ARTICLE pubs.acs.org/Langmuir
Evaluation of Substrata Effect on Cell Adhesion Properties Using Freestanding Poly(L-lactic acid) Nanosheets Toshinori Fujie,*,†,‡ Leonardo Ricotti,†,§ Andrea Desii,†,§ Arianna Menciassi,†,§ Paolo Dario,†,§ and Virgilio Mattoli*,† †
Center for MicroBioRobotics IIT@SSSA, Istituto Italiano di Tecnologia, Viale Rinaldo Piaggio 34 Pontedera, Pisa, 56025, Italy European Biomedical Science Institute (EBSI), Organization for European Studies, Waseda University, 2-2 Wakamtsu-cho, Shinjuku, Tokyo 162-8480, Japan § The BioRobotics Institute, Polo Sant’Anna Valdera, Scuola Superiore Sant’Anna, Viale Rinaldo Piaggio 34 Pontedera, Pisa, 56025, Italy ‡
bS Supporting Information ABSTRACT: Investigation of the interactions between cells and material surfaces is important not only for the understanding of cell biology but also for the development of smart biomaterials. In this study, we investigated the substrate-related effects on the interaction between cell and polymeric ultrathin film (nanosheet) by modulating the mechanical properties of the nanosheet with a metal substrate or mesh. A freestanding polymeric nanosheet with tens-of-nanometers thickness composed of poly(L-lactic acid) (PLLA nanosheet) was fabricated by combination of a spin-coating technique and a water-soluble sacrificial layer. The freestanding PLLA nanosheet was collected on a stainless steel mesh (PLLAmesh) and subsequently used for cell adhesion studies, comparing the results to the ones on a control SiO2 substrate coated with an ultrathin layer of PLLA (PLLAsubstrate). The adhesion of rat cardiomyocytes (H9c2) was evaluated on both samples after 24 h of culture. The PLLAmesh with the tens-of-nanometers thick nanosheets induced an anisotropic adhesion of H9c2, while H9c2 on the PLLAsubstrate showed an isotropic adhesion independent from the nanosheet thickness. Interestingly, an increment in the nanosheet thickness in the PLLAmesh samples reduced the cellular anisotropy and led to a similar morphology to the PLLAsubstrate. Considering the huge discrepancy of Young’s modulus between PLLA nanosheet (3.54.2 GPa) and metal substrate (hundreds of GPa), cell adhesion was mechanically regulated by the Young’s modulus of the underlying substrate when the thickness of the PLLA nanosheet was tens of nanometers. Modulation of the stiffness of the polymeric nanosheet by utilizing a rigid underlying material will allow the constitution of a unique cell culture environment.
’ INTRODUCTION Investigation of the interactions between cells and material surfaces plays an important role in the development of smart biomaterials. There have been several attempts to construct engineered scaffolds or matrices paying attention to their chemical and physical surface properties, in order to obtain suitable interactions with cells.13 These studies have been undertaken on solid substrates such as plastics, glass and SiO2. However, such microenvironments are substantially different from those surrounding the living systems from the mechanical point of view (frequently called “mechanobiology”).4 For example, mechanotaxis is one of the most important aspects for cell mechanobiology, as cells sense matrix stiffness and preferentially adhere onto harder regions.5,6 Besides, the matrix stiffness was also unveiled to have effects on proliferation, migration and even differentiation. These approaches have been undertaken by integrating soft materials, such as elastic gels,4 rubbers,7 and functional membranes,8 thanks to their mechanical tunability, achieved by changing the degree of chemical cross-linking.9,10 r 2011 American Chemical Society
Freestanding polymeric ultrathin films (nanosheets) can be raised as a new category of the quasi-two-dimensional soft materials. Typical features of the polymeric nanosheet are tens-of-nanometers thickness, a huge size-aspect ratio (>106), and unique interfacial and mechanical properties, such as tunable flexibility, noncovalent adhesiveness, and high transparency.11 From the structural point of view, the quasi-two-dimensional structure of the polymeric nanosheet could represent an ideal interface to mimic a native extracellular matrix (ECM). Freestanding polymeric nanosheets have been fabricated using different approaches, including simple spin-coating,12 layer-by-layer (LbL) method,13,14 LangmuirBlodgett method with cross-linkable amphiphilic copolymers,15 and solgel method with organicinorganic interpenetrating networks.16 Up to date, clinical benefits of the polymeric nanosheets have been investigated for biomedical applications, Received: August 11, 2011 Revised: September 5, 2011 Published: September 13, 2011 13173
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Figure 1. Preparative steps of “PLLAmesh” by using a freestanding PLLA nanosheet. “PLLAsubstrate” is prepared by direct spin-coating of a PLLA solution on a SiO2 substrate (not shown).
