Micropatterns Based on Deformation of a Viscoelastic Honeycomb

Wako, Saitama 351-0198, Japan, Local Spatio-Temporal Functions Laboratory, Spatio-Temporal Function ..... Jung Hak Kim , Myungeun Seo , Sang Youl ...
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Micropatterns Based on Deformation of a Viscoelastic Honeycomb Mesh Takehiro Nishikawa,*,† Makiko Nonomura,†,§ Keiko Arai,† Junko Hayashi,‡ Tetsuro Sawadaishi,† Yasumasa Nishiura,| Masahiko Hara,‡ and Masatsugu Shimomura*,†,⊥ Dissipative-Hierarchy Structures Laboratory, Spatio-Temporal Function Materials Research Group, Frontier Research System, The Institute of Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako, Saitama 351-0198, Japan, Local Spatio-Temporal Functions Laboratory, Spatio-Temporal Function Materials Research Group, Frontier Research System, The Institute of Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako, Saitama 351-0198, Japan, Graduate School of Science, Hiroshima University, 1-3-1, Kagamiyama, Higashi-Hiroshima 739-8526, Japan, Research Institute for Electronic Science, Hokkaido University, Kita 12, Nishi 6, Kita-ku, Sapporo 060-0812, Japan, and Nanotechnology Research Center, Research Institute for Electronic Science, Hokkaido University, Kita 12, Nishi 6, Kita-ku, Sapporo 060-0812, Japan Received January 16, 2003. In Final Form: May 7, 2003 We report that various geometric patterns can be formed upon mechanical deformation of hexagonal micro polymer mesh. The patterning of micromesh can be applied to the fabrication of micropatterned soft-materials for cell culturing. A microporous film was prepared from a viscoelastic polymer, poly(caprolactone). The film was a hexagonal mesh of 4 µm diameter. Plastic deformation of the film was caused by loading tensile force in one direction. Geometrical patterns such as elongated hexagons, rectangles, squares, and triangles were found in the stretched microporous film. These four types of deformation were reproduced by computer simulations using a viscoelastic network of hexagonally connected viscoelastic bonds. On the stretched hexagonal mesh, cardiac myocytes formed fibrous tissue where cells were aligned along the direction of the long axis of micropores. The hierarchical structure of blood vessels could be modeled by the coculture of endothelial cells and smooth muscle cells using a stretched honeycomb film as a micropatterned substrate.

Introduction The “bottom-up approach” is a novel strategy for the fabrication of mesoscopic scale structures ranging from nanometer to micrometer.1,2 The characteristic feature of the bottom-up fabrication is that the hierarchical mesoscopic structures can be spontaneously formed by the selforganization of unit molecules and pattern formation of the organized molecules at the mesoscopic scale. Of such mesoscopic scale structures, microporous material is a well-known structure and feasibly fabricated by the template method using inorganic particles,3 polymer beads,4 emulsions,5 vesicles,6 micelles,7 bacteria,8 and * To whom correspondence should be addressed. Phone: +8148-462-1111, ext 6333. Fax: +81-48-462-4695. E-mail: tnishi@ postman.riken.go.jp. † Dissipative-Hierarchy Structures Laboratory, Spatio-Temporal Function Materials Research Group, Frontier Research System, The Institute of Physical and Chemical Research (RIKEN). ‡ Local Spatio-Temporal Functions Laboratory, Spatio-Temporal Function Materials Research Group, Frontier Research System, The Institute of Physical and Chemical Research (RIKEN). § Graduate School of Science, Hiroshima University. | Research Institute for Electronic Science, Hokkaido University. ⊥ Nanotechnology Research Center, Research Institute for Electronic Science, Hokkaido University. (1) Organic Mesoscopic Chemistry; Masuhara, H., De Schryver, F. C., Eds.; Blackwell Science Ltd.: Oxford, 1999. (2) Shimomura, M.; Sawadaishi, T. Curr. Opin. Colloid Interface Sci. 2001, 6, 11. (3) Matsushita, S.; Miwa, T.; Fujishima, A. Chem. Lett. 1997, 925. (4) Park, S. H.; Xia, Y. Adv. Mater. 1998, 10, 1045. (5) Imhof, A.; Pine, D. J. Nature 1997, 389, 948. (6) Ozin, G. A. Acc. Chem. Res. 1997, 30, 17. (7) Walsh, D.; Mann, S. Nature 1995, 377, 320.

