Patterned Silk Films Cast from Ionic Liquid Solubilized Fibroin asScaffolds for Cell Growth

Maneesh K. Gupta,†,‡ Shama K. Khokhar,† David M. Phillips,‡ Laura A. Sowards,‡

Lawrence F. Drummy,‡ Madhavi P. Kadakia,*,† and Rajesh R. Naik*,‡

Department of Biochemistry and Molecular Biology, Wright State UniVersity, Dayton, Ohio 45432, andMaterials and Manufacturing Directorate, Air Force Research Laboratory,

Wright-Patterson Air Force Base, Dayton, Ohio 45433

ReceiVed July 14, 2006. In Final Form: October 26, 2006

Silk is an attractive biomaterial for use in tissue engineering applications because of its slow degradation, excellentmechanical properties, and biocompatibility. In this report, we demonstrate a simple method to cast patterned filmsdirectly from silk fibroin dissolved in an ionic liquid. The films cast from the silk ionic liquid solution were foundto support normal cell proliferation and differentiation. The versatility of the silk ionic liquid solutions and the abilityto process large amounts of silk into materials with controlled surface topography directly from the dissolved silkionic liquid solution could enhance the desirability of biomaterials such as silk for a variety of applications.

Introduction

Of late, a considerable amount of interest has developedregarding the use of patterned and textured biomaterial surfacesfor applications in tissue engineering and drug delivery. Recentresearch has shown the importance of surface topography andtexture in controlling the shape, orientation, and gene expressionof adherent cells.1

Historically, silk fibers have been widely used in biomedicalapplications as a suture material for repairing wound injuries.The recent ability to fabricate silk-based materials of variousgeometries including films, sponges, mats, and fibers, frompurified silk fibroin solution has resulted in the reemergence ofsilk as a biomaterial. Silk is an attractive tissue engineeringscaffold because of its slow degradation, excellent mechanicalproperties, and biocompatibility.2 Numerous studies have proventhe biocompatibility of silk substrates in supporting the adhesionand proliferation of mammalian cells.3

Silk fibroin protein, isolated from the cocoon fiber of theBombyx morisilkworm, consists of a 391 kDa heavy chain and26 kDa light chain covalently linked through a disulfide bond.4

The heavy chain portion of the protein is divided into twelve

crystalline regions separated by amorphous regions 30-40 aminoacids in length. The crystalline regions are primarily composedof the GAGAGS amino acid motif which assumes a hydrogen-bonded antiparallelâ-sheet structure in the native fiber. Thehighly crystalline secondary structure of silk is thought to beresponsible for the excellent mechanical characteristics. Nativefibers are coated in gluelike sericin proteins which hold the fiberstogether in the shape of the cocoon.4

Unfortunately, due to its stabilized secondary protein structure,silk fibroin has limited solubility characteristics, resulting insignificant limitations regarding the fabrication of tissue engi-neering scaffolds. Previous work in our laboratory has shownthe ability to dissolve silk fibroin in di- and trialkylimidazolium-based ionic liquids and the use of the resulting solutions tofabricate silk films and fibers.5,6 This holds many advantagesover conventional methods. Traditionally, ionic aqueous solutionssuch as 9.3 M LiBr have been used to dissolve silk fibers. Thesesolutions require extensive dialysis, yielding aqueous silk fibroinsolutions with a shelf life of 1-2 days. Alternatively, aqueoussolutions of silk fibroin have been lyophilized and redissolvedin organic solvents such as 1,1,1,3,3,3-hexafluoroisopropanol(HFIP). These solvents are typically extremely corrosive andtoxic, requiring considerable care in handling. Ionic liquids, onthe other hand, have long been recognized as “green” solvents7

and have been used in the dissolution of silk,5,6 cellulose,8 andkeratin.9 The resulting solution has extended stability and easeof processing of biopolymers into fibers and films.5,6,10 In thiswork, we have extended the versatility of the silk ionic liquidsolutions and demonstrate the ability to fabricate silk fibroinfilms with controlled surface topography directly from thedissolved silk ionic liquid solution as scaffolds for cell growth.

