Three-dimensional nanofibrillar surfaces covalentlymodified with tenascin-C-derived peptides enhanceneuronal growth in vitro

Ijaz Ahmed,1,* Hsing-Yin Liu,1,* Ping C. Mamiya,2 Abdul S. Ponery,1 Ashwin N. Babu,1 Thom Weik,3

Melvin Schindler,4 Sally Meiners1

1Department of Pharmacology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey 088542Department of Psychology, Busch Campus, Rutgers University, New Brunswick, New Jersey 089033Donaldson Company, Inc., P.O. Box 1299, Minneapolis, Minnesota 554404Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824

Received 22 July 2005; revised 18 August 2005; accepted 19 August 2005Published online 12 December 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.30587

Abstract: Current methods to promote growth of culturedneurons use two-dimensional (2D) glass or polystyrene sur-faces coated with a charged molecule (e.g. poly-l-lysine(PLL)) or an isolated extracellular matrix (ECM) protein (e.g.laminin-1). However, these 2D surfaces represent a poortopological approximation of the three-dimensional (3D) ar-chitecture of the assembled ECM that regulates neuronalgrowth in vivo. Here we report on the development of a new3D synthetic nanofibrillar surface for the culture of neurons.This nanofibrillar surface is composed of polyamide nano-fibers whose organization mimics the porosity and geometryof the ECM. Neuronal adhesion and neurite outgrowth fromcerebellar granule, cerebral cortical, hippocampal, motor,and dorsal root ganglion neurons were similar on nanofibersand PLL-coated glass coverslips; however, neurite genera-

tion was increased. Moreover, covalent modification of thenanofibers with neuroactive peptides derived from humantenascin-C significantly enhanced the ability of the nanofi-bers to facilitate neuronal attachment, neurite generation,and neurite extension in vitro. Hence the 3D nanofibrillarsurface provides a physically and chemically stabile cellculture surface for neurons and, potentially, an exciting newopportunity for the development of peptide-modified ma-trices for use in strategies designed to encourage axonalregrowth following central nervous system injury. © 2005Wiley Periodicals, Inc. J Biomed Mater Res 76A: 851–860,2006

Key words: nanofiber; neuron; tenascin-C; peptide; extracel-lular matrix

INTRODUCTION

Neurobiologists have predominantly employedtwo-dimensional (2D) surfaces composed of polysty-rene or glass for the culture of primary neurons. Thesesurfaces must first be coated either with a chargedmolecule, commonly poly-l-lysine (PLL), or with pro-

teins derived from the extracellular matrix (ECM),such as laminin-1 or fibronectin. In the absence of oneof these coatings, primary neurons will not adhere tothe polystyrene or glass. However, there is increasingrecognition that these coated 2D surfaces have distinctlimitations. For example, the coatings themselves areexpensive and time-consuming to prepare. Further-more, PLL can demonstrate considerable variation inits secondary structure, which could correspondinglyaffect its activity, depending on the temperature, pH,and solvent polarity of the dissolving solution.1,2

Moreover, laminin-1 and fibronectin are instructivesubstrates that can activate neuronal receptors andsignal transduction pathways,3 potentially interferingwith the study of other neuroactive molecules. Finally,the flatness of these 2D surfaces provides a poor ap-proximation of the more complex three-dimensional(3D) architecture of the astrocyte-derived ECM4 andthe Schwann cell-derived basement membrane5 thatregulate neuronal growth and regeneration in vivo.

*These authors contributed equally to this manuscript.One or more of the authors, has received or will receive

remuneration or other perquisites for personal or profes-sional use from a commercial or industrial agent in direct orindirect relationship to their authorship.

Correspondence to: S. Meiners, Department of Pharmacol-ogy, UMDNJ-Robert Wood Johnson Medical School, 675Hoes Lane, Piscataway, New Jersey 08854, USA; e-mail:meiners@umdnj.edu

Contract grant sponsor: National Institutes of Health; con-tract grant number: R01 NS40394

Contract grant sponsor: New Jersey Commission on Spi-nal Cord Research; contract grant number: 04–3034 SCR-E-O

© 2005 Wiley Periodicals, Inc.

In this report, we evaluated a completely synthetic3D matrix of electrospun polyamide nanofibers as aculture surface for neurons. Nanofibers produced viathe process of electrospinning6 have unprecedentedporosity (�70%), a high surface-to-volume ratio, andhigh interconnectivity, all physical properties ideal forpromoting cell attachment and growth.7 Furthermore,the nanotopography of electrospun nanofibers closelyresembles the nanofibrillar and nanoporous 3D geom-etry of the ECM and basement membrane.8,9 We pre-viously used the electrospun polyamide nanofibersdescribed herein as a culture surface for NIH 3T3fibroblasts and normal rat kidney (NRK) cells.10,11 TheNIH 3T3 fibroblasts and NRK cells readily adhered tothe nanofibers and adopted a migratory-like pheno-type that was more characteristic of their in vivo cel-lular counterparts. Therefore, we hypothesized thatthe 3D nanofibrillar surface might also support neu-ronal adhesion and subsequent neurite extension evenin the absence of PLL or proteins derived from theECM.

Because of the potential of nanofibrillar substratesto be used as grafts in neuroregenerative medicine,12

we also developed a simple method to covalentlymodify polyamide nanofibers with neuroactive pep-tides. Two peptides were used in this study, the firstderived from alternatively spliced fibronectin type IIIrepeat D of human tenascin-C, called the D5 peptide(VFDNFVLKIRDTKKQ), and the second an extendedversion of the D5 peptide, called the D5� peptide (AD-EGVFDNFVLKIRDTKKQ). The D5 peptide was pre-viously identified as a neurite outgrowth promotingmotif for cerebellar granule neurons, with the aminoacids FD and FV required for activity.13,14 In the cur-rent report, we hypothesized that the extra aminoacids at the N-terminus of the D5� peptide might allowfor better access to the active site by neuronal growthcones. The ability of the polyamide nanofibers to stim-ulate neuronal growth was evaluated in the absenceand presence of the tenascin-C-derived peptides. Theresults indicate that polyamide nanofibers provide anew culture surface for neurons and further suggestthat they may have important applications in peptide-based approaches to central nervous system (CNS)repair.