such as tissue-defect repair without surgical adhesion,17,18 local delivery of antibiotics by implantation,19 remote-controllable tissue sealing,20,21 and other applications in different fields.22 We currently focused on the flexible structure of the polymeric nanosheet and pursued biomedical research in the development of tissue engineering scaffold and biomedical devices.2325 These studies revealed the influence of the surface chemistry on cell adhesion properties, including morphology, adhesion area, and elongation ratio. However, the entire complexity of cell activity on the polymeric nanosheet has not been fully understood because several factors other than surface chemistry could also influence the cellular activity (e.g., surface topology and mechanical properties). In particular, the substrate-related effect on the interaction between cells and a polymeric nanosheet is not negligible, because the mechanical flexibility of the nanosheet can be impaired by the stiffness of the underlying material during cell culture. In this study, we investigated the effect of an underlying substrate on the interaction between cells and nanosheet, by coupling the flexible nanosheet with rigid materials, such as SiO2 substrates and metal meshes. A polymeric nanosheet composed of biocompatible poly(L-lactic acid) (referred to as “PLLA nanosheet”) was fabricated by combination of a spin-coating technique and a water-soluble sacrificial layer method, which facilitated the aqueous exfoliation of a freestanding PLLA nanosheets showing a thickness from tens to hundreds of nanometers. These nanosheets were collected on stainless steel meshes (i.e., PLLA mesh) and subsequently used for cell adhesion analysis on the PLLA nanosheet. On the other hand, we prepared PLLA-coated SiO2 substrates by directly spin-coating a PLLA solution (i.e., PLLAsubstrate) as a control sample. Then, cell activity and adhesion of rat cardiomyocytes (H9c2) were compared for both sample typologies after 24 h of culture. H9c2 is a permanent cell line derived from rat cardiac tissue,26 sensitive to matrix topography and substrate stiffness. Therefore, adhesion, proliferation, and differentiation rate of H9c2 myoblasts are strongly affected by the matrix properties.27,28 Furthermore, we characterized surface morphology and mechanical properties of the PLLA nanosheets using scanning electron microscope (SEM), atomic force microscopy (AFM), and strain-induced elastic buckling instability for mechanical measurement (SIEBIMM) test. The role of the underlying substrate on the biointerfacial properties was thus clarified, evaluating how it modulated the topological and mechanical characteristics of the PLLA nanosheets.
’ MATERIALS AND METHODS Materials. SiO2 substrates (Si-Mat Silicon Materials, Kaufering, Germany), used as substrates for film deposition, were cut (2 2 cm2), treated with an acid mixture [SPM 96% H2SO4:30% H2O2 = 4:1 (v/v)] at 120 °C for 10 min, and then thoroughly rinsed with deionized (DI) water (18 MΩ cm) in order to remove impurities from the surface. Poly(vinyl alcohol) (PVA), average Mw 13 00023 000, 98% hydrolyzed, was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Poly(L-lactic acid) (PLLA), Mw 80 000100 000, was obtained from Polysciences Inc. (Warrington, PA). A stainless steel mesh with a 0.5 mm 0.5 mm lattice composed of 150-μm-wide stainless steel wires was obtained from Tokyu Hands, Co. Ltd. (Tokyo, Japan). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) and used without further treatment, unless noted. Preparation of Freestanding PLLA Nanosheets. Freestanding PLLA nanosheets were fabricated by spin-coating, applying a PVA sacrificial layer method (Figure 1). The film deposition process was achieved by the following steps: (1) to obtain a water-soluble sacrificial layer, an aqueous solution of PVA (1 wt %) was spin-coated (WS-650 spin processor, Laurell Technologies Corp., North Wales, PA) on a SiO2 substrate (4000 rpm, 60 s) and baked (120 °C for 90 s); (2) a chloroform solution of PLLA (5, 10, 20, 30, and 50 mg/mL) was spin-coated on the PVA layer (4000 rpm, 60 s) and dried at room temperature overnight; (3) the ultrathin PLLA layered substrate was immersed in water in order to dissolve the PVA sacrificial layer, which allowed the release of freestanding PLLA nanosheet. Then, the freestanding PLLA nanosheet was collected on the SiO2 substrate, poly(dimethyl siloxane) (PDMS) slab, or stainless steel mesh for material characterization and/or cell culture studies. All the routines for the PLLA nanosheets fabrication were conducted in a clean-room (class 1000) to avoid contamination. Surface Characterization of PLLA Nanosheets. Macroscopic images of the PLLA nanosheets sustained by the stainless steel mesh were taken by using an optical microscope (Hirox KH7700 digital microscope, Hirox Co Ltd., Tokyo, Japan), equipped with a MX(G)-10C zoom lens and OL-700II objective