* E-mail: rajesh.naik@wpafb.af.mil; madhavi.kadakia@wright.edu.† Wright State University.‡ Wright-Patterson AFB.(1) Dunn, G. A; Brown, A. F.J. Cell Sci.1986, 83, 313-340. Curtis, A. S.

G.; Clark, P.Crit. ReV. Biocompat.1990, 5, 343-362. Chou, L.; Firth, J. D.;Uitto, V. J.; Brunette, D. M.J. Cell Sci.1995, 108, 1563-1573. den Braber, E.T.; de Ruijter, J. E; Smits, H. T. J.; Ginsel, L. A.; von Recum, A. F.; Jansen, J.A. J. Biomed. Mater Res.1995, 2, 511-518. Grinnell, F.Trends Cell Biol.2003,13, 264-269. Wen-Ta, S.; Chu, I.-M.; Yang, J.-Y.; Lin, C.-D.Microndoi:10.1016/j.micron.2006.03.007.

(2) Altman, G. H.; Diaz, F.; Jakuba, C.; Calabro, T.; Horan, R. L.; Chen, J.;Lu, H.; Richmond, J.; Kaplan, D. L.Biomaterials2003, 24, 401-416. Unger,R. E.; Wolf, M.; Peters, K.; Motta, A.; Migliaresi, C.; Kirkpatrick C. J.Biomaterials2004, 24, 1069-1075.

(3) Servoli, E.; Maniglio, A.; Motta, A.; Predazzer, R.; Migliaresi, C.Macromol.Biosci.2005, 5,1175-1183. Kim, H. J.; Kim, U. J.; Vunjak-Novakovic, G.; Min,B. H.; Kaplan, D. L.Biomaterials2005, 26, 4442-4452. Li, C.; Vepari, C.; Jin,H.-J.; Kim, H. J.; Kaplan, D. L.Biomaterials2006, 27, 3115-3124. Inouye, K.;Kurokawa, M.; Nishikawa, S.; Tsukada, M.J. Biochem. Biophys. Methods1998,37, 159-164.

(4) Zhou, C.-Z.; Confalonieri, F.; Jacquet, M.; Perasso, R.; Li, Z.-G.; Janin,J.Proteins2001, 44(2), 119-122. Asakura, T.; Yao, J.; Yamane, T.; Umemura,K.; Ulrich, A. S. J. Am. Chem. Soc. 2002, 124, 8794-8795. Canetti, M.; Seves,A.; Secundo, F.; Vecchio, G.Biopolymers1989, 28, 1613-1624.

(5) Phillips, D. M.; Drummy, L. F.; Conrady, D. G.; Fox, D. M.; Naik, R. R.;Stone, M. O.; Trulove, P. C.; De Long, H. C.; Mantz, R. A.J. Am. Chem. Soc.2004, 126 , 14350-14351.

(6) Phillips, D. M.; Drummy, L. F.; Naik, R. R.; De Long, H. C.; Fox, D. M.;Trulove, P. C.; Mantz, R. A.J. Mater. Chem.2005, 15, 4206-4208.

(7) Rogers, R. D.; Seddon, K. R.Science2003, 302, 792-793.(8) Swatloski, R. P.; Spear, S. K.; Holbrey, J. D.; Rogers, R. D.J. Am. Chem.

Soc.2002, 124, 4974-4975.(9) Xie, H.; Li, S.; Zhang, S.Green Chem.2005, 7, 606-608.(10) Turner, M. B.; Spear, S. K.; Holbrey, J. D.; Rogers, R. D.Biomacro-

molecules2004, 5, 1379-1384.

