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Micropatterned Thermoresponsive Polymer Brush Surfaces for Fabricating Cell Sheets with Well-Controlled Orientational Structures Hironobu Takahashi, Masamichi Nakayama, Kazuyoshi Itoga, Masayuki Yamato, and Teruo Okano* Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University (TWIns), 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan ABSTRACT: Newly developed fabrication technique of thermoresponsive surface using RAFT-mediated block copolymerization and photolithography achieved stripelike micropatterning of poly(N-isopropylacrylamide) (PIPAAm) brush domains and poly(N-isopropylacrylamide)-b-poly(N-acryloylmorpholine) domains. Normal human dermal fibroblasts were aligned on the physicochemically patterned surfaces simply by one-pot cell seeding. Fluorescence images showed the well-controlled orientation of actin fibers and fibronectin in the confluent cell layers with associated extracellular matrix (ECM) on the surfaces. Furthermore, the aligned cells were harvested as a tissue-like cellular monolayer, called “cell sheet” only by reducing temperature below PIPAAm’s lower critical solution temperature (LCST) to 20 °C. The cell sheet harvested from the micropatterned surface possessed a different shrinking rate between vertical and parallel sides of the cell alignment (approximately 3:1 of aspect ratio). This indicates that the cell sheet maintains the alignment of cells and related ECM proteins, promising to show the mechanical and biological aspects of cell sheets harvested from the functionalized thermoresponsive surfaces.
’ INTRODUCTION Over the past decade, tissue-like cellular monolayers, called “cell sheets” have been developed using proprietary thermoresponsive cell culture substrates. Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm) grafted cell culture substrates with nanoscale thicknesses allow confluently cultured cells to be harvested as a cell sheet with intact associated extracellular matrix (ECM) simply by reducing culture temperature below PIPAAm’s lower critical solution temperature (LCST) of 32 °C through the thermal switchable hydrophobic-to-hydrophilic and conformational changes of the polymer surfaces.1,2 Our group has opened up a new field in regenerative medicine using a unique strategy for establishing a tissue reconstruction technology, “cell-sheet engineering”.3,4 For example, oral mucosal epithelial cell sheets have already been applied to human clinical studies for corneal reconstruction and esophageal ulceration treatment after endoscopic submucosal dissection.5,6 In addition, for applying cell-sheetbased regenerative medicine to multiple damaged tissues and organs, various types of cell sheets are now in progress of fabrication.4,79 For example, treatment with cardiomyocyte sheets is expected as a new class technique for myocardial tissue reconstruction.10 It is well known that muscle tissues in heart and skeletal muscle require orientational structures for expressing their functions effectively in vivo.11 Therefore, for the next-generation cell-sheet technology, the controlled structural organization of cells and their related ECM in cell sheets must be a critical issue for giving desirable mechanical and biological functions to the cell sheets.1215 From this viewpoint, “on-demand” and “tailor-made” functionalized thermoresponsive surfaces are interesting candidates for controlling cell alignment in cell sheets as an advanced cell-sheet technology. r 2011 American Chemical Society
To data, for cell-sheet fabrication, PIPAAm-grafted cell-culture substrates have been prepared via electron beam (EB)induced graft polymerization,16 surface-initiated living radical polymerization,17 and other techniques. As a recently developed living radical polymerization, reversible additionfragmentation chain transfer radical (RAFT) polymerization process is known to enable to control polymer molecular weights precisely, applicable to the wide range of monomers under various experimental conditions.18 Therefore, our group has previously demonstrated that RAFT-mediated PIPAAm grafting to solid surfaces using dithiobenzoate (DTB) compounds as a chain tranfer agent (CTA) can be applied to the preparation of thermoresponsive polymer brushes with controlled chain lengths.19 Whereas PIPAAm brush surfaces prepared by this grafting technique allow cell sheets to be harvested effectively by adjusting the chain lengths and graft densities of PIPAAm brushes, the technique also gives chain-transfer active DTB groups to grafted PIPAAm termini. In recent years, utilizing these “living” terminal groups produced in RAFT process, the various kinds of block copolymer architectures have been produced via multistep RAFT polymerization process.20,21 This unique property has been expected to provide additional functions to thermoresponsive polymer brush surfaces. In this work, poly(N-isopropylacrylamide)-b-poly(N-acryloylmorpholine) (PIPAAm-b-PAcMo) block copolymer was grafted on glass substrates through a two-step RAFT polymerization process. A hydrophilic polymer, poly(N-acryloylmorpholine) (PAcMo), shows a repellent property for protein adsorption and cell adhesion, Received: January 20, 2011 Revised: March 7, 2011 Published: March 08, 2011 1414
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Scheme 1. Schematic Representation of Photolithographically Patterned Thermoresponsive Brush Surface through Selective Grafting of Poly(N-acryloylmorpholine) (PAcMo) Segments from Poly(N-isopropylacrylamide) (PIPAAm) Blocksa
a
Red termini indicate chain-transfer active DTB groups. PAcMo segments shown as the blue brushes were grafted through the second-step RAFT polymerization.
