Geometric Control of Cell Alignment and Spreading within the

Article pubs.acs.org/journal/abseba

Geometric Control of Cell Alignment and Spreading within the Confinement of Antiadhesive Poly(Ethylene Glycol) Microstructures on Laser-Patterned Surfaces Christine Strehmel,†,‡,§ Heidi Perez-Hernandez,‡,⊥,# Zhenfang Zhang,§ Axel Löbus,§ Andrés F. Lasagni,*,⊥,# and Marga C. Lensen*,§ §

Department of Chemistry, Technische Universität Berlin, Straße des 17. Juni 124, D-10623 Berlin, Germany Institute of Manufacturing Technology, Technische Universität Dresden, George-Bähr-Straße 3c, D-01062 Dresden, Germany # Fraunhofer Institute for Material and Beam Technology IWS, Winterbergstraße 28, D-01277 Dresden, Germany

Downloaded by UNIV OF SUSSEX on August 25, 2015 | http://pubs.acs.org Publication Date (Web): August 5, 2015 | doi: 10.1021/ab5001657

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ABSTRACT: In this study, a mask-less laser-assisted patterning method is used to fabricate welldefined cell-adhesive microdomains delimited by protein-repellent poly(ethylene glycol) (PEG) microstructures prepared from multiarm (8-PEG) macromonomers. The response of murine fibroblasts (L-929) toward these microdomains is investigated, revealing effective cell confinement within the celladhesive areas surrounded by nonadhesive 8-PEG microstructures. Moreover, the spatial positioning of cells in microdomains of various sizes and geometries is analyzed, indicating control of cell density, size, and elongated cell shape induced by the size of the microdomains and the geometric confinement. KEYWORDS: cell adhesion, laser-assisted patterning, microstructure, poly(ethylene glycol), geometry

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with maleic anhydride polymers.6 In addition, PEG microstructures have been fabricated on silicon, glass, or poly(dimethylsiloxane) surfaces modified with a 3-(trichlorosilyl) propyl methacrylate (TPM) monolayer, using mask-based photolithography, enabling cell adhesion onto the adhesive surfaces and minimal cell adhesion onto the PEG microstructures.7a,b In principle, PEG microstructures are an effective barrier to protein adsorption and cell adhesion, since this well- established nontoxic and non- immunogenic polymeric material has been demonstrated to prevent nonspecific protein adsorption and unspecific prokaryotic and eukaryotic cell adhesion.8a−c Because of this versatile bioinertness, PEG-derived polymers have been widely used to passivate surfaces in order to reduce inflammatory response at the biomaterial interface.9 Intriguingly, we did, however, observe some protein adsorption as well as significant cell adhesion and spreading onto intrinsically inert PEG-based substrates when they were topographically or elastically patterned.1c,10,11 In the present study, we created unique polymer microstructures from our recently developed 8-arm, star-shaped PEGprecursors (8-PEG)12 on poly(ethyl maleic anhydride) (PEMA) coated glass surfaces and transparent Cyclic Olefin Polymer films (COP films) using the laser-assisted micro lens array patterning technique that we have previously applied to make PEGmicrostructures on functionalized glass substrates. Cross-like patterns yielding square, rectangular and rhomboidal shaped celladhesive microdomains were obtained in few processing steps upon cross-linking by UV light induced free radical mechanism

