Mechanical Regulation of Cellular Adhesion onto Honeycomb

Mar 19, 2013 - ... Adhesion onto Honeycomb-. Patterned Porous Scaffolds by Altering the Elasticity of Material. Surfaces. Takahito Kawano,. †. Yuki ...
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Mechanical Regulation of Cellular Adhesion onto HoneycombPatterned Porous Scaffolds by Altering the Elasticity of Material Surfaces Takahito Kawano,† Yuki Nakamichi,‡ So Fujinami,† Ken Nakajima,† Hiroshi Yabu,*,‡ and Masatsugu Shimomura†,‡,§ †

WPI-Advanced Institute for Materials Research, Tohoku University, 2-1-1, Katahira, Aoba-ku, Sendai, 980-8577, Japan Institute of Multidisciplinary Research for Advanced Materials (IMRAM), Tohoku University, 2-1-1, Katahira, Aoba-ku, Sendai, 980-8577, Japan § Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), 4-1-8, Kawaguchi, Saitama, 332-0012, Japan ‡

ABSTRACT: In this report, we show the preparation of honeycomb scaffolds for cell culturing by using “breath figure” method, and we found that their mechanical and topographical properties strongly affect the adhesion of fibroblasts. By photocross-linking of the poly(1,2-butadiene), the hardness of the honeycomb scaffold can be successfully controlled without any surface chemical changes, and detail modulus values of scaffolds were measured by atomic force microscopy. We found that only small numbers of the cells adhered on the softer honeycomb scaffolds, which has even higher modulus value than conventional gels, comparing with flat films and a hard honeycomb scaffold. These results indicate that the elastomeric honeycomb substrates are useful for evaluating the effect of the mechanical signal-derived geometry on the transduction system of cells.

1. INTRODUCTION Cell culture scaffolds act as a template for tissue regeneration, and encourages cells to form healthy and functional tissues. Cell behaviors, including cellular adhesion, proliferation, migration, and differentiation, are regulated by the interactions between cells and the microenvironment of the cells; therefore, the chemical, topological, and mechanical properties of scaffold surfaces are significant for regulating the cell behavior.1,2 The hydrophobicity of the scaffold surface changes the adhesion of the extracellular matrices, and controls the cell adhesion to the scaffold.3 Cell-adhesive molecules, such as the arginine-glycineaspartic acid amino acid sequence which binds cell-surface integrin receptors, attach cells to surfaces.4,5 Surface topography is important for aligning cells and regulating cell locomotion through contact guidance.6,7 Twodimensional topographies, such as grooves, pits, and posts, have been examined for modeling the structures of in vivo cell microenvironments. The height, width, and micrometer scale spacing of these topographic features affect the cell growth and functions.8−10 However, surface topography studies have often been conducted with solid, rigid substrates, which are not the same as mechanical environments. Less rigid hydrogel and elastomer scaffolds have been used to examine the effect of mechanical properties of the scaffold on cell function.11,12 The cell function was strongly influenced by the mechanical properties of the scaffold through the focal adhesions of the cells, which anchor the cytoskeleton to the © 2013 American Chemical Society

scaffold surface, and through numerous mechanotransduction signaling processes in fibroblasts and in stem cells.13−15 Patterned elastomers and hydrogels have been used to determine the effect of surface topography and mechanical properties simultaneously. However, the most previous studies have used the micropost arrays to evaluate the mechanical properties, such as elasticity, flexibility and deformation, of the patterned soft substrates.16,17 Moreover, because soft scaffolds are sometimes deformed by the contraction of the cultivated cells, further studies for understanding the interaction between cells and soft scaffolds with micrometer-scale features are required. We have previously reported that honeycomb-patterned porous polymer films can be prepared by casting a polymer solution under humid conditions.18 By using this breath figure (BF) technique, microporous films with hexagonal pores ranging from submicrometer to micrometer scale can be prepared. These honeycomb films have a unique double-layered structure with spherical pores that are formed around template water droplets ranging from cellular to subcellular scale.19 The films are suitable for cell culture scaffolds. Various types of cells, including endothelial cells and cardiac myocytes, have been cultured on honeycomb films.20−22 However, the mechanical Received: February 7, 2013 Revised: March 14, 2013 Published: March 19, 2013 1208

