, using TRICT filter (λex/λem=550/600 nm).

Results and discussion Whereas many different chemical systems have been proposed for the elaboration of porous films by the Breath Figures approach, the use of polymer blends has received limited attention in spite of several important advantages. First of all, blending permits the compartmentalization of the different components and functional groups. As a result, it is possible to prepare materials with different chemical functions inside and outside the pores. Moreover, depending on the amount of each component in the precursor solution, the density of functional groups present at the interface can be varied. Finally, blending allows us to create surfaces with a large variety of chemical functionalities with one or more different functional groups at the surface.9, 34 According to precedent studies, the chemical composition at the surface plays a key role on both the bacterial and cell adhesion. In particular, surfaces with hydrophobic and highly hydrophobic (fluorinated) groups and those containing PEGMA moieties demonstrated to prevent bacterial adhesion, at least during the initial incubation process.31 On the contrary, polypeptides sequences or hydrophilic groups such as poly(acrylic acid) show extensive bacterial immobilization.30 In order to fabricate polymeric microstructured surfaces with both, antifouling properties against bacteria and adhesive properties towards cells, we have selected the polymers depicted in Figure 1. Fluorinated moieties were introduced in the porous films by using a blend of P5FS-b-PS with PS (2). The blends with variable amount of the components were dissolved in CS2 and cast under humid atmosphere. PEGMA containing porous films were prepared by mixing PS-b-PPEGMA with PS (3). In this case, the solvent employed was THF. Control


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experiments were carried out with PS; in this case the porous structure was obtained using CHCl3 as solvent (1).

Figure 1. Three components were employed for the preparation of the porous films: (1) homopolystyrene, (2) poly(2,3,4,5,6-pentafluorostyrene)-b-polystyrene (P5FS-b-PS) and (3) polystyrene-b-poly[poly(ethylene glycol) methyl ether methacrylate] i.e. PS-b-PPEGMA. Blends with variable composition of polymers (1) and (2) and polymers (1) and (3) were employed for the preparation of functional porous films with different chemical composition.

Characterization of the pores and surface chemical distribution In Figure 2 are depicted the SEM images of the porous films obtained upon solvent casting of the above mentioned blends using either 75% RH (blends containing PS-b-PPEGMA) or 100% RH (blends containing P5FS-b-PS). The polymer concentration employed was 30 mg/ml for all the experiments. The porous surfaces obtained using exclusively the fluorinated block copolymer (PS/ P5FS-b-PS= 0/100) lead to surfaces with hexagonal arrays of pores, honeycomb structures, having diameters of around 5 µm. The order and the average pore size (around 5 µm) 11 ACS Paragon Plus Environment


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are maintained in surfaces prepared using blends of 50% of polystyrene and 50% of fluorinated block copolymer. On the contrary, the order clearly decreases in the porous films prepared using only 25% of P5FS-b-PS block copolymer. In the cases of porous surfaces prepared using the amphiphilic block copolymer in a percentage of 50% and 25%, the SEM micrographs indicated the formation of pores with narrow size distribution but lacking of the perfect hexagonal structuration. Nevertheless, the average pore size, of around 5µm, was similar for both blends and for the films prepared using exclusively PS (employed as reference). It has to be mentioned that the use of PS-b-PPEGMA copolymer alone (instead of a blend) does not induce the formation of structured breath figures films. According to previous studies, the optimal pore size to favor cell adhesion and growth is comprised in the range of 3.5 to 5µm.17,

19-20, 28

Therefore, in this study the experimental conditions were optimized to obtain pore sizes within this range, independently of the chemical groups employed to fabricate the porous films.


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Figure 2. SEM images of the porous films: (1), (2) and (3) blends composed of P5FS-b-PS and PS. (4) and (5) porous films prepared from blends of PS-b-PPEGMA and PS using two different compositions. (6) corresponds to a porous surface obtained using exclusively PS. In addition to the pore size and regularity, a key factor investigated in this work is the distribution of the chemical functional groups of the different systems over the surface. An interesting tool to obtain this information is TOF-SIMS, which permits imaging of regions with distinct chemical composition. For this purpose it is possible to generate images corresponding to specific atoms or molecular fragments from the total mass spectrum collected by the TOFSIMS software from every pixel of the area of interest. For instance, the porous surfaces prepared using blends of P5FS-b-PS can be characterized taking into account the particular mass segments originated from the poly(pentafluorostyrene) block. As it is shown in the spectrum depicted in Figure 3, two peaks characteristic of the poly(pentafluorostyrene) block (m/z=19 and 167 for the F- ion and the C8F5- ion) can be clearly identified. These two peaks have been chosen to generate the images, besides the image of all the detected ions. Consequently, TOF-SIMS provides information not only about the film morphology but also about the chemical distribution of the functional groups at the top surface.