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10.1021/la062047p CCC: $37.00 © 2007 American Chemical SocietyPublished on Web 12/14/2006

Experimental Section

Fibroin Solution Preparation. Silk was obtained fromB. morisilkworms raised on a diet of silkworm chow (Mullberry Farms,Fallbrook, CA). Live pupae were extracted from the cocoons priorto sericin removal in order to avoid potential contamination ordegradation of the fibroin protein. Silk fibers were prepared aspreviously described.5,6,11 Briefly, silkworm cocoons were soakedat 3.3% (w/v) in a solution of 8 M urea, 40 mM Tris-SO4, and 0.5M â-mercaptoethanol and heated to 90°C for 1 h. The silk fiber wasthen extensively washed with ultrapure distilled water (18 MΩ‚cm)and dried overnight under vacuum. Dried silk fibers were subse-quently dissolved in 1-butyl-3-methylimidazolium chloride (BMIC)ionic liquid (io-li-tec GmbH and Co., Denzlingen, Germany) toform a 10% (w/w) solution. Dissolution was accomplished by heatinga mixture of the silk fiber and BMIC powder to 90°C for 1 h inorder to melt the ionic liquid. The fiber and ionic liquid mixture wasthen mixed for 30 s in a speed mixer (FlackTek, Inc., Landrum, SC)and returned to heating at 90°C for 1 h. The mixing was repeatedfour times until a solution with uniform consistency was obtained.In order to lower the viscosity and melting point of the solution,25% (w/w) water was added to the heated solution. Care was takento incrementally add the water (5% w/w increments) to the solutionso as to avoid excessive localized dilution and potential precipitationof silk from the solution. The final composition of the solution is7.5% (w/w) silk fibroin, 25% (w/w) water, and 67.5% (w/w) BMIC.

Patterned Film Fabrication. Films were cast from the silk ionicliquid solution using a methanol rinse bath. When submerged inmethanol, the ionic liquid is extracted out of the silk solution intothe methanol bath, resulting in crystallization of the silk fibroinprotein into the antiparallelâ-sheet secondary structure.5,6 Poly-dimethylsiloxane (PDMS) stamps with channels fabricated frompatterned masters were used as micromolds for creating patternedsilk films. The silk solution was spin-coated on top of the PDMSmold and submerged in methanol for 10 min, resulting in crystal-lization of the silk film. The optically clear silk film can then bereleased from the PDMS mold (Figure 1). Alternatively, TEM gridswere used as molds to pattern the silk film. Here, the TEM grid isplaced on top of the silk solution layered over a glass slide or siliconwafer. After rinsing with methanol, the TEM grid is lifted off, leavingbehind a patterned film.

Film Characterization. Films were characterized using opticaland scanning electron microscopy. Profiles of patterned films weremeasured using white light interferometry. Secondary structure andionic liquid removal were monitored using attenuated total reflectionFourier transformed infrared spectroscopy (ATR-FTIR).

Cell Culture. Primary keratinocytes were purchased from CascadeBiologics (Portland, OR) and maintained in EpiLife media supple-

mented with EpiLife defined growth supplement (Cascade Biologics).For cell proliferation and differentiation assays, keratinocytes weregrown on normal plastic, silk-coated, and collagen-coated (BDBiosciences) tissue culture plates. Silk-coated plates were preparedby evenly coating 6-cm tissue culture plates with 150µL of silkionic liquid solution. The plates were then rinsed two times with 5mL of methanol for 10 min each time. Silk-coated plates are thenrinsed twice with 5 mL of distilled water, after which they are driedand exposed under UV for 15 min. Before seeding cells, plates arerinsed once each with PBS and growth media. For cell proliferationstudies, cells were seeded and counted at 6, 24, 48, and 72 h timepoints.

Cell Differentiation. For cell differentiation studies, upon reaching80% confluence cells were treated with 1R,25-dihydroxyvitaminD3 (VD3) (Sigma, St. Louis, MO). Differentiation was monitoredby measuring mRNA transcript levels of known differentiationmarkers.12 For RNA studies, cells were lysed directly on the cultureplate using the RNAeasy method as per manufacturer’s protocol(Qiagen, Valencia, CA). Reverse transcription of RNA was performedusing the TaqMan Reverse transcription kit (Applied Biosystems,Foster City, CA). Quantitative real-time PCR analysis was performedin a 96-well microtiter plate format on an ABI Prism 7900 sequencedetection system using TaqMan Universal Mastermix and Assay onDemand reagents specific for human gene Serpin B9 and PADI III(PE Applied Biosystems, Foster City, CA). Each well was monitoredfor fluorescent dye, and signals were considered significant if thefluorescence intensity significantly exceeded the standard deviationof the basic fluorescence, defined as the threshold cycle (CT). RelativemRNA quantitation was performed using the comparative∆∆Ctmethod.13