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properties of the honeycomb films and their effect on the cell behavior have not yet been examined in detail. Recently, elastomeric honeycomb-patterned microporous films have been prepared from poly(1,2-butadiene) (PB), which is a synthetic rubber.23 The double bond in the side chain means that PB honeycomb films can be cross-linked by ultraviolet (UV) irradiation.24 The UV irradiation process can be used to control the mechanical properties of the PB honeycomb films without chemically altering the film surface. In this paper, we report the characterization of the mechanical properties of non-cross-linked and UV-cross-linked PB honeycomb films with atomic force microscopy (AFM) and structural mechanics simulations. Furthermore, we discuss the effect of the mechanical properties of PB honeycomb films on fibroblast adhesion.

The surface elasticity (E) of the substrates was measured by analyzing the force−distance curves according to JKR theory. JKR theory is expressed by eqs 3 and 4, a3 =

R (F + 3πWR + K

δ=

a 2F + 3R 3aK

K=

4E 3(1 − ν 2)

K=

W=−

1.27F1 R(δ0 − δ1)3 2F1 3πR

(6) (7)

where δ0, δ1, and F1 were measured from the force−distance curves. Thus, the surface elasticity can be calculated from the force−distance curves. Structural Mechanics Simulation. Finite element analysis was performed with the structural mechanics module of COMSOL Multiphysics Ver. 4.2 (COMSOL AB, Stockholm, Sweden) to simulate the mechanical properties of a honeycomb substrate under an applied horizontal traction force. The honeycomb substrate was modeled as a linear elastic material and discretized into a tetrahedral mesh. The elastic modulus values from the AFM force volume measurement were used for the PB honeycomb substrates. The Poisson ratio and the PB density were 0.46 and 9100 kg/m3, respectively. To simulate the deformation of the honeycomb substrates, a horizontal traction force of 30 nN was applied to the top surface of the pillar, and the bottom surface of the substrate was assigned as a fixed boundary condition. Cell Culture. The honeycomb substrates were washed in 1propanol to remove polymer 1 and then rinsed in ethanol and water. The substrates were immersed in Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Gibco BRL) overnight. Mouse fibroblast cells (3T3-Swiss albino, Health Science Research Resources Bank, Tokyo, Japan) were seeded at density of 5 × 103 cells/cm2 on the honeycomb substrates, and cultured in DMEM containing 10% FBS for 24 h under 5% CO2 in a humidified incubator. Fluorescent Microscopy. The amount of fibronectin adsorbed onto the honeycomb substrates, the cell morphology, and the distribution of focal adhesions were determined using fluorescence microscopy (FSX 100, Olympus). The amount of fibronectin was measured by adsorbing rhodamine-fibronectin (10 μg/mL; Cytoskeleton Inc., Denver, CO) onto the honeycomb substrates for 24 h and measuring the fluorescence intensity. The cell morphology and distribution of the focal adhesion on the honeycomb substrates of cells cultured on the honeycomb substrates for 24 h were characterized by fixing the cells with 4% paraformaldehyde (WAKO) for 20 min, blocking them in 10% goat serum (Invitrogen, Carlsbad, CA), and permeabilizing them with 0.1% Triton-X 100 (MP Biomedicals, Irvine, CA) at room temperature for 45 min. The cells were incubated with antivinculin (Chemicon, Temecula, CA; diluted 1:50) for 1 h, and then with Alexa Fluor 488 antimouse IgG (Invitrogen; diluted 1:500)