lens. Thickness, topography, and surface roughness of the PLLA nanosheets were evaluated by AFM (Veeco Innova Scanning Probe Microscope, Veeco Instuments Inc., Santa Barbara, CA) operating in tapping mode, using an RTESPA Alcoated silicon probe (Veeco Instruments Inc.) with a spring constant of 2080 N/m, a resonance frequency of 235317 kHz, and an average tip radius of 8 nm. All the measurements were performed in air at room temperature. For thickness measurements, PLLA nanosheets on the SiO2 substrate were scratched with a needle. The sample was scanned across the scratched edge over a 20 μm 20 μm area, recording 64 64 samples. The resulting scan data were elaborated using Gwyddion SPM analysis tool (free download from http://gwyddion.net). Raw scan data 13174
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were leveled with a facet level tool to remove sample tilt, and then the sample profile was extracted in a direction perpendicular to the scratch edge. Height data were averaged and the thickness was evaluated from the difference between horizontal lines fitting to the SiO2 substrate and the PLLA nanosheet. For roughness (root mean square, rms) measurement, the surface of the PLLA nanosheets recollected on the SiO2 substrates was scanned in tapping mode over 2 μm 2 μm area (512 512 samples). Then, the rms values were obtained from the topographical images. Microscopic surface morphology of the PLLAmesh samples was investigated by SEM (EVO/MA10, Carl Zeiss SMT, Oberkochen, Germany) without metal sputtering. Mechanical Characterization of PLLA Nanosheets. The mechanical properties of the PLLA nanosheet were evaluated by means of the SIEBIMM technique. The SIEBIMM test is based on the buckling metrology and allowed the calculation of Young’s modulus for polymeric nanosheets. As reported previously in the literature,2931 the elastic modulus can be calculated by measuring the buckling wavelength of the nanosheet on a mechanically compressed or stretched matrix. A freestanding PLLA nanosheet was collected from water onto a prestretched (∼3% strain of the original size) PDMS slab (1.5 cm 4.0 cm). The prepared sample was dried in a desiccator overnight prior to the SIEBIMM test. Then, the strain of the PDMS substrate was relaxed, producing a relative compression of the PLLA nanosheet and generating a characteristic corrugation. Immediately, the wavelength (λ) of the buckling pattern on the PLLA nanosheet was analyzed using an optical microscope. Then, the Young’s modulus (EPLLA) of the PLLA nanosheets with different thickness (h) was individually obtained using the following formula 1 EPLLA ¼ 3
EPDMS ð1 vPLLA 2 Þ λ 3 1 vPDMS 2 2πh
ð1Þ
where E and v represented the Young’s modulus and Poisson’s ratio of PLLA nanosheet and PDMS (i.e., EPDMS = 1.8 MPa). In this calculation, we assumed Poisson’s ratios for the PLLA nanosheets and for the PDMS equal to 0.33 and 0.50, respectively, in accordance with previous reports.30,31 In addition, the stiffness of the PLLA nanosheet was also evaluated by using tapping mode AFM scans, where the phase images (15 μm 15 μm range) of the PLLAmesh samples were collected with a scanning direction perpendicular to a metal wire. The recorded data were averaged on columns and displayed as average profiles in order to evaluate the gradient of topographical and mechanical properties across the metal wire border. Cell Culture. H9c2 cells (embryonic myocardium rat cell line, ATCC, CLR-1446, Milan, Italy) were cultured in expansion medium, constituted by Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 100 μg/mL gentamycin, and 2 mM L-glutamine. Cells were maintained at 37 °C in a saturated humidity atmosphere (95% air, 5% CO2). Before reaching confluent, the cells were detached from a polystyrene culture plate by means of a 0.05 wt % trypsin, 0.53 mmol/L EDTA-4Na solution with phenol red. The aliquot was purified by centrifugation and suspended in fresh culture medium. Then, a small quantity of a cell suspension (200 μL) was gently deposited on the sample surface with a seeding density of 1.5 104 cells/cm2 and incubated for 30 min in the cell incubator. This initial treatment facilitated the stable tethering of H9c2 on the PLLA surface. Afterward, additional culture medium was added, and the sample was cultured under standard conditions for 24 h. Cell Activity Assay. Cell activity of H9c2 on PLLAmesh and PLLAsubstrate was evaluated at 24 h after cell seeding using two tests. First, a qualitative viability assay was performed by using the LIVE/ DEAD Viability/Cytotoxicity kit (Invitrogen Co., Carlsbad, CA). The kit contains calcein AM (4 mM in anhydrous DMSO) and ethidium