MATERIALS AND METHODS

Polyamide nanofibers

Coverslips coated with electrospun polyamide nanofibers(Ultra-Web�) were obtained from Donaldson Co. (Minneap-olis, MN) (http://www.donaldson.com/en/filtermedia/nanofibers/index.html or www.synthetic-ecm.com). Theelectrospinning process uses an electric field to create nano-

fibers, as follows. A polymer solution is injected with anelectrical potential. This creates a charge imbalance, whichleads to the ejection of a polymer stream from the tip of aneedle. As the polymer stream is ejected from the needle, thesolvent flash evaporates, resulting in the formation of acontinuous fiber that collects as a nonwoven fabric. Thenanofibers used in this study were electrospun onto 12-mmglass coverslips in a controlled thickness (4–5 �m) and fiberdensity from a blend of two polymers, (C28O4N4H47)n and(C27O4.4N4H50)n. The nonwoven nanofibrillar fabric wascrosslinked in the presence of an acid catalyst. Samples forscanning electron microscopy (SEM) were sputter-coatedwith gold and examined under high-vacuum using a JEOLmodel JSM-5900. Other coverslips were placed into 24-wellplates, sterilized under ultraviolet light for 15 min, and usedas a 3D nanofibrillar surface for neuronal culture.

Peptides and antibodies

Synthetic peptides (fluorescein-labeled and unlabeled)were prepared by BioSynthesis Inc. (Lewisville, TX). Thepeptides were characterized by the company using bothmass spectral analysis and HPLC tracing. Monoclonalmouse antibodies against neurofilament-M and peripherinwere from Chemicon (Temecula, CA). A Cy3-conjugatedgoat anti-mouse secondary antibody was from Jackson Im-munoResearch (West Grove, PA).

Neuronal culture

Cerebellar granule neuronal cultures were prepared frompostnatal day 8 (P8) rat pups as described previously.14

Cerebral cortical neuronal cultures were prepared from em-bryonic day 15 (E15) rat pups as described previously,13 andhippocampal and ventral spinal cord neuronal cultures wereprepared from E15 rat pups as described previously.15,16

Dorsal root ganglion neuronal cultures were prepared fromadult rats using methods adapted from Davies et al.17 Na-tional Institutes of Health (NIH) guidelines for the care anduse of laboratory animals (NIH Publication No. 85–23 Rev.1985) were strictly observed.

The culture media used were as follows: cerebellar gran-ule neurons were cultured in neurobasal medium (GIBCOBRL) (Rockville, MD) supplemented with B27 (GIBCO BRL)and 25 mM KCl. Cerebral cortical, hippocampal, ventralspinal cord, and dorsal root ganglion neurons were culturedin neurobasal medium supplemented with B27. Cerebellargranule, cerebral cortical, hippocampal, and ventral spinalcord neurons were plated at a density of 30,000 neurons/well onto glass coverslips in 24-well plates coated with PLL(Sigma Chemical Co., St. Louis, MO) (MW 361,850–574,500;100 �g/mL) or nanofibers. Dorsal root ganglion neuronswere plated at a density of 10,000 neurons/well.

Neurite outgrowth assay

To assess the ability of the 3D nanofibrillar surface tosupport neurite outgrowth in vitro, neuronal process exten-

852 AHMED ET AL.

sion was first compared on PLL- and nanofiber-coated glasscoverslips in the absence of neuroactive peptides. Cerebellargranule, cerebral cortical, hippocampal, and ventral spinalcord neurons were allowed to extend processes on the 2Dand 3D surfaces for 24 h, at which time they were fixed withparaformaldehyde (4%). After fixation, cerebellar granule,cerebral cortical, and hippocampal neurons were immuno-stained with monoclonal mouse antibody against neurofila-ment-M (1:400 dilution overnight at room temperature), fol-lowed by a Cy3-conjugated goat anti-mouse secondaryantibody (1:500 dilution for 1 h at room temperature). Ven-tral spinal cord neurons were immunostained with a mono-clonal mouse antibody against peripherin to identify motorneurons (1:100 dilution overnight at room temperature),followed by a Cy3-conjugated goat anti-mouse secondaryantibody (1:500 dilution for 1 h at room temperature).

Images of the cultures were captured using a Zeiss Axio-plan microscope equipped with an epifluorescence illumi-nator. Image analysis was then performed using NIH ImageJ Software.18 For image analysis, only neurons with pro-cesses equal to or longer than the diameter of one cell somawas considered for each condition. The length of each pri-mary process and its branches was measured for each neu-ron, and the total neurite length was calculated as the sum ofthe lengths of individual neurites. The percentage of neu-rons with one or more neurites equal to or longer than thediameter of one cell soma was also determined for a sampleof 100 neurons for each condition.

Covalent modification of polyamide nanofiberswith tenascin-C-derived peptides

To assess whether the ability of the polyamide nanofibersto support neurite outgrowth in vitro could be increased bythe addition of neuroactive peptides, we covalently modi-fied the 3D nanofibrillar surface with the tenascin-C-derivedpeptides D5 and D5�. Each peptide was synthesized with anextra cysteine at the N-terminus. The D5 peptide (aminoacid sequence � VFDNFVLKIRDTKKQ) was previouslyshown to promote neurite outgrowth from cerebellar gran-ule neurons and required the FD and FV amino acids foractivity.13 Although the 8 amino acid peptide VFDNFVLKpromoted neurite outgrowth to the same extent as the D5peptide,13 we chose to use the D5 peptide in the currentstudy because it supports substantially more neuronal ad-hesion than does VFDNFVLK (S. Meiners, unpublisheddata). Furthermore, because the covalent modificationmethod (below) links the N-terminal cysteine of the D5peptide to the surface of the nanofibers, we also employedthe D5� peptide (amino acid sequence � ADEGVFDNFV-LKIRDTKKQ). We hypothesized that the proximity of theFD/FV active site to the nanofiber surface in the D5 peptidemight obstruct its ability to promote neurite outgrowth, andthat the four additional amino acids at the N-terminus of theD5� peptide would allow for better presentation of epitopeto neurons.

