Spatially Controlled Cell Engineering on Biomaterials Using

Nov 7, 2003 - Girish Kumar, Yu Chi Wang, Carlos Co, and Chia-Chi Ho*. Department of Chemical and Materials Engineering, University of Cincinnati,...
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Spatially Controlled Cell Engineering on Biomaterials Using Polyelectrolytes Girish Kumar, Yu Chi Wang, Carlos Co, and Chia-Chi Ho* Department of Chemical and Materials Engineering, University of Cincinnati, 497 Rhodes Hall, Cincinnati, Ohio 45221 Received July 18, 2003. In Final Form: September 24, 2003

Controlling the spatial organization of cells is a critical step toward engineering tissues with distributed networks of blood vessels or nerve cells. Here we report a new soft-lithography-based approach for micropatterning proteins and cells on the surface of biodegradable chitosan substrates that are more applicable to engineering tissues than the gold, silver, glass, or silicone substrates currently used in cell micropatterning studies. In this approach, we use random copolymers of oligo(ethylene glycol) methacrylate (OEGMA), which resists protein and cell adsorption, and methacrylic acid (MA), which adheres strongly onto the chitosan substrate via acid-base interactions, to form stable protein and cell resistant micropatterns on chitosan surfaces. At optimal ratios of OEGMA to MA, copolymers are formed that exhibit superior long-term resistance to protein adsorption and cell adhesion even under cell culture conditions. Spatial control of cell organization and alignment using OEGMA/MA micropatterned chitosan is demonstrated using human microvascular endothelial cells.

Introduction Controlling the spatial organization of cells is vital to many tissue engineering applications. For example, functional nerves or blood vessels form only when groups of cells are organized and aligned in very specific geometries.1,2 Spatial patterning of cells is also a generally useful tool for understanding and controlling cell behaviors3-5 with a multitude of applications in biosensors and high-throughput screening. A variety of techniques have been developed to pattern cells by creating micron-sized cell adhesive and cell resistant regions on substrates. These techniques include photolithographic patterning with silanes,6 photochemical immobilization of biomolecules,7 and microcontact printing of self-assembled monolayers.4 Among these techniques, microcontact printing of self-assembled monolayers (SAMs) has been used most widely because of its simplicity. In this method, self-assembled monolayers of alkanethiolate and oligo(ethylene glycol)-terminated thiols are transferred onto gold substrates using a reusable poly(dimethylsiloxane) (PDMS) micropatterned stamp. The alkanethiolate SAM promotes the adhesion of extracellular matrix proteins (e.g., fibronectin, laminin, collagen, etc.) and attachment of cells, while the background region, * To whom correspondence should be addressed. Phone: (513) 5562438. Fax: (513) 5563473. E-mail: [email protected]. (1) Pollock, M. Curr. Opin. Neurol. 1995, 8, 354-358. (2) Dike, L. E.; Chen, C. S.; Mrksich, M.; Tien, J.; Whitesides, G. M.; Ingber, D.E. In Vitro Cell Dev. Biol.: Anim. 1999, 35, 441-448. (3) Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Science 1997, 276 (5317), 1425-1428. (4) Singhvi, R.; Kumar, A.; Lopez, G. P.; Stephanopoulos, G. N.; Wang, D. I.; Whitesides, G. M.; Ingber, D. E. Science 1994, 264 (5159), 696698. (5) Parker, K. K.; Brock, A. L.; Brangwynne, C.; Mannix, R. J.; Wang, N.; Ostuni, E.; Geisse, N. A.; Adams, J. C.; Whitesides, G. M.; Ingber, D. E. FASEB J. 2002, 16 (10), 1195-1204. (6) Kleinfeld, D.; Kahler, K. H.; Hockberger, P. E. J. Neurosci. 1988, 8, 4098-4120. (7) Matsuda, T.; Sugawara, T. J. Biomed. Mater. Res. 1995, 29, 749756.