microspheres of water.9-12 The porous structure is molded from two-dimensional and three-dimensional arrays of the small particles. For example, polymer films with honeycomb-like regular arrays of micropores have been reported by several research groups since the mid1990s.9-12 The microporous films can be obtained when a spread polymer solution is evaporated on a substrate in a humid atmosphere. In situ observation of the film formation process revealed that microspheres of water were formed by the condensation of atmospheric water at the air-polymer solution interface.13 The template for the honeycomb-like porous structure is a two-dimensional array of hexagonally packed water microspheres. The template itself evaporates gradually and is finally replaced with a honeycomb-like mesh structure of polymer. The pore size and thickness of the mesh structure can be controlled by the experimental parameters such as solvent volatility, polymer concentration, and atmospheric conditions (temperature and humidity).10,14 The regular alignment of microspheres in the templates enables repro(8) Davis, S. A.; Burkett, S. L.; Mendelson, N. H.; Mann, S. Nature 1997, 385, 420. (9) Widawski, G.; Rawiso, B.; Francois, B. Nature 1994, 369, 387. (10) Maruyama, N.; Koito, T.; Nishida, J.; Sawadaishi, T.; Cieren, X.; Ijiro, K.; Karthaus, O.; Shimomura, M. Thin Solid Films 1998, 327329, 854. (11) Jenekhe, S.; Chen, X. Science 1999, 283, 372. (12) Srinivasarao, M.; Collings, D.; Philips, A.; Patel, S. Science 2001, 292, 79. (13) Karthaus, O.; Maruyama, N.; Cieren, X.; Shimomura, M.; Hasegawa, H.; Hashimoto, T. Langmuir 2000, 16, 6071. (14) Nishikawa, T.; Ookura, R.; Nishida, J.; Arai, K.; Hayashi, J.; Kurono, N.; Sawadaishi, T.; Hara, M.; Shimomura, M. Langmuir 2002, 18, 5734.

10.1021/la0300129 CCC: $25.00 © 2003 American Chemical Society Published on Web 06/26/2003

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ducible fabrication of a microporous structure with a relatively narrow size distribution. The shapes of microspheres are faithfully imprinted in the shapes of micropores. These are common features of the template method. Though the shape imprinting is an advantage of the template method, this advantage restricts pursuing the versatility of the geometrical shape of micropores. We have fabricated micropatterned surfaces for microelectrodes,15 photonic crystals,16 and cell culture substrates17-19 by using the honeycomb-like microporous films. We reported controlled cell adhesion on honeycombpatterned substrates.14,17-19 The extent of cell movement including spreading, migrating, and shape forming was affected by the pore size of the microporous film.14,19 We are now studying a self-supporting microporous film of a biodegradable polymer for tissue regeneration.20 Cellular orientation is one of the typical features of living tissues. However, cellular orientation along a specific direction cannot be expected for the cell culture on the microporous film, since the microporous film shows an isotropic pattern of hexagons which is molded from a two-dimensional array of water microspheres. Cell culture substrates with various geometric micropatterns have been fabricated by the conventional microfabrication methods such as photolithography and related techniques. Such micropatterned substrates have been extensively studied in terms of cellsurface interaction and coculture systems of different types of cells.21 It was reported that anisotropic micropatterns of lines and grooves can guide cell movement in a specific direction.22-24 Most of the micropatterns were fabricated on hard substrates such as metal and glass unsuitable for tissue scaffolds. Meanwhile, a number of soft materials for tissue scaffolds are not suitable for the conventional patterning technique. Though anisotropic patterns do not appear in the two-dimensional array of water microspheres, various kinds of polymers are applicable to the fabrication of the microporous film. To pursue the broader application of the microporous films, we propose a new fabrication method for the anisotropic patterns based on the mechanical stretching of a viscoelastic polymer film. In this report, we describe the fabrication of anisotropic patterns from a viscoelastic microporous polymer film, the verification of the pattern formation process using numerical simulation, and the cell culture experiment on a stretched microporous film providing anisotropic arrays of stretched micropores. Experimental Section Materials. Poly(-caprolactone) (Figure 1A, PCL) and a copolymer of dodecylacrylamide and ω-carboxyhexylacrylamide (Figure 1B, polymer 1) were used in the fabrication of microporous films. PCL was purchased from Birmingham Polymers, Inc. The molecular weight of PCL is Mw ) 6.7 × 104, which is estimated (15) Shimomura, M.; Koito, T.; Maruyama, N.; Arai, K.; Nishida, J.; Gråsjo¨, L.; Karthaus, O.; Ijiro, K. Mol. Cryst. Liq. Cryst. 1998, 322, 305. (16) Kurono, N.; Shimada, R.; Ishihara, T.; Shimomura, M. Mol. Cryst. Liq. Cryst. 2002, 377, 285. (17) Nishikawa, T.; Nishida, J.; Ookura, R.; Nishimura, S.; Wada, S.; Karino, T.; Shimomura, M. Mater. Sci. Eng., C 1999, 8-9, 495. (18) Nishikawa, T.; Nishida, J.; Ookura, R.; Nishimura, S.; Wada, S.; Karino, T.; Shimomura, M. Mater. Sci. Eng., C 1999, 10, 141. (19) Nishikawa, T.; Nishida, J.; Nishikawa, K.; Ookura, R.; Ookubo, H.; Kamachi, H.; Matsushita, M.; Todo, S.; Shimomura, M. Stud. Surf. Sci. Catal. 2001, 132, 509. (20) Nishikawa, T. Paper presented at the 48th American Vacuum Society international symposium, San Francisco, CA, Nov 1, 2001. (21) Curtis, A.; Wilkinson, C. Biomaterials 1997, 18, 1573. (22) Kane, R. S.; Takayama, S.; Ostuni, E.; Ingber, D. E.; Whitesides, G. M. Biomaterials 1999, 20, 2363. (23) den Braber, E. T.; de Reijter, J. E.; Ginsel, L. A.; von Recum, A. F.; Jansen, J. A. J. Biomed. Mater. Res. 1998, 40, 291. (24) Folch, A.; Toner, M. Biotechnol. Prog. 1998, 14, 388.