INTRODUCTION Controlling cellular behavior through biomolecular, chemical, physical, and topographical cues has attracted great interest in tissue engineering and applied biomaterials research. Particularly, the control of cell shape, distribution and orientation by physical microstructures (grooves, pillars, ridges, pores, and wells), and chemical cues (functionalized patches) using inorganic materials, metals, or polymers have been reported.1a−c One of the main advantages of polymers versus inorganic materials is their versatility in chemical composition, mechanical properties, and processing. Thus, polymers are widely used in microfabrication technologies, such as soft-, photo-, and electron beam lithography.2a,b However, polymer micropatterning of tissue-like polymeric materials by fast large-area mask or stamp techniques remains a challenge. Furthermore, microfabrication of microstructures with sharp angles and thin barriers to delimit cell-sized domains is difficult, for example by soft lithography (e.g., microcontact printing), due to the inherent limitations of these techniques such as lateral diffusion, surface tension of the ink, characteristics of the stamp, and the target surface.3 Therefore, alternative surface patterning techniques, e.g., laserassisted patterning, have been proposed for rapid, mask-less, and large-area microfabrication of well-defined periodic structures.4a−c Such micropatterns are ideal microenvironments for highthroughput parallel screening of soluble biomolecules, permitting a systematic analysis of cell phenotypes under certain conditions as well as providing a robust intermediate bioassay platform for drug discovery, diagnostics, and other related biomedical applications.5 Recently, we reported a mask-less, laser-assisted micro lens array (MLA) patterning procedure to fabricate periodic protein and cell adhesive microdomains using inert poly(ethylene glycol) (PEG) microstructures on cell adhesive glass surfaces coated © XXXX American Chemical Society

Received: December 14, 2014 Accepted: July 22, 2015

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DOI: 10.1021/ab5001657 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering to form polymeric networks.6 By changing the displacement distances and rotation angles before every subsequent laser exposure in the laser-assisted patterning procedure used, different geometries (square, rectangle or rhomboidal shaped) with a graded periodicity of 40, 60, 80, and 120 μm were fabricated to study the spatial confinement of cells in the adhesive areas and the geometric influence on cellular behavior.


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droxy-2methyl-1-propane-1-one, Irgacure 2959, BASF) and 8-PEG (35 wt %) was deposited on PEMA-functional glass coverslip or COP film surfaces, see Figure 1a. To pattern the 8-PEG, the substrate was irradiated with a 10 ns pulsed Nd:YAG laser (Quanta- Ray PRO 290, Spectra Physics) at 355 nm (10 Hz repetition rate) through a MLA (fused silica, from Suss Micro Optics, ROC 3.9 mm, pitch 300 μm), Figure 1b. After a single exposure (10 laser pulses at 6 mJ/cm2, total exposure: 60 mJ/cm2) through the MLA, parallel stripes separated by 300 μm (the pitch of the microlens array) were produced. To fabricate smaller graded micropatterns (stripes separated by 40, 60, 80, and 120 μm, respectively, see Figure 2), the surface was translated and/or rotated (at 90° for rectangles and 45° for rhomboidal micropatterns) and exposed several times (1 s (10 laser pulses) per position), Figure 1c. After patterning, the substrates were briefly heated on a hot plate at 80 °C, then left to cool to room temperature and developed by rinsing repeatedly with water to eliminate residues of non-cross-linked 8-PEG precursor, see Figure 1d. Micropattern Characterization. White Light Interferometry. The height of dry 8-PEG micropatterns on PEMA-modified glass surfaces was measured after drying using a white light interferometer (WLI, Zygo New view 7000 with 5, 10, or 20× objectives). All data were further processed with MetroPro software. Atomic Force Microscopy. An atomic force microscope (JPK instruments, Nanowizard II) was used to measure the height of in water swollen 8-PEG micropatterns on PEMA modified glass surfaces. Prior to atomic force microscopy (AFM) measurements, micropatterned samples were swollen for at least 12 h in deionized water. The imaging was conducted in intermittent contact mode using silicon nitride cantilevers (PNP TR tips, k ≈ 0.08 N/m, f 0 ≈ 17 kHz, Nanoworld Innovative Technologies) with a chromium−gold coating. Images were edited with the NanoWizard IP Version 3.3a (JPK instruments). Protein Adsorption. Water-swollen samples (1 cm in diameter) were placed in 24-microwell plates (Beckton Dickinson) and incubated with 1 mL of protein solution (50 μg/mL isothiocyanate conjugated albumin in phosphate buffered saline, Sigma, or 20 μg/mL bovine fibronectin in phosphate buffered saline, Calbiochem) for 1 h in a thermostated incubator (37 °C and 5% of CO2). Subsequently, samples incubated with albumin were washed twice with Dulbecco’s phosphate buffered saline (DPBS, PAA Laboratories GmbH) and analyzed by laser scanning confocal microscopy (LSCM, Leica SP 5), whereas samples incubated with bovine fibronectin were analyzed by fluorescence microscopy shortly after immunological staining. In this case, samples were incubated for 60 min with primary antibodies (antibovine Fibronectin from rabbit, 1:200 in DPBS, Millipore), 45 min with secondary antibodies (rhodamine-conjugated goat antirabbit, 1:100 in DPBS, Millipore) and rinsed twice with DPBS. Images were analyzed with the ImageJ software (1.440, Wayne Rasband, National Institutes of Health, USA). Cell Culture. Mouse connective tissue fibroblasts (L-929) were grown in 75 cm2 cell culture flasks (Greiner Bio-One) in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin/ streptomycin (all PAA Laboratories GmbH). For the following experiments, L-929 cells were used between passages 12 and 20. Prior to cell experiments, micropatterned samples were washed with ethanol (70 vl %), then rinsed in DPBS and placed in a 24-microwell plate (Becton Dickinson). Subsequently, L-929 cells were washed with DPBS and harvested using Trypsin-EDTA (PAA Laboratories GmbH). One milliliter of cell suspension containing 50.000 cells was seeded on top of every substrate and incubated for 24 h in a humidified incubator with 5% CO2 and 37 °C (CB 150, Binder GmbH). Following incubation, samples were washed with DPBS to remove unattached cells and remaining medium components. Subsequently, cells were fixed with formaldehyde (4%, Carl Roth GmbH & Co. KG) for 30 min. Optical, Scanning Electron, and Fluorescence Microscopy. Optical images were taken with the Axio Oberserver.Z1 (Carl Zeiss) and analyzed using the Axio Vision software (V4.8.2 Carl Zeiss). For scanning electron microscopy (SEM) of L-929 cells on distinct micropatterned samples, samples were dehydrated in a graded acetone series, dried with critical point drying (CPD 030, Baltec) and sputtered

