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Biomaterials 26 (2005) 5624–5631

www.elsevier.com/locate/biomaterials

A synthetic nanofibrillar matrix promotes in vivo-like organizationand morphogenesis for cells in culture

Melvin Schindlera, Ijaz Ahmedb, Jabeen Kamalb, Alam Nur-E-Kamalb,Timothy H. Grafec, H. Young Chungc, Sally Meinersb,�

aDepartment of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USAbDepartment of Pharmacology, Robert Wood Johnson Medical School-UMDNJ, 675 Hoes Lane, Piscataway, NJ 08854, USA

cDonaldson Company, Inc., P.O. Box 1299, Minneapolis, MN 55440, USA

Received 12 November 2004; accepted 8 February 2005

Available online 18 April 2005

Abstract

The purpose of this study was to design a synthetic nanofibrillar matrix that more accurately models the porosity and fibrillar

geometry of cell attachment surfaces in tissues. The synthetic nanofibrillar matrices are composed of nanofibers prepared by

electrospinning a polymer solution of polyamide onto glass coverslips. Scanning electron and atomic force microscopy showed that

the nanofibers were organized into fibrillar networks reminiscent of the architecture of basement membrane, a structurally compact

form of the extracellular matrix (ECM). NIH 3T3 fibroblasts and normal rat kidney (NRK) cells, when grown on nanofibers in the

presence of serum, displayed the morphology and characteristics of their counterparts in vivo. Breast epithelial cells underwent

morphogenesis to form multicellular spheroids containing lumens. Hence the synthetic nanofibrillar matrix described herein

provides a physically and chemically stable three-dimensional surface for ex vivo growth of cells. Nanofiber-based synthetic matrices

could have considerable value for applications in tissue engineering, cell-based therapies, and studies of cell/tissue function and

pathology.

r 2005 Elsevier Ltd. All rights reserved.

Keywords: Biomimetic material; Nanotopography; ECM; Cell culture

1. Introduction

Cell development, organization, and function intissues are regulated by interactions with a diversegroup of macromolecules that comprise the extracellularmatrix (ECM). For various mesenchymal and tumorcells [1,2], the ECM provides a surrounding coat offibrils that is in contact with both apical and basal cellsurfaces. The basement membrane, a structurallycompact form of ECM, uniquely makes contact withthe basal surfaces of cells that form tissues, e.g. epithelialand endothelial cells [1,2]. The three-dimensionalstructure of the basement membrane/ECM has been

e front matter r 2005 Elsevier Ltd. All rights reserved.

omaterials.2005.02.014

ing author. Tel.: +1732 235 2890.

ess: meiners@umdnj.edu (S. Meiners).

shown to be as important as the chemistry in itsinfluence on cellular processes such as tumor develop-ment and drug sensitivity [3,4].The vast majority of culture work has been performed

using two-dimensional plastic and glass cell culturesurfaces. However, such culture media do not reflect thethree-dimensional geometry and porosity observed forthe cell attachment sites and migration pathways withinand between tissues. Nonetheless, two-dimensionalculture surfaces have the advantage that they areuniform and can be reproducibly manufactured toprecise tolerances. It is now generally acknowledgedthat the movement from cell to true tissue culture willrequire new three-dimensional environments, preferablysynthetic, to faithfully recapitulate cell-basement mem-brane/ECM interactions within tissues [4–6].

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To pursue the goal of creating chemically andphysically stable synthetic three-dimensional surfacesthat mimic the structural geometry and porosity ofbasement membrane/ECM, we have utilized the techni-que of electrospinning to produce nanofibers that self-assemble into three-dimensional nanofibrillar networks[7–9]. The physical form of the nanofibrillar matricesprovides unprecedented porosity (470%), high spatialinterconnectivity, and a high surface to volume ratio forcell attachment. In the current study, we demonstratethat nanofibrillar matrices promote in-vivo-like cellmorphology and organization of cytoskeletal compo-nents in NIH 3T3 fibroblasts and normal rat kidney(NRK) cells. In addition, these nanofibrillar surfaces arepermissive for the morphogenesis of breast epithelialcells (T47D) into multicellular spheroids containinglumenal cavities.Most significantly, nanofibers can be incorporated

into: (a) large-scale cell culture protocols such as rollercultures and cell reactors/fermentors; (b) high through-put drug screening protocols; and (c) scaffolds for exvivo culturing of cells for use in various applications oftissue engineering and cell-based therapies. In addition,the fibrillar structure of the nanofiber matrices structu-rally resembles the connective tissue and basementmembrane through which tumor cells migrate in theprocesses of metastasis and intravasation [10,11]. Thisphysical similarity suggests a potential role for nanofi-brillar surfaces in studies of amoeboid tumor cellmigration [10]. Thus polyamide nanofibers provide anew three-dimensional cell culture surface for morephysiologically relevant cell growth that can be incor-porated into a variety of applications.