synthesized according to a literature method (Mw = 84 kg/mol, Mw/ Mn = 4.94).25 PB (180 mg) and polymer 1 (18 mg) were dissolved in chloroform (40 mL). The PB honeycomb scaffolds were fabricated by casting the chloroform solution of PB and polymer 1 under a flow of humid air (10 L/min). The PB honeycomb scaffolds were prepared by osmium sputtering and the surface structures were observed with a field emission-scanning electron microscope (FE-SEM; S-5200, Hitachi, Japan). The flat planar substrates were fabricated by spincasting a chloroform solution of PB (60 mg/mL) using a spin-caster (MS-A100, Mikasa, Co. Ltd., Japan). The PB was photo-cross-linked by irradiating the film with UV light for 3 min in vacuo by using a UV lamp (30 mW cm−2, λmax = 365 nm, OCA-150L-D, EYE Graphics Co. Ltd., Japan). AFM Measurement. Maps of the elastic modulus of the substrates were produced using a procedure based on a combination of JohnsonKendall-Roberts (JKR) contact mechanics and a two-point method together with AFM force volume measurements.26 Force volume experiments were performed by AFM (Nanoscope V, Bruker AXS, Santa Barbara, CA) under ambient conditions. The elastic modulus measurements were obtained by using a cantilever (OMCLTR800PSA-1, Olympus, Tokyo, Japan) with a nominal spring constant of 0.57 N/m. An actual spring constant was measured by the thermal tune method with a spring constant of 0.63 N/m. The force−distance curves were measured at a resolution of 64 × 64 pixels. The scan sizes of the planar control substrates and the PB honeycomb substrates were 5 × 5 μm and 10 × 10 μm, respectively. The sample deformation (δ) was calculated by using eqs 1 and 2, (2)

(5)

where ν is Poisson’s ratio. However, these equations cannot be converted to a function of sample deformation (δ) and load force (F). Therefore, we used the two-point method proposed by Sun.27 In this method, two points of the force−distance curve are analyzed; one is where the attractive force and the repulsive force are equivalent, and the other is where the adhesive force reaches its maximum (F1). Using these two points, the JKR equations were converted to the following functions,

Scheme 1. Chemical Structure of Polymer 1 (m/n = 4:1)

δ=z−Δ

(4)

where a is the contact radius, R is the tip radius, W is the adhesive energy, F is the load force, and δ is the deformation of the sample. K is the combined elastic modulus and is related to the elastic modulus (E) by eq 5,

Preparation of PB Honeycomb Scaffolds. PB (RB-820, Mw = 250 kg/mol, Mw/Mn = 2.50) was supplied by JSR Co. Ltd., Japan. Chloroform was purchased from Wako Chemical Industries Ltd., Japan. The amphiphilic copolymer (polymer 1, Scheme 1) was

(1)

(3)

2

2. EXPERIMENTAL SECTION

F = kΔ

6πWRF + (3πWR )2 )

where F is the load force, k is the spring constant of the cantilever, Δ is the deflection of the cantilever, δ is the sample deformation, and z is the piezo-scanner displacement. The value of F was 10 nN and z was measured from the force−distance curves. 1209

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for 1 h at 37 °C. The cell morphology was visualized by labeling the actin filaments with rhodamine-phalloidin (Cytoskeleton). The samples were mounted on a slide glass and observed under a 40× objective lens (NA = 0.95). The microscopic images of the cell morphology visualized by fluorescent phalloidin and the focal adhesions visualized by indirect vinculin immunofluorescence were quantitatively analyzed using ImageJ software (National Institute of Health, Bethesda, MD). Statistical tests were performed for three times using different honeycomb scaffolds with washing the scaffolds with PBS for three times before immunofluorescence staining to avoid counting dead cells.

21.8 ± 1.6 and 25.4 ± 7.2 MPa, respectively. These values indicate that surface elasticity was not altered by the honeycomb structure. However, the elastic modulus of the photo-cross-linked PB honeycomb film was 371.2 ± 76.0 MPa, which was at least 15-fold larger than the non-cross-linked film and similar to that of some engineering plastics.28 3.2. Cell Adhesion on PB Honeycomb Scaffolds. The BF technique and the photo-cross-linking process were used to fabricate four cell culturing substrates: photo-cross-linked and non-photo-cross-linked flat and honeycomb films. The surface properties of the PB honeycomb films were not altered by cross-linking, because photo-cross-linking only occurs at the double bonds of the PB side chains. The water contact angles of the PB honeycomb films and the flat films were similar before and after cross-linking.21 Before cell culturing, protein absorption on the PB honeycomb films was observed. Figure 3 shows the fluorescent

3. RESULTS AND DISCUSSION 3.1. Characterization of PB Honeycomb Scaffolds. The SEM images of top-view and tiled PB honeycomb films are shown in Figure 1. Hexagonally packed 5 μm pores and