homodimer-1 (EthD-1, 2 mM in DMSO/H2O 1:4 v/v), which identifies live versus dead cells on the basis of membrane integrity and esterase activity. After 24 h of cell culture, the culture medium was removed, and the cellular layers on the sample surface were rinsed with phosphate buffered saline (PBS), which was then treated for 10 min at 37 °C with 2 μM calcein AM and 4 μM EthD-1. Cells were finally observed under an inverted fluorescent microscope (TE2000U, FITC-TRITC filters, Nikon Co., Tokyo, Japan) equipped with a cooled CCD camera (DS-5MC USB2, Nikon Co., Tokyo, Japan) and with NIS Elements Imaging Software. Second, the cell population was quantitatively evaluated, measuring the DNA concentration for each type of sample after 24 h of culture. The samples were removed from the original cell culture wells and placed in new wells, which were treated with appropriate volumes of DI water. Cell lysates were then obtained by two freeze/thaw cycles of the samples —overnight freezing at 80 °C and 15 min thawing at 37 °C in an ultrasonication bath—to enable the DNA to go into the aqueous media. The DNA content in the cell lysates was measured by using the PicoGreen kit (Invitrogen Co., Carlsbad, CA). The PicoGreen dye binds to DNA, and the resulting fluorescence intensity is directly proportional to the DNA concentration. Standard solutions of DNA in DI water at concentrations from 0 up to 6 μg/mL were prepared and 50 μL of standard or sample were loaded for quantification in a 96-well black microplate. Working buffer and PicoGreen dye solution were prepared and added according to the manufacturer’s instructions (100 and 150 μL/well, respectively). After 10 min of incubation in the dark at room temperature, fluorescence intensity was measured on a microplate reader (Victor3, PerkinElmer Inc., Waltham, MA) using an excitation wavelength of 485 nm and an emission wavelength of 535 nm. Cell Adhesion Properties. The adhesion properties of H9c2 on the samples were evaluated by measuring the adhesion area and elongation ratio of a representative sample (more than five images for each material). First, cells were fixed with a 4 wt % paraformaldehyde solution after 24 h of culture in the expansion medium, and their membrane became permeabilized by means of a 0.1% Triton-X solution in PBS. Second, the fixed cells were stained with Alexa Fluor 594 phalloidin and Hoechst (Invitrogen Co., Carlsbad, CA). Finally, the cell adhesion area and the elongation ratio were analyzed using ImageJ, a software for image analysis (free download from NIH, http://rsbweb.nih.gov/ij/). Individual cells were identified by the fluorescent signals of nuclei, and the contour of cells was distinguished by fluorescent contrast of actin filaments after converting the color image into gray scale mode. The cell elongation ratio was defined by the following formula 223,32 ε ¼ rmax =rmin
ð2Þ
where ε represents the cell elongation ratio, and rmax and rmin represent the maximum and minimum distance of adhered pseudopodia from the median point of the cell. Cell adhesion was compared between PLLA mesh and PLLAsubstrate samples (excluding cell adhesive region on the metal wire from the analytical region of the PLLAmesh) and between the inside and outside regions of the individual mesh lattice in the PLLAmesh samples. According to these evaluations, the substrate effect on cell adhesion properties could be investigated for different materials: plain nanosheet, nanosheet supported by SiO2 substrate, and nanosheet supported by steel mesh. Statistical Analysis. All data are presented as mean values ( SD. Statistical analysis of the cell adhesion area and the cell elongation ratio was performed using the unpaired t test, with *p < 0.05 and **p < 0.01 set as the level of statistical significance.
’ RESULTS AND DISCUSSIONS Surface Properties of PLLA Nanosheets. The thickness of the PLLA nanosheets was easily controlled by changing the PLLA concentration and keeping the same spin-coating conditions 13175
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Figure 2. AFM characterization of PLLA nanosheets recollected on the SiO2 substrates. (a) Thickness profiles as the function of PLLA concentration, and surface morphologies for different PLLA thickness of (b) 29, (c) 54, (d) 318, and (e) 703 nm. All images were obtained in the 2 μm 2 μm scan range.
(4000 rpm, 40 s), thus resulting in tens- to hundreds-of-nanometers thicknesses (Figure 2a). The obtained thickness was in good agreement with a previous report.33 Unlike in the previous study, we avoided heating treatments after the PLLA spincoating step, in order to obtain a uniform surface. Heating process beyond the glass transition temperature of PLLA (56 °C) sometimes causes crystallization and provides a rougher surface.24 In particular, cell adhesion is affected by the presence of submicrometer structures on the material surface, as widely evidenced for many cell types.3440 Thus, surface morphology and roughness of PLLA nanosheets were evaluated by AFM scans. Each sample surface appeared homogeneous across 2 μm 2 μm areas, without large outliers, as evidenced by the range of each color bar in the AFM scans (