EXPERIMENTAL SECTION

Acrylation of 8-PEG Macromonomers. 8-arm PEG octaol (15 kDa) was purchased from Jenkem Technology USA. Potassium carbonate (K2CO3), sodium chloride (NaCl) and magnesium sulfate (MgSO4) as well as dry dichloromethane (CH2Cl2), petroleum ether and acryloyl chloride were obtained from Sigma-Aldrich. All solvents were of analytical grade. The PEG-based macromonomers were functionalized to yield the acrylated derivative 8-PEG as previously described elsewhere.12 Briefly, 8-arm PEG octaol and potassium carbonate were mixed in dry dichloromethane under nitrogen-based atmosphere. After cooling the solution to 0 °C, acryloyl chloride was added and the mixture stirred at 60 °C for 4 days. Subsequently, the solution was filtered and poured into cold petroleum ether. Following stirring for 10 min, the petroleum ether was decanted. Finally, the crude product was dissolved in dichloromethane and washed with saturated sodium chloride solution. The organic layer was collected and dried with magnesium sulfate overnight. After filtration, the solvent was removed under reduced pressure to obtain the product as a white solid in moderate yield (72%). NMR analysis revealed complete conversion of hydroxyl-groups into acrylate functions. Surface Modification. Poly(ethylene-alt-maleic anhydride)Coated Glass Coverslips. Glass coverslips (1 cm in diameter, Menzel GmbH) were oxidized in a mixture of ammonia (Acros organics) and hydrogen peroxide (Merck KGaA) and modified with 3-aminopropyldimethyl ethoxy-silane (ABCR GmbH). Afterward, a solution of poly(ethylene-alt-maleic anhydride) (PEMA, Mw 125 000 g/mol, Aldrich) in acetone (0.3 wt %), was spin-coated on the substrates as previously described elsewhere.13 Surfaces were further heated for 2 h at 120 °C to bind covalently PEMA to the glass substrate. The PEMAcoated glass surfaces were heated for 2 h at 120 °C before patterning. Poly(ethylene-alt-maleic anhydride)-Coated Cyclo Olefin Polymer Films. Cyclo olefin polymer films (COP film, Zeonor transparent plastic film ZF 16−188, Zeon GmbH) were treated in a pulsed low pressure ammonia plasma to create free amino groups on the surface as previously described.14 Subsequently, a solution of PEMA in acetone (0.3 wt %) was spin-coated on the treated surfaces, dried under nitrogen, and heated for 2 h at 120 °C. Micropattern Fabrication. A micro lens array (MLA) was used to fabricate simple periodic micropatterns on PEMA-coated glass and COP substrates.6 The MLA patterning procedure is schematically depicted in Figure 1. A uniform layer of a photoreactive aqueous solution containing photoinitiator (0.175 wt % 1-[4-(2-Hydroxyethoxy)-phenyl]-2-hy-