covered with an oligo(ethylene glycol)-terminated SAM, resists protein adsorption. Complementary patterns of these two SAMs allow for the spatially controlled attachment of cells. One restriction of the self-assembled monolayer patterning approach is that it requires the use of gold- or silver-coated substrates, which limits its use in tissue culture applications. The objective of this work is to develop a new procedure for patterning cells directly on the surface of biomaterials, such as chitosan, that intrinsically support cell attachment and naturally degrade in the body. We have achieved this objective by replacing the alkanethiols used in the SAM/microcontact printing technique with random copolymers of oligo(ethylene glycol) methacrylate (OEGMA) and methacrylic acid (MA) that adhere to chitosan substrates via acid-base/electrostatic interactions. Polyelectrolyte assembly has been widely used to encapsulate micron- and nanometer-sized particles and coat substrates with nanometer thick thin film coatings. The flexible linear polyions can alternately adsorb onto oppositely charged surfaces via electrostatic interactions to form films 5-500 nm in thickness. Uniform nanometer thick coatings in two dimensions have been achieved using a wide range of polyelectrolyes including synthetic polymer,8 proteins,9,10 DNA,11,12 and dyes.13,14 Hammond et al.,15,16 for example, have developed an elegant technique (8) Lo¨sche, M.; Schmitt, J.; Decher, G.; Bouwman, W. G.; Kjaer, K.; Macromolecules 1998, 31, 8893-8906. (9) Pommersheim, R.; Schrezenmeir, J.; Vogt, W. Macromol. Chem. Phys. 1994, 195, 1557-1567. (10) Onda, M.; Lvov, Y.; Ariga, K.; Kunitake, T. Biotechnol. Bioeng. 1996, 51, 163-167. (11) Sukhorukov, G. B.; Lvov, Y. M.; Mo¨hwald, H.; Decher, G. Thin Solid Films 1996, 284-285, 220-223. (12) Lvov, Y. M.; Lu, Z.; Schenkman, J. B.; Zu, X.; Rusling, J. F. J. Am. Chem. Soc. 1998, 120, 4073-4080. (13) Jiang, H.; Su, W.; Hazel, J.; Grant, J. T.; Tsukruk, V. V.; Cooper, T. M.; Bunning, T. J. Thin Solid Films 2000, 372, 85-93. (14) Ariga, K.; Lvov, Y.; Kunitake, T. J. Am. Chem. Soc. 1997, 119, 2224-2231.

10.1021/la035309l CCC: $25.00 © 2003 American Chemical Society Published on Web 11/07/2003

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to pattern charged thiol compounds on gold surfaces that subsequently guide the spatial adsorption of polyelectrolytes. Here, we report a new approach that foregoes the use of gold and thiol compounds and instead directly pattern random copolymers of OEGMA and MA onto chitosan substrates. By optimizing the ratio of OEGMA and MA, we have found copolymers that adhere reliably to the chitosan substrate, via the acid-base interaction between MA and chitosan, but still have a sufficient number of oligo(ethylene glycol) units to effectively resist protein and cell adsorption. The resulting copolymers can be used to form two-dimensional patterns that limit cell attachment and spreading within the unprinted chitosan regions over long periods of time. Using this approach, we have precisely controlled the spatial distribution and spreading of human capillary endothelial cells with subcellular resolution. Compared to other micropatterning approaches, this technique is simple and inexpensive and can be generally applied to a large variety of proteins and cells without the need to coat the substrate with gold, thiols, or specific ligands and receptors. Materials and Methods Materials. Chitosan of medium molecular weight (Mw ∼ 400 000) was purchased from Fluka (St. Louis, MO). PDMS (Sylgard 184) was obtained from Dow Corning (Midland, MI). Random block copolymers of OEGMA/MA of varying molar ratios were prepared in our laboratory using the procedure described later. Bovine serum albumin (BSA) fluorescein conjugate, Alexa 488-phalloidin, and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Molecular Probes (Eugene, OR). Phosphatebuffered saline (PBS) solution (137 mM NaCl, 2.7 mM KCl, 10 mM KH2PO4, 10 mM Na2HPO4, and 10 mM NaOH) was prepared from PBS pellets purchased from Fluka. Microvascular endothelial cell growth medium and fetal bovine serum (FBS) were purchased from Cambrex Bioscience (Walkersville, MD). Preparation of OEGMA/MA Random Copolymers. Random copolymers of OEGMA and MA (Scientific Polymer Products, NY) were prepared by free radical polymerization of the respective monomers in methanol at 60 °C. Polymerizations were initiated with 1 wt % 2,2′-azobis(2-amidinopropane) dihydrochloride (Wako, VA) and allowed to react for 16 h. Polymerizations, starting with 10 wt % monomer in methanol and monomer mass ratios ranging from 90:10 to 30:70 OEGMA:MA, were carried out to prepare random copolymers of varying OEGMA to MA ratio. Similar polymerizations when carried out in water resulted in precipitation of the copolymer product. Microfabrication of the Silicon Master Pattern and Transfer of the Topological Patterns onto PDMS. Micropatterns with parallel grooves 60 µm wide and ridges of varying widths (10, 20, and 30 µm) were fabricated on silicon wafers using standard photolithographic techniques. From this silicon master pattern, complementary PDMS replicas were formed by pouring PDMS prepolymer (mixed in a 10:1 ratio with a crosslinking catalyst) over the Si master and cured at 56 °C for 2 h. The PDMS replicas were used as stamps in subsequent microcontact printing steps. Preparation of Chitosan Films. Chitosan films were prepared on glass slides by spreading uniformly 0.187 g of 2.5% chitosan solution with 0.2 M acetic acid as solvent over an area of 18.75 cm2. The wet chitosan film was dried in an oven at 56 °C for 30 min and then neutralized with 2 N NaOH (in 70% ethanol). Excess NaOH was washed off by immersing the chitosan film in an excess of 70% ethanol for 2 h followed by soaking in deionized water overnight. Before use in cell cultures, the chitosan film was immersed in Hank’s buffered saline solution (HBSS, Life Technologies). (15) Hammond, P. T.; Whitesides, G. M. Macromolecules 1995, 28, 7569-7571. (16) Clark, S. L.; Montague, M. F.; Hammond, P. T. Macromolecules 1997, 30, 7237-7244.