similar to a biocompatible poly(ethylene glycol).22,23 As new type of functionalized thermoresponsive surfaces, stripe-like micropatterned surfaces comprising PIPAAm-b-PAcMo brush domains and PIPAAm brush domains were fabricated, and cellular orientations on the physicochemically patterned surfaces were investigated for developing advanced cell-sheet technology.
’ EXPERIMENTAL SECTION Preparation of PIPAAm Brush Surfaces. PIPAAm was grafted from initiator-immobilized surfaces by a surface-initiated RAFT polymerization process, as previously reported.19 First, a silane coupling agent, 3-aminopropyltriethoxysilane (APTES) (Shin-Etsu Chemical, Tokyo), was reacted on glass surfaces through silane coupling reaction, and then a carboxylated azoinitiator, 4,40 -azobis (4-cyanovaleric acid) (V-501) (Wako Pure Chemical Industries, Osaka), was immobilized on the APTES-surface using 1-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) (Tokyo Chemical Industries, Tokyo) as a condensing agent. The V-501-immobilized substrates were immersed in 1,4-dioxane containing IPAAm (1.0 mol/L) (kindly gifted by Kohjin, Tokyo) and 4-cyanopentanoic acid dithiobenzoate (0.5 mmol/L) as CTA, and the solution was reacted at 70 °C for 20 h in a nitrogen atmosphere. Photolithographic Patterning of PIPAAm Brush Termini. Schematic illustration of procedures for obtaining the desired patterns of DTB groups produced by our proprietary maskless photolithography method using liquid crystal display projector (LCDP) is shown in Scheme 1.24 Positive photoresist (OFPR-800 LB, 34 cP) (Tokyo Ohka Kogyo, Kanagawa) was spin-coated at 8000 rpm for 30 s by a spin coater ACT-300D (ACTIVE, Saitama) onto the PIPAAm brush surfaces. After being prebaked in a heating chamber for 1 h at 80 °C, the photoresistcoated substrates were placed the LCDP apparatus, and visible-light irradiation (20 20 mm) from the LCDP was performed for making stripe patterns (25, 50, and 100 μm in width). The photoresist at the irradiated areas was removed selectively in developer solution (2.38% tetramethylammonium hydroxide solution) (NMD-3) (Tokyo Ohka Kogyo). For the control sample, the entire area of the photoresist-coated surface was irradiated with visible light; then, the photoresist was removed in the developer solution. As the second control, photoresist-coated surface without light irradiation was also obtained. After postbaking, the exposed DTB groups of PIPAAm brushes were converted to maleimide groups for preventing further polymerization. The
patterned surfaces (exposed DTB groups and protected DTB groups by patterned photoresist), nonpatterned surface with exposed DTB on all regions, and the photoresist-coated surface with no exposed DTB groups were immersed in Dulbecco’s phosphate-buffered saline (PBS) solution (pH 7.4) containing 2-aminoethanol (10 mmol/L) (Wako Pure Chemical), maleimide (30 mmol/L) (Sigma-Aldrich, St. Louis, MO), and sodium hydrosulfite (1 mmol/L) (Wako Pure Chemical) for 20 h at 20 °C in a nitrogen atmosphere.20 After being washed with MilliQ water (an electrical resistance of 18.3 MΩ) (Millipore, Billerica, MA), the residual photoresist was washed off by soaking in acetone with sonication; then, the PIPAAm brush surfaces with patterned DTB and maleimide groups (Mal/DTB-PIPAAm) were obtained (Scheme 1). In addition, nonpatterned maleimide-terminated PIPAAm brush surfaces (Mal-PIPAAm) and nonpatterned DTB-terminated PIPAAm surfaces (DTB-PIPAAm) were also prepared as the controls.