Figure 1. Schematic representation of the mask-less micro lens array patterning technique to fabricate 8-PEG micropatterns on PEMAcoated glass and COP film surfaces: (a) deposition of a photoreactive precursor solution on the surface; (b) irradiation with 355 nm laser beam through a micro lens array; (c) irradiation and subsequent translation and rotation of the sample to obtain different geometries after several irradiation steps; (d) developing of microstructures by rinsing, and sterilizing the samples for application in cell culture. B


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Figure 2. Optical micrographs and SEM image of 8-PEG microstructures on different substrates; (a) on the COP polymer substrate (dry state); (b, c) on PEMA-coated glass (b, hydrated state; c, dry state).

Figure 3. (a.1) Representative WLI surface profile of the topography of an ∼150 μm × 200 μm patterned area and (a.2) a corresponding cross-section through 3 lines of dry 8-PEG microstructures on PEMA coated glass. 3D AFM height profiles of water-swollen (b) 90° and (c) 45° 8-PEG microstructures on PEMA-modified glass. Samples measured in the swollen state were immersed for at least 12 h in deionized water prior to AFM measurements. with gold using a sputter coater (SCD 030, Balzers). Scanning electron images were taken with a Hitachi S-520 using an acceleration voltage of 20 kV and a working distance of 10 mm. Pictures were taken using the Digital Image Processing System (2.6.20.1, Point Electronic). Fluorescence micrographs of the nucleus and the cells̀ cytoskeleton were recorded by confocal laser scanning microscopy (CLSM, Leica TCS SP5 II, Leica Microsystems). Prior to fluorescence microscopy, fixed cells were permeabilized with 0.1% Triton X-100 (Sigma- Aldrich), and blocked in 1% bovine serum albumin (BSA) in DPBS. Subsequently, cells were labeled with tetramethylrhodamine B isothiocyanate (TRITC)-conjugated phalloidin (2 μg/mL in 1% BSADPBS solution) for 45 min followed by DNA staining with chromomycin A3 (CMA3 50 μg/mL in 1% BSA-DPBS solution) for 15 min (Applichem GmbH). After repeated DPBS rinsing steps, samples were imaged by CLSM using an excitation wavelength of 458 nm (CMA3) and 561 nm (TRITC-phalloidin), and emission was recorded in the range of 495−525 nm (CMA3) and 567−642 nm (TRITC-phalloidin).

microstructures of 8-PEG on PEMA-coated glass or COP surfaces, see Figure 1. The quality of the obtained microstructures was verified by optical and scanning electron microscopy, Figure 2. Wavy and deformed 8-PEG microstructures, which detached partially from the surface when hydrated, were observed on COP films by optical microscopy (Figure 2a). The partial detachment observed can be attributed to poor physisorption of the 8-PEG material to the PEMA-modified, plastic COP film, as well as to stresses at the interface between the microstructure and the support surface due to swelling and shrinking while processing the substrate. On the other side, uniform 8-PEG microstructures were observed on PEMA-coated glass (Figure 2b, c); where good physisorption between the cross-linked 8-PEG material and the PEMA-coated glass surface held the 8-PEG microstructures in place. The microstructures of 8-PEG fabricated on PEMA functional glass were very stable even in hydrated state up to 5 days, which is of critical importance for further application in cell culture (i.e., in aqueous medium). These observations are in accordance with our recently investigated PEG structures on various maleic anhydride copolymer prelayers. Furthermore, the best physisorption and pattern resolution was also observed on PEMA-