2. Materials and methods

2.1. Materials

The reagents and dilutions employed in this studywere as follows. Phalloidin-Alexa Fluor 488 (1:100),monoclonal cellular fibronectin antibody (1:500), mono-clonal beta tubulin antibody (1:500), monoclonal actinantibody (1:500), and monoclonal vinculin antibody(1:400) were from Sigma Chemical Co. (St. Louis, MO).CY3-goat anti-mouse IgG (H+L) (1:500), CY3-donkeyanti-rabbit IgG (H+L) (1:500), CY3-streptavidin(1:500), normal goat serum (1:10), and normal donkeyserum (1:10) were from Jackson Labs (West Grove, PA).FAK (PY397) rabbit polyclonal antibody (1:500) wasobtained from Biosource (Camarillo, CA). Biotin-mouse anti-hamster IgM (monoclonal) (1:100) wasobtained from Pharmingen (San Diego, CA). Hamsteranti-rat/mouse CD29 (Integrin beta 1) IGM (1:100) wasobtained from RDI (Flanders, NJ). Gel/Mount (aqu-eous mounting medium with antifading agents) was

obtained from Biomeda (Foster City, CA). Dulbecco’sModified Eagle’s Medium (DMEM) (high glucose), calfserum, and fetal calf serum were all obtained fromInvitrogen (Carlsbad, CA). Coverslips (18mm, No.1)were purchased from Fisher Scientific (New Brunswick,NJ). NRK cells were a gift from Dr. Sanford Simon(Rockefeller University, New York, NY), and T47Dbreast epithelial cells were a gift from Dr. Leroy Liu(Robert Wood Johnson Medical School-UMDNJ,Piscataway, NJ). Cell culture plates were purchasedfrom Corning (Corning, NY). Nanofibrillar matrices arecommercially available as Ultra-WebTM Synthetic ECMand were obtained from Donaldson Co. (Minneapolis,MN).

2.2. Nanofiber production by electrospinning

The nanofibers were electrospun using an adaptedindustrial electrospinning process with a field strength of30 kV as described [12]. The nanofibers were electrospunonto glass coverslips (18mm, No.1) in a controlledthickness and fiber density. The nanofibers are com-posed of two kinds of polyamide polymers, A(C28O4N4H47)n and B (C28O4.4N4H47)n, that have beencross-linked in the presence of an acid catalyst.

2.3. Cell culture and fluorescence staining

NIH 3T3 and NRK cells were seeded at 5� 104 cells/ml and cultured in DMEM (4.5 g/l glucose) in thepresence of 10% calf serum at 5% CO2 and 37 1C, whileT47D breast epithelial cells and MCF-7 cells wereseeded at 5� 104 cells/ml and cultured in DMEM (4.5 g/lglucose) in the presence of 10% fetal calf serum at 5%CO2 and 37 1C. All cell types were grown on either glassor nanofiber-coated glass coverslips in 12-well cellculture plates.Staining for F-actin was performed in the following

manner. Cells were rinsed once with phosphate-bufferedsaline (PBS), fixed with 4% paraformaldehye in PBS(15min), washed with PBS, treated with 0.5% Triton X-100 (5min), washed with PBS, incubated with phalloi-din-Alexa Fluor 488(diluted with PBS containing 0.3%Tween-20) for 1 h, washed with PBS (3� , 5min perwash), and then mounted on a slide with Gel/Mount.Imaging was performed with a Zeiss Axioplan Epi-Fluorescent Microscope.Mouse monoclonal antibody staining was performed

as described for phalloidin-Alexa Fluor 488 except afterthe treatment with Triton X-100, cells were washed withPBS, blocked with normal goat serum (diluted withPBS/0.3% Tween-20) for 30min at room temperature,washed with PBS (3� , 5min per wash), incubatedovernight with primary antibody, washed with PBS(3� , 5min per wash) followed by incubation for 1 hwith the secondary antibody goat anti-mouse IGg*CY3

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(diluted with PBS/0.3% Tween-20), washed with PBS(3� , 5min per wash), and then mounted on a slide withGel/Mount. Staining with rabbit polyclonal antibodiessubstituted the following steps: blocking was performedwith normal donkey serum (diluted with PBS containing0.3% Tween-20) for 30min at room temperature, andthe incubation with secondary antibody utilized donkeyanti-rabbit IGg*CY3 (diluted with PBS/0.3% Tween-20) for 1 h. Confocal imaging was performed with aZeiss LSM-410 microscope. Routine controls for stain-ing were performed.

