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Biomaterials 29 (2008) 4665–4675

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Biomaterials

journal homepage: www.elsevier .com/locate/biomateria ls

The effect of lactose-conjugated silk biomaterials on the developmentof fibrogenic fibroblasts

Chitrangada Acharya a, Boris Hinz b,*, Subhas C. Kundu a,**

a Department of Biotechnology, Indian Institute of Technology, Kharagpur 721302, Indiab Laboratory of Cell Biophysics (LCB), Ecole Polytechnique Federale de Lausanne (EPFL), Batiment SG – AA-B 143, Station 15, CH-1015 Lausanne, Switzerland

a r t i c l e i n f o

Article history:Received 17 July 2008Accepted 20 August 2008Available online 19 September 2008

Keywords:FilmScaffoldLactoseSilk fibroinMyofibroblast

Abbreviations: a-SMA, a-smooth muscle actin; Conuric chloride; ECM, extracellular matrix; FA, focal acyanuric chloride–silk fibroin conjugates; Lac, lactosetemperature; SF, silk fibroin.

* Corresponding author. Tel.: þ41 21 693 9703; fax** Corresponding author. Tel.: þ91 3222 283764; fax

E-mail addresses: boris.hinz@epfl.ch (B. Hinz)(S.C. Kundu).

0142-9612/$ – see front matter � 2008 Elsevier Ltd.doi:10.1016/j.biomaterials.2008.08.033

a b s t r a c t

Surface properties of implanted biomaterials can cause fibrotic tissue reactions by stimulating differ-entiation of host fibroblasts into contractile myofibroblasts. Silk fibroin (SF) protein has been used asbiomaterial in pure and blended form. however, its effect on myofibroblast differentiation remainselusive. We here conjugated SF with lactose using cyanuric chloride as coupling spacer. NMR spectros-copy and the conjugates ability to agglomerate Abrus precatorius agglutinin verified efficient conjugation.Two-dimensional films and three-dimensional scaffolds produced from pure and lactose-conjugated SFsolutions were tested as culture substrates for subcutaneous fibroblasts and myofibroblasts. Lactose-conjugated SF substrates mediated higher adhesion, proliferation and viability of fibroblastic cells thanpure SF. This SF film composition promotes better attachment of fibroblasts than myofibroblasts. Pro-fibrotic cytokine TGFb1 was ineffective in inducing fibroblast-to-myofibroblast differentiation on suchsubstrates. Pre-differentiated myofibroblasts lost their contractile phenotype within a few days of beingcultured on lactose-conjugated SF. Myofibroblast differentiation was also suppressed by growth in three-dimensional lactose-conjugated SF scaffolds that, however, support population with fibroblasts. Wepropose that this biomaterial will promote tissue integration without causing a fibrotic host reaction.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

When severely damaged tissues cannot be regenerated by theroutine repair mechanism of the body or when physiologicalhealing is imperfect then tissue engineering is considered. Variousbiomaterials have been developed to substitute damaged struc-tures by delivering reparative and regenerative cell populations,e.g. to cover and support large surface area after burn or to healchronic wounds. Ideal biomaterial is the one that is non-immu-nogenic, biocompatible and biodegradable and allows itsfunctionalization with bioactive proteins and chemicals. It supportsattachment of reparative cells that are to be delivered and of cellsresiding within the host tissue to promote its acceptance. This cell-adhesive property can cause severe complications when host cellsof mesenchymal origin get activated by the biomaterial to attain

nA, concanavalin A; CY, cya-dhesion; Lac–CY–SF, lactose–; RGD, Arg-Gly-Asp; RT, room

: þ41 21 693 8305.: þ91 3222 278433.

, kundu@hijli.iitkgp.ernet.in

All rights reserved.

a fibrogenic character. Such activation leads to tissue deformationand fibrosis [1].

Natural fibrous silk fibroin (SF) protein, obtained from thecocoons of mulberry silkworm Bombyx mori, is increasingly usedas biomaterial for tissue engineering [2] in the form of films [3–5],membranes [6], gels [7], sponges [8], powders [9], and scaffolds[2,10,11]. Applications include burn-wound dressings [12], enzymeimmobilization matrices [13], nets [14], vascular prostheses andstructural implants [15,16]. SF protein constitutes a supportingmatrix for a variety of cell types including fibroblasts [3–5],osteoblasts [17–19], keratinocytes [20], chondrocytes [21], endo-thelial cells [22] and mesenchymal stem cells [2,10,23]. Theadhesive properties of SF-based materials for cells are generallyenhanced by sulphonation [24], blending [19,23,25–27] or func-tionalization with adhesive peptides. Of those, the Arg-Gly-Asp(RGD) motif and its derivates are most widely used because itrepresents the cell integrin ligand in a large number of extracel-lular matrix (ECM) proteins, including collagen and fibronectin[28]. A variety of RGD-containing peptides (linear or cyclic) weregrafted to different biomaterials surfaces and were tested in termsof cell behavior [29–31].

Sugars like mannose, lactose (Lac) and galactose are also usedfor SF functionalization. This is done because glycoproteins presenton the cell membrane facilitate interaction with the substrate.