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Figure 1. Polymers used in this study: (A) poly(-caprolactone) and (B) amphiphilic polymer 1. from the inherent viscosity of 1.13 dL/g given by the supplier using the relation between the viscosity ([η]) and molecular weight (Mw): [η] ) 1.30 × 10-4Mw0.83.25 The polymer 1 was obtained by radical polymerization using AIBN as an initiator in benzene. The monomer unit ratio of hydrophobic part to hydrophilic part of the copolymer is 4:1, which was determined by proton NMR analysis and elemental analysis. The molecular weight of the copolymer is Mw ) 2.2 × 104 and Mn ) 1.5 × 104 (Mw/Mn ) 1.48), which were measured by size exclusion chromatography. Water was purified by a Millipore system (Milli-Q, Millipore). Benzene was spectroscopy grade (Dojindo) and was used without further purification. Film Preparation. Microporous films were fabricated by applying moist air to a spread polymer solution on the water surface.14 A Petri dish (diameter, 9 cm) was filled with 50 mL of Milli-Q grade water. Benzene solution containing 1 g/L of PCL and 0.1 g/L of polymer 1 was prepared for the film fabrication. To ensure that a polymer solution for film preparation can form a stable liquid film on the water surface, 20 µL of the polymer solution was spread on the water surface in advance. The water surface was covered with a mixed monolayer of PCL and polymer 1 and deposited film which was formed from the unspread polymer solution. Fifty microliters of the polymer solution was deposited onto the water surface for film fabrication. The polymer solution formed a lens-shaped liquid film on the water surface. Humidified air (20 °C and 75% relative humidity (RH)) was supplied from a 250 mL gas washing bottle filled with 200 mL of Milli-Q water by an electric motor pump and was blown to the deposited polymer solution from a glass pipe nozzle (diameter, 5 mm) at the flow rate of 1200 mL/min. The flow rate was controlled using a flow meter. The nozzle was set at 15 mm above the water surface. The polymer solution started to evaporate, and the surface turned turbid due to the condensation of water vapor of the humidified air. After solvent evaporation, a thin opaque film remained on the water surface. The film was clipped by a pair of tweezers with 1 cm wide carbon tips and stretched in one direction. The stretched film was transferred onto a solid substrate and left in an air-conditioned room (20 °C and 33% RH) to dry. Observation of Surface Morphology. A honeycomb film transferred onto a glass plate was characterized by optical microscopy (BX-60, Olympus), atomic force microscopy (AFM; Explorer SPM, ThermoMicroscopes), and scanning electron microscopy (SEM; S-3500N, Hitachi). The surface topographic images of microporous films were obtained by noncontact mode AFM. Cell Culturing. Cardiac myocytes (CMYs) were isolated by enzyme treatment of minced heart tissues of 19-day rat embryos (Sprague Dawley rats; Japan SLC, Inc).26 CMYs were seeded onto cell culture substrates (a glass plate, a microporous film on a glass plate, and a stretched microporous film on a glass plate) at the density of 4.0 × 105 cells/cm2. CMYs were cultured with a Hepes-buffered Hams F10 medium containing 0.5% insulintransferrin-selenium-X (Gibco) and 3% fetal calf serum (Gibco). For quantitative analysis of the spreading behavior of cells, filamentous actin of CMYs was stained by rhodamine-conjugated phalloidin (Molecular Probes) after fixation with 4% paraform(25) Biodegradable Polymers as Drug Delivery Systems; Chasin, M., Langer, R., Eds.; Mercel Dekker: New York, 1990. (26) Denyer, M. C. T.; Riehle, M.; Scholl, M.; Sproessler, C.; Britland, S. T.; Offenhaeusser, A.; Knoll, W. In Vitro Cell. Dev. Biol.: Anim. 1999, 35, 352-356.