PAcMo Grafting from Patterned PIPAAm Brush Surface. PAcMo was polymerized on the PIPAAm brush surfaces for forming the second blocks from the DTB-terminated PIPAAm chains as macrochain transfer agents (macro-CTAs). The patterned surfaces (Mal/ DTB-PIPAAm) and nonpatterned surfaces (Mal-PIPAAm and DTBPIPAAm) were immersed in 1,4-dioxane containing distilled 4-acryloylmorpholine (1 mol/L) (Tokyo Chemical) and V-501 (1 mmol/L) (Wako Pure Chemical); then, the reaction solution was heated to 70 °C for 20 h. The substrates were washed with N,N-dimethylformamide, water, and methanol.
Thickness of Polymer Brush Layer Determined by Ellipsometry. Ellipsometry was carried out for determining the dry thickness of the PIPAAm and the PIPAAm-b-PAcMo layers by a spectroscopic ellipsometer (M-2000D instrument) (J. A. Woollam, Lincoln, NE) at wavelengths ranging from 193 to 1000 nm.17 For ellipsometric analysis, PIPAAm and PIPAAm-b-PAcMo were grafted on silicon wafers with the same manner as described above.
Adhesion and Proliferation of Fibroblasts on the Patterned Polymer Brush Surfaces. Normal human dermal fibroblasts (NHDFs) (Lonza, Walkersville, MD) were cultured in fibroblast growth medium (FGM-2) (Lonza) on tissue culture polystyrene (TCPS) dishes at 5% CO2, 37 °C. The polymer-grafted glass coverslips were cut into half size (24 25 mm) and placed on TCPS dishes (35 mm in diameter). Cultured cells (at passage 36) were trypsinized and seeded onto the patterned or nonpatterned polymer brush surfaces at a density of 2 104 cells/cm2. The media was changed every 2 days for 35 days. 1415
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Figure 1. Microscopic photographs of adherent fibroblasts on (A) nonpatterned PIPAAm brush surfaces and (B,C) PIPAAm/PIPAAm-b-PAcMo patterned brush surfaces (stripe pattern width: 50 μm). The photographs were taken at (A,B) 24 and (C) 48 h after the cell seeding. Scale bar: 100 μm.
Figure 2. Phase contrast microscopic photographs of adherent cells on PIPAAm/PIPAAm-b-PAcMo patterned brush surfaces (stripe patterns: (A) 25 μm and (B,C) 100 μm in width). NHDFs were incubated for (A,B) 24 and (C) 48 h after the cell seeding. Scale bar: 100 μm. Phase contrast microscopic observations were carried out with a microscope ECLIPSE TE2000-U (Nikon, Tokyo).