C. Acharya et al. / Biomaterials 29 (2008) 4665–46754666

Sugars, glycated proteins and sugar residues in lipid molecules bindcell surface lectins and allow cell–cell interactions [32]. Cost anddifficulty of grafting sugar molecules such as b-galactose are low incomparison to synthetic peptides. Glycated cationic polymers havemostly been used in cell transfection and internalization studieslike endocytosis and phagocytosis in epithelial cells, liver cells,dendritic cells, and macrophages [33]. Synthetic polymersendowed with b-galactose residues as biological recognitionsignals were developed as cell-specific culture substrata [34]. SFfunctionalization with b-galactose residue bearing Lac was previ-ously shown to promote attachment and growth of hepatocyteswhen cast as two-dimensional (2D) films [35]. The potential of Lac-conjugated SF to form three-dimensional (3D) scaffold is yet to beexplored. Moreover, the adhesive properties of such films forfibroblastic cells are yet to be elucidated.

Fibroblast attachment is an important yet neglected parameterdefining the suitability of a biomaterial for clinical applications asmost of the tissue grafts comes into contact with the connectivetissue cells that are part of the wound healing process. Duringphysiological tissue repair, acquisition of high contractile activityand increasing ECM synthesizing activity by fibroblasts contributeto wound closure and tissue regeneration [1,36–38]. This pheno-typic transition of fibroblasts into highly contractile myofibroblastsis hallmarked by de novo expression of a-smooth muscle actin(a-SMA) in stress fibers [39] and by the development of excep-tionally large focal adhesions (FAs) with the ECM [40]. Fibroblast-to-myofibroblast differentiation is driven by cytokines of whichTGFb1 is the most potent, and by the biophysical properties of themicroenvironment, including ECM stiffness and adhesiveness[41–44]. Persistent myofibroblast activity leads to the pathologicalcontractures and ECM deformations that are characteristic ofvirtually all fibrotic diseases and disorders, including fibrotic reac-tions to tissue implants [45]. Contraction and deformation of theECM by myofibroblasts impede normal function of the organ inseveral cases [1,46].

We here produce SF-based scaffolds and films that wereconjugated with Lac by using a cyanuric chloride (CY) linker. Wecan adjust the properties of this material to support attachment offibroblastic cells. At the same time, development of a fibrogenicphenotype is suppressed even in presence of the pro-fibroticcytokine TGFb1; growth on this material can reverse fibrogenicmyofibroblasts into normal fibroblasts.

2. Materials and methods

2.1. Chemicals

We used CY, a-Lac monohydrate, TGFb1, 1,4-dioxanelithium bromide, conca-navalin A (ConA), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide(MTT), and other chemicals from Sigma (St. Louis, USA), Dulbecco’s Modified Eagle’sMedium (DMEM), trypsin–EDTA, antibiotic solution and FCS from GibcoBRL (GrandIsland, NY, USA), cellulose dialysis tubing with 3.5 and 12 kDa cut-off range fromPierce (Rockford, IL, USA) and tissue culture ware from Nunc (Rochester, NY, USA).Fresh live silk cocoons of B. mori were obtained from Debra Sericulture Complex(Directorate of Sericulture, West Midnapore District, West Bengal, India).

2.2. Preparation of Lac–CY–SF protein conjugates

SF protein was extracted from mulberry silk cocoons by a standard extractionprocedure [47]. The cocoons were cut into small pieces (approximately 1�1 cm)and boiled for 30 min in an aqueous solution of 0.02 M Na2CO3 (w10% w/v of silkcocoons and Na2CO3) for complete degumming of the shells and removal of sericin.The pieces were then washed in distilled water and dried for 1 h at 50 �C. Thedegummed shell pieces were dissolved in 9.3 M lithium bromide for 1 h at 55 �C andthen centrifuged to remove insoluble material. The supernatant was dialyzed for 3 dagainst deionized water with frequent changes.

Lac–CY–SF conjugates were prepared by improving a previously establishedprotocol [35]. CY-activated Lac (Lac–CY) was prepared by reacting Lac with equiv-alent molar quantity of CY. Lac monohydrate (60 mg) was dissolved in 4.5 mldistilled water on ice. To this solution, 30 mg CY in 1.5 ml 1,4-dioxane was addedover 15 min at 4 �C maintaining the pH at 9.0 by buffering it with a 20% (w/v) Na2CO3

solution. Lac–CY modifier was processed with constant stirring of the mixture at4 �C and pH 9.0 for 2 h, followed by the coupling of Lac and SF. To every 4.5 ml of theLac–CY solution, SF aqueous solution containing 60 mg SF was added. The mixturewas then incubated at 37 �C and pH 8–9 for 3 h to facilitate reaction. The resultingmixture was neutralized by adding 1 N HCl and followed by dialyzing it againstdistilled water with a cut-off of 12 kDa overnight to remove non-reacted reagentsfrom the Lac–CY–SF product. Ultra filtration with 10 kDa Centricon (Millipore, USA)was done to concentrate the dialyzed solution to about 4 ml. Further purificationwas carried out by gel filtration. Sephadex G-100 was used for gel filtration after pre-equilibration with 2 M urea/0.02 M Tris–HCl (pH 8.0) and eluted with the same buffer.Protein-containing fractions were combined and dialyzed against distilled water at4 �C for a few days. The resultant Lac–CY–SF solution (w2% w/v) was used forspectrometric analysis. It was diluted to be cast as films, or was concentrated bydialyzing against 15% polyethylene glycol (PEG) 6000 using a 3500 kDa dialysismembrane (Pierce, USA) for making scaffolds.