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Figure 3. TOF-SIMS negative spectrum of the pure P5FS-b-PS analyzed with a Bi3+ beam. Two characteristic peaks can be clearly identified at m/z of 19 and at m/z of 167 assigned respectively to F- and C8F5-.

Figure 4. Images of: the sum of all negative ions, F- (m/z 19), and C8F5- (m/z 167). (a) Porous films prepared from pure P5FS-b-PS (30 x 30 µm2). (b) Porous films prepared from blends 50:50 wt% of PS/P5FS-b-PS (50 x 50 µm2). The color scale goes from black (lack of emission) to white (saturated emission). We can clearly observe in Figure 4 that in the case of films prepared exclusively from the block copolymer P5FS-b-PS, the selected segments are distributed homogenously in the surface. This indicates a homogeneous distribution of the fluorinated block copolymer. On the contrary, for the case of the PS/P5FS-b-PS blends (50/50 wt %), the images obtained for the F- and the C8F5ions (Figure 4b) show ring-like patterns suggesting that the block copolymer segregates to the edges of the cavities. Most probably, the enrichment of the fluorinated diblock within the pores is mainly due to the phase separation between the P5FS-b-PS diblock and the PS homopolymer occurring during the film formation. Whilst it is not possible to determine the exact position of each block, it is likely that a homogeneous chemical composition would be observed since the water condensation process is very fast in comparison with the kinetics required to obtain a regular microphase separated system. 14 ACS Paragon Plus Environment

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The other relevant information for the next step of this study results from the comparison between the pore sizes obtained in the films prepared from the pure diblock and the blends. In spite of the partial loss of the perfect hexagonal array, the average pore sizes are similar, around 5 µm. Thus, blending the PS homopolymer with the fluorinated diblock copolymer, drives the latter to migrate toward the pore edges (as represented in Figure 4), creating a variable chemistry on the film surface and around the cavities without critical modification of the dimensional parameters of the microstructures. Interestingly, when the highly hydrophobic block, i.e. P5FS, is replaced by a hydrophilic block (PEGMA) not only the chemical composition but also the surface distribution completely changes. As illustrated in Figure 5 for the case of PS/PS-b-PPEGMA blend (75:25), the emission of the C2H3O- ions clearly emerges from the cavities. Assuming that this ion is a fingerprint of the PEGMA block, we can deduce that this hydrophilic block is preferentially oriented inside the pores (Figure 5). Such a phase separation can be predicted taking into account the breath figures mechanism. During the formation and growth of the condensed water droplets, there is still solvent in the polymer solution that permits the reorientation of the hydrophilic block towards the water droplets. As a result, upon complete evaporation of solvent and water droplets the amphiphilic block copolymer is arranged around the inner part of the pore.



Figure 5. 100 x 100 µm2 images of the total negative ions, and the C2H3O- ions emitted from the PS-b-PPEGMA diblock copolymer blended with polystyrene (PS) with a ratio of 25:75. As a result, by combination of amphiphilic or double hydrophobic block copolymers within blends it is possible to largely vary the interfacial composition that in turn, will modify the cell adhesion, as illustrated in Figure 6. Hydrophobic or highly hydrophobic interfaces with homogeneous surface chemical composition were prepared using either PS or P5FS. The preparation of porous films by combination of PS and P5FS-b-PS resulted in surfaces in which the pore edge was surrounded with highly hydrophobic P5FS whereas the rest of the surface is composed of PS. This particular surface composition and distribution is interesting from a biological point of view, since previous studies evidenced the role of the pore edge on the cell adhesion.27 Finally, when amphiphilic block copolymers were employed in the blend, the pores were fully enriched with the hydrophilic block.

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PS/P5FS-b-PS

(d) PS/PS-b-PPEGMA

Figure 6. Schematic representation of the chemical distribution of the blend components at the surface, as evidenced by TOF-SIMS. The porous films described here were prepared by using (a) exclusively PS (red), (b) the block copolymer P5FS-b-PS (green), (c) a blend of PS and


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P5FS-b-PS (50:50 wt.% blend) and (d) blends of PS and PS-b-PPEGMA (75:25 wt.% blend). (Note that in the cross sectional profile the PEGMA chains are depicted in blue). Bacterial adhesion assays The bacterial adhesion onto microporous films having different chemical composition was evaluated against S. aureus. The bacterial adhesion tests were carried out by incubation of S. aureus during 1 h at room temperature at an optical density of 1. After incubation with the bacterial suspension the surface was thoroughly washed. The results of the assay recorded using fluorescence microscopy are presented in Figure 7. As shown in the figure, a higher bacterial concentration was observed in the microporous surfaces obtained using exclusively PS. Interestingly, the incorporation of either fluorinated moieties or PEG functional groups clearly decreased the adhesion at short-medium incubation times. This interesting result is the base of the following studies related to the analysis of adhesion and proliferation using both premyoblastic C2C12 cells as well as Swiss 3T3 cells.