Image Analysis.In order to threshold the image and isolate thecells from the background, a number of filters were applied. Asshown in the Supporting Information, the patterned silk backgroundwas removed by performing a fast Fourier transform (FFT) on theoriginal image, selecting the reflections corresponding to the periodicspacings in the film, performing an inverse FFT, and subtracting theinverse FFT from the original image. The image was then invertedand flattened, using a high-pass filter, so that large-scale variationsin background grayscale were removed. The image was thresholded,a Gaussian blur was applied to remove noise, and the image wasthen thresholded again to produce the final black and white imageof the cells (Figure S1). NIH Image was used for calculation of theFFTs, and theAdobe Photoshopsoftware package with Fovea Proplug-ins was used for application of the various filters as well as forparticle counting and analysis. The data were then graphicallyrepresented as the angle of the cell with respect to the vertical axisof the image.

Results and Discussion

We have previously shown that silk fibroin can be dissolvedin ionic liquids (ILs) such as 1-butyl-3-methylimidazoliumchloride (BMIC) to form a stable silk fibroin solution.5,6 Thesolubility of silkworm silk fibroin in ILs is attributed to theability of the anion (Cl) to disrupt the hydrogen bonding in thesilk fibroin, and thus ILs are excellent solvents in which to dissolvesilk. The silk ionic liquid (SIL) solution obtained is clear andviscous. The viscosity of the solution can be decreased by addingwater to the SIL solution. In most experiments, 7% (w/w) SILsolution was used to generate optically clear and mechanicallyrobust silk films. In order to imprint features on silk films,elastomeric molds or metal masks were used as templates togenerate surface features. The SIL solution is spin-coated directlyonto a PDMS mold, or a metal mask can be placed on substratecoated with the SIL solution. We have previously demonstratedthat acetonitrile or methanol can be used to extract out the IL

(11) Yamada, H.; Nakao, H.; Takasu, Y.; Tsubouchi, K.Mater. Sci. Eng., C2001, 14, 41-46.

(12) Lu, J.; Goldstein, K. M.; Chen, P.; Huang, S.; Gelbert, L. M.; Nagpal,S. J. InVest. Derm.2005, 124 (4), 778-785.

(13) Pfaffl, M. W. Nucleic Acids Res.2001, 29, 2002-2007.

Figure 1. Schematic illustration of fabricating patterned silk films.(A) PDMS stamp of the desired design is cast. (B) The silk solutionis spin-coated onto the PDMS stamp. (C) The coated stamp issubmerged in a methanol bath to extract the ionic liquid solvent,causing the silk film to crystallize. (D) The crystallized silk film ispeeled from the stamp.

1316 Langmuir, Vol. 23, No. 3, 2007 Gupta et al.

as well as cause the silk to crystallize.5,6The resulting crystallizedfilm is optically clear and mechanically robust, and can be releasedfrom the substrate after methanol treatment. The patterned filmswere analyzed by scanning electron microscopy (SEM). Figure2A,B shows the surface features on the silk films generatedusing PDMS or TEM grid as molds, respectively. Numerouspatterns with varying feature sizes were generated using bothPDMS and TEM patterning techniques. A PDMS mold inconjunction with a metal mask can also be used in patterningboth the top and bottom surfaces of a film. The features obtainedon the silk films were well-defined and retained the features ofthe mask or mold used. We used white light interferometry inorder to characterize the surface features of the patterned silkfilms (Figure 2C). The size features imprinted on the silk filmwere found to correspond to the size features of the mold with15-20% decrease in the overall height when the films are allowedto dry for extended periods.