Fluorescence Staining of Aligned Cells on PIPAAm Brush Surface. Confluently cultured cells were fixed with 4% paraformaldehyde
in PBS for 15 min at 37 °C and were permeabilized with 0.5% Triton X-100 in PBS for 5 min at 37 °C.25 The fixed cells were blocked with 2% bovine serum albumin in PBS (BSA-PBS) for 30 min at 37 °C. After being washed with PBS, the cells were incubated with AlexaFluor568 conjugated phalloidin (Invitrogen, Carlsbad, CA) (a dilution of 1:200 in BSA-PBS) for 1 h at 37 °C. For nuclei staining, the cells were treated with Hoechst 33258 (1:500) (Dojindo Laboratories, Kumamoto) for 5 min at RT. For fibronectin staining, the blocked cell layers were incubated with antifibronectin antibody (Abcam, Cambridge, MA) (1:200) for 2 h at 37 °C and then incubated with fluorescein-labeled secondary antibody (Abcam) (1:800) for 1 h at RT.13,19 After being washed with PBS, the fluorescence photographs of stained cells were obtained by a fluorescence microscope (ECLIPSE TE2000-U) and processed with AxioVision 4.6 software. Harvesting and Fluorescence Staining of Cell Sheet. After being confluent, NHDFs adhering on the patterned polymer surfaces were incubated at 5% CO2, 20 °C and then observed microscopically and visually. For fibronectin staining, the cell sheet and the polymer brush surface were treated in the same manner as that previously reported.19
’ RESULTS AND DISCUSSION Scheme 1 demonstrates the use of a combination of surfaceinitiated RAFT polymerization and photolithography technique for preparing micropatterned polymer brush surface.24 First, PIPAAm brush surfaces were prepared through the surface-initiated RAFT polymerization using azoinitiator-immobilized glass substrates, as previously reported.19 Dry thickness of the PIPAAm brush layer was determined to be 1.62 nm by ellipsometry. The thickness agreed with the grafted amount of the PIPAAm (0.41 μg/cm2) allowing the PIPAAm brush surface to achieve an effective cell-sheet harvest, as previously reported.17,19 After photoresist coating and development process, the RAFT-related DTB groups of grafted PIPAAm termini were partially substituted with inert maleimide groups for preventing
further polymerization (PIPAAm brush regions).20 After removing the residual photoresists from all regions, PAcMo was grafted as the “second block” from the termini of grafted PIPAAm chains, resulting in block copolymer brushes (PIPAAm-b-PAcMo regions). As the control samples, nonpatterned polymer brush surfaces of both PIPAAm and PIPAAm-b-PAcMo were also prepared in the same manner. The dry thickness of PIPAAm-b-PAcMo layer was determined to be 3.13 nm on the nonpatterned copolymer brush surface. NHDFs adhered randomly on the nonpatterned PIPAAm brush surfaces (Figure 1A), whereas they adhered site-specifically on the patterned polymer brush surfaces (PIPAAm/PIPAAm-bPAcMo surfaces) (Figure 1B). For the PIPAAm/PIPAAm-bPAcMo patterned brush surfaces with 50 μm stripes, NHDFs adhered only on the PIPAAm regions and formed stripe patterns as expected. These results indicated that the grafting of PAcMo segments suppressed an initial cell adhesion on the brush surfaces, even in serum-containing culture media. In fact, no cells adhered on the nonpatterned PIPAAm-b-PAcMo brush surface, even after 5 days of incubation (data not shown). The adherent NHDFs on the patterned brush surfaces were found to show the same orientation on the PIPAAm regions at 24 h after the cell seeding (Figure 1B), and subsequent further incubation at 37 °C allowed the patterned cells to migrate and proliferate on all regions of PIPAAm/PIPAAm-b-PAcMo patterned surfaces with maintaining the cell alignment (Figure 1C). This indicated that the cell-repellent property of PIPAAm-b-PAcMo regions were insufficient for inhibiting cell invasion from the neighboring celladhesive PIPAAm regions, although the property of the surface was found to be effective for initially seeded cells at the start of incubation. Therefore, the presence of adherent cells on the patterned PIPAAm regions allowed cells to migrate and proliferate on the patterned PIPAAm-b-PAcMo regions with the same orientation. This unique phenomenon for controlling cell alignment was confirmed on the patterned surfaces with 50 μm wide stripes. With reducing the stripe width to 25 μm, NHDFs adhered randomly on the PIPAAm/PIPAAm-b-PAcMo patterned brush 1416
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Figure 3. (A,B) Phase contrast microscopic images and (CF) the fluorescence images of adherent fibroblasts on patterned polymer brush surfaces (A, C, and E on the left column) and nonpatterned PIPAAm brush surfaces (B, D, and F on the right column). Actin (red) and nuclei (blue) in the aligned cells on patterned surface (C) and randomly adhering cells on PIPAAm brush surface (D) were stained with AlexaFluor568-phalloidin (red) and Hoechst 33258 (blue), respectively. (E,F) Fibronectin in the cell layers was also stained with fluorescein (green). Scale bar: 100 μm.