2.3. Fabrication of SF-based 2D films and 3D scaffolds

To produce SF and Lac–CY–SF films for cell culture studies we used 1%, 0.1% and0.01% solutions. These films were cast onto the surfaces of cover glasses placedwithin the wells of a 12-well plate. The plates were kept under laminar flow for 6–8 h for drying, and the films were rendered insoluble and sterilized by ethanoltreatment before being washed with phosphate buffered saline (PBS). Films wereblocked using 0.02% BSA in sterile PBS for 1 h, excess BSA was removed by washingthree times in PBS. On drying, the films had a thickness between 0.04 and 0.07 mm(estimated using a Micro Hardness tester fitted with a CCD camera, Leco, St. Joseph,MI). To produce Lac–CY–SF scaffolds a concentration of 6% Lac–CY–SF was preparedby dialyzing 2% Lac–CY–SF against 15% PEG 6000; 300 ml of this solution was coatedon the wells of a 96-well tissue culture plate and was frozen at �20 �C for 24 h.Thereafter, it was lyophilized at�40 �C for 16 h until all the water had been removedfrom the porous structure.

2.4. Lac conjugation efficiency tests

1H NMR measurements were carried out for SF protein films (2%) to confirm theincorporation of Lac after conjugation. 1H NMR spectra for SF and Lac–CY–SF filmswere recorded in D2O at 600 MHz in a Bruker spectrometer. Lectin agglutinin, iso-lated from Abrus precatorius (gift from Dr. T. K. Maiti, Indian Institute of Technology,Kharagpur) was used to study lectin-induced agglutination of Lac–CY–SF [35].Briefly, 750 ml of 0.1% Lac–CY–SF was taken in both reference and sample cuvettes ofa UV/VIS spectrophotometer (Lambda 25, Perkin–Elmer) and baseline was recordedat 350 nm. Then, 50 ml of 2 mg/ml agglutinin was added to the sample. Agglutinationof Lac–CY–SF was recorded with time as absorbance increases at 350 nm; controlswere carried out using 0.1% pure SF.

Finally, dot blots onto nitrocellulose membrane were carried with solutions of SFand Lac–CY–SF, with ConA and PBS as positive and negative controls, respectively.Samples were dried on the membrane and blocked using 4% casein in PBS for 1 h.After three PBS (pH 7.4) washes, the samples were probed with biotinylatedagglutinin at a concentration of 0.1 mg/ml for 1 h. After 3 more PBS washes thesamples were probed with streptavidin–HRP conjugate and was developed using3,3-diamino benzidine tetrahydrochloride substrate with hydrogen peroxide. After15 min the reaction was stopped by repeated washing in ice-cold distilled water.

2.5. Mechanical properties of SF films

The tensile strength of SF and Lac–CY–SF films was tested on an universal testingmachine (H25kS, Hounsfield, Surrey, UK) fitted with a 50 kN load cell using specificfilm-grips according to the ASTMD 638 standard. The extension maximum was set at10 mm and the test speed was 0.05 mm/min. Data were recorded as an average ofthree replicates and were analyzed using QMAT 3.1 software.

2.6. Cell culture

Primary subcutaneous fibroblasts were isolated from the subcutaneous layer ofskin of Wistar rats as described previously [39] and were cultured in DMEMsupplemented with 10% FCS and antibiotics. To induce myofibroblast differentia-tion, fibroblasts were treated with 10 ng/ml of TGFb1 for at least 4 d either before(de-differentiation tests) or during culture in/on SF and Lac–CY–SF matrices(differentiation tests); TGFb1 treatment was continued during culture on SF for de-differentiation tests. Cells were seeded at 5000 cells/film or 30,000 cells/scaffoldthat were pre-equilibrated in medium for 1 h before cell seeding.

2.7. Cell adhesion

To measure attachment strength, 5000 cells were seeded on 1%, 0.1% and 0.01%SF and Lac–CY–SF films in a 96-well plate for 2, 3, 6 and 9 h. Thereafter, thesupernatant was removed, the plate was upturned and centrifuged for 5 min at1000 rpm. Remaining adherent cells were stained with 0.1% crystal violet in 20%methanol/water for 10 min [40]. Unbound dye was removed by aspiration and theplates were washed in a water bath for 2 h with repeated water changes. The plates

C. Acharya et al. / Biomaterials 29 (2008) 4665–4675 4667

were then air-dried and the bound dye was eluted in methanol. Absorbance wasmeasured at 570 nm using a multiplate spectrophotometric reader (Wallac Viktor,Perkin–Elmer).

2.8. Cell proliferation, viability and apoptosis

Fibroblastic cells were grown for 4 d (�TGFb1) on 1%, 0.1% and 0.01% SF andLac–CY–SF films, cast on 24-well plates. Cell viability and proliferation were eval-uated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT)colorimetric assay [48]. 0.5 ml of 5 mg/ml MTT was added to each well and plateswere incubated at 37 �C for 4 h. Finally, tissue culture plates were centrifuged at2500�g for 5 min, supernatant was discarded and the blue formazan reactionproduct was dissolved in 0.5 ml DMSO. Each sample (0.2 ml) was transferred toa 96-well plate and absorbance was measured at 570 nm using a multiplate spec-trophotometric reader in triplicates (Wallac Viktor, Perkin–Elmer Life Sciences).Fibroblasts and myofibroblasts grown in the conditions described above were testedfor apoptosis using Caspase-Glo 3/7 assay kit (Promega, Wallisellen, Switzerland)and a luminometer (Centro LB, Berthold Technologies, Regensdorf, Switzerland).Initially seeded cell numbers were adjusted to obtain similar cell counts at the timeof apoptosis test.