While ionic liquids have been considered “green solvents”,they are known to have toxic effects when in contact withbiological systems.14 As such, the removal of ionic liquid iscritical in the fabrication of silk films for cell growth experiments.Methanol can be used to extract out ILs from SIL-cast films,resulting in a transparent film, and the removal of ILs can bemonitored using attenuated total reflectance FTIR (ATR-FTIR)spectroscopy. BMIC has a characteristic peak at 1463 cm-1, andthe loss of this peak upon methanol treatment indicates removalof BMIC from the silk film (Figure 2D, graph II).15 Methanol

treatment also results in a conformational change in the secondarystructure of the silk fibroin protein from random coil toâ-sheet;this renders the silk film water-insoluble. This transition wasalso confirmed via FTIR spectroscopy (Figure 2D, graph I). Thistransition in secondary structure is indicated by the shift in theamide I peak from∼1650-1622 cm-1 wavenumbers.4a,16

In addition to the mechanical stability, biocompatibility, andbiodegradability of cell scaffolds, adequate cell adhesion andproliferation is also essential (see ref 17 for review). Since thetreatment with methanol results in the removal of BMIC andcrystallization of the silk film, we next tested the patterned silkfilms for their ability to support cell growth. Tissue culture plateswere first coated with the SIL solution, treated with methanol,rinsed extensively in distilled water and sterilized by a briefexposure to UV radiation. Then, equal numbers of primary humanepidermal keratinocytes cells were seeded onto the tissue culturedish coated with the silk film as well as onto bare plastic andcollagen-coated tissue culture dishes for comparison. Afterallowing the cells to attach, we monitored cell growth by countingthe number of cells that attached to the tissue culture plate andexhibited normal cell morphology at indicated time points (Figure3). The cells were found to adhere to tissue culture plates coatedwith silk, similar to the control and collagen-coated plate. In allcases, the cells on three different substrates exhibited similar cellmorphology (Figure 3A,B).

Furthermore, no significant differences were observed in cellproliferation under the three different conditions (Figure 3C),

(14) Swatloski, R. P.; Holbrey, J. D.; Memon, S. B.; Caldwell, G. A.; Caldwell,K. A.; Rogers, R. D.Chem. Commun.2005, 1,668-669. Dochert, K. M.; Kupla,C. F. Green Chem. 2005, 7, 185-189. Stepnowski, P.; Skladanowski, A. C.;Ludwiczak, A.; Laczynska, E.Hum. Exp. Toxicol.2004, 23, 513-517.

(15) Zhou, Z.; Wang, T.; Xing, H.Ind. Eng. Chem. Res.2006, 45, 525-529.

(16) Chen, X.; Shao, Z.; Marinkovic, N. S.; Miller, L. M.; Zhou, P.; Chance,M. R. Biophys. Chem.2001, 89, 25-34.

(17) Rosso, F.; Marino, G.; Giordano, A.; Barbarisi, M.; Parmeggiani, D.;Barbarisis, A.J. Cell. Phys.2005, 203, 465-470.

Figure 2. Patterned silk films. SEM micrographs of (A) PDMS stamp patterned silk film with a peak-to-peak periodicity of 6.6µm and(B) hexagonal patterns obtained using a TEM grid as a mask. (C) White light interferometry scan of hexagonal patterned silk film. (D)ATR-FTIR spectra of amide I region of silk (I) and BMIC (II) before (solid line) and after (dashed line) methanol treatment.

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though a slight increase in cell proliferation was observed oncells grown on collagen, which is not surprising, since collagen17

is widely used as an extracellular matrix (ECM) for cell growth;however, it lacks the inherent mechanical properties of silk.Nonetheless, our results indicate that the silk film cast from theSIL solution is able to serve as a cell scaffold and allows fornormal cell adhesion and proliferation.

We tested the effect of differentiation on gene expression incells grown on silk films. 1R,25-Dihydroxyvitamin D3 (VD3),the hormonally active form of vitamin D3, has been shown toinduce differentiation in primary keratinocytes. As a result, VD3and its analogs show great promise as therapeutics in skinabnormalities. VD3 induces the vitamin D receptor (VDR)signaling pathway that results in the induction of several genesinvolved in cellular differentiation.12 We examined whetherkeratinocytes grown on the silk films generated from the SILsolution were able to undergo normal differentiation behaviorupon exposure to VD3 by monitoring the expression of thedifferentiation markers. The expression of peptidylargininedeiminase (PADI), kallikrein (KLK), and serine proteinaseinhibitor (SERPIN B) gene family members in primary normalhuman epidermal keratinocytes have been shown to be vitaminD dependent and are induced when cells undergo differentiation.12