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RESULTS AND DISCUSSION Fabrication and Characterization of the Micropatterned Surfaces. A mask-less laser-assisted patterning technique (micro lens array patterning) was used to fabricate C


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ACS Biomaterials Science & Engineering coated surfaces, probably because of sufficient wetting and uniform spreading of the photoreactive PEG mixture over the surface before irradiation.6 The scanning electron micrographs of the treated samples (Figure 2c) revealed that the microstructures exhibit a substantial height, in the micrometer range. For cell culture studies, notably, the height of in water swollen structures is relevant: micrometerhigh structures are assumed to be more effective boundaries than nanometer-high patterns. To verify the heights of dry and waterswollen 8-PEG microstructures on PEMA-coated glass surfaces, we performed white light interferometry (WLI) and atomic force microscopy (AFM) measurements. Representative (WLI and AFM) images of the 8-PEG structures are shown in Figure 3. From the WLI image it is apparent that the structures are indeed micrometer-sized (around 5 μm in height), while height fluctuations are obvious in the patterned area of ∼150 μm × 200 μm. The AFM images, which are recorded on a smaller area (maximally 100 μm × 100 μm) reveal more details of the topography. Whereas the WLI image is useful for illustrative purposes and already gives an indication of the height of the topographic structures, the real height was determined more accurately by AFM. The height of the lines was determined in triplicate on a selected area, before and after swelling in deionized water. Analysis of one representative profile by AFM demonstrated that the microstructures had an effective height of 5.0 ± 0.4 μm in air and 7.3 ± 0.2 μm in water. This result indicates that the 8-PEG microstructures grafted to PEMA coated glass surfaces increased in height by approximately 46% due to swelling. Similar results were reported in an earlier study for PEG microstructures on silicon and glass surfaces, exhibiting height changes of nearly 30%.15 Nevertheless, those microstructures were fabricated using a linear PEG (Mw 575 Da) precursor, whereas a multiarm PEG (Mw 15 kDa) was used in our present research. This is consistent with our experience that the longer chains of 8-PEG result in a lower cross-linking density and correspondingly larger swelling degree. Protein Adsorption. Because protein adsorption due to nonspecific interactions between the protein and the surface is largely dependent upon the chemical characteristics of the surface, plasma and extracellular matrix protein adsorption onto micropatterned samples were examined by fluorescence microscopy using fluorescently labeled proteins and immunological staining techniques, respectively. In line with our expectation, 8-PEG microstructures were observed to resist protein adsorption, whereas proteins did adsorb onto the adhesive PEMA surfaces within the microdomains, see Figure 4. PEG surfaces are well-known to effectively inhibit nonspecific adsorption of proteins and other biomolecules from biological fluids such as cell culture media, and the protein repellent effect is mainly attributed to the hydrophilicity of the PEG substrate.16 In another recent study, we have also confirmed the protein- and cell-repellent effect of (smooth) 8-PEG substrates, showing round-shaped cells, indicative of the absence of spreading and cell clustering.1717 Cell Behavior within Adhesive Microdomains. The microengineered substrates containing patterns of 8-PEG on PEMA-modified glass surfaces were tested in cell culture in order to explore cell morphology, distribution and orientation. In order to achieve an effective cell confinement, we employed the cell antiadhesive PEG material to fabricate cell adhesive microdomains on the cell adhesive PEMA-coated glass surface. As can be clearly seen in Figure 5, cells exclusively attach to the adhesive

Figure 4. Fluorescence micrographs revealing albumin and fibronectin adsorption onto well-defined microdomains delimited by protein repellent 8-PEG micropatterned structures (black stripes on the figure) fabricated on PEMA-modified glass. Previously hydrated samples were incubated with isothiocyanate conjugated albumin (50 μg/mL) or bovine fibronectin (20 μg/mL). Subsequently, fibronectin adsorption was elucidated by immuno-staining with specific primary antibodies and fluorescently labeled (rhodamine) secondary antibodies.