To modify the nanofiber surface with peptides, polyamidenanofibers electrospun onto glass coverslips were first co-valently coated with a proprietary polyamine polymer usingPhotoLink® chemistry by Surmodics (Eden Prairie, MN). A

solution containing the heterobifunctional crosslinker Sulfo-LC-SDPD (Pierce Biotechnology, Rockford, IL) (100 �g/mLin phosphate-buffered saline (PBS)) was incubated with theresulting amine-coated nanofibers for 1 h. Excess crosslinkerwas rinsed away with PBS. D5 or D5� peptide (50 �g/mL inPBS) was incubated with the nanofibrillar matrix for 1 h, andexcess peptide was rinsed away with PBS. Substrate coatingefficiencies were determined by incubating nanofiber-coatedcoverslips with peptides labeled with fluorescein. After 1 h,excess peptide was rinsed away with PBS, and �-mercapto-ethonal (150 mM in PBS) was added to reduce the disulfidelinkage and free the bound peptide. The fluorescence of thebound peptide was then assessed as we have describedpreviously.13,14

To evaluate the activity of tenascin-C-derived peptides ina 3D environment, the D5 and D5� peptides were covalentlybound to polyamide nanofibers as described above. Alter-natively, the peptides were passively adsorbed to the surfaceof either nanofiber- or PLL-coated glass surfaces. Cerebellargranule or ventral spinal cord neurons were then platedonto the 2D and 3D peptide-modified surfaces at a density of50,000 neurons/well and cultured for 24 h. Dorsal rootganglion neurons were plated at a density of 10,000 neu-rons/well and cultured for 24 h. At that time, the neuronswere fixed with paraformaldehyde, immunostained as de-scribed above using an antibody against neurofilament-Mfor cerebellar granule and dorsal root ganglion neurons andan antibody against peripherin for ventral spinal cord neu-rons, and used for neurite outgrowth assays.

RESULTS

Polyamide nanofibers form a fibrous scaffold forneuronal growth in vitro

Polyamide nanofibers were electrospun onto 12-mmglass coverslips to create nanofiber-coated coverslips.The 3D nanofibrillar surface formed a network of fil-aments interspersed with pores approximately 100–800 nm in diameter (Fig. 1). The distribution of nano-fiber diameters ranged from �40 to 400 nm with amedian diameter of 180 nm.10 The nanofiber networkstructurally resembles the ECM and the basementmembrane, a more compact form of the ECM.8 How-ever, polyamide nanofibers are completely syntheticand lack native ECM or basement membrane mole-cules. As such, this 3D surface was designed to sepa-rate chemical cues of the ECM/basement membranefrom geometric cues.

To investigate whether neurons could attach andextend neurites on polyamide nanofibers, rat cerebel-lar granule, cerebral cortex, hippocampal, or ventralspinal cord neurons were plated onto nanofiber-coated coverslips and cultured for 24 h. The cerebellargranule, cerebral cortical, and hippocampal neuronswere stained with an antibody against neurofila-ment-M, and the ventral spinal cord neurons were

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stained with an antibody against peripherin to iden-tify motor neurons. The amount of neuronal attach-ment (as assessed by counting the number of neuronsper field), the percentage of neurons that extendedneurites, and the length of the extended neurites werecompared for neurons cultured on nanofibers andneurons cultured on glass coverslips coated with PLL,our standard laboratory control for neuronal cul-ture.13,14

We first evaluated the mean number of neurons perfield in 15 nonoverlapping fields of the coverslip(equivalent to �0.5 mm2) on PLL- and nanofiber-coated coverslips. As shown in Figure 2(A), neuronalattachment was similar on both surfaces. In contrast,the number of neurons per nonoverlapping field of thecoverslip was zero when a flat membrane of poly-amide was substituted for nanofibers. Hence poly-amide nanofibers supported the initial attachment ofneurons, and the nanotopography of the surface wasessential for this function. The significance of nanoto-pography is also supported by the fact that the poly-amide composition of the nanofibers, unlike PLL, isnot highly charged and cannot support neuronal ad-hesion through a charge-dependent mechanism.

The mean number of neurons per field reflected allattached neurons, regardless of whether those neu-rons extended neurites. Therefore, the percentage ofneurons with neurites was next evaluated on 2D PLL-and 3D nanofiber-coated coverslips [Fig. 2(B)]. A sam-ple of 100 neurons per condition was considered. Theneurons were scored as either having one or moreneurites equal to or longer than the diameter of onecell soma, or as having no neurites. For all four neu-ronal types, we observed that approximately 40–50%of the neurons generated neurites on PLL-coated cov-

erslips, whereas approximately 60–70% of the neuronsgenerated neurites on nanofiber-coated coverslips[Fig. 2(B)]. These results demonstrate that the 3D

Figure 2. Polyamide nanofibers support neuronal attach-ment and increase neurite generation in vitro. Cerebellargranule, cerebral cortical, hippocampal, and ventral spinalcord neurons were plated onto PLL- or nanofiber-coatedglass coverslips. After 24 h, cerebellar granule (CGN), cere-bral cortical (CCN), and hippocampal (HN) neurons werefixed and immunostained with an antibody to neurofila-ment-M. Ventral spinal cord neurons were immunostainedwith an antibody against peripherin to identify motor neu-rons (MN). A: The number of neurons per field in 15 non-overlapping fields of the coverslip (equivalent to approxi-mately 0.5 mm2) was then counted. Bars represent mean �the standard error of the mean (SEM) (n � 4). Neuronaladhesion to PLL- and nanofiber-coated coverslips was sim-ilar for all four neuronal types. B: The percentage of neuronswith neurites on PLL- and nanofiber-coated coverslips wasevaluated. Bars represent mean � the SEM (n � 4). Neuritegeneration was significantly enhanced on the 3D nanofibril-lar surface (asterisks, p � 0.01, two-tailed Mann-Whitney).

Figure 1. 3D network of electrospun polyamide nanofi-bers. A view of nanofibers electrospun onto glass coverslipsobtained using SEM reveals a fibrous network of filamentsinterspersed with pores. Bar, 5 �m.

854 AHMED ET AL.

nanofibrillar surface significantly enhanced the neu-rite generation in comparison to the 2D surface.