The expression of PADI 3, Serpin B9, and KLK10 in keratinocyteswas analyzed by real-time RT-PCR (Figure 3D). We observedthat the cells grown on patterned silk films cast from ionic liquidsexhibited a normal gene expression pattern when treated withVD3; expression of PADI 3, Serpin B9, and KLK10 was inducedafter VD3 treatment in all growth conditions. The cells grownon the silk films exhibited similar expression levels to thosegrown on standard tissue culture plates or on collagen-coated

(18) Lam, M. T.; Sim, S.; Zhu, X.; Takayama, S.Biomaterials2006, 27,4340-4347.

Figure 3. (A,B) Optical micrographs of keratinocytes growing on silk or collagen coated tissue culture Petri dishes after 24 h of incubation(100× magnification). (C) Proliferation of keratinocytes on bare (control), collagen-coated, or silk-coated tissue culture Petri dishes. Cellcount is represented as the number of cells/field of view. (D) Real-time RT-PCR showing the expression of known differentiation makersPADI 3, Serpin B9, and KLK10 in keratinocytes grown on standard tissue culture plates (control), silk-coated, and collagen-coated film tissueculture plates after 6 h exposure to VD3. They-axis is the relative fold change in expression compared to untreated cells.

Figure 4. (A) Optical micrographs of keratinocytes growing onpatterned silk films at 6 and 24 h. (B) Optical micrograph of cellsgrowing on control (collagen-coated) tissue culture plates. Histogramsdisplaying cell alignment on (C) patterned films and (D) controlsubstrates (collagen-coated). Thex-axis represents cell angle(measured as the angle between the longest axis of the cell and thevertical axis of the image), and they-axis is cell count. The datashow alignment of cells on patterned films as compared to the control.

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tissue culture plates. In addition, expression of several othermarkers was found to be normal in cells grown on silk films castfrom ionic liquids (data not shown).

Surface topography such as wavy features, grooves, and pillarshas been shown to impact cell behavior.1,18 For example, 6µmwave features generated on an elastomeric mold were found toalign myoblasts.18 Silk films from the SIL solution patternedwith periodic grooves with a spacing of 10µm and 2µm in depthwere able to support the growth and differentiation of primarykeratinocytes. We observed that the primary keratinocytes adheredto the micropatterned silk film within a few hours of seeding.The alignment of the primary keratinocytes with the patternobserved at the 6 h and 24 h time points is shown in Figure 4A.

Preliminary analysis using optical images of keratinocytesgrown on the patterned and unpatterned silk films indicates thata significant number of cells grown on the patterned silk filmsexhibit preferential alignment along the surface pattern. This isdemonstrated by the histograms showing the cell angle data forthe patterned and unpatterned silk films (Figure 4C,D). The cellangle is the measured angle between the long axis of the cell andthe vertical direction of the image (see Experimental Section).On the basis of the data, the cells appear to orient and elongatealong the direction of the pattern, as seen by a large number ofcells clustering around 0° or 180° to the vertical axis of theimage (Figure 4C), and this behavior was retained until the cells

begin to divide. In contrast, cells on the unpatterned films do notexhibit any preferential orientation (Figure 4D). We believe thatour method of introducing surface features onto the silk filmswould allow us in the future to further study the effect of surfacetopography on cell behavior.

Together, the data suggests that silk films cast from the ionicliquid solution did not have any detrimental effect on cell viability,differentiation, or gene expression, and the use of ILs as a solventfor silk fibroin provides an avenue for the fabrication of silkscaffolds for tissue engineering applications. Our future studieswill focus on the introduction of molecular signals by physicalentrapment during the film processing step, coupling of smallmolecules onto the silk films after casting, and the study ofvarious surface features in order to guide cellular behavior.

Acknowledgment. Funding for this research was providedby the Air Force Office of Scientific Research. We thank membersof the Biotech Group at AFRL and the Kadakia Lab forsuggestions and technical assistance.

Supporting Information Available: Additional experimentaldata as described in the text. This material is available free of chargevia the Internet at http://pubs.acs.org.

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