Figure 5. (a−d) Scanning electron micrographs showing L-929 cell adhesion and spreading on the PEMA-coated glass surfaces exposed within the cell-repellent 8-PEG microstructures, and revealing the different morphology of L-929 cells in (a, b) big and (c, d) small microdomains after 24 h. Prior to SEM, L-929 cells were fixed with formalin and the samples were dehydrated in a graded acetone series.

PEMA modified glass and completely avoid the surrounding 8PEG microstructures. It is remarkable to note that the cellrepellent effect of the microstructures is manifest also several micrometers besides the obvious, topographic structures; a certain margin next to the walls remains undecorated by cells. The reason for this could be a chemical one; one would tentatively attribute this observation to the presence of a thin layer of 8-PEG residues that could not be detected topographically. However, comparison with the protein adsorption in Figure 4 implies that these areas are not protein-repellent, making it unlikely to be a PEG coating. It is more feasible that the remarkable observation in the SEM images is due to a drying effect. In the sample preparation procedure for electron microscopy, the samples from cell culture must be dehydrated in order to be investigated in the vacuum chamber of the microscope. The dehydration leads to shrinking of the PEG hydrogel, and the topographic structures appear thinner on the SEM image than they used to be in aqueous cell culture medium. The cell adhesive area is not affected by this procedure, and the cell coverage reflects the real size of the microcontainers under physiological conditions. The number of cells inside the adhesive microwells was controlled by changing the geometry of the 8-PEG micropatterns D


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ACS Biomaterials Science & Engineering

Figure 6. (a) Scanning electron micrographs showing L-929 cells confined within 8-PEG microstructures on PEMA-coated glass surfaces of different geometry, i.e., in rhomboidal microdomains exhibiting an interior angle of 90, 45, or 20°, respectively; (b) analysis of cell dimensions in dependence of the geometry (values measured for the medium-sized cell-adhesive areas); (c) corresponding fluorescence micrographs revealing the more or less elongated morphology of the cells; the staining shows the nuclei in green and the cells’ cytoskeleton (actin) in red. Cells were cultured for 24 h.

on the PEMA-functional cell-adhesive glass surface and their lateral dimensions. As expected, larger adhesive containers were observed to accommodate larger number of cells. Intriguingly, a similar trend was observed for the geometric control of cell spreading; analysis of the cell spreading area revealed remarkable differences in cell size depending on the size of the adhesive area. Cells positioned in larger cell-adhesive domains spread more (the observed cell diameter, or rather the long axis of the cells, was 39.1 ± 3.5 μm) as compared to cells confined in small celladhesive domains (where the average cell size was 18.6 ± 0.6 μm). Thus, it seems that the cells in smaller microdomains feel an effective geometrical restriction imposed by the protein repellent 8-PEG microstructures and this is reflected in their adhesion and spreading behavior. It should be noted that the cell size could appear to be larger in case cells elongate their cell shape. This aspect will be discussed next, in relation to the geometrical form of the microcontainers (vide infra). Moreover, the geometry of the 8-PEG micropatterns was modified in order to further investigate the geometric influence on the cellular behavior. For this purpose, we explored three different geometries: square and rectangular (90°) as well as rhomboidal (45, 20°) microdomains. Interestingly, we observed partially adapted cell shapes in square, rectangular and rhomboidal domains (90, 45, 20°), as observed by both scanning electron microscopy and fluorescence microscopy, see Figure 6 (values listed in b apply for the medium-sized containers). Particularly, cells were observed to elongate more when confined within areas delimited by sharp angles (

Geometric Control of Cell Alignment and Spreading within the

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