Figure 1. SEM images of the PB honeycomb scaffold.

approximately 1.5 μm polymer frames were observed. The thickness of the film was also about 5 μm. The tilted image shows that the honeycomb film had a double-layered structure supported with pillars. The pores of the honeycomb films were spherical and connected. The photo-cross-linking process made the PB honeycomb film more stable. Non-cross-linked PB honeycomb films can be easily melted when they are annealed above the glass transition temperature and are soluble in chloroform, whereas crosslinked films are not.24 The chemical cross-linking of polymers should stiffen the PB films. Figure 2 shows the AFM mapping of the elastic modulus of the flat and PB honeycomb films before and after photo-cross-linking. Note that the elastic modulus was measured only at the surface of the film (approximately 5 nm of depth), the elastic modulus was not affected by 3D structures of honeycomb scaffolds. The modulus values of the flat and non-cross-linked honeycomb films were

Figure 3. Microscopic images (a) and fluorescence intensity profiles (b) of rhodamine-labeled fibronectin in non-cross-linked honeycomb substrate (black line) and UV-cross-linked honeycomb substrate (gray line). Fluorescence intensity profiles are plotted along the broken white lines in (a).

micrographs of the PB honeycomb films before and after the photo-cross-linking. The films were soaked in rhodaminelabeled fibronectin solutions for 24 h. The fluorescence intensities of both films were similar and were not affected by photo-cross-linking. These results also indicate that the surfaces of the PB honeycomb films was not affected by photo-crosslinking, and photo-cross-linking does not affect the adsorption of proteins, which is an important biochemical process during cell culturing.

Figure 2. Distribution of the surface elasticity of flat (a), non-cross-linked honeycomb substrate (b), and UV-cross-linked honeycomb substrate (c). 1210

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Figure 4. Characterization of the fibroblast response to the experimental substrates. Plots of adhesive cell count (a) and cell area (b). *Statistically significant level: P < 0.01. Fluorescence microscope images of cell morphology, visualized with fluorescence-labeled phalloidin (c).

Figure 5. Fluorescence images (a) and distribution profile (b) of the focal adhesions on the planar control substrates, the non-cross-linked honeycomb substrates, and the UV-cross-linked honeycomb substrates. Representative images are shown for each microscopic observation of the focal adhesions, visualized with immunofluorescence-labeled vinculin.

Fibroblast 3T3 cells were cultured on the four substrates at low seeding densities in order to exclude the effects of cell−cell interactions, which could conceal the cell−substrate interactions. Figure 4a,b shows the number and the area of cells after they were cultivated for 24 h on each scaffold. No difference was observed between the cross-linked and non-cross-linked flat films. In contrast, the number of cells cultured on the noncross-linked honeycomb film was significantly smaller than that on the cross-linked honeycomb film. The cell area on the noncross-linked honeycomb film was also smaller than that of the cross-linked film. Figure 4c shows the fluorescent images of the fibroblast 3T3 cells with actin filaments stained with rhodamine-phalloidin. Because the cells on the flat films showed well spread shapes and highly organized actin stress fibers, the difference in rigidity caused by cross-linking did not affect the cell adhesion. The cells on the photo-cross-linked honeycomb film also exhibited similar shapes and stress fibers. However, the cell adhesion was very weak on the non-cross-linked honeycomb film, which had a similar modulus to the non-cross-linked flat film. The weak adhesion on the non-cross-linked honeycomb film was investigated by observing the focal adhesions of the cells on the flat and honeycomb substrates by fluorescent microscopy. The focal adhesions are integrin-based cellular structures which link the cells to underlying substrates at the tip of lamellipodia and filopodia, and they can be used as an index of cell adhesion strength. Figure 5a shows fluorescent (upper column) and close-up fluorescent (bottom column) image of vinculin-stained fibroblast 3T3 cells on the non-cross-linked flat and honeycomb films, and the cross-linked honeycomb scaffolds. Vinculin is also one of the constituents of focal adhesions. The fluorescent images of the cells on the flat film show that the

Figure 6. Deformation analysis of honeycomb substrates calculated for an applied horizontal traction force of 30 nN.