Micropatterns Based on a Viscoelastic Mesh aldehyde (Sigma) in phosphate-buffered saline (PBS; Gibco) and treatment with 0.1% Triton X-100 (Sigma) in PBS at 20 °C. Fluorescence images of cells were taken by an inverted microscope (IX70, Olympus) equipped with a mercury lamp (USH-102D, USHIO) and a CCD camera (DC-330, DAGE-MTI Inc.). Projected areas of CMYs were measured using computerized image analysis (Image-pro Plus ver. 4.0, Media Cybernetics). Bovine aortic endothelial cells (ECs) and smooth muscle cells (SMCs) were purchased as cryopreserved samples of primary cultures (lot no. 9F0373 (ECs) and lot no. 7CC002 (SMCs)) from BioWhittaker. After the frozen cells were thawed at 37 °C, the cells were resuspended into a supplemented culture medium (MCDB-131, BioWhittaker). A stretched microporous film fixed on a 5 mm diameter hole in a Teflon plate was soaked in the supplemented medium for 30 min before cell seeding. ECs and SMCs were cocultured by seeding SMCs on one side of a stretched honeycomb film and then seeding ECs on the other side of the film at 6 h after the initial plating of SMCs. The initial cell density was 2.0 × 104 cells/cm2. ECs and SMCs were cultured with the supplemented medium. To visualize the cocultured tissue of ECs and SMCs, Von Willebrand factor of ECs and R smooth muscle actin of SMCs were stained by the immunological method using primary antibodies (rabbit anti-Von Willebrand factor IgG, DAKO; and mouse anti-R smooth muscle actin IgG, Sigma) for each antigen and fluorescence-labeled secondary antibodies (fluorescein-labeled goat anti-rabbit IgG (Cappel) and rhodaminelabeled goat anti-mouse IgG (Cappel)). For immunostaining, cells were fixed by immersing into cold methanol (Wako) (-20 °C) for 10 min and permeated with 0.1% PBS solution of Triton X-100 for 5 min at 20 °C. Fluorescence images of cells were taken by confocal laser scanning microscope (FV300, Olympus).

Results and Discussion Viscoelastic Microporous Film. We focused on the fabrication of microporous structure exhibiting geometrical shapes which are topologically equivalent to a hexagon: elongated hexagon, rectangle, square, and triangle. These shapes can be obtained in principle by deformation of hexagons.27 If the microporous film is made of a viscoelastic matter, the pores can be deformed to various shapes by mechanical stretching. To demonstrate the mechanically induced shape formation, we prepared a viscoelastic microporous film from a viscoelastic polymer, PCL, which is a viscoelastic degradable polymer extensively used in the medicinal field.28 Due to its low glass transition temperature (-65 °C), PCL exhibits rubber elasticity at room temperature. Various amphiphilic compounds such as polyion complexes and amphiphilic copolymers have been utilized in our fabrication method for microporous films.10 Recently we found that microporous films of nonamphiphilic polymers such as polystyrene and poly(L-lactic acid) can be obtained by using amphiphiles as additives for stabilization of water microspheres.13,14 For the fabrication of a microporous film of PCL, a mixed benzene solution of PCL and amphiphilic polymer 1 (Figure 1) (total concentration: 1 g/L, 10 to 1 by weight ratio) was spread onto the water surface (20 °C). Moist air (70% RH at 20 °C) was blown onto a droplet of the spread polymer solution at the flow rate of 1200 mL/min. The surface of the droplet became turbid due to the condensation of water vapor of the moist air. After solvent evaporation, a thin opaque film was floating on the water surface. The cast film exhibited a disk shape with a diameter of 2 cm, when 150 µL of the polymer solution was spread on the water surface (Figure 2A, right). The microporous film composed of PCL and 1 in Figure 2A was a single layered array of micropores. The average (27) Hyde, S.; Andersson, S.; Larsson, K.; Blum, Z.; Landh, T.; Lidin, S.; Ninham, B. W. The Language of Shape; Elsevier Science B. V.: Amsterdam, 1997. (28) Engelberg, I.; Kohn, J. Biomaterials 1991, 12, 292.