Neurite outgrowth was measured for the popula-tions of neurons that did have neurites. Figure 3(A)shows distributions of total neurite length for at least40 process-bearing neurons per condition, and Figure3(B) provides a view of process-bearing cerebellargranule neurons obtained using epifluorescence mi-croscopy. Neurite outgrowth was not significantly dif-ferent whether neurons were cultured on PLL ornanofibers. Thus, polyamide nanofibers were theequivalent of PLL in terms of neurite outgrowth pro-motion.

Covalent modification of polyamide nanofiberswith tenascin-C-derived peptides increases neuriteoutgrowth promotion

We next evaluated whether the neurite outgrowthpromoting abilities of the 3D nanofibrillar surfacecould be increased through the introduction of the D5or D5� peptide. Such a result would be of particularimportance given the recent attention that nanofibril-lar materials have received as potential grafts tobridge brain and spinal cord injuries.12 The D5 andD5� peptides are derived from the alternativelyspliced fibronectin type III repeat D of human tenas-cin-C [Fig. 4(A)]. These peptides facilitate neuronalprocess extension from cerebellar granule neurons viainteractions with the 7�1 integrin receptor.14 As dis-cussed above, the sequences of the D5 and D5� pep-tides are the same, except that D5� peptide has fouradditional amino acids at the N-terminus to optimizeepitope presentation of the FD/FV active site. Pep-tides were first adsorbed to 2D PLL-coated coverslipsor to 3D nanofiber-coated coverslips. Cerebellar gran-ule neurons were then plated onto the coverslips andallowed to extend processes for 24 h, at which timethey were fixed and immunostained with an antibodyagainst neurofilament-M.

Distributions of total neurite length are presented inFigure 4(B) and demonstrate that the D5 and D5�peptides each promoted neurite outgrowth to a simi-lar extent whether adsorbed to PLL-coated coverslips(labeled PLL-D5, PLL-D5�) or to nanofiber-coated cov-erslips (labeled nanofibers-D5, nanofibers-D5�). Thiswas likely due to the fact that similar amounts of thepeptides were adsorbed to the PLL and to the nano-fibers. The substrate coating efficiency of the D5 pep-tide was 7.8 � 0.9 pmol/cm2 on PLL and 8.6 � 0.6pmol/cm2 on nanofibers, and the substrate coatingefficiency of the D5� peptide was 8.2 � 0.7 pmol/cm2

on PLL and 9.4 � 1.1 pmol/cm2 on nanofibers. Sincenanofiber-coated coverslips have a larger surface areathan PLL-coated coverslips,10 this suggests that ad-sorption of the peptides to polyamide was relativelyinefficient. The D5 and D5� peptides also demon-strated similar activity to each other on the 2D and 3Dsurfaces, suggesting that nonspecific absorption doesnot obscure or expose the FD/FV active site to agreater or lesser extent in either peptide.

The amount of D5 peptide adsorbed to PLL-coatedglass (and hence to polyamide nanofibers) was previ-ously determined to be biologically limiting to neuriteoutgrowth.13 Therefore, we reasoned that increasingthe amount of peptide on the nanofibrillar surfacethrough covalent attachment might result in an en-hanced neuronal response, in agreement with thework of Woo et al.,19 who demonstrated that nanofi-brous scaffolding selectively enhanced protein ad-sorption and increased cell attachment. The poly-

Figure 3. The 3D nanofibrillar surface supports neuriteoutgrowth in vitro. A: Quantification of neurite outgrowth.Distributions of total neurite length are presented as a box-and-whisker plot. One representative experiment of four isshown. Boxes enclose 25th and 75th percentiles of eachdistribution and are bisected by the median; whiskers indi-cate 5th and 95th percentiles. Cerebellar granule, cerebralcortical, hippocampal, and ventral spinal cord neurons wereplated onto PLL-coated or nanofiber-coated glass coverslips.After 24 h, cerebellar granule, cerebral cortical neurons, andhippocampal were immunostained with an antibody againstneurofilament-M, whereas ventral spinal cord neurons wereimmunostained with an antibody against peripherin. PLL-coated glass coverslips and polyamide nanofibers allowed asimilar extent of neurite outgrowth. B: A view of process-bearing cerebellar granule neurons after 24 h of culture onPLL- and nanofiber-coated glass coverslips. Bar, 10 �m.

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amide nanofibers were first covalently coated with aproprietary polyamine polymer (Surmodics, EdenPrairie, MN). A heterobifunctional crosslinker wasthen employed to covalently bond the amines on thesurface of the nanofibers to the cysteine at the N-terminus of the D5 or D5� peptide, as described inMaterials and Methods.

Figure 4(B) demonstrates that the extent of out-growth from cerebellar granule neurons was signifi-cantly higher on peptide-modified nanofibers (labelednanofibers amines-D5, nanofibers amines-D5�)than on unmodified nanofibers. It was also signifi-cantly higher than on nanofibers or PLL with nonspe-cifically adsorbed peptides. The amount of D5 and D5�peptide covalently bound to the nanofibrillar surfacewas correspondingly higher as well: approximately5-fold more of each peptide was covalently bound asopposed to adsorbed to the nanofibers. In addition tothe increased amount of peptide, it is also possible thatan optimized epitope presentation of the FD/FV ac-tive site, resulting from covalent modification as op-posed to nonspecific adsorption, may have contrib-uted to the increased neuronal response. Whatever thecase, it is clear that the increased neurite outgrowthwas not due to the activity of the amines, as a nano-fiber amines control, which consisted of amine-coated nanofibers reacted with heterobifunctionalcrosslinker but not with peptide, did not promote

neuronal process extension. These results suggest thatcovalent modification of polyamide nanofibers withtenascin-C-derived peptides contributes to a more invivo-like phenotype for cultured neurons, as neuritesin the brain and spinal cord are significantly longerthan they are when cultured on 2D surfaces.20

We found that nanofibers modified with the D5�peptide supported more neurite outgrowth than didnanofibers modified with the D5 peptide [Fig. 4(B)],even though a similar amount of each peptide wascovalently bound to the nanofibers. As suggestedabove, the most likely explanation is that the FD/FVepitope is partially obscured in the D5 peptide, whereonly two amino acids separate FD from the nanofibersurface, whereas six amino acids separate FD from thenanofiber surface in the D5� peptide. Blocking the7�1 integrin with function-blocking antibodies

