Letter pubs.acs.org/Langmuir
Fabrication of Poly(N‑isopropylacrylamide) Films Containing Submicrometer Grooves for Constructing Aligned Cell Sheets Szu-Wei Fu, Hsiu-Wen Chien, and Wei-Bor Tsai* Department of Chemical Engineering, National Taiwan University, Taipei 106, Taiwan ABSTRACT: Transplantation of cell sheets including an intact extracellular matrix is one tissue-engineering strategy for tissue regeneration. Temperature-responsive substrates based on poly(Nisopropylacrylamide) (PNIPAAm) have been used to harvest intact cell sheets by temperature change. In this work, we immobilized PNIPAAm on plastic substrates by a UV-activated azide-based crosslinking mechanism. We demonstrated that the UV-cross-linked PNIPAAm films could respond to temperature changes and be used for cell-sheet fabrication. Next, grooved PNIPAAm substrates were fabricated by imprinting from grooved poly(dimethylsiloxane) (PDMS) molds (800 nm in groove width and 500 nm in depth). C2C12 cells formed aligned cell sheets on the grooved PNIPAAm surface. The aligned cell sheet could be transferred to a gelatin substrate without losing cell alignment. We expect that this simple time-saving technique for the fabrication of grooved PNIPAAm substrates will benefit from the application of cellular alignment in tissue-engineering products. line) (PNIPAAm-b-PAcMo) domains.12 Human dermal fibroblasts initially adhered only to the PNIPAAm regions and formed striped patterns along the orientation of PNIPAAm stripes. The cells later migrated to and proliferated on the regions of PNIPAAm-b-PAcMo while maintaining their alignment. In another example, Lin et al. grafted PNIPAAm onto microtextured poly(dimethylsiloxane) (PDMS) to produce aligned vascular smooth muscle cell sheets.13 These approaches create striped patterns with widths accommodating only one or two cells in order to confine the cell orientation. In this study, we tried to develop a simple technique that combines two of our previous techniques for fabricating grooved PNIPAAm surfaces to create aligned cell sheets. One is using a UV-activated azide-based cross-linking method, which was used previously in our laboratory to conjugate polymers on substrates,14,15 to immobilize PNIPAAm on polymeric substrates. We first verified that UV-immobilized PNIPAAm could be used to release cell sheets. Second, we previously demonstrated that nanogrooved surfaces direct cell alignment.16,17 In this study, we fabricated grooved PNIPAAm substrates by imprinting from poly(dimethylsiloxane) molds containing submicrometer grooves. We then evaluated whether aligned myoblast sheets could be formed on grooved PNIPAAm surfaces and be released at a temperature below the LCST.
1. INTRODUCTION Transplantation of cell sheets containing intact extracellular matrices is one tissue-engineering strategy.1,2 To this end, Okano’s group developed a cell-culture substrate composed of brushes of thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm) via surface polymerization by electron beam irradiation.3 When the culture temperature is above the lower critical solution temperature (LCST) of PNIPAAm (∼32 °C), the polymer becomes hydrophobic and insoluble. Thus, cells could attach and proliferate on the PNIPAAm substrate at a conventional cell culture temperature of 37 °C. When the culture temperature drops below the LCST, the polymer becomes more hydrophilic, chain-swelling, and cell-resistant, leading to the detachment of intact cell sheets.4 In recent years, this technique has been widely applied in tissue engineering of ligament,5 heart,6,7 and corneal epithelia.8 The cell organization of cell sheets on conventional PNIPAAm substrates is disoriented, in contrast to the anisotropic cell arrangement in some tissues. For example, skeletal muscle cells and cardiomyocytes are arranged in alignment in vivo.9 Myoblast alignment is suggested as a critical step in myotube formation.10 The alignment of cardiomyocytes is associated with the electrical stability of signal transmission, and arrhythmias are one of the potential consequences of cardiomyocyte sheets without alignment.11 Therefore, isotropic cell arrangement on ordinary PNIPAAm substrates may not mimic the organization of such native tissues, thus damaging the functions of tissue-engineered products. Several strategies have been developed to create PNIPAAm stripes for creating aligned cell sheets. For example, Takahashi et al. used reversible addition−fragmentation chain-transfer radical-mediated block copolymerization and photolithography to fabricate alternating microstripes of PNIPAAm domains and poly(N-isopropylacrylamide)-block-poly(N-acryloylmorpho© 2013 American Chemical Society