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length of diagonal lines in each hexagon (pore size) was measured to be 4.5 ( 0.3 µm by surface analysis using an atomic force microscope. The thickness of the microporous film was 2.0 ( 0.1 µm. Geometric Patterns in a Stretched Microporous Film. Deformation of micropores was studied by manual stretching of the microporous film of PCL. As shown in Figure 2, a microporous film (size, 2 cm in diameter) floating on the water surface was carefully held by using two pairs of tweezers (Figure 2A, left) and was stretched outward in one direction until the length of the long axis of the stretched film was approximately twice as long as that of the short axis of the film (Figure 2B, left). In the stretching process, the tensile force was applied to the microporous film for 10 s and then loosened for 5 s to relax the internal stress. This stretching process was repeated four times. Thus it took 60 s to reach the final length of the film. The stretched film was held for several seconds to relax the internal stress remaining in the film. A rectangular sheet of microporous film (Figure 2B, right) was obtained from the disk shape of the microporous film (Figure 2A, right). The microporous film was extended in the direction of the tensile force and slightly compressed in the normal direction to the tensile force. The dimensions of the stretched film were 3.6 cm in the long axis and 1.8 cm in the short axis. The film was not ruptured by the stretching. The shape of the deformed film appeared to be preserved after unloading the force and removing the tweezers. This indicates that plastic deformation is caused in the stretched microporous film. The stretched film transferred onto a glass plate was observed by an optical microscope. Four geometric shapes of micropores, elongated hexagons (Figure 3E), rectangles (Figure 3F), squares (Figure 3G), and triangles (Figure 3H), were confirmed in the stretched film. Figure 3B,C,D shows each one of these regions where micropores with each geometric shape were observed. If the honeycomb pattern is perfect, single domains of rectangles or elongated hexagons can be obtained by stretching the film. However, the domain of elongated hexagons and the domain of rectangles are aligned side by side in the stretched honeycomb film (Figure 3B). Coexistence of elongated hexagons and rectangles indicates that our honeycomb film contains some defects and grain boundaries in the array of micropores. Square shape is not expected to form upon the uniaxial stretching. The domain of square-like micropores is located between the domains of elongated hexagons or rectangles (Figure 3C). Distorted hexagons and large irregular defects (indicated by * in Figure 3C) were observed at the boundary between the domain of squares and the domain of elongated hexagons. This indicates the second stretching force which acted locally on the small domain of hexagons in addition to the original stretching force. Biaxial stretching is expected to occur on a local domain of hexagons. The domain of triangular micropores was found in a region where elongated hexagons and rectangles were distorted, twisted, or collapsed by the compressing force (Figure 3D). Numerical Simulation of Geometric Pattern Formation. To explain the formation of the four shapes in the stretched microporous film, we introduced a model of a viscoelastic network and carried out computer simulations of the mechanical deformation of the viscoelastic network. The viscoelastic network was described as hexagonally connected viscoelastic bonds. The motion of each vertex of hexagons was analyzed under the deformation. It was assumed that each bond of hexagons has the viscoelastic property given by the Maxwell model,

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Figure 2. Stretching of a microporous film on the water surface. (A) Before stretching. (B) After stretching. Scale bar: 1 cm.

1 σ˘ Rβ ) - σRβ + G˘ Rβ λ

(1)

where σRβ(t) is the stress tensor, Rβ(t) is the strain tensor, λ is the relaxation time, G is the elastic coefficient, and the dot means the time derivative. The tensile force F loaded at both ends of a bond is written as F ) Sσ11, where the subscript 1 stands for the direction along the cylindrical axis of the bond. The cross-sectional area of the bond S is related to the bond length R as V ) SR since the volume of each bond is conserved. Assuming that the deformation occurs homogeneously along the bond, the force F is obtained from eq 1 as the differential form

F˙ ) -

R˙ (1λ + RR˙ )F + GV R R

(2)

0

and as the integral form

F(t) )

GV R0R(t)

∫-∞t e-(t-s)/λR˙ (s) ds

(3)

The position vector of each vertex of hexagons, b xi(t), is determined by the following equation,

bj b x i -x

∑j Fij(t) R (t)

γx b˙ i ) -

(4)

ij

where γ is the friction constant between the vertex and

the surrounding medium. The force Fij(t) is loaded on a bond between the nearest neighbor vertexes i and j as shown in Figure 4A and varies with changing the bond bi - b xj| according to eqs 2 and 3 where Rij length Rij(t) ) |x (Fij) is substituted for R (F). The summation in eq 4 is over three neighboring vertexes. The dimensionless parameter D ≡ GVλ/(R02γ) ) 1000 was utilized in the present simulations where the bond length of the undeformed hexagon was set to R0. Figure 4B-E displays four geometrical patterns which were obtained numerically by solving eqs 2-4. The simulation was started from the regular hexagonal network (Figure 4A) as the initial pattern. The system boundary was represented by the bold rectangular frame in Figure 4A. To deform the network homogeneously, a force was applied to a pair of the parallel sides of the boundary such that the vertexes on them moved at a constant speed, 0.2R0/λ. An excess speed of stretching (e.g., 100R0/λ) caused inhomogeneous deformation of the network resulting in the coexistence of stretched pores and undeformed pores (Figure 4F). The vertexes on these boundaries were allowed to move only along the boundaries. The network was stretched or compressed in two B and B ΓM, which were parallel and vertical directions ΓK to the initial hexagonal sides (Figure 4A). The uniaxial stretching in the ΓK B and B ΓM directions caused stretched hexagons (Figure 4B) and rectangles (Figure 4C), respectively. These structures were obtained when the system was extended to 3 times as large as the initial size. The triangle-like structure was obtained by applying a com-