Langmuir, Vol. 19, No. 25, 2003 10551 Chemical Patterning of Nonadhesive OEGMA/MA Regions. Microcontact printing was used to pattern random block copolymers of OEGMA and MA over chitosan. The PDMS stamp was first cleaned with ethanol and then air-dried. Lint-free Kimwipes were dipped into the OEGMA/MA solution, partially air-dried, and then applied gently over the stamp to coat the plateaus of the stamp with OEGMA/MA copolymer without overfilling the troughs. Patterns of OEGMA/MA copolymer were then transferred onto chitosan films by contact printing. Gentle pressure was applied to ensure conformal contact between the stamp and chitosan film. After 15 s, the stamp was removed from the chitosan and the substrate was air-dried. Spatial control of protein adsorption onto the OEGMA/MA-patterned chitosan films was tested by incubating the substrates with fluorescently labeled BSA visualized using a Nikon TE-2000 inverted microscope. Culture of Endothelial Cells. Human microvascular endothelial cells (HMVEC-d, purchased from Cambrex Bioscience, MD) were cultured in endothelial basal medium containing 5% fetal bovine serum, 1 µg/mL of hydrocortisone, 10 µg/mL of epidermal growth factor (EGF), 10 µg/mL of bovine brain extract, 50 µg/mL of gentamycin, and 50 µg/mL of amphotericin-B under 5% CO2. Prior to incubation with the micropatterned biomaterials, cells were dissociated from the culture dish with trypsin, resuspended in endothelial basal medium containing 10% serum, and allowed to attach onto micropatterned chitosan. Immunostaining. After 72 h of incubation, the attached cells were fixed with 3.7% paraformaldehyde for 10 min, washed in phosphate-buffered saline, and then permeabilized with 0.2% Triton X100 for 5 min. Samples were then rinsed with PBS and incubated with Alexa 488-phalloidin and DAPI to stain F-actin and the nuclei. Immunofluorescence images were obtained with the Nikon TE-2000 inverted microscope. Image Analysis. Images of BSA adsorbed on patterned chitosan were acquired using a SPOT II CCD camera (SPOT Diagnostic Instruments Inc., version 3.5.1, Sterling Heights, MI) with Metamorph (Universal Imaging, version 6.0r4, Westchester, PA) image analysis software. The boundaries outlining the bare chitosan or OEGMA/MA-coated regions were set by manually tracing the two regions based on the width of each pattern. To quantify the resistance of the OEGMA/MA-coated regions against protein adsorption, the relative fluorescence intensity of BSA adsorbed on adjacent regions of OEGMA/MA and bare chitosan was measured. Intensity measurements were averaged from multiple images taken at each tested condition.