2.9. Immunofluorescence, microscopy and image analysis

5000 fibroblastic cells were seeded on films and incubated for 3 h, 6 h, 9 hand 4 d. Scaffolds were populated with 30,000 fibroblasts and pre-differentiatedmyofibroblasts and incubated for 4 d. For immunostaining processing, cells wererinsed with serum-free medium, fixed for 10 min at room temperature (RT) with3% paraformaldehyde in PBS, followed by permeabilization for 5 min with 0.2%Triton X-100. Primary antibodies were applied for 60 min at RT directed againstvinculin (hVin-1, mouse IgG1, Sigma) and a-SMA (aSM-1, mouse IgG2a) [49].Secondary antibodies TRITC- and FITC-conjugated goat anti-mouse IgG1 andIgG2a (Southern Biotechnology Associates Inc., Birmingham, AL) were applied for60 min. F-actin was probed with phalloidin-Alexa 647 (Molecular Probes, Eugene,OR) and DNA with DAPI (Fluka, Buchs, CH). Confocal images were acquired usinga 40� oil immersion objective (HC PL APO, NA 1.25-0.75, Leica, Glattbrugg, CH),mounted on an upright confocal microscope (DM IRE2 with a laser scanningconfocal head TCS SP2 AOBS, Leica); scaffolds were observed with waterimmersion objectives (Leica 63�, HCX APO L, NA 0.9). After acquisition, theimages at different depths were deconvoluted using Huygens’ principle andstacked using IMARIS software (Bitplane AG, Zurich, CH). Figures were assembledusing Adobe Photoshop.

Fig. 1. SF conjugation with Lac using a CY linker. (A) Schematic representation of Lac–CY–

2.10. Statistical analysis

All data were expressed as mean� standard deviation (SD) for n¼ 6 (viabilityassays) and n¼ 3 (adhesion and apoptosis analysis). Single factor analysis of variance(ANOVA) technique was used to assess statistical significance of results. Student’st-test was carried out at a level of 95% significance.

3. Results

3.1. Lac conjugation of SF using a CY linker

To conjugate SF with Lac we employed the reaction strategy(Fig. 1) adapted from Gotoh and coworkers [35,50]: first, Lac(Fig. 1A, oligosaccharide) was activated with equivalent molarquantities of CY using dioxane at pH 9.0 and 4 �C. Second, Lac–CYwas coupled to SF by warming the solution to 37 �C and the reac-tion was stopped by neutralization. An important intermediate stepis the synthesis of a Lac–CY modifier by the reaction of the terminalanomeric hydroxyl group in oligosaccharides with a chlorine atomof CY (Fig. 1B). A second chlorine atom of the activated oligosac-charide–CY reacts with either the phenolic hydroxyl group of thetyrosine residue or the 3-amino group of the lysine residue in SF(Fig. 1B).

To verify effective conjugation of SF with Lac residues we firstperformed 1H NMR spectroscopy (Fig. 2). Analysis of both SF(Fig. 2A) and Lac–CY–SF (Fig. 2B) revealed peaks that were specificfor each condition. The spectrum of Lac–CY–SF exhibited broadpeaks between 3.4 and 3.7 ppm, at 4.0, 2.45 and 1.3 ppm (Fig. 2B)that were not detected in SF spectra (Fig. 2A). With Lac conjugation,the original peak at 7.1 ppm in the SF spectrum, attributed to theprotons in tyrosine residues [51] and to other aromatic residues at6.6 and 6.9 ppm in the SF spectrum decreased in intensity andamplitude (Fig. 2B). Similarly, between 2.7 and 3.0 ppm, there wasa broadening of peaks in the Lac–CY–SF spectrum due to aliphaticamino acids like lysine present in the SF spectrum.

SF conjugate synthesis. (B) Chemical structure of the resulting Lac–CY–SF conjugate.

Fig. 2. 1H NMR spectra of Lac–CY–SF conjugate. (A) Spectrum of SF in D2O. (B) Spectrum of Lac–CY–SF in D2O.

C. Acharya et al. / Biomaterials 29 (2008) 4665–46754668

To confirm that SF–CY-covalently linked Lac still bearsfunctional b-galactose, we evaluated the interactions between Lac–CY–SF and the agglutinin lectin of A. precatorius. Lectin-inducedagglutination was measured as increased turbidity in a photometer(Fig. 3A). Agglutinin moderately increased the turbidity in SFsolutions but strongly and rapidly in Lac–CY–SF solutions (Fig. 3A).We assessed the presence of b-galactose in dot blot experimentswith biotinylated agglutinin. SF, being a glycoprotein [52], and Lac–CY–SF both showed positive staining (Fig. 3B) demonstrating theiraffinity for lectin. However, staining intensity was higher in the caseof Lac–CY–SF due to the presence of Lac residues. Strongest stainingwas obtained for the positive control ConA, which effectively bindsto this lectin; PBS control was negative (Fig. 3B). These experiments

demonstrate that our method produces solutions of functional Lac–CY–SF conjugates that are recognized by cell lectins.

3.2. Reduction of the tensile strength of 2D SF films afterconjugation with Lac–CY

One possible application of Lac-conjugated fibroin is delivery ofcells on a sheet or film of biomaterial, e.g. for skin wound repair. Tohandle these films, mechanical stability is needed. We conductedtests to confirm whether Lac conjugation altered the tensilestrength of films produced with different fibroin concentrations.Measurement at a constant force of 50 kN showed that the tensilestrength of SF films was higher compared with that of Lac–CY–SF.