2. MATERIALS AND METHODS 2.1. Materials. Poly(N-isopropylacrylamide) (cat. no. 21458-10, MW 40 000) was purchased from Polysciences Inc. (USA). Porcine gelatin (cat. no. 9000-70-8) was purchased from Sigma (USA). Received: August 23, 2013 Revised: November 6, 2013 Published: November 8, 2013 14351
dx.doi.org/10.1021/la403129c | Langmuir 2013, 29, 14351−14355
Langmuir
Letter
Figure 1. Schematic illustration of the fabrication of (A) flat and (B) grooved PNIPAAm films. (C) Transfer process of an aligned cell sheet from a grooved PNIPAAm film to a gelatin film. Poly(acrylic acid-graf t-azidoaniline), abbreviated as PAA-g-Az, was synthesized by using a previously published procedure.15 The content of azido groups in PAA-g-Az, estimated by 1H nuclear magnetic resonance (Avance-500 Hz, Bruker), was 6.0 mol % according to the ratio of peak areas of the azidophenyl protons at 6.5−7.5 ppm and the methylene protons of the polymer main chain at 1.3−2.5 ppm. The others chemicals were received from Sigma-Aldrich unless otherwise stated. Murine skeletal muscle C2C12 cells (ATCC no. CRL-1772) were obtained from Food Industry Research and Development Institute (Hsinchu, Taiwan). The cell culture medium consisted of 90% (v/v) Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1.5 g/L sodium bicarbonate, 0.5% (v/v) fungizone, 0.25% (v/v) gentamycin, and 10% (v/v) fetal bovine serum. 2.2. Fabrication of PNIPAAm Films. PNIPAAm and PAA-g-Az were dissolved in methanol to 0.67 and 0.067 (w/v)%, respectively. The polymer mixture (600 μL) was added to 24-well plate containing tissue culture polystyrene (TCPS) and allowed to air dry. PNIPAAm films were fixed on TCPS after exposure to a UV lamp (wavelength range 280−380 nm, intensity about 65 mW/cm2) for 5 min (Figure 1A). We previously found that C2C12 cells aligned and elongated on the grooved surfaces with a ridge/groove width in the range of 450−900 nm,16 so a grooved pattern with 800-nm-wide ridges/grooves was used in this study. For the fabrication of grooved PNIPAAm films, the PNIPAAm/PAA-g-Az solution was placed on a polyethylene terephthalate film and then pressed with a grooved PDMS mold with 100 g weight (Figure 1B). The PDMS molds with submicrometer grooved patterns (ridges/grooves were 800 nm in width and 500 nm in depth) were fabricated from a silicon wafer master, according to a previously reported process.17 After the PNIPAAm/PAA-g-Az solution was dried, the PDMS mold was peeled off. The dried PNIPAAm film was then exposed to UV light for 5 min for cross-linking. The grooved structure of dried PNIPAAm films was characterized by atomic force microscopy using tapping mode (AFM, NanoScope IIIa, Digital Instruments, Plainview, NY). Water contact angle measurements on PNIPAAm films were carried out at 20 and 37 °C
using a contact angle goniometer (100-26-TH, Rame-Hart Inc., Mountain Lakes, NJ). 2.3. Fabrication of Myoblast Sheets. Prior to cell seeding, the PNIPAAm substrates were placed in 24-well plates, sterilized by soaking in 70% ethanol for 30 min, and then rinsed with PBS twice. C2C12 cells were seeded at a density of 2 × 105 cell/cm2 and then cultured for 4 days. The reason for using a high cell-seeding density is that we want to shorten the time for cell confluency in order to perform the cell detachment experiment. Takahashi et al. previously showed that directly detached aligned myoblast sheets would shrink anisotropically, leading to a loss of cell alignment.18 They prevented the anisotropic shrinkage of an aligned myoblast sheet by placing a gelatin gel-coated plunger onto a cell sheet for the harvest and transfer of the cell sheet. The myoblasts on the