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Figure 3. Geometrical patterns found in a stretched microporous film of PCL. (A) Hexagonal pattern before stretching. Scale bar: 5 µm. (B-D) Geometrical patterns induced by stretching. Scale bar: 25 µm. (E) Elongated hexagons. (F) Rectangular shape. (G) Square-like shape. (H) Triangle-like shape. Scale bar: 5 µm.

pressive force to the regular hexagons (Figure 4A) in the ΓK B direction until the network was shrunken to 0.35 times the initial system size (Figure 4D). The square-like structure was formed when the rectangular hexagons B (Figure 4C) were stretched uniaxially further in the ΓK direction (Figure 4E), while a simultaneous biaxial stretching caused a simple expansion of hexagons. The present viscoelastic model of the hexagonal network successfully simulated the formation process of the four geometric patterns in the stretched microporous film. The remarkable outcome of the simulation is that all the structures, Figure 4B-E, were preserved after removing the external force. The preservation of the structure was observed in the stretched microporous film of PCL. On

the other hand, the formation of the four geometric patterns obtained numerically needed different deformations in terms of the directions of applied force and the number of stretching axes, while the real microporous film exhibits all of the four geometric patterns concurrently by uniaxial stretching. This difference was attributed to the facts that the porous structure of the real PCL film contained defects and grain boundaries and that the force was not loaded uniformly to the whole part of the PCL film. In the case of the real microporous PCL film, the formation of the rectangular and the elongated hexagonal structures would depend on the orientation of the initial unloaded hexagonal grains. The triangle-like structures were observed in a limited area between the stretched

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Figure 4. Numerical analysis of the deformation of microporous film. (A) Regular hexagons. The bold lines depict the system boundaries. The arrows indicate the symmetry axes (ΓK B and ΓM B) of the hexagons. (B) Elongated hexagons (stretching in the B ΓK direction). (C) Rectangles (stretching in the ΓM B direction). (D) Triangle-like structure (compressing in the ΓK B direction). (E) Square-like structure (stretching in the ΓM B direction and then in the ΓK B direction). (F) Network structure containing stretched pores and undeformed pores (stretching in the B ΓM direction at the speed of 100R0/λ).

part and the unstretched part of the film. This observation suggests that compressive force occurs when two neighboring grains are in different mechanical environments. Furthermore, the square-like structures were observed not always but occasionally in the stretched microporous PCL film. This might be related with the fact that the secondary force was necessary to obtain the square-like structures in the numerical simulations. It is believed that the secondary force was applied to certain grains from the surrounding grains during the corrective relaxation process of the primary stress. Cell Culture on a Stretched Microporous Film. We have proposed that microporous films of biodegradable polymers are applicable to tissue engineering.20 Regulated cell alignment is a common feature of tissue structure. For example, heart muscle is a striated tissue in which cardiac myocytes are connected together in a straight line and the aligned cells are arrayed side by side in parallel.29 However, artificial extracellular matrixes of soft matter that can induce the cell alignment have not been achieved yet except in some cases in which external stimuli are applied to the cell culture.30 To achieve the regulated cell alignment in vitro, we used a uniaxially stretched microporous film as a cell culture substrate. For the cell (29) Young, B.; Heath, J. W. Weater’s Functional Histology, 4th ed.; Churchill Livingstone: New York, 2000. (30) Fink, C.; Ergu¨n, S.; Kralisch, D.; Remmers, U.; Weil, J.; Eschenhagen, T. FASEB J. 2000, 14, 669.

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culture experiment, three types of substrates were used. Substrate-1 was a flat cast film of PCL-1, which covered a surface of a glass plate. Substrate-2 was a hexagonal patterned surface, which was fabricated by transferring a microporous film of PCL-1 (pore size, 4.5 ( 0.3 µm; thickness, 2.0 ( 0.1 µm (Figure 5A,C,E)) onto a glass plate. Substrate-3 was an anisotropic micropattern, which was fabricated by transferring a stretched microporous film of PCL-1 onto a glass plate (Figure 5B). Topographical features of the stretched micropores were 5.0 ( 0.2 µm in the short axis and 20 ( 1.0 µm in the long axis (Figure 5D). The depth of the micropores (the thickness of the stretched film) was 1.4 ( 0.1 µm (Figure 5F,G). Cardiac myocytes of rat embryo were cultured on these substrates. The cells were attached onto the substrates. On substrate1, the cells could extend their shape in any direction (Figure 6A). Most cells formed a triangular spread shape whose size was 5000 ( 1300 µm2/cell. On the other hand, the spreading behavior of the cells was regulated by the hexagonal pattern (substrate-2) and the elongated hexagonal pattern (substrate-3) (Figure 6B,C). The cell sizes were 1900 ( 800 µm2/cell (substrate-2) and 1400 ( 500 µm2/cell (substrate-3), which were less than half the size of the cardiac cells on substrate-1. The cells on these patterned surfaces extended their body especially along the alignment of hexagons. However, the cells on substrate-2 (hexagonal pattern) could not extend in a specific direction since the regular hexagonal pattern is an isotropic pattern. Shape index and cell orientation angle were used in order to characterize the cell morphology quantitatively. The shape index (S) for the degree of cell elongation was calculated by the following equation:31