2.4. Physical characterization of nanofibers

For scanning electron microscopy (SEM), sampleswere sputter coated with gold and examined under highvacuum using a JEOL model JSM-5900. Scanning forcemicroscopy (SFM) imaging of the nanofiber-coatedsurfaces was performed in ambient air using a DigitalInstruments Nanoscope IIIa operated in tapping modewith etched silicon probes, each with a nominal tipradius of curvature of 5–10 nm.

3. Results and discussion

3.1. Scanning electron and scanning force microscopy of

nanofibers

SEM of nanofiber-coated glass surfaces demonstratedan integrated network of overlapping fibers and pores(Fig. 1A,B). Characterization of fiber diameters demon-strated a distribution centered at approximately 180 nm(Fig. 1C). The fibers are structurally continuous witheach other at crossing points. Analysis of individualnanofibers using SFM showed a fiber with a diameter ofapproximately 180 nm (Fig. 1Da) and a pore diameter ofapproximately 700 nm (Fig. 1 Db). The surface smooth-ness was measured to be within 5 nm over a length of1.5 mm (unpublished results). A similar three-dimen-sional organization of fibers has been demonstrated forbasement membranes [13,14].

3.2. Organization of the actin cytoskeleton and focal

adhesion components in NIH 3T3 cells cultured on glass

and nanofiber-coated glass surfaces

A number of cell types were employed to test theeffect of nanofibrillar surfaces on cell structure andgrowth. We first examined intracellular components(actin, focal adhesion components) whose organizationand structure change as a function of adhesion on two-vs. three-dimensional substrates [5,15,16]. The F-actindistribution within NIH 3T3 fibroblasts (Fig. 2) andNRK cells (Fig. 3) was first observed after 24 h ofgrowth utilizing phalloidin-Alexa Fluor 488 (phalloidin-

Alexa Fluor). As shown in Fig. 2A, fibroblasts plated onglass were well spread with an elaborate checkerboardpattern of stress fibers, as previously described [5,16]. Incontrast, cells on nanofiber-coated surfaces showed achange in shape, becoming more elongated and bipolarwith thinner actin fibers arranged parallel to the longaxis of the cell. A notable increase was observed in theformation of actin-rich lamellipodia, membrane ruffles,and cortical actin (Fig. 2B).Fibroblasts and NRK cells cultured on nanofiber-

coated surfaces also demonstrated about 40–50% lessactin than did cells cultured on glass (data not shown).In addition, fibroblasts and NRK cells demonstratedenhanced rates of proliferation after 24 h (1.9� forfibroblasts and 1.5� for NRK cells) (data not shown).Similar changes in actin organization, total actinamount, and rates of proliferation have been describedfor cells cultured on cell-free three-dimensional matricesderived either from detergent-extracted mouse embryosections or from naturally deposited three-dimensionalECMs of NIH 3T3 fibroblasts that were denuded of cells[5]. These three-dimensional matrices, in the experimentsdescribed [5], resemble the basement membrane in thatthey only contact the basal cell surface.Vinculin is a prominent component of focal com-

plexes and focal adhesions [17,18] that links thecytoskeleton, plasma membrane, and ECM [19]. Asshown in Fig. 2C, immunocytochemical techniquesdemonstrated a pattern of vinculin staining after 24 hof growth that was arranged in parallel streaks,characteristic for adhering fibroblasts cultured on two-dimensional glass or tissue culture plastic [15–17]. Incontrast, streaked staining for vinculin within fibroblastscultured on nanofibers was limited to the edge oflamellipodia with a more diffuse staining throughout thecell cytoplasm (Fig. 2D). As with actin, such changes inthe pattern of vinculin labeling have been shown tocorrelate with cellular differentiation and morphogenesisin vivo [20,21].Focal adhesion kinase (FAK) has been proposed to

function as a central mechano-sensing transducer in cells[22]. In particular, phosphorylation of FAK at tyrosine397 (FAK PY397) in fibroblasts and breast epithelialcells has been demonstrated to be a key signaling event[5,20]. We utilized an antibody specific for FAK PY397

to examine its distribution in fibroblasts 24 h afterseeding cells [5,20]. As observed in Fig. 2E, cells culturedon glass demonstrated a streaky pattern of labeling,which was similar to vinculin staining in cells culturedon glass (Fig. 2C). The localization of FAK PY397 forcells on nanofibers was more punctate and less welldefined (Fig. 2F) and was also similar to that observedfor vinculin in cells on nanofibers (Fig. 2D). Likevinculin labeling, the FAK PY397 labeling observed onglass is characteristic of localization at focal adhesions[5,15,17]. Loss of FAK PY397 localization at focal