Fig. 3. Recognition of galactose residues by Abrus precatorius agglutinin. (A) Timedependant changes in turbidity of SF and Lac–CY–SF conjugate after addition ofagglutinin. (B) Dot blot for qualitative assay of recognition of b-galactose residues usingbiotinylated agglutinin.

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Lac conjugation lead to w2-fold higher deformability (Table 1). Thiscan be explained by the incorporation of Lac in the SF side chainwhich may have lowered the SF chain strength. However, tensilestrength of Lac-conjugated films was sufficiently high to bemanipulated with a fibroin concentration of 0.1% and higher.

3.3. Proliferation and viability of fibroblastic cells on Lac–CY–SFfilms

To test their proliferation and viability, subcutaneous fibroblastswere seeded on 1%, 0.1%, 0.01% Lac–CY–SF and on pure SF films andcultured for 4 d� TGFb1. Treatment with TGFb1 was previouslyshown to significantly up-regulate the low baseline expression ofa-SMA and the percentage of a-SMA-positive myofibroblasts fromw20% to w90% in this cell type in standard culture [39]. Thenumber of viable cells was quantified using an MTT colorimetricassay (Fig. 4A,B). Generally, Lac–CY–SF films yielded higher numberof viable fibroblastic cells compared with pure SF films; thisdifference was most pronounced (w2-fold) on films produced with1% SF protein and was greater for fibroblasts (Fig. 4A) than formyofibroblasts (Fig. 4B). The cell yield from almost all Lac–CY–SFfilms was comparable to tissue culture polystyrene (TCPS) controls.When compared with fibroblasts (Fig. 4A), the number of viablemyofibroblasts (Fig. 4B) on Lac–CY–SF films and on TCPS controlwas w1.4-times lower; this was due to the well known anti-proliferative effect of TGFb1 that accompanies myofibroblastdifferentiation [53,54]. Notably, this TGFb1 effect was not observedon 1% Lac–CY–SF films; on these, myofibroblast numbers werehigher than the respective TCPS control (Fig. 4A) and reached thecorresponding number of fibroblasts (Fig. 4B).

Table 1The tensile strength of unconjugated (SF) and of Lac–CY conjugated SF protein (Lac–Cy–SF) was measured using a tensile tester

1% SF 0.1% SF 0.01% SF

SF 20.31� 2.21 3.13� 0.33 1.02� 0.02Lac–CY–SF 16.67� 1.19 1.9� 0.08 0.7� 0.01

Data are represented in MPa as mean� standard deviation for n¼ 3.

To further evaluate the apoptosis rate of fibroblastic cells in thesame growth conditions, we quantified Caspase 2/3 activity witha luminescence assay (Fig. 4C,D). No significant difference wasobserved between fibroblasts (Fig. 4C) and myofibroblasts (Fig. 4D).Growth on 1% SF films induced w1.6 higher number of apoptoticcells compared with TCPS control; this rate decreased withdecreasing concentration of pure SF protein to that observed onTCPS (Fig. 4C,D). In contrast, on Lac–CY–SF films apoptosis wasalways lower compared with TCPS and was not altered by theconcentration of SF protein (Fig. 4C,D). Together, these resultsdemonstrate that conjugation of SF with Lac–CY enhances prolif-eration and viability of fibroblastic cells cultured on SF proteinfilms. The anti-proliferative effect of TGFb1 on fibroblast growth isstrongly reduced on 1% Lac–CY–SF.

3.4. Differential attachment of fibroblasts and myofibroblasts toLac–CY conjugated SF

Both, cell proliferation and survival are dependent onsubstrate adhesion [55]. To test the cell-adhesive properties of SF-based films, subcutaneous fibroblasts and pre-differentiatedmyofibroblasts were seeded on 1%, 0.1% and 0.01% Lac–CY–SF andSF films (Fig. 5). The number of cells firmly attaching after 2, 3, 6and 9 h was evaluated by measuring 570 nm light absorptionafter cell staining with crystal violet. Generally, conjugation of SFwith Lac–CY augmented fibroblast adhesion by about 2-fold(Fig. 5A) and myofibroblast adhesion by 2–4-fold (Fig. 5B)compared with pure SF produced with the same proteinconcentration. Cell attachment to all films increased withincreasing protein concentrations with the exception of myofi-broblast adhesion to 1% Lac–CY–SF films (Fig. 5B). Notably,myofibroblast adhesion (Fig. 5A) was always lower than fibroblastadhesion (Fig. 5B) in comparable conditions; this difference wasstrongest on 1% Lac–CY–SF, ranging from w6-fold higherfibroblast adhesion after 2 h to w4-fold after 9 h spreading. Inaddition, fibroblasts attached more rapidly to all films, showingstrongest adhesion already 2 h after spreading (Fig. 5A) comparedwith myofibroblasts that reached strongest attachment not before6 h. Slower attachment of myofibroblasts was a generalphenomenon also observed on TCPS controls.