surfaces (Figure 2A). This shows that the stripe width was a key factor for cell patterning, and >25 μm stripe pattern was required for NHDF patterning in this system. Although NHDFs also formed patterns on 100 μm wide stripe patterns (Figure 2B), some of them proliferated and invaded without maintaining the orientation to the block copolymer regions (Figure 2C). In addition, the cell migration from the 100 μm wide patterns caused difference in the density of adherent cells between PIPAAm and PIPAAm-b-PAcMo regions. The aligned cells on 50 μm stripe patterns proliferated uniformly (with the same orientation) on all regions of the patterned surfaces regardless of the difference in the cell adhesive properties between the two kinds of regions. These indicated that the pattern width was quite important and necessary for achieving well-defined cell alignment on culture surfaces. The aligned cells proliferated and finally reached to confluent with maintaining the same orientation on PIPAAm/PIPAAm-bPAcMo patterned surfaces 5 days postseeding (50 μm in width) (Figure 3A). It is noteworthy that this aligned cell layer was prepared simply by one-pot cell seeding. Adherent cells proliferated randomly on the nonpatterned PIPAAm brush surfaces as the control (Figure 3B). Therefore, both confluent cell layers obviously showed absolute differences in cellular orientation. After immunostaining both cell layers (aligned cells and randomly adhering cells), the fluorescence photographs also showed the orientation of actin fibers and fibronectin in the cell layers with associated ECM on the surfaces (Figure 3CF), indicating that the alignments of cytoskeleton and ECM proteins were also
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Figure 4. (A) Microscopic image of cell detachment on patterned polymer brush surface within 30 min after reducing temperature to 20 °C. (B) Photograph of a cell sheet showing orientation. The cell sheet was harvested only by temperature change to below PIPAAm’s LCST (20 °C). The table represents the width, the length, and the aspect ratio of the cell sheets harvested from patterned and nonpatterned PIPAAm brush surfaces. (C) Phase contrast microscopic and (D) the fluorescence photographs of the cell sheet harvested by temperature change. Adherent cells were incubated on the patterned surface (stripe pattern width: 50 μm) for 5 days at 37 °C before reducing temperature (20 °C). Fibronectin in the harvested cell sheet and on the PIPAAm brush surface were stained with fluorescein (green). Cell nuclei were stained with Hoechst 33258 (blue).