Figure 4. Covalent modification of polyamide nanofiberswith tenascin-C peptides increases their ability to promoteneurite outgrowth from cerebellar granule neurons. A:Multi-domain structure of human tenascin-C. The N-terminiof three arms are joined to form a trimer, and two trimers areconnected at the central nodule via a disulfide bond to forma hexamer. Each arm consists of 14 domains with homologyto epidermal growth factor (EGF), 8–17 fibronectin type III(FN-III) repeats, depending on alternative RNA splicing,and a single fibrinogen (fbg) domain. The universal FN-IIIrepeats (fn1–5 and fn6–8) are present in all tenascin-C splicevariants. The largest tenascin-C splice variant contains ninealternatively spliced FN-III repeats (designated A1, A2, A3,A4, B, AD2, AD1, C, and D, or fnA–D), which are missing inthe shortest splice variant. The D5 and D5� peptides used inthis study (see text for details) are found within alternativelyspliced FN-III repeat D (asterisk). B: Quantification of neu-rite outgrowth. One representative experiment of four isshown. Cerebellar granule neurons were plated onto PLL- ornanofiber-coated coverslips; PLL or nanofibers with ad-sorbed D5 or D5� peptide (PLL-D5, PLL-D5�, nanofibers-D5,or nanofibers-D5�); nanofibers with polyamine coating plusheterobifunctional crosslinker (nanofibers amines); nano-fibers with polyamine coating plus heterobifunctionalcrosslinker and covalently bound peptides (nanofibers amines-D5� or nanofibers amines-D5); or nanofibers withpolyamine coating plus heterobifunctional linker and co-valently bound mutant D5� peptide (nanofibers amines-D5�-mt). The neurons were fixed 24 h later and stained withan antibody against neurofilament-M. Neurite outgrowthwas similar on PLL- and nanofiber-coated coverslips. Thepeptides promoted neurite outgrowth to a similar extentwhen adsorbed to PLL or the nanofibers; the increase inoutgrowth in comparison to outgrowth on PLL or nanofi-bers alone was significant (asterisks, p � 0.01, Kolmogorov-Smirnov test). The addition of amines plus heterobifunc-tional crosslinker to the nanofibers had no effect on neuronalprocess extension, while covalent modification of the nano-fibers with the D5� or D5 peptide resulted in significantlymore neurite outgrowth in comparison to outgrowth onnanofibers with nonspecifically adsorbed peptides (doubleasterisks, p � 0.01, Kolmogorov-Smirnov test). Nanofiberscovalently modified with a mutant version of the D5� pep-tide (D5�*) did not promote neurite outgrowth in compari-son to the unmodified nanofibers.

856 AHMED ET AL.

against the 7 or �1 integrin chains eliminated theresponse to the peptide-modified nanofibers (data notshown), demonstrating the involvement of the inte-grin in the neurite outgrowth response. In contrast tothe results with the D5 and D5� peptide, a mutantversion of the D5� peptide (D5�*) had no effect onneuronal process extension. (FD/FV in the mutantpeptide was changed to SP/GS, a change previouslyshown to render the epitope inactive.13) Therefore,nanofibers in combination with the D5� peptide mayhave more applicability than nanofibers in combina-tion with the D5 peptide for strategies to increaseaxonal regeneration in vivo.

Nanofibers covalently modified with tenascin-Cpeptides also supported more neurite generation andneuronal adhesion than did unmodified nanofibers.The percentage of cerebellar granule neurons withneurites was 80 � 6.3 on nanofibers modified with theD5 peptide, 92 � 9.3 on nanofibers modified with theD5� peptide, and 70 � 6.2 on unmodified nanofibers.The mean number of neurons per field was 28.6 � 4.5on nanofibers modified with the D5 peptide, 25.1 � 3.2on nanofibers modified with the D5� peptide, and7.8 � 1.5 on unmodified nanofibers (Fig. 2). Thus theobserved increase in neurite generation, but not theincrease in neuronal adhesion, was greater on nanofi-bers with covalently bound D5� peptide in comparisonto nanofibers with covalently bound D5 peptide.Taken together with these neurite outgrowth data[Fig. 4(B)], this suggests that neuronal adhesion to thepeptide-modified surface was mediated through a siteother than the FD/FV neurite outgrowth promotingepitope. This is in agreement with our unpublishedwork, demonstrating that the eight amino acid pep-tide VFDNFLK did not increase neuronal adhesionwhen adsorbed to PLL-coated glass, while the D5peptide did increase adhesion.

Because grafting approaches to nervous system re-pair are of clear relevance to spinal cord injuries, wenext evaluated the response of motor neurons anddorsal root ganglion neurons to the peptide-modifiednanofibers. Both neuronal types express the 7�1 in-tegrin receptor21,22 and thus have the potential to re-spond to the D5� peptide. The peptide was adsorbedto the surface of PLL-coated glass coverslips or co-valently bound to nanofiber-coated coverslips. Ventralspinal cord or dorsal root ganglion neurons were thenplated onto the coverslips and allowed to extend pro-cesses for 24 h. They were then fixed with paraformal-dehyde. Ventral spinal cord neurons were immuno-stained with an antibody against peripherin to labelthe motor neurons, and dorsal root ganglion neuronswere immunostained with an antibody against neuro-filament to label primarily large diameter propriocep-tive and mechanoreceptive neurons.