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(a)

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(b)

(c)

Figure 7. honeycomb

Fluorescence

images of

microstructured

films

the after

incubation with S. aureus during 1 h on microporous surfaces prepared using (a) PS, (b) P5FS-b-PS and (c) PS/PS-b-PEGMA.

Cell adhesion/spreading/growth experiments onto the functional porous films In order to test the suitability of the micropatterned surfaces for in vitro tissue engineering, preliminary cytocompatibility tests were made using pre-myoblastic C2C12-GFP cells. In particular, adhesion, spreading and growth of smooth muscle cells have been explored. As depicted in Figure 8A, pre-myoblastic cells proliferated extensively at day 7 of culture, almost forming a monolayer in those porous films prepared using exclusively PS-b-PPEGMA. Despite this, the growth rate occurs slowly at the initial stage or even during the first days, in comparison with control cells. The labelling of actin of the cytoskeleton showed an outstretched and myoblast-like phenotype. An inmunocytochemistry for vinculin was performed to analyze the focal adhesions, i.e. the closest contacts between cells and the underlying porous substrate.


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As shown in Figure 8C, a high number of cellular anchoring junctions over these surfaces can be identified. In addition, Alamar Blue (AB) assays were performed as a cell health indicator by evaluating the metabolic state to measure quantitatively the proliferation of cell culture on different stages. This technique allows detecting any possible cytotoxic molecules released from biomaterial or provided by the biomaterial itself. The graph inserted in Figure 8B, illustrates the metabolically active cells on top of porous surfaces prepared from blends of PS/PS-b-PPEGMA varying the amount of block copolymer in the blend. Interestingly, independently of the amount of PS-b-PPEGMA employed to prepare the surfaces, there is a larger amount of proliferating cells in comparison with the porous films prepared exclusively using PS. Moreover, by comparing different PPEGMA surfaces with variable hydrophilic group density, it is clearly shown that an increase of the PPEGMA content improves the metabolic levels accordingly. As shown in Figure 6, PPEGMA hydrophilic blocks are preferentially oriented inside the pores. These groups promote specific cell adhesion into the pore cavity (25% and 50% PPEGMA containing copolymer) or over the entire film surface (in the case of 100% PS-b-PPEGMA). In fact, vinculin staining (Figure 7C) shows an active and strong cell adhesion over the 50% PS/PS-b-PPEGMA surface, including the pore cavity where all the PEGMA groups are mainly localized (Figure 7D). This junction by focal adhesion complex allows a good spreading behavior, extracellular matrix formation and cell growth. In summary, we can conclude that PPEGMA promotes adhesion, proliferation and cell culture activity in the porous films studied.



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Figure 8. C2C12-GFP cells seeded on different porous platforms of PEGMA-PS copolymer (A) From left to right: self-fluorescence of C2C12-GFP cells (green), actin staining (red), and Hoechst (blue), respectively, after fixation at 168 hours of culture. (B) Evaluation of the mitochondrial metabolic activity (Alamar Blue) at 96 hours of cell seeding on different PEGMA copolymers and PS control. (C) Anti-vinculin inmunocytochemistry of C2C12-GFP cells on 50% PEGMA at 168 h.

Besides the PS-b-PPEGMA copolymer, P5FS-b-PS was employed as additive for the preparation of porous films and these were tested as cell culture surfaces for myoblastic cells. C2C12-GFP cells have proliferated on the polymer surfaces (Figure 9A). Nevertheless, in comparison with platforms containing PEGMA, cells have a less expanded phenotype. It is observed that cell attachment is slower: cells were slightly adhered at first hours after seeding; later, they started to grow in aggregates instead along the whole surface. This fact could be due to the hydrophobic character of the polymeric surface. The actin staining of cytoskeleton 20 ACS Paragon Plus Environment

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(Figure 9B) pointed out a rounded morphology on P5FS-b-PS surfaces, proving that cells are weaker adhered. In Alamar Blue assays (Figure 9C), films with 25% P5FS-b-PS reached higher levels of metabolic activity than other P5FS-b-PS compositions, and similar to the films with 100% PS without fluorinated groups. When fluorinated groups are augmented in the surface composition, metabolic activity levels decreased (Figure 9C). As we have analyzed before in PS/P5FS-b-PS surfaces, fluorinated groups were found in the pore edges (Figure 6C), resulting in highly hydrophobic areas that markedly interfered with cellular adhesion mechanism. In P5FS-b-PS films (100% F), P5FS groups are distributed over the entire surface (Figure 6B), conferring a general hydrophobic behavior where cells are unable to adhere or proliferate. It is known that a high level of hydrophobicity results in a negative cell response.35 As a result, a decrease of the amount in fluoro-containing block copolymer improves both, cellular adhesion and proliferation.