Results and Discussion The key element of this cell patterning approach is the identification and optimization of polyelectrolytes that remain adsorbed on chitosan surfaces over extended periods of time while preventing the attachment of cells. With this polyelectrolyte, we can make micron-sized protein or cell adhesive regions on adhesive biomaterial by coating the background regions with the cell resistant polyelectrolytes. In this work, we chose to use random copolymers of OEGMA/MA. The charged acid (-COOH) group of MA was expected to bind the copolymer strongly to the basic amine groups of the chitosan while the oligo(ethylene glycol) units resist protein and cell attachment. Previous studies have shown that ethylene glycol groups are inert to nonspecific protein adsorption.17 The resistance of poly(ethylene glycol) (PEG) to the adsorption of proteins is generally considered as inherent in its hydrophilicity, molecular conformation,18-20 and tightly bound water on the oligo(ethylene glycol) moieties.21 (17) Mrksich, M.; Dike, L. E.; Tien, J.; Ingber, D. E.; Whitesides, G. M. Exp. Cell Res. 1997, 235, 305-313. (18) Taunton, H. J.; Toprakcioglu, C.; Fetters, L. J.; Klein, J. Nature 1988, 332, 712-714. (19) Jeon, S. I.; Andrade, J. D. J. Colloid Interface Sci. 1991, 142, 159-166. (20) Harder, P.; Grunze, M.; Dahint, R.; Whitesides, G. M.; Laibinis, P. E. J. Phys. Chem. B 1998, 102, 426-436.

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Figure 1. Schematic outline of the use of polyelectrolytes to pattern proteins and mammalian cells on chitosan.

To form micrometer-size nonadhesive regions, we used microcontact printing to pattern OEGMA/MA on chitosan substrates. The schematic of this procedure is shown in Figure 1. Briefly, a silicon test pattern with parallel lines of varying widths was fabricated using traditional photolithographic techniques. From this silicon master, reusable elastomeric PDMS stamps were prepared by soft lithography.22 After the PDMS stamp is “inked” with OEGMA/MA copolymers, the stamp is applied onto chitosan films, thereby forming protein and cell resistant OEGMA/MA patterns that correspond to the raised regions of the stamp which contact the chitosan surface. The use of a random copolymer OEGMA/MA as the ink and its immobilization on the chitosan surface via electrostatic acid/base interaction are unique to this method, eliminating the need for gold-coated substrates. To engineer OEGMA/MA with optimized cell resistance and adhesiveness to the chitosan substrate, copolymers with different ratios of two monomers were evaluated. Figure 2A,C,E,G,I shows phase contrast images of 60 µm wide line patterns spaced 30 µm apart printed over the surface of chitosan using OEGMA/MA of varying monomer ratios. In each case, the OEGMA/MA-patterned chitosan substrates were air-dried without rinsing and form rough layers over the patterned region with dark and white spots visible in phase contrast images. To examine the ability of OEGMA/MA to resist nonspecific protein adsorption, patterned chitosan films were incubated with fluorescently labeled BSA. BSA selectively adsorbs to the 30 µm spaced areas that are not coated with OEGMA/MA for copolymers with OEGMA molar ratios of 0.9, 0.8, 0.7, and 0.5 (Figure 2B,D,F,H). This is a clear demonstration of OEGMA’s effectiveness in resisting protein adsorption. However, MA, which is necessary for binding to the chitosan surface, can induce protein adsorption, as is evident in Figure 2J. The OEGMA/MA patterns of Figure 2J, with a low OEGMA (21) Wang, R. L. C.; Kreuzer, H. J.; Grunze, M. J. Phys. Chem. B 1997, 101, 9767-9773. (22) LeDuc, P.; Ostuni, E.; Whitesides, G.; Ingber, D. Methods Cell Biol. 2002, 385-401.