S ) 4π × (projected area of a cell)/ (perimeter of a cell)2 The index S can vary from 0 (straight line) to 1 (circle) depending on the alignment of the cells. The orientation angle (θ) is defined as an angle formed between the long axis of the cells and the direction of reference. The cardiac cells on substrate-2 had a shape index of 0.6 ( 0.3 suggesting that various cell shapes such as triangle, rectangle, and spindle were formed. Their major axis was oriented at 50° ( 44° with the direction of the reference depicted in Figure 6B. This orientation angle indicates a random orientation of cells. Cardiac cells could be oriented in a specific direction, which was denoted by the long axis of the elongated hexagons (substrate-3). The cardiac cells on substrate-3 were oriented at 4° ( 8° with the direction of the long axis of the stretched film and exhibited an elongated morphology with a shape index of 0.4 ( 0.1. Thus by using the stretched microporous film, it is possible to guide cell orientation as well as to regulate the spreading behavior of cells. Three-dimensional culture and coculture of different types of cells are key strategies for reconstruction of hierarchical tissue structure.32 Blood vessels have a characteristic hierarchical tissue structure consisting of three layers: the intima of endothelial cells, the media of smooth muscle cells, and the adventitia of smooth muscle cells and fibroblasts.29 Various porous membranes are produced from many types of polymers and are commercially available at present. The porous membranes have been used for the coculture of different types of (31) Levesque, M. J.; Nerem, R. M. J. Biomech. Eng. 1985, 176, 341. (32) Principles of Tissue Engineering; Lanza, R., Langer, R., Vacanti, J., Eds.; Academic Press: San Diego, 2000.

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Figure 5. Scanning electron microscope images of a microporous film of PCL-1 (A) and a stretched microporous film of PCL-1 (B). Scale bar: 5 µm. Surface topographic images of a microporous film of PCL-1 (C) and a stretched microporous film of PCL-1 (D). (E) Cross-sectional profile measured along the depicted line in (C). (F,G) Cross-sectional profiles measured along the depicted lines in (D).

Figure 6. Guided cell alignment of cardiac myocytes on a stretched microporous film. (A) On a flat film (substrate-1). (B) On a microporous film (substrate-2). (C) On a stretched microporous film (substrate-3). These images were taken after staining filamentous actins of cardiac myocytes with rhodamine-conjugated phalloidin. The arrows depicted in (B) and (C) are the direction of reference. Scale bar: 40 µm.

cells33,34 in addition to the use for filtration, separation, and dialysis. However, the conventional porous mem-

branes are not appropriate for studying the micropattern effects on the cell behavior because of a lack of regularity

(33) Weber, E.; Ha¨mmerle, H.; Vatti, R.; Berti, G.; Betz, E. Vessels Appl. Pathol. 1986, 4, 246.

(34) Fillinger, M.; Sampson, L.; Cronenwett, J.; Powell, R.; Wagner, R. J. Surg. Res. 1997, 67, 169.

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were aligned in one direction mainly dictated by the long axis of the elongated micropores (Figure 7, X-Y plane image). On the contrary, in the case of real blood vessels, both cell types are aligned in one direction in each tissue layer, but the directions are not parallel. In the intima layer, endothelial cells are exposed to a shearing force imposed by blood flow and are aligned along the direction of blood flow. The media layer of smooth muscle cells surrounds the intima layer of endothelial cells helically to resist pulsatile blood pressure.29 Our three-dimensional cell culture system using a stretched microporous film can be applied to the study of the micropattern effect and the external force effect on the cellular orientation in vascular tissue formation. Conclusion

Figure 7. Fluorescence image of three-dimensional culture of bovine aortic ECs (yellow-green) and SMCs (red). Each cell type was separately cultured on each side of stretched microporous films of PCL-1. Von Willebrand factor of ECs and R smooth muscle actin of SMCs were stained by the immunological method using primary antibodies for each antigen and fluorescence-labeled secondary antibodies. The image was taken by confocal laser scanning microscopy. Cross-sectional images were obtained along the lines depicted in the X-Y plane image. Scale bar: 10 µm.