regulated by the designed micropatterning of polymer domains and the resultant cell orientation. This cell alignment must provide mechanical and biological aspects to the cell assemble, promising advanced cell-sheet engineering.12,13 To apply the cell alignment technique for cell-sheet technology, we would need to harvest the cell layers with intact orientational structures and associated ECM. Because patterned surfaces were fabricated on the basis of thermoresponsive PIPAAm brushes, cell sheets were harvested only by reducing temperature below PIPAAm’s LCST to 20 °C (Figure 4A). It should be noted that the harvested cell sheet showed a distinctive aspect ratio (Figure 4B). As previously reported, cell sheets detached from nonpatterned PIPAAm surfaces shrank two-dimensionally with the original aspect of the thermoresponsive surfaces.19 Of significant interest, cell sheets harvested from the patterned polymer brush surfaces possessed quite different shrinking rates between the vertical and parallel sides of the cell alignment, as shown in the table in Figure 4B. (The aspect ratio of width/length of a cell sheet was ∼3.) Because cell morphology changes from spread to round after the low-temperature treatment, cytoskeleton organization influences directly the shrinking rate of cell sheets. Therefore, this result agreed with the well-defined alignment of cytoskeleton (Figure 3C). Moreover, this shrinking rate also 1417
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Biomacromolecules indicated that the controlled orientational structure of sheetforming cells was maintained after the cell-sheet harvesting. This distinctive shrinking rate clearly showed the unique mechanical aspect of cell sheet prepared on patterned polymer brush surfaces (50 μm stripe). Cell layers on 100 μm stripe patterned surfaces were scarcely harvested, suggesting that micropattern design also influenced thermoresponsive cell-sheet detachment process. This is probably due to longer incubation time required to be confluent, the difference in the density of adherent cells between PIPAAm and PIPAAm-b-PAcMo regions, or both. In this study, 50 μm wide stripe patterning was successfully achieved to fabricate NHDF sheets with well-controlled orientational structures. Furthermore, harvested cell sheet was able to be detached with its associated ECM from the patterned polymer brush surfaces (Figure 4C,D). Because the deposited ECM as a glue facilitates to allow cell sheet to be transplanted to host tissues and organs effectively,26 engineered cell sheets harvested from the patterned surfaces should give a significant benefit to cell-sheet transplantation technology.
’ CONCLUSIONS In summary, RAFT-mediated block copolymerization achieved the stripe-like micropatterning of thermoresponsive polymer domains and cell-repellent polymer domains. NHDFs were aligned on PIPAAm brush surfaces simply by one-pot cell seeding. The alignments of cells and ECM proteins can promise to show the mechanical and biological significance of cell sheets harvested from thermoresponsive patterned surfaces. Compared with other techniques previously reported, this technique for the advanced cell-sheet preparation has significant advantages. In particular, the present method gave aligned cell layers by solely single-step cell seeding without any other sophisticated procedure. In addition, covalently designed surface was able to be used repeatedly for cell sheet preparation. Furthermore, the RAFTmediated grafting method is achieved to control precisely and independently the chain length of PIPAAm segments and PAcMo segments for designed cell sources composing cell sheets.19 Therefore, this functionalized thermoresponsive surface is useful for realizing the simple and tailor-made preparation of cell sheets with well-controlled orientational structures. Engineered cell sheets obtained in this study are believed to be able to create tissuemimicking structures with specific biological functions in cell-sheet engineering. ’ AUTHOR INFORMATION Corresponding Author