As for motor neurons (Fig. 3), Figure 5 demonstratesthat polyamide nanofibers supported neurite out-

growth from dorsal root ganglion neurons to the sameextent as PLL-coated glass. However, the ability of thenanofibers to promote neurite outgrowth from bothneuronal types was considerably enhanced when they

Figure 5. Peptide-modified nanofibers enhance neurite ex-tension from spinal cord motor and dorsal root ganglionneurons. One representative experiment of three is shown.Ventral spinal cord or dorsal root ganglion neurons wereplated onto PLL- or nanofiber-coated coverslips; PLL-coatedcoverslips with adsorbed D5� peptide (PLL-D5�); nanofiberswith polyamine coating plus heterobifunctional crosslinker(nanofibers amines); or nanofibers with polyamine coat-ing plus heterobifunctional crosslinker and covalentlybound D5� peptide (nanofibers amines-D5�). Ventral spi-nal cord neurons were fixed 24 h later and stained with anantibody against peripherin, and dorsal root ganglion neu-rons were fixed 24 h later and stained with an antibodyagainst neurofilament-M. Neurite outgrowth from both neu-ronal types was similar on PLL- and nanofiber-coated cov-erslips. The D5� peptide promoted neurite outgrowth to asignificant extent when adsorbed to PLL (PLL-D5�) (aster-isks, p � 0.01, Kolmogorov-Smirnov test). However, cova-lent modification of the nanofibers with the peptide (nano-fibers amines-D5�) resulted in significantly more neuriteoutgrowth (double asterisks, p � 0.01, Kolmogorov-Smirnovtest). The addition of amines plus heterobifunctionalcrosslinker alone to the nanofibers (nanofibers amines)had no effect on neuronal process extension from motorneurons (shown) or dorsal ganglion neurons (not shown) inthe absence of the D5� peptide.

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were covalently modified with the D5� peptide (nano-fibers amines-D5�). Similar to the results with cere-bellar granule neurons [Fig. 4(B)], the covalently mod-ified nanofibers were a significantly better substratefor neuronal process extension than were PLL-coatedcoverslips with nonspecifically adsorbed peptide(PLL-D5�) (Fig. 5) or nanofibers with nonspecificallyadsorbed peptide (data not shown). Nanofibers withpolyamine coating and heterobifunctional linker butno peptide (nanofibers amines) did not promoteneurite outgrowth from motor neurons (Fig. 5) orfrom dorsal root ganglion neurons (data not shown) incomparison to unmodified nanofibers, indicating thatthe D5� peptide, and not the amine coating, was re-sponsible for the observed increase in neurite out-growth.

DISCUSSION

In one of the first studies to address the importanceof three-dimensionality on cellular architecture andfunction, Cukierman et al.23 demonstrated that fibro-blasts cultured on a 3D cell-free ECM derived frommouse embryos had significant differences in thestructure and composition of focal adhesions in com-parison to fibroblasts cultured on a 2D surface. Inaddition, the fibroblasts cultured on the mouse em-bryo-derived 3D surface were elongated and hadhigher rates of migration. Evidence was provided thatthese differences more accurately mimicked in vivocellular physiology.

In a previous study,10 we demonstrated that manyof the consequences of culturing fibroblasts on animal-derived 3D matrices, including the introduction of amigratory phenotype, could be recapitulated by cul-turing the cells on a matrix composed of an intercon-nected, highly porous network of electrospun poly-amide nanofibers. Cellular extensions from migratoryfibroblasts can be considered to be analogous to neu-rite extensions from developing or regenerating neu-rons, with similar proteins (e.g., the Ena/VASP familyof adaptor proteins) regulating the cytoskeleton dur-ing fibroblast motility and axon outgrowth and guid-ance.24 Therefore, we now evaluated whether poly-amide nanofibers could similarly support neuronalattachment, neurite generation, and neurite out-growth. We also evaluated the impact of incorporatingneurite outgrowth promoting peptides derived fromthe ECM molecule tenascin-C onto the nanofibrillarsurface. The results indicate that the 3D nanofibrillarmatrix can indeed support neuronal growth from avariety of neuronal types in vitro. Moreover, the abilityof the nanofibers to promote neuronal attachment andsubsequent neurite generation and extension was sig-nificantly enhanced through the addition of the neu-

roactive peptides. As such, our results suggest thatpolyamide nanofibers provide a novel 3D culture sur-face for neurons. This surface can be modified to pro-mote more in vivo-like growth patterns of long, wellelaborated neuronal processes, suggesting that pep-tide-modified nanofibers may find therapeutic appli-cations in the repair of spinal cord injury and otherCNS disorders.

The electrospun 3D nanofibrillar surface describedin this study offers a number of benefits for neuronalculture. For example, unlike PLL, it demonstrates littlebatch-to-batch variation and requires no prior prepa-ration by the investigator. The nanofibrillar surface isstabile for months at room temperature and, beingcompletely synthetic, contains no pathogens. Neuronsneed not be embedded within the nanofibers; they areinstead cultured on the nanofibrillar surface, makingthe nanofibers an ideal substrate for immunocyto-chemisty (illustrated in Fig. 3). In addition, fiber di-ameter and density of electrospun nanofibers canreadily be altered,25 thus modifying the compliance ofthe nanofibrillar matrix, a parameter shown to influ-ence neurite elongation.26 Moreover, fiber alignmentcan be altered to influence the direction of neuriteoutgrowth.25 Therefore, the advantage of electrospin-ning for production of 3D nanofibrillar culture sur-faces is that there are adjustable parameters that canbe controlled to optimize neuronal growth in vitro andutilized to optimize scaffolds for tissue engineering.

Underscoring the importance of three-dimensional-ity, a number of additional novel 3D biomaterials forneuronal culture have recently been introduced. Forexample, Yang et al.25 demonstrated that perfectlyaligned electrospun polylactic acid nano/microfibersfacilitated guided neurite outgrowth, which was notobserved on 2D surfaces or on 3D nano/microfibrillarsurfaces comprised of a random array of nanofibers.Such aligned nano/microfibrillar surfaces are of par-ticular interest for applications in tissue engineeringfor guided axonal regrowth (e.g. spinal cord injury).Furthermore, Hayman et al.27 reported that a highlymicroporous 3D scaffold prepared from polystyrenesupported differentiation of human pluripotent stemcells into neurons with excellent neurite extension,although the scaffold first had to be coated with poly-d-lysine or laminin-1 to allow the stem cells to attach.The extent of neurite outgrowth observed was fargreater than that observed on 2D surfaces coated withpoly-d-lysine or laminin-1, again demonstrating theimpact of 3D geometry on neuronal growth.