in size and alignment of micropores. As mentioned above, our microporous film and stretched microporous film can be regarded as micropatterns in terms of structural regularity. Therefore, endothelial cells and smooth muscle cells were cultured on both sides of a stretched microporous film of PCL in order to mimic the structure of blood vessels. To fabricate a cell culture substrate, a stretched microporous film was sandwiched by two Teflon plates (size, 15 mm × 15 mm) with a 5 mm hole punched in the center of the plate. Smooth muscle cells were initially plated onto the 5 mm diameter area of the stretched microporous film, and then endothelial cells were seeded on the opposite side of the film at 6 h after the initial plating. Both cell types proliferated and reached confluence on the stretched microporous film in 7 days. Von Willebrand factor and R smooth muscle actin are marker molecules of endothelial cells and smooth muscle cells, respectively.35,36 Immunological staining of these cells with antibodies for the markers clarified that endothelial cells and smooth muscle cells were spatially separated by the stretched microporous film (Figure 7, cross-sectional images). The porous structure of the stretched honeycomb film may facilitate material exchanges and physiological contacts between the cells in addition to the separation of cells. Furthermore, basement membrane may be reconstructed at the interfacial space between the endothelial cell layer and the smooth muscle cell layer. In fact, the cell-cell interaction and the cell-extracellular matrix interaction are required for the redifferentiation of smooth muscle cell.36,37 The stretched microporous film with the anisotropic pattern enabled these cell types to form monolayers where cells (35) Jaffe, E. N. Engl. J. Med. 1977, 296, 377. (36) Chamley-Campbell, J.; Campbell, G.; Ross, R. Physiol. Rev. 1979, 59, 1. (37) Thyberg, J.; Blomgren, K.; Roy, J.; Tran, P.; Hedin, U. J. Histochem. Cytochem. 1997, 45, 837.

We summarize the results obtained in the present study. Geometric patterns, which are topologically equivalent to a regular hexagonal pattern, can be obtained by deformation of the hexagonal network. We applied the geometric pattern formation to the fabrication of a micropatterned surface. We succeeded in the fabrication of porous polymer thin films with various geometric patterns. When a microporous film of a viscoelastic polymer, poly(-caprolactone), was stretched, the isotropic array of hexagonal micropores was transformed into anisotropic alignment of stretched micropores. Geometric patterns observed on the stretched film were elongated hexagons, rectangles, squares, and triangles. The formation of the patterns was simulated by the model of a viscoelastic network. From the numerical simulations, it was found that uniaxial stretching or compression of the network gave the structures of elongated hexagons, rectangles, and triangles and a secondary force was necessary for the formation of the squares. By comparison of the numerical results of the viscoelastic network with the real microporous film, the secondary forces arose probably from existence of the grains and the nonuniformity of the external force acting on the film. The parameters to be studied for further improvement of the simulation are the strain rate, the direction of the forces, the number of stretching axes, and the inhomogeneity of the network. We are now studying the morphology and dynamics of microporous films experimentally and by computer simulations in detail. The stretched microporous films can work as micropatterned cell culture substrates. The arrays of stretched micropores (elongated hexagons and rectangles) could guide spreading of cardiac myocytes along the long axis of the stretched pores. Three-dimensional tissue cultures consisting of endothelial cells and smooth muscle cells were also realized by culturing each cell type separately on each side of the stretched microporous film. Both cells were aligned in the parallel direction to the stretch direction of the microporous film. In such a coculture system, heterotypic cell-cell interaction which is necessary to achieve unified tissue functions is expected to occur in addition to the regulated cell alignment. Mechanical stretching of polymer materials such as threads and films has been conventionally performed to enhance the strength of the materials. However, it has not been imagined that the stretching produces various geometric patterns. Recently it was reported that a threedimensional lattice of ellipsoidal microhollows could be fabricated by stretching a hollow structure of poly(methyl methacrylate).38 Our method, stretching of a twodimensional polymer mesh, can be applied to the formation of various geometrical patterns in any types of stretchable (38) Jiang, P.; Bertone, J. F.; Colvin, V. L. Science 2001, 291, 453.

Micropatterns Based on a Viscoelastic Mesh

microporous films. In addition to the biological application of our microporous films, we are studying the application of the porous film to optical devices. The porous film exhibits an infrared absorption band that is not derived from the chemical bonds of the constituent polymer.16 The absorption peak is determined by the spatial pitch of the micropore arrays. We expect that the anisotropic patterns appearing in the stretched porous film can be utilized as devices exhibiting anisotropic optical properties.

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Acknowledgment. This study was partially supported by the Industrial Technology Research Grant Program in ’02 from the New Energy and Industrial Technology Development Organization (NEDO) of Japan. The author (M.N.) thanks Professor Takao Ohta and Dr. Tohru Okuzono of Hiroshima University for valuable discussions in computer simulation. LA0300129