molar ratio of 0.3, not only fail to resist protein adsorption but actually induce more protein adsorption compared to the uncoated regions of chitosan. This result is evident in Figure 3 which shows the fluorescence intensity of BSA adsorbed on the OEGMA/MA-printed regions relative to that on the bare chitosan regions. As the molar ratio of OEGMA is reduced from 0.8, increasingly more protein adsorbs to the OEGMA/MA regions. This effect can be attributed to the decreased entropic repulsions between the protein and OEG as the OEG content is reduced. Indeed, when the OEGMA content is reduced to 0.3, the protein preferentially adsorbs on the OEGMA/MA-coated region compared to the bare chitosan regions. Random OEGMA/MA copolymers with an OEGMA molar ratio of 0.8 are most effective in resisting protein adsorption, with a relative adsorbed BSA fluorescence intensity of 0.29. As the OEGMA ratio is increased to 0.9, the OEGMA/MA becomes less resistant to protein adsorption. It appears that as the number of methacrylic acid groups, which bind to the chitosan amine groups, is reduced, the adsorption of OEGMA/MA to the chitosan surface is also reduced, leading to diminished resistance against protein and cell attachment. To investigate the stability of these polyelectrolyte patterns, the patterned chitosan films were immersed in deionized (DI) water (Figure 4A) and PBS solution (Figure 4B) over prolonged periods of time and then re-evaluated for resistance to protein and cell attachment. The stability of the pattern in solution is critical since patterned substrates used to control the attachment and spreading of cells will be challenged by culture media which are composed of various ions and proteins. In performing these tests, we were surprised to find that patterned chitosan films, annealed for several days or weeks after stamping with the OEGMA/MA polyelectrolyte, exhibited improved stability in solution. Figure 4 shows the stability of OEGMA/MA patterns with an OEGMA molar ratio of 0.8. Line shape patterns (Figure 2) were printed on chitosan, and the patterned dry chitosan films were kept at room temperature while exposed to air

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Figure 2. Optical micrographs (A,C,E,G,I) of OEGMA/MA patterned on chitosan film and the corresponding fluorescence micrographs (B,D,F,H,J) showing selective adsorption of BSA. Selective deposition of BSA to bare chitosan was observed for the patterns formed with OEGMA/MA with OEGMA molar ratios of (B) 0.9, (D) 0.8, (F) 0.7, and (H) 0.5. Reverse deposition of BSA to OEGMA/MA-coated regions was observed for polyanions with 30% OEGMA (J). Wider 60 µm stripes are the OEGMA/MA-coated regions, and 30 µm stripes are the bare chitosan. The scale bar applies to all the images.

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Figure 3. Effect of polyanion composition (molar ratio of OEGMA in OEGMA/MA) on protein micropatterning. The fluorescence intensity of adsorbed BSA over OEGMA/MA-coated regions, normalized to the intensity over the chitosan region, is plotted as a function of OEGMA molar ratio in OEGMA/MA.

for 2, 5, and 15 days before immersion in DI water (Figure 4A) or PBS (Figure 4B) for 0, 5, 24, and 120 h at 37 °C. Afterward, the patterned chitosan films were incubated with fluorescent BSA to test whether the protein resistant patterns remain adsorbed after immersion in solution. The normalized fluorescence intensity shown in Figure 4

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is calculated in the same way as in Figure 3 and represents the fluorescence intensity of BSA adsorbed on the OEGMA/ MA patterns relative to that of the bare chitosan regions. For patterns annealed for 2 days before BSA adsorption, the normalized intensity is below 0.5. The normalized intensity increases after the pattern is left in water or PBS for 5 h, and the pattern is nearly lost after 24 h. When the polyelectrolyte-patterned chitosan is annealed for 15 days, the stability of the patterns is improved very significantly with only a slight increase in normalized intensity after 24 h in DI water or PBS. Even after 5 days in PBS, the normalized intensity remains below 0.7, which is from our experience the critical threshold for effectively patterning cells. The increase in normalized intensity with increasing immersion time in DI water or PBS is most likely due to desorption of OEGMA/MA from the chitosan surface. The stability of patterns in PBS (Figure 4B) is similar to that in water (Figure 4A) and is apparently unaffected by the increased ionic strength, which typically reduces the stability of polyelectrolyte multilayers.23 The nature of OEGMA/MA adsorption onto chitosan is likely dominated by macroscopic adsorption rather than monolayer adsorption, which explains why the patterns are

Figure 4. Pattern stability in (A) DI water and (B) PBS improves with time. The fluorescence intensity of adsorbed BSA over OEGMA/MA regions after immersing in solution for 0, 5, 24, and 120 h plotted as the function of time after patterning. The intensity was normalized to the intensity of BSA adsorbed to bare chitosan regions. OEGMA/MA with an OEGMA molar ratio of 0.8 was used. Studies were repeated independently at least three times, and three areas were randomly chosen for analysis of each sample.