3D gels as well as 3D scaffolds have been used forneuronal culture. Matrigel�, a secretion derived fromtumors and composed of laminin-1, collagen IV, andgrowth factors, is perhaps the best known of these andhas been successfully employed to culture dorsal rootganglion neurons.28 Collagen 3D gel matrices havealso been successfully employed for the culture of rat

858 AHMED ET AL.

cortical progenitor cells,29 rat cortical neurons,30 andrat hippocampal neurons20 and demonstrated superi-ority to 2D surfaces in promoting neuronal processextension. In addition, a new class of biomaterials hasbeen developed that is comprised of self-assemblingpeptide amphiphiles12,31–33 and presents a 3D nano-structured gel for cell interactions.31 Self-aggregationof amphiphiles containing the laminin-1-derived pep-tide IKVAV yielded a material that supported differ-entiation of murine neural stem cells into neurons12

and entrapped migrating hippocampal neural cells32

in vitro. Thus all of these studies, in agreement withthe results of the current study, demonstrate that 3Dgeometry, especially in combination with chemicalcues derived from the ECM, allows for a more invivo-like environment for cultured neurons, with thecapacity to provide more physiologically relevant re-sults. To this point, 3D nanofibrillar surfaces preparedfrom naturally occurring ECM polymers (e.g., colla-gen), while used for the growth of other cell types34,35

but not, to our knowledge, for neurons, may similarlybe quite advantageous for neuronal culture.

As mentioned above, synthetic scaffolds that mimicthe 3D nanotopography and porosity of the ECM areof crucial importance to applications of regenerativemedicine as well as to the development of more phys-iologically relevant cell culture systems. We are cur-rently evaluating the utility of polyamide nanofibersmodified with the tenascin-C-derived D5� peptide inspinal cord injury models. Whereas polyamide is not,per se, biodegradable, the polymer formulation can bereadily altered to introduce this feature (Thom Weik,Donaldson Co., personal communication). Moreover,nonwoven fabrics of polyamide nanofibers are ex-tremely flexible,36 allowing for excellent incorporationinto the damaged spinal cord. The tenascin-C-derivedD5� peptide to be used in the spinal cord studies is aligand for the 7�1 integrin,14 which is expressed byboth motor neurons21 and dorsal root ganglion neu-rons,22 allowing these key neuronal populations in thespinal cord to extend longer processes in response tothe peptide (Fig. 5). In addition, the nanofibers can be“customized” with other neuroactive peptides to tar-get additional specific CNS regions.

The hippocampus, location of the CA2–CA4 pyra-midal neurons that degenerate in lysosomal storagediseases,37 is a case in point. We found that hippocam-pal neurons attached to polyamide nanofibers andextended neurites, with significantly enhanced neuritegeneration in comparison to neurite generation onPLL-coated coverslips (Fig. 3). This suggests that thenanofibers might provide a good grafting surface forneuronal replacement therapies (e.g., with hippocam-pal progenitor cells) even in the absence of neuroac-tive peptides. However, Neiiendam et al.38 demon-strated that a synthetic peptide derived from secondfibronectin type III module of the Neural Cell Adhe-

sion Molecule, called the FGL peptide, promoted neu-rite outgrowth from hippocampal neurons through adirect interaction with fibroblast growth factor recep-tor. Thus, nanofibers modified with the Neural CellAdhesion Molecule-derived FGL peptide might be aneven more efficacious growth surface for replacementof hippocampal neurons than unmodified nanofibers.

CONCLUSIONS

The 3D polyamide nanofibrillar surface described inthis study supported neuronal attachment and neuriteoutgrowth to a similar extent as 2D PLL-coated glass.However, neurite generation was significantly en-hanced. Covalent modification of the nanofibers withneuroactive tenascin-C peptides further enhancedtheir ability to support neuronal attachment, neuritegeneration, and neurite outgrowth. These results sug-gest that the 3D nanofibrillar matrix mimics the geom-etry and topography of the ECM to provide a more invivo-like environment for neurons, particularly in con-junction with ECM-derived chemical cues.

References

1. Grigsby JJ, Blanch HW, Prausnitz JM. Effect of secondarystructure on the potential of mean force for poly-l-lysine in the-helix and �-sheet conformations. Biophys Chem 2002;99:107–116.

2. Chittchang M, Alur HH, Mitra AK, Johnston TP. Poly (l-lysine) as a model drug macromolecule with which to inves-tigate secondary structure and membrane transport, part I:Physiochemical and stability studies. J Pharm Pharmacol 2002;54:315–323.

3. Meiners S, Mercado MLT. Functional peptide sequences de-rived from extracellular matrix glycoproteins and their recep-tors: Strategies to improve neuronal regeneration. Mol Neuro-biol 203;27:177–196.

4. Silver J, Miller JH. Regeneration beyond the glial scar. Nat RevNeurosci 2004;5:146–156.

5. Chernousov MA, Carey DJ. Schwann cell extracellular matrixmolecules and their receptors. Histol Histopathol 2000;15:593–601.

6. Chung HY, Hal JRB, Gogins MA, Crofoot DG, Weik TM.Polymer, polymer microfiber, polymer nanofiber and applica-tions including filter structures. US Patent No 6,743,273 B2,2004.

7. Li W-J, Laurencin CT, Caterson EJ, Tuan RS, Ko FK. Electro-spun nanofibrous structure: A novel scaffold for tissue engi-neering. J Biomed Mater Res 2002;60:613–621.

8. Abrams GA, Goodman SL, Nealey PF, Franco M, Murphy CJ.Nanoscale topography of the basement membrane underlyingthe corneal epithelium of the rhesus macaque. Cell Tissue Res2000;299:39–46.

9. Tsiper MV, Yurchenco PD. Laminin assembles into separatebasement membrane and fibrillar matrices in Schwann cells.J Cell Sci 2002;115:1005–1015.

10. Schindler M, Ahmed I, Nur-E-Kamal A, Kamal J, Grafe TH,Chung HY, Meiners S. Synthetic nanofibrillar matrix promotes

3D NANOFIBRILLAR SURFACES MODIFIED WITH TENASCIN-C-DERIVED PEPTIDES 859

in vivo-like organization and morphogenesis for cells in cul-ture. Biomaterials 2005;26:5624–5631.

11. Nur-E-Kamal A, Ahmed I, Kamal J, Schindler M, Meiners S.Three dimensional nanofibrillar surfaces induce activation ofRac. Biochem Biophys Res Commun 2005;331:428–434.

12. Silva GA, Czeisler C, Niece KL, Harrington D, Kessler JA,Stupp SI. Selective differentiation of neural progenitor cells byhigh-epitope density nanofibers. Science 2004;303:1352–1355.