Figure 9. Cell culture of C2C12 GFP on surfaces containing P5FS-b-PS. (A) Growth of cells over 50% P5FS-b-PS platform at 48, 96 and 168 hours of culture. (B) Actin and Hoechst staining of C2C12-GFP cells on 100% P5FS-b-PS after fixation at 96 hours of culture. (C) Evaluation of the mitochondrial metabolic activity of cells seeded on different P5FS-b-PS platforms (100%, 50% and 25%) and on control 100% PS at 96 hours.



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In addition to the C2C12-GFP muscle cells, Swiss 3T3 cells were seeded to evaluate the adhesion and growth of fibroblasts on the different porous films. Figure 10 shows Swiss 3T3 actin and Hoechst stainings at 168 hours after the cells were cultured on porous films prepared from PS-b-PEGMA and P5FS-b-PS. The fluorescence microscope images (Figure 10) showed how fibroblasts adhered and grew onto these surfaces. In particular, similarly to C12C12-GFP cells, Swiss 3T3 cells exhibited an outstretched morphology on PEGMA surfaces. Bright field inverted microscope (Supporting Information) also showed a good visualization through 25% PEGMA surfaces. The actin filaments of cells on P5FS-b-PS platforms were highly contracted, and as a result, cells appeared as small spheres (Figure 10), while cells on the PEGMA surfaces spread better and exhibit well-defined stress fibers and protrusions. Furthermore, Hoechst assay performed at 168 hours showed a higher number of nuclei on PEGMA films than in P5FS films, in particular with higher P5FS compositions. Taking these results into account, both eukaryotic cell lines studied (C2C12-GFP and Swiss 3T3) behave similarly over these surfaces, a fact that can be related to a parallel fibroblastic-like adhesion mechanism.


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Figure 10. Actin and Hoechst staining of Swiss 3T3 fibroblasts at 168 hours of culture on three different micropatterned porous films prepared using the following polymer components. Above: blend of PS and PS-b-PEGMA (75:25 wt%), center: P5FS-b-PS and down: a mixture of PS and P5FS-b-PS (50:50 wt%).

Conclusions We evaluated the preparation of functional porous surfaces that provided antifouling behavior against bacteria and permitted the adhesion and growth of both pre-myoblastic cells as well as fibroblast cells. In addition, we fabricated porous surfaces with variable chemical composition while maintaining a similar pore size of around 5 µm. For this purpose, we employed blends of a diblock copolymer in a homopolymer matrix. Blending allowed us to precisely control the position of the chemical functional groups inside, at the edge or outside the pores. For the fabrication of the functional porous films, two functional groups were selected, i.e. a hydrophobic P5FS and a hydrophilic PEGMA that are known to repel bacterial adhesion at least during the initial stages. We have shown that the position of the functional groups, distributed over the entire surface or at the edge of the pore, did not play a significant role when using the P5FS-b-PS block copolymer. However, the hydrophobicity of the P5FS segment appears to be determinant in the non-adhesion of both pre-myoblastic as well as fibroblast cells explored. On the other hand, when using PS-b-PPEGMA as additive the hydrophilic functional groups were encapsulated inside of the pores. Interestingly, in this case the cell lines explored were able to adhere, grow and even form a monolayer on top of the porous films. We believe that these results can serve as starting point to explore novel approaches to obtain selective surfaces to cell adhesion while preventing the contamination by microorganisms at the initial stages.



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Supporting Information. Optical images of the Swiss 3T3 fibroblast culture on the porous films prepared with variable chemical composition is provided as Supporting Information. Acknowledgments In memory of Professor José Luis López Lacomba, head of Tissue Engineering Group (IEB/UCM). This work was financially supported by the MINECO (MAT2013-47902-C2-1-R (JRH), and CONSOLIDER project; CSD2009-00088). A. Muñoz-Bonilla gratefully acknowledges the MINECO for her Ramon y Cajal. We also thank Noricum S.L. for its support in the cell studies.

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Using antifouling porous surfaces this manuscript describes the improvement of the cell growth (premyoblastic and fibroblastic) on surfaces decorated with poly(ethylene glycol methacrylate) (PEGMA). 396x169mm (96 x 96 DPI)

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