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Figure 5. Spatially defined attachment of endothelial cells on linear patterns. Phase contrast micrographs of human microvascular endothelial cells on (A) 10 µm wide lines, (B) 20 µm wide lines, (C) 30 µm wide lines, and (D) unpatterned regions on the chitosan film. Magnification, ×100.

visible under phase contrast microscopy (Figure 2). Patterns annealed while exposed to air at room temperature for more than 15 days remain clearly visible under phase contrast microscopy, even after being immersed in PBS solution for 5 days. The long-term stability of OEGMA/MA even in solutions of high ionic strength is likely due to the fact that the copolymers are insoluble in water. The mechanism through which the stability of OEGMA/MA patterns on chitosan improves upon annealing is not clear but possibly related to the alteration of the molecular conformation of OEGMA/MA. As a demonstration that OEGMA/MA patterns can be used to control the spatial distribution of cells, human microvascular endothelial cells were seeded onto patterned chitosan films. Figure 5 shows phase contrast images of the cells cultured over OEGMA/MA-patterned chitosan after 72 h. Cells on neighboring unpatterned regions on the same chitosan film are shown in Figure 5D. Line patterns consisting of multiple 60 µm wide OEGMA/MA lines, separated by 10, 20, and 30 µm spacing, were formed on the chitosan film by microcontact printing. OEGMA/ MA patterns, with an OEGMA molar ratio of 0.8, annealed for 2 weeks on chitosan were used in the study. Endothelial cells plated on these patterns selectively attached and spread exclusively within the 10, 20, and 30 µm lines separated by the 60 µm wide lines of OEGMA/MA (Figure 5A-C). Cells adapt to the line shape, adopting high aspect ratios to spread within the narrow lines. Cells spreading on the 10 µm lines were highly confined and, on occasion, bridge across the OEGMA/MA regions. The 10 and 20 µm wide lines of chitosan allowed for only one single cell to span their widths, whereas two cells were sometimes observed stretching across wider 30 µm lines. Visualization of the actin cytoskeletons of the patterned cells using fluorescence microscopy (Figure 6) reveals the (23) Hoogeveen, N. G.; Stuart, M. A. C.; Fleer, G. J.; Bohmer, M. R. Langmuir 1996, 12, 3675-3681.

preferential alignment of the cytoskeleton (F-actin labeled with Alexa 488 linked phalloidin) along the axis of the 10, 20, and 30 µm wide chitosan line patterns. The nuclei of the patterned cells, stained with DAPI, also align to the main axis of the chitosan lines. In comparison, cells on the unpatterned control chitosan substrate (Figure 6C,G) exhibit no preferential alignment of either cytoskeleton or nuclei. These results demonstrate the efficacy of this patterning approach in controlling the spreading, alignment, and spatial organization of cells. Extending this approach to form three-dimensional scaffolds can be accomplished by stacking multilayers of patterned sheets or by cutting and folding the sheets to form three-dimensional objects. Future work will examine the feasibility of these approaches and the application of this method to pattern cells on other biomaterials. The use of readily available polyelectrolytes and biodegradable chitosan substrates should encourage the use of this approach in fabricating a new generation of micropatterned scaffolds for tissue engineering applications. Conclusions We have reported a new approach for controlling the spatial organization, spreading, and orientation of cells on chitosan substrates. Unlike traditional cell patterning techniques that make use of gold, silver, palladium, or silicone substrates, cells patterned on the biodegradable chitosan substrates have much broader tissue engineering applications. This technique exploits the electrostatic interactions of OEGMA/MA random copolymers with chitosan to create stable micron-size patterns. Optimization of the OEGMA to MA ratio is critical to establish the correct balance of resistance against protein or cell attachment and electrostatic acid/base binding between the copolymer and the chitosan substrate. The approach can be used to create a variety of other micron-sized

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Figure 6. Cytoskeletal alignment in microvascular endothelial cells cultured on (A) 20 µm lines (magnification, ×200), (B) 30 µm lines (magnification, ×200), (C) unpatterned regions on the chitosan film (magnification, ×200), (D) 10 µm lines (magnification, ×400), (E) 20 µm lines (magnification, ×400), (F) 30 µm lines (magnification, ×400), and (G) unpatterned regions on the chitosan film (magnification, ×400). Microfilaments aligned parallel to the axis of the line patterns within 72 h. Actin microfilaments (green) were visualized by Alexa 488 labeled phalloidin. Cell nuclei were visualized by DAPI (blue).

patterns on biomaterials to suit the specific tissue engineering applications. Acknowledgment. This work was supported in part by a grant from the university research council at the

University of Cincinnati. The authors acknowledge Professor Ian Papautsky and Erik Peterson for providing the silicon master. LA035309L