13. Meiners S, Nur-E-Kamal MS, Mercado MLT. Identification of aneurite outgrowth promoting motif within the alternativelyspliced region of tenascin-C. J Neurosci 2001;21:7215–7225.

14. Mercado MLT, Nur-E-Kamal A, Liu H-Y, Gross S, Movahed R,Meiners S. Neurite outgrowth by the alternatively spliced re-gion of tenascin-C is mediated by neuronal 7�1 integrin.J Neurosci 2004;24:238–247.

15. Akbum BF, Chen M, Gunderson SL, Riefler GM, Scerri-HansenMM, Firestein BL. Cypin regulates dendrite patterning in hip-pocampal neurons by promoting microtubule assembly. NatNeurosci 2004;7:145–152.

16. Escurat M, Djabali K, Gumpel M, Gros F, Portier MM. Differ-ential expression of two neuronal intermediate-filament pro-teins, peripherin and the low-molecular-mass neurofilamentprotein (NF-L) during the development of the rat. J Neurosci1990;10:764–784.

17. Davies JE, Tang X, Denning JW, Archibald SJ, Davies SJ.Decorin suppresses neurocan, brevican, phosphacan, and NG2expression and promotes axon growth across adult rat spinalcord injuries. Eur J Neurosci 2004;19:1226–1242.

18. Abramoff MD, Magelhaes PJ, Ram SJ. Image processing withimage. J Biophoton Int 2004;11:36–42.

19. Woo KM, Chen VJ, Ma PX. Nano-fibrous scaffolding architec-ture selectively enhances protein adsorption contributing tocell attachment. J Biomed Mater Res A 2003;67:531–537.

20. Edelman D, Keefer EW. A cultural renaissance: In vitro cellbiology embraces three-dimensional context. Exp Neurol 2005;192:1–6.

21. Hammarberg H, Wallquist W, Piehl F, Risling M, Cullheim S.Regulation of laminin-associated integrin subunit mRNAs inrat spinal motoneurons during postnatal development andafter axonal injury. J Comp Neurol 2000;428:294–304.

22. Gardiner NJ, Fernyhough P, Tomlinson DR, Mayer U, von derMark H, Streuli CH. 7 integrin mediates neurite outgrowth ofdistinct populations of adult sensory neurons. Mol Cell Neu-rosci 2005;28:229–240.

23. Cukierman E, Pankov R, Stevens DR, Yamada KM. Takingcell-matrix adhesions to the third dimension. Science 2001;294:1708–1712.

24. Lebrand C, Dent EW, Strasser GA, Lanier LM, Krause M,Svitkina TM, Borisy GG, Gertler FB. Critical role of Ena/VASPproteins for filopodia formation in neurons and in functiondownstream of netrin-1. Neuron 2004;42:37–49.

25. Yang F, Murugan R, Wang S, Ramakrishna S. Electrospinningof nano/micro scale poly(l-lactic acid) aligned fibers and theirpotential in neural tissue engineering. Biomaterials 2005;26:2603–2610.

26. Willits RK, Skornia SL. Effect of collagen gel stiffness on neu-rite extension. J Biomater Sci Polym 2004;15:121–1531.

27. Hayman MW, Smith KH, Cameron NR, Przyboski SA. Growthof human stem cell-derived neurons on solid three-dimen-sional polymers. J Biochem Biophys Methods 2005;62:231–240.

28. Khan Z, Ferrari G, Kasper M, Tonge DA, Steiner JP, HamiltonGS, Gordon-Weeks PR. The non-immunosuppressive immu-nophilin ligand GPI-1046 potently stimulates regeneratingaxon growth from adult mouse dorsal root ganglia cultured inMatrigel. Neuroscience 2002;114:601–609.

29. Ma W, Fitzgerald W, Liu Q-Y, O’Shaughnessy TJ, Maric D, LinHJ, Alkon DL, Barker NJ. CNS stem and progenitor cell differ-entiation into functional neuronal circuits in three-dimensionalcollagen gels. Exp Neurol 2004;190:276–288.

30. O’Conner SM, Stenger DA, Shaffer KM, Maric D, Barker JL, MaW. Primary neuronal precursor expansion, differentiation andcytosolic Ca2 response in three-dimensional collagen gel.J Neurosci Methods 2000;102:187–195.

31. Zhang S. Fabrication of novel biomaterials through molecularself-assembly. Nat Biotechnol 2003;21:1171–1177.

32. Semino CE, Kasahara J, Hayashi H, Zhang S. Entrapment ofmigrating hippocampal neural cells in three-dimensional pep-tide nanofiber scaffold. Tissue Eng 2004;10:643–655.

33. Genove E, Shen C, Zhang S, Semino CE. The effect of function-alized self-assembling peptide scaffolds on human aortic en-dothelial cell function. Biomaterials 2005;26:3341–3351.

34. Venugopal J, Ramakrishna S. Biocompatible nanofibers matri-ces for the engineering of a dermal substitute for skin regen-eration. Tissue Eng 2005;11:847–854.

35. Kidoaki S, Kwon IK, Matsuda T. Mesoscopic designs of nano-and microfiber meshes for tissue-engineering matrix and scaf-fold based on newly devised multilayering and mixing ofelectrospinning techniques. Biomaterials 2005;26:37–46.

36. Moeschel K, Nouaimi M, Steinbrenner C, Bisswanger H. Im-mobilization of thermolysin to polyamide nonwoven materi-als. Biotechnol Bioeng 2002;82:190–199.

37. Tyynela J, Cooper JD, Khan MN, Shemilts SJ, Haltia M. Hip-pocampal pathology in the human neuronal ceroid-lipofusci-noses: Distinct patterns of storage deposition, neurodegenera-tion and glial activation. Brain Pathol 2004;14:349–357.

38. Neiiendam JL, Kohler LB, Christensen C, Li S, Pedersen MV,Ditlevsen DK, Kornum MK, Kiselyvov VV, Berezin V, Bock E.An NCAM-derived FGF-receptor agonist, the FGL-peptide,induces neurite outgrowth and neuronal survival in primaryrat neurons. J Neurochem 2004;91:920–935.

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