*Tel: þ81-3-5367-9945 (6201). Fax: þ81-3-3359-6046. E-mail:
[email protected].
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’ REFERENCES (1) Yamada, N.; Okano, T.; Sakai, H.; Karikusa, F.; Sawasaki, Y.; Sakurai, Y. Makromol. Chem., Rapid Commun. 1990, 11, 571–576. (2) Akiyama, Y.; Kikuchi, A.; Yamato, M.; Okano, T. Langmuir 2004, 20, 5506–5511. (3) Yang, J.; Yamato, M.; Kohno, C.; Nishimoto, A.; Sekine, H.; Fukai, F.; Okano, T. Biomaterials 2005, 26, 6415–6422. (4) Yamato, M.; Okano, T. Mater. Today 2004, 7, 42–47. (5) Nishida, K.; Yamato, M.; Hayashida, Y.; Watanabe, K.; Yamamoto, K.; Adachi, E.; Nagai, S.; Kikuchi, A.; Maeda, N.; Watanabe, H.; Okano, T.; Tano, Y. N. Engl. J. Med. 2004, 351, 1187–1196. (6) Ohki, T.; Yamato, M.; Murakami, D.; Takagi, R.; Yang, J.; Namiki, H.; Okano, T.; Takasaki, K. Gut 2006, 55, 1704–1710. (7) Arauchi, A.; Shimizu, T.; Yamato, M.; Obara, T.; Okano, T. Tissue Eng. 2009, 15, 3943–3949. (8) Kanzaki, M.; Yamato, M.; Yang, J.; Sekine, H.; Kohno, C.; Takagi, R.; Hatakeyama, H.; Isaka, T.; Okano, T.; Onuki, T. Biomaterials 2007, 28, 4294–4302. (9) Ohashi, K.; Yokoyama, T.; Yamato, M.; Kuge, H.; Kanehiro, H.; Tsutsumi, M.; Amanuma, T.; Iwata, H.; Yang, J.; Okano, T.; Nakajima, Y. Nat. Med. 2007, 13, 880–885. (10) Shimizu, T.; Yamato, M.; Kikuchi, A.; Okano, T. Biomaterials 2003, 24, 2309–2316. (11) Ross, M. H.; Kaye, G. I.; Pawlina, W. Histology: a Text and Atlas: With Cell and Molecular Biology, 4th ed.; Lippincott Williams & Wilkins: Baltimore, MD, 2003. (12) Isenberg, B. C.; Tsuda, Y.; Williams, C.; Shimizu, T.; Yamato, M.; Okano, T.; Wong, J. Y. Biomaterials 2008, 29, 2565–2572. (13) Williams, C.; Tsuda, Y.; Isenberg, B. C.; Yamato, M.; Shimizu, T.; Okano, T.; Wong, J. Y. Adv. Mater. 2009, 21, 2161–2164. (14) Ahmed, W. W.; Wolfram, T.; Goldyn, A. M.; Bruellhoff, K.; Rioja, B. A.; Moller, M.; Spatz, J. P.; Saif, T. A.; Groll, J.; Kemkemer, R. Biomaterials 2010, 31, 250–258. (15) Lu, J.; Rao, M. P.; MacDonald, N. C.; Khang, D.; Webster, T. J. Acta Biomater. 2008, 4, 192–201. (16) Tsuda, Y.; Kikuchi, A.; Yamato, M.; Nakao, A.; Sakurai, Y.; Umezu, M.; Okano, T. Biomaterials 2005, 26, 1885–1893. (17) Mizutani, A.; Kikuchi, A.; Yamato, M.; Kanazawa, H.; Okano, T. Biomaterials 2008, 29, 2073–2081. (18) Moad, G.; Rizzardo, E.; Thang, S. H. Aust. J. Chem. 2005, 58, 379–410. (19) Takahashi, H.; Nakayama, M.; Yamato, M.; Okano, T. Biomacromolecules 2010, 11, 1991–1999. (20) Nakayama, M.; Okano, T. Biomacromolecules 2005, 6, 2320–2327. (21) McCormick, C. L.; Sumerlin, B. S.; Lokitz, B. S.; Stempka, J. E. Soft Matter 2008, 4, 1760–1773. (22) Jo, Y. S.; van der Vlies, A. J.; Gantz, J.; Antonijevic, S.; Demurtas, D.; Velluto, D.; Hubbell, J. A. Macromolecules 2008, 41, 1140–1150. (23) Jo, Y. S.; van der Vlies, A. J.; Gantz, J.; Thacher, T. N.; Antonijevic, S.; Cavadini, S.; Demurtas, D.; Stergiopulos, N.; Hubbell, J. A. J. Am. Chem. Soc. 2009, 131, 14413–14418. (24) Itoga, K.; Kobayashi, J.; Tsuda, Y.; Yamato, M.; Okano, T. Anal. Chem. 2008, 80, 1323–1327. (25) Takahashi, H.; Emoto, K.; Dubey, M.; Castner, D. G.; Grainger, D. W. Adv. Funct. Mater. 2008, 18, 2079–2088. (26) Yamato, M.; Konno, C.; Kushida, A.; Hirose, M.; Utsumi, M.; Kikuchi, A.; Okano, T. Biomaterials 2000, 21, 981–986.
’ ACKNOWLEDGMENT This work was partially supported by Formation of Innovation Center for Fusion of Advanced Science and Technologies in the Special Coordination Funds for Promoting Science and Technology “Cell Sheet Tissue Engineering Center (CSTEC)” and Grant-in-Aid for Scientific Research (B) (20300169) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. 1418
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