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Fig. 1. Physical characterization of nanofibers. (A,B) SEM images of a glass coverslip coated with nanofibers. Samples were sputter coated with gold

and examined under high vacuum using a JEOL model JSM-5900. (C) A histogram of nanofiber diameters within the network. (D) AFM imaging of

the nanofiber surfaces was performed in ambient air using a Digital Instruments Nanoscope IIIa operated in tapping mode with etched silicon

probes, each with a nominal tip radius of curvature of 5–10 nm. AFM analysis shows the diameter (D(a)) of a single fiber and (D(b)) shows the

diameter of a pore within the network. Scale bar in (A), 10 mm and (B) 5mm.

M. Schindler et al. / Biomaterials 26 (2005) 5624–5631 5627

adhesions has been correlated with morphogenesis anddifferentiation in breast epithelial cells [20]. Mostnotably, Cukierman et al. [5] reported a loss of FAKPY397 staining at adhesion sites and a decrease in theamount of phosphorylated FAK for fibroblasts culturedon cell-free three-dimensional matrices derived eitherfrom detergent-extracted mouse embryo sections orfrom naturally deposited three-dimensional ECMs ofNIH 3T3 fibroblasts [5]. They named such sites, whichare more characteristic for cells in tissue, ‘‘3D-matrixadhesions’’ [5,15].Importantly, neither cell type adhered to films with

smooth surfaces that were made from the identicalformulation of polyamide (data not shown). Thisindicates that the phenotype observed for fibroblastsand NRK cells cultured on nanofibers is a directconsequence of the three-dimensional nanotopographyof the nanofibers as opposed to the chemistry of thepolyamide.

3.3. Organization of fibronectin on NIH 3T3 fibroblasts

following 24 h of culture on glass and nanofiber-coated

glass surfaces

We next examined the distribution of fibronectin atthe cell surface. After 24 h of culture on glass, fibroblastsdisplayed a classic linear pattern of fibrils (Fig. 2G)[5,23,24]. This is contrasted with the formation of athicker network of more randomly deposited apicallylocalized fibrils for fibroblasts grown on nanofibers(Fig. 2H). This later pattern of fibronectin deposition issimilar in structure and fibrillar dimensions to the three-dimensional matrices employed in the study by Cukier-man et al. [5] and may suggest that in contrast to two-dimensional surfaces, the nanofiber-based matrices usedin our study are permissive for the assembly of a matrixthat can promote the formation of 3D-matrix adhesions.Isolated cells were also observed to produce an intenselystained network of fibronectin fibrils after 24 h (Fig. 2H,

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(A) (B)

(D)(C)

(E) (F)

(G) (H)

Fig. 2. A comparison of the F-actin network, focal adhesion components, and fibronectin organization for NIH 3T3 fibroblasts cultured on glass

and nanofibers. Fluorescent image of phalloidin-Alexa Fluor staining on glass (A) and nanofibers (B). Indirect immunofluorescence of fibroblasts on

glass (C,E,G) and nanofibers (D,F,H) stained with vinculin (C,D), FAK PY397 (E,F), and fibronectin (G,H) antibodies. The extent of nuclear

labeling was variable. Scale bar, 10mm.

M. Schindler et al. / Biomaterials 26 (2005) 5624–56315628

insert), a condition not observed after 24 h of growth onglass (not shown).

3.4. Organization of the actin cytoskeleton and focal

adhesion components in normal rat kidney (NRK) cells

cultured on glass and nanofiber-coated glass surfaces

Results with NRK cells (Fig. 3) closely paralleled theobservations for fibroblasts (Fig. 2) when staining foractin (Fig. 3A,B), vinculin (Fig. 3C,D), and FAK PY397

(Fig. 3E,F) were compared for cells grown on glass andnanofiber-coated surfaces. There were, however, somenoteworthy exceptions. For example, the extent offibronectin staining appeared to be less for NRK cellsthan for fibroblasts on both glass and nanofibers

(compare Fig. 2G and H with Fig. 3G and H).Furthermore, the distribution of fibronectin on theNRK cell surface was distinctly different from that onfibroblasts, with a more peripheral localization for NRKcells on both substrates (Fig. 3G,H). However, as withfibroblasts, there appeared to be more fibril formationfor NRK cells grown on nanofibers (Fig. 3H). Hence,matrix production and organization for one cell typecannot necessarily be extrapolated to another cell type.