transferred cell sheet remained well aligned. Therefore, in this study, after the cells grew as a confluent cell sheet on the PNIPAAm film, the cell sheet was transferred to a gelatin film, which was fabricated according to a previous procedure.19 The gelatin films were cast on a poly(ethylene terephthalate) film from a 1.5% (w/v) gelatin solution in deionized water20 and were held at 4 °C prior to use. The gelatin film was placed on the cell sheet and then kept at 0 °C for 2 h to allow the transfer of the cell sheet from the PNIPAAm film (Figure 1C). For the fluorescent staining of F-actin, the samples were rinsed in PBS twice, fixed with 4% (w/v) paraformaldehyde for 15 min, and then permeabilized with 0.1% Triton-X 100 in PBS for 15 min. Nuclei and actin filaments were stained with 100 nM 4,6-diamidino-2phenylindole (DAPI, Invitrogen) and 500 nM phalloidin, respectively, for 1 h. Fluorescent images were captured with a confocal spectral microscope imaging system (TCS SP5, Leica, Mannheim, Germany). The alignment of actin filaments was analyzed by fast Fourier transform (FFT) of the fluorescent images using National Institutes of Health (NIH) Image J software. The detailed procedure has been described in the literature.21 Briefly, a fluorescent image of F-actin was first converted to a frequency diagram by FFT, and the intensity in each direction was determined by the Oval Profile plugin. A groove direction of 90° is indicated. 14352
dx.doi.org/10.1021/la403129c | Langmuir 2013, 29, 14351−14355
Langmuir
Letter
3. RESULTS AND DISCUSSION 3.1. Cell Detachment from UV-Cross-Linked PNIPAAm Surfaces. We previously integrated PAA-g-Az in polyelectrolyte multilayer films for UV-initiated covalent conjugation of polyelectrolytes on polymeric substrates.14,15 Under UV irradiation, phenyl azido groups are activated to nitrenes that react readily with neighboring C−N or C−H bonds and form stable covalent bonds.22 The UV-cross-linked polyelectrolyte multilayer films are very stable under acidic, alkaline, and ultrasonic treatment. In this study, the same mechanism was applied to immobilize PNIPAAm on polymeric substrates. We first evaluated whether the immobilized PNIPAAm films possess a thermoresponsive property. The static water contact angles on flat PNIPAAm films were measured below and above the LCST of PNIPAAm (32 °C). The static water contact angles on the flat PNIPAAm films were 45.1 ± 5.4 and 60.9 ± 4.8° at 20 and 37 °C, respectively (p < 0.05, n = 3) (Table 1). The difference in the water contact
sheets. Our method is simpler and more time-saving for preparing temperature-responsive PNIPAAm surfaces, compared to conventional electron beam irradiation-initiated polymerization. 3.2. Fabrication of Aligned Cell Sheets on Grooved PNIPAAm Films. We previously demonstrated that nanogrooved substrates of rigid polymers such as polystyrene and poly(lactic-co-glycolic acids) direct cell alignment and elongation.16,17 In this study, we tried to fabricate grooved PNIPAAm films by using the UV-cross-linking method and investigated whether cells could comply with the surface topography. The water contact angles on the grooved PNIPAAm films in the direction parallel to the grooves were 32.7 ± 7.2 and 60.3 ± 2.8° at 20 and 37 °C, respectively (p < 0.001, n = 3) (Table 1). However, the water contact angles on the grooved PNIPAAm films in the direction perpendicular to the grooves were slightly higher, 50.1 ± 4.7 and 71.3 ± 1.7° at 20 and 37 °C, respectively (p < 0.001, n = 3) (Table 1). The thermoresponsive property of the grooved PNIPAAm films was also demonstrated by the contact angle measurement. It is noted that the water contact angles on the grooved surface differ from the measurement directions, indicating that water spread more in the direction along the grooves than in the direction against the grooves. The surface topography of the dried PNIPAAm grooved substrate was characterized by AFM (Figure 3). The depth of
Table 1. Static Water Contact Angles at 20 and 37 °C surface type
T < LCST (20 °C)
T > LCST (37 °C)
p valuec
flat grooved (=)a grooved (⊥)b
45.1 ± 5.4° 32.7 ± 7.2° 50.1 ± 4.7°
60.9 ± 4.8° 60.3 ± 2.8° 71.3 ± 1.7°