To qualitatively compare the different attachment kinetics ofboth cell types on Lac–CY–SF films, subcutaneous fibroblasts andTGFb1 pre-differentiated myofibroblasts were fixed after 3, 6 and9 h of spreading and stained for a-SMA (red), F-actin (phalloidin,green) and nuclei (blue) (Fig. 6). Morphological assessment indi-cated that Lac–CY conjugation of SF promoted comparable initialspreading (3 h) but faster enlargement and polarization (6–9 h) ofa-SMA-negative fibroblasts (Fig. 6A) than that of a-SMA-positivemyofibroblasts (Fig. 6B) at all SF protein concentrations. Notably,incorporation of a-SMA into stress fibers of myofibroblasts wasimpaired on Lac–CY–SF films after 9 h of spreading (Fig. 6B, 9 h)compared with TCPS control (Fig. 6D). This is appreciated frompredominant green (F-actin) stress fiber staining and diffuse red(a-SMA) staining around the nucleus (Fig. 6B, 1%); colocalization ofF-actin and a-SMA produced yellow stress fiber staining in TCPScontrol (Fig. 6C). Efficiency of cell spreading increased withincreasing SF protein concentrations from 0.01% to 1% in fibroblastand from 0.01% to 0.1% in myofibroblasts. Myofibroblast spreadingand a-SMA incorporation into stress fibers were impaired on 1%Lac–CY–SF (Fig. 6D).

3.5. Myofibroblast differentiation on SF films and scaffolds

The insensitivity of fibroblasts to the anti-proliferative effect ofTGFb1 (Fig. 4B) and the impaired incorporation of a-SMA in stressfibers (Fig. 6B) suggest that Lac–CY–SF substrates affect fibroblast-

Fig. 4. Viability and proliferation of subcutaneous fibroblasts is higher on Lac–CY–SF compared with pure SF films. Viable cell numbers were assessed with a colorimetric MTT assay(570 nm absorbance) of (A) fibroblasts and (B) myofibroblasts grown for 4 d on 1%, 0.1% and 0.01% SF and Lac–CY–SF films. Apoptosis of (C) fibroblasts and (D) myofibroblasts wasquantified using a luminometric Caspase3/7 assay. All results are represented as mean� SD from three independent experiments.

C. Acharya et al. / Biomaterials 29 (2008) 4665–46754670

to-myofibroblast differentiation. To further elucidate this point,subcutaneous fibroblasts were grown for 4 d in the absence(Fig. 7A–E) and presence of TGFb1 (Fig. 7F–J) on 1%, 0.1% and 0.01%Lac–CY–SF films, 1% pure SF (Fig. 7D,I) and TCPS control (Fig. 7E,J).After staining for a-SMA (red) and F-actin (phalloidin, green),confocal microscopy revealed that TCPS control culture supporteddifferentiation of fibroblasts to myofibroblasts under the influenceof TGFb1 (Fig. 7E,J, turquoise stress fiber staining). In contrast, SFfilm cultures clearly suppressed a-SMA expression and thus myo-fibroblast differentiation (Fig. 7A–E, green stress fiber staining).This effect was strongest on 1% pure and Lac–CY–SF films thatalmost completely abolished a-SMA expression in stress fibers(Fig. 7H,I).

One possibility of how growth on SF substrates inhibits myofi-broblast differentiation is by inhibiting the formation of large FAs.

We have previously shown that a critical FA size is the prerequisitefor generating the high intracellular tension that drives expressionof a-SMA [43]. Confocal laser scanning microscopy of vinculin-positive FAs (Fig. 7, insets) elucidated that growth on films with 1%SF protein restricted FA length of TGFb1-treated subcutaneousfibroblasts (Fig. 7H) to �6 mm length and thus to the level ofuntreated cells (Fig. 7C). This effect was less pronounced on filmswith lower SF protein concentration (Fig. 7F–G); growth on controlTCPS demonstrated formation of large FAs (�20 mm) in response toTGFb1 (Fig. 7E,J). Together, these results suggest that Lac–CY–SFfilms promote attachment, proliferation and survival of fibroblasticcells but do not support development of the fibrogenic myofibro-blast phenotype.

Fibroblast-populated 2D Lac–CY–SF films are applicable tosupport healing of burn wounds and/or chronic wounds, whereas

Fig. 5. Lac–CY–SF conjugated films promote differential adhesion for fibroblasts and myofibroblasts. (A) Fibroblasts and (B) myofibroblasts were seeded at a density of 5000 cells/film on 1%, 0.1% and 0.01% SF and Lac–CY–SF films and on control TCPS. The number of adherent cells was assayed with crystal violet staining (570 nm absorbance) after 2, 3, 6 and9 h. All results are represented as mean� SD from at least three independent experiments.

C. Acharya et al. / Biomaterials 29 (2008) 4665–4675 4671

scaffolds can be used for tissue repair in a 3D environment. Wefinally tested, whether Lac–CY–SF scaffolds, which are produced athigher SF protein concentration (6%), retain the capacity to sup-porting adherence, proliferation, and survival of fibroblasts as wellas suppress myofibroblast differentiation. For this, 30,000 subcu-taneous fibroblasts were seeded per Lac–CY–SF scaffold. After 4 dgrowth in control medium, fibroblasts showed regular spreadingmorphology within the scaffold (Fig. 8A). When treated with TGFb1during the growth in scaffolds, fibroblasts did not express a-SMA instress fibers (Fig. 8B). Moreover, TGFb1 pre-differentiated myofi-broblasts lost their fibrogenic phenotype and did not show stainingfor a-SMA in scaffolds (Fig. 8C). Even at the high SF proteinconcentrations of scaffolds Lac–CY–SF retained the anti-fibroticpotential.