3.5. Distribution of b-1 integrins on the surface of NRK

cells grown on glass and nanofiber-coated glass surfaces

Patterns of integrin organization and expression havebeen demonstrated to change in response to cell

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(A) (B)

(D)(C)

(E) (F)

(G)

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Fig. 3. A comparison of the F-actin network, focal adhesion components, fibronectin, and b1 integrin organization in NRK cells. Fluorescent image

of phalloidin-Alexa Fluor staining on glass (A) and nanofibers (B). Indirect immunofluorescence images of NRK cells on glass (C,E,G,I) and

nanofibers (D,F,H,J) stained with vinculin (C,D), FAK PY397 (E,F), fibronectin (G,H), and b1 integrin antibodies. The extent of nuclear labeling wasvariable. Scale bar, 10 mm.

M. Schindler et al. / Biomaterials 26 (2005) 5624–5631 5629

interactions with two-dimensional substrates vs. three-dimensional matrices [3–5,15,25] and also in response tothe flexibility of these matrices [26]. For example, b1integrin was localized at classical focal adhesions in cellscultured on two-dimensional substrates and at themorphologically distinct, long and slender 3D-matrixadhesions for cells cultured on three-dimensionalmatrices [5,15]. In the current study, staining for b1integrin was punctate in NRK cells cultured on glass(Fig. 3I) and organized in long, slender aggregates for

cells cultured on nanofibers (Fig. 3J). This may reflectlocalization of b1 integrin in focal adhesions and 3D-matrix adhesions.

3.6. Morphogenesis of T47D breast epithelial cells

cultured on nanofibers

To determine whether nanofiber matrices can func-tion as permissive surfaces for morphogenesis, wecultured T47D breast epithelial cells on glass and

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(A) (B) (D)(C)

(E) (F) (G) (H)

Fig. 4. A series of confocal sections of a multicellular spheroid composed of T47D breast epithelial cells grown on nanofibers and stained with

phalloidin-Alexa Fluor(A–D). Note the lumen extending through the spheroid. Sections were taken at 0 (A), 20 (B), 34 (C), and 48 (D) micron steps

from the top. A fluorescent image is shown of a tubule (E). T47D cells after 10 days of culture on glass (F). MCF-7 cells grown on glass and

nanofibers, respectively stained with phalloidin-Alexa Fluor 488 (G and H). Scale bar, 10 mm.

M. Schindler et al. / Biomaterials 26 (2005) 5624–56315630

nanofiber-coated glass coverslips. These cells have beendemonstrated to form duct-like tubular structures andspheroids under conditions that promote three-dimen-sional interactions with collagen or matrigel [4,20].Cultures were stained with phalloidin-Alexa Fluor andexamined at 5, 8, and 10 days. After 5 days of growth onnanofibers, we observed a mixed population of multi-cellular structures comprised of tubules and spheroids(Fig. 4). A decrease in the number of tubules wasobserved after 8 days (data not shown). Following 10days in culture, the multicellular spheroids weredominant (Fig. 4A–D), although some tubules persisted(Fig. 4E). Fig. 4A–D shows a confocal series through amulticellular spheroid demonstrating a lumen. Thelowest portion of the spheroid, in contact with thenanofibers (Fig. 4D), appears to have an opening. Amulticellular tubule with an elongated lumen is shown inFig. 4E. In contrast, growth of T-47D cells on glassdemonstrated a monolayer with groups of F-actin fibers(Fig. 4F). Interestingly, while MCF-7 cells (a humanbreast tumor line) proliferated as a monolayer on glass(Fig. 4G), these cells grew as more complex multilayerson nanofibers (Fig. 4H).

4. Conclusions

We have used a number of published criteria [5,15] todemonstrate that a chemically and physically stablesynthetic three-dimensional surface composed of elec-trospun polyamide nanofibers allows NIH 3T3 fibro-blasts and NRK cells to form more in vivo-likemorphologies. This surface is also permissive for

epithelial cells to undergo morphogenesis. We anticipatethat such nanofibrillar 3D surfaces will have majorbenefits for applications in tissue engineering [27,28],high throughput drug screening [29,30], and regenerativemedicine.

Acknowledgements

This work was supported by National Institutes ofHealth Grant R01 NS40394 and New Jersey Commis-sion on Spinal Cord Research Grants 04-3034-SCR-E-Oto S.M. and a grant from The Center for Plant Productsand Technology (MSU, E. Lansing, MI) to M.S. (NewJersey grant was inadvertently left out).

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