green fluorescence from the stained 3T3 fibroblasts was localized at the edge of the cells. The large number of large (ca. 9 μm) focal adhesions at the edge of a cell indicated that strong integrated adhesive sites were formed on the substrate surface underneath the cells. Similar structures were observed for the cross-linked honeycomb scaffold. Many focal adhesions were located on the polymer frames of the honeycomb scaffolds. In contrast, very weak fluorescence was observed for the non-cross-linked honeycomb scaffold. Figure 5b shows the histograms of focal adhesion lengths and numbers on noncross-linked flat, honeycomb, and cross-linked honeycomb scaffolds. For the non-cross-linked honeycomb scaffolds, there were few focal adhesions and they were unusually small. These results indicate that there were few adhesive sites formed beneath the cells on the non-cross-linked honeycomb film. 3.3. Relationship between the Mechanical Properties of the Honeycomb Scaffolds and Cell Adhesion. The PB honeycomb scaffolds generally had elastic moduli in the 1211

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Figure 7. SEM images of fibroblasts on a flat substrate, a non-cross-linked honeycomb substrate, and a photo-cross-linked honeycomb substrate.

This report demonstrates that the honeycomb scaffolds can regulate the cell adhesion by altering the microtopography, not only by changing the elasticity of the scaffold materails. The microtography effect is one of the important factors, because in vivo cells usually grow on the various topographically regulated spaces provided by other cells and extracellular matrices. Additionally, the topological regulation of the cells is a biologically noninvasive strategy without the chemical factors. The microtopography effect of honeycomb scaffolds offer understanding of mechanotransduction between cells and material interface and scaffolds for tissue engineering and medical implant devices.

megapascal range, which were greater than those of the hydrogels and silicone elastomers previously used to control cell behaviors. To explain why the high modulus of the substrate changed the cell adhesion, we performed structural mechanics simulations of the non-cross-linked and cross-linked honeycomb scaffolds under a traction force caused by the cell adhesion. Figure 6 shows the simulation results for non-crosslinked and cross-linked honeycomb scaffolds under a 30 nN lateral force, which is similar to the cell traction force.29 For the non-cross-linked honeycomb scaffold, the mainframe of the scaffold was deformed and a high strain was observed in the same direction as the traction force. However, the cross-linked honeycomb scaffold did not show any changes. Figure 7 shows the SEM and close-up SEM images of cells on the non-cross-linked and cross-linked honeycomb scaffolds, respectively. In the cross-linked honeycomb scaffold, the cells adhered tightly to the frame of the honeycomb structure, and no deformation was observed on the honeycomb scaffold. In contrast, in the non-cross-linked honeycomb scaffold the honeycomb structure was deformed and elongated against the cell edges. These results indicate that the deformation properties of the non-cross-linked honeycomb scaffold strongly affected the adhesion of cells. When cells adhere to substrates they form focal adhesions, which link the extracellular matrix to the cell cytoskeletons via membrane-bound receptors. The cells generate traction forces with the contractile actin cytoskeleton at the focal adhesion, which reinforces the extracellular matrix.30,31 Figure 7 shows that the deformation of the scaffold polymer frames prevents the cells from spreading on the noncross-linked honeycomb scaffold because the cells cannot generate enough traction force. The deformation of the substrate has been exploited in studies where elastomeric magnetic micropost arrays with different post heights were used to control the flexibility of the substrate and force was applied to the cells by magnetic field induced torque. Increasing the substrate rigidity increased the traction force, which pulled and spread the substrate. The cell spreading and the focal adhesion formation produced the traction force.16,17,32 The traction force of the cell cytoskeleton contraction is necessary for the cell adhesion, and our findings is identical with the reports by Zouani et al.10,33 Our observations suggest that the fibroblasts are useful for understanding the mechanical properties of the honeycomb scaffold and how the scaffold affects the traction force.

4. CONCLUSION Photo-cross-linking PB honeycomb cell culture substrates can modulate their deformation behavior. We demonstrated that the deformation behavior on a scale of several micrometers affects the cell morphology and focal adhesion formation. These results indicate that the elastomeric honeycomb substrates are useful for evaluating the effect of the mechanical signal-derived geometry of transduction system of cells.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work has been partially supported by Grant-in-Aid for Challenging Exploratory Research (No. 23651100).

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