4. Discussion

Efficiency of biomaterials to support physiological tissue repairrelies on a good balance between (a) being sufficiently adhesive topromote tissue integration and proliferation of delivered cells and(b) exhibiting surface properties that prevent fibroblast activation

and development of fibrosis in host tissue. We tested whether filmsand scaffolds produced of pure SF protein and Lac-conjugated SFprotein could satisfy these requirements for fibroblastic cells.Conjugation with Lac supports cell attachment, proliferation andlow rates of apoptosis, whereas pure SF promotes poor adhesionand survival of fibroblastic cells. Subcutaneous fibroblasts thatexhibit low expression levels of a-SMA in normal culture up-regulate a-SMA expression levels in response to TGFb1 whengrown in TCPS and 3D collagen substrates [39]. We here show thatmyofibroblast differentiation is suppressed by growth on bioma-terials produced from pure SF and Lac–CY–SF. Moreover, fibroblaststhat have been pre-differentiated into myofibroblasts by treatmentwith TGFb1 on TCPS lose a-SMA expression when cultured on SFbiomaterials.

On pure SF films, fibroblastic cells adhere and spread within 9 hafter seeding whereas assessment of cell morphology revealed poorFA formation and cell spreading after 4 d. This is possibly due to theabsence of RGD recognition sites for integrin-mediated cell adhe-sion in the SF of B. mori [56]. Low attachment to pure SF protein canaccount for the poor viability and proliferation as well as increasedapoptosis of fibroblastic cells because both cell survival and division

Fig. 6. Cell spreading morphology on SF films. (A) Fibroblasts and (B) myofibroblasts were seeded onto Lac–CY–SF films, produced with 0.01%, 0.1% and 1% SF protein and on TCPScontrol (C). Confocal images were produced of cells that have been stained for nuclei (blue, DAPI), filamentous actin (green, Phalloidin) and a-SMA (red) after 3, 6, and 9 h. Scale bar:25 mm. (D) The scheme summarizes the effect of increasing SF protein concentration on fibroblast/myofibroblast morphology.

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depend on cell–ECM adhesion [55]. High SF protein concentrationpromoted efficient adhesion and spreading of fibroblast and myo-fibroblasts after 9 h; it also induced significant apoptosis and poorcell proliferation after longer cell growth. Thus we conclude thatpure SF is an unsuitable biomaterial for tissue repair and func-tionalization is required.

SF has previously been conjugated to various polymers likePEG [51,57], N-acetyl oligosaccharides [50] and insulin [58].Because PEG-conjugation inhibits L929 fibroblast attachment toSF [57], we here followed a Lac functionalization strategy usingCY as linker [35]. PEG 6000 (15%) was taken as the basis for ourstudy for utilization of glycated SF as a matrix for tissue repairapplications.

Glycated polymers from SF protein have previously beenproduced by conjugating the oligosaccharide Lac, bearing ab-galactose residue, to tyrosine and lysine residues of SF using CY.Such functionalized SF films were shown to promote adhesion ofhepatocytes [35]. Several tests confirmed that our conjugation

procedure delivered comparable incorporation of Lac/b-galactoseresidues in SF films and scaffolds. First, Lac–CY–SF aggregated thelectin A. precatorius agglutinin significantly stronger than pure SF.We attribute this reaction to the recognition of b-galactose residuesin Lac–CY–SF by agglutinin [59]; its low reaction with pure SF isexplained because SF is essentially a glycoprotein [52]. Second, inNMR spectroscopy the incorporation of new peaks between 3.5 and4.5 ppm in Lac–CY–SF as compared to pure SF can be attributed tothe presence of additional methine and methylene protons of Lac inthe conjugated chain. This is consistent with the previous obser-vation of a very close association between the observed andexpected values of reacting molar ratios of tyrosine and lysineresidues with the b-galactose of Lac [35,50]. We also recorded twobroad peaks between 6.6 and 7.2 ppm in the Lac–CY–SF spectrum,overlapping with previously reported peaks of aromatic phenylal-anine residues [51]. The broadening of peaks between 2.7 and3.0 ppm could be due to the unshielding of the heterocyclic triazinering of the CY spacer linker [51]. This shows that the phenolic

Fig. 7. Growth on Lac–CY–SF films reduces the fibrogenic character of fibroblastic cells. Fibroblasts were cultured for 4 d in (A–E) control medium and (F–J) in the presence ofmyofibroblast-inducing TGFb1 and stained for filamentous actin (green, Phalloidin), a-SMA (blue) and focal adhesion protein vinculin (red and insets). Culture substrates were Lac–CY–SF films, produced with (A,F) 0.01%, (B,G) 0.1%, and (C,H) 1% SF protein, (D,I) 1% pure SF films and (E, J) TCPS control. Confocal images were produced. Scale bar: 50 mm for imagesand 20 mm for insets.

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hydroxyl group of tyrosine and the 3-amino group of lysine reactwith the chlorine of CY. Extra peaks were observed at 2.45 ppm aswell as at 1.3 ppm which could be attributed to the presence ofmethyl protons of acetyl-like groups of lysine residues [51]. In eachspectrum, the integral value of the b-methyl protons of the alanineresidue in SF at 1.38 ppm was used as an integration referencemaintained at 1.0. Integration of peaks between 3.5 and 4.5 ppm inthe SF spectrum was 1.87 (Fig. 2A), while that for Lac–CY–SF was2.53. This shows that the integral value ascribed to Lac in theinterval between 3.5 and 4.5 ppm was 0.66. This was in accordancewith comparison of NMR spectra of SF, Lac–CY–SF and a mixture ofSF and Lac [35].

Lac-conjugated SF promoted significantly better initial adhe-sion and long-term attachment of fibroblasts as well as high cell

Fig. 8. Growth in three-dimensional Lac–CY–SF scaffolds suppresses myofibroblast deveincubated for 4 d. Cells were stained for filamentous actin (green, phalloidin) and a-SMA (reauto-fluorescence of Lac-conjugated SF. (A) Scaffold culture in control medium. (B) ScaffoldTGFb1 pre-differentiated fibroblasts. Note that cells are a-SMA-negative in all conditions. S

yields and low apoptosis compared with pure SF. This can beexplained by the presence of b-galactose residues in the Lac whichenhance cell adhesion, proliferation and viability through cellsurface lectin binding [60]. It remains speculative whetherdifferent levels or types of lectin surface expression may accountfor the fact that fibroblasts in the presence of myofibroblast-inducing TGFb1 show lower initial attachment to Lac–CY–SFcompared with control conditions. Notably, after 4 d culture on 1%Lac–CY–SF in the presence of TGFb1 fibroblast proliferation wasenhanced compared to TCPS controls. This is likely due to thereducing effect of this material on myofibroblast differentiationwhich is associated with reduced proliferation rates [53,54].Hence, Lac-conjugated SF films provide satisfying conditions forfibroblast survival and attachment.

lopment. 30,000 cells were seeded into scaffolds produced with 6% Lac–CY–SF andd). Upon excitation with 405 nm and 543 nm laser light, scaffolds appear purple due to

culture in the presence of TGFb1. (C) Scaffold culture in the presence of TGFb1 withcale bar: 25 mm.

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One possible application of silk films and scaffolds revolvesaround the repair of skin wounds where they promote re-epithe-lialization [61]. The mechanical strength of 0.1% and 1% SF films inour study was sufficient to be applicable for delivering cells despiteLac–CY conjugation moderately decreased the tensile strength. Thishas also been observed in conjugations with other polymers likePEG [57] and insulin [58]. Healing of large skin wound areas such asafter burn injury is characterized by hypertrophic scarring andconsiderable tissue deformation [1]. This is mainly caused bydifferentiation of fibroblasts into myofibroblasts which contributeto physiological wound healing. However, the excessive contractileand ECM synthesizing activities of myofibroblasts are associatedwith hypertrophic scarring, skin disorders including sclerodermaand Dupuytren’s disease and organ fibrosis [1,46]. The main factorsdriving myofibroblast development are TGFb1 and mechanicalstress [41,42]. Growth on 1% Lac–CY–SF films and in 6% SF scaffoldsinhibits myofibroblast differentiation even in the presence ofexogenous TGFb1. Thus, we propose that the surface properties ofLac-conjugated SF do not allow generation of the high intracellularstress that is required to generate the contractile phenotype. Weand others have previously shown that ECM stiffness needs tosurpass a critical value to promote a-SMA expression and itsincorporation into stress fibers [43,44,62]. However, the measuredextension stiffness of SF films (MPa) is of higher magnitude thanthe minimum ECM stiffness of w15 kPa determined for myofibro-blast differentiation in culture and wound granulation tissue [43].

Another possibility to reduce intracellular tension and myofi-broblast differentiation is to reduce cell adhesion. Indeed, the levelof a-SMA expression and stress fiber organization grossly corre-lated with the attachment strength to SF films in our experiments.Films produced with pure SF in various concentrations promotedpoor myofibroblast adhesion and spreading and did not supportmyofibroblast differentiation. The most adhesive 0.1% Lac–CY–SFconjugate promoted significant levels of myofibroblast differenti-ation. Morphological analysis revealed that myofibroblast FAsformed on Lac–CY–SF films did not surpass the average length of6 mm, characteristic for a-SMA-negative fibroblasts. However,myofibroblast differentiation requires longer FAs of �8 mm [41,43].Hence, it is conceivable that FA size restriction accounts for theinhibition of myofibroblast differentiation on Lac–CY–SF, concom-itant with previous findings using micropatterned surfaces [43].

5. Conclusion

The ability of conjugated Lac–CY–SF matrices in causing de-differentiation of myofibroblasts and inhibiting de novo myofibro-blast differentiation can play an important role in the functionalapplication of silk matrices in skin wound healing and as tissueimplant. We propose that Lac–CY–SF will prevent a-SMA expres-sion by fibroblastic cells in the presence of pro-fibrotic cytokineslike TGFb1 therefore improving wound healing without inducinghypertrophic scar tissue formation. It remains to be shown whetherLac–CY–SF materials are appropriate to deliver other mesenchymalcell types that undergo myofibroblast differentiation in patholog-ical conditions, such as smooth muscle cells, hepatic stellate cellsand mesenchymal stem cells [45].

Acknowledgements

We are grateful to J. Smith-Clerc for excellent technical assis-tance, to the staff of the BioImaging and Optics platform of the EPFLfor providing image facilities and training, and to J.-J. Meister forproviding laboratory facilities. This work was financially supportedby the Department of Biotechnology, Council of Scientific andIndustrial Research, Government of India, New Delhi. CA and SCKare grateful for receiving a short-term research fellowship from the

IIT-Kharagpur–EPFL-Lausanne exchange program which was kindlycoordinated by Dr. P. Rastogi (EPFL). BH acknowledges support fromthe Swiss National Science Foundation (grants #3100A0-102150/1and #3100A0-113733/1) and the Gebert-Ruf Stiftung, Switzerlandfor this work.

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