Cell-Fiber Interactions on Aligned and Suspended Nanofiber Scaffolds

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Copyright: American Scientific Publishers

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Copyright © 2013 American Scientific PublishersAll rights reservedPrinted in the United States of America

Journal ofBiomaterials and Tissue Engineering

Vol. 3, 355–368, 2013

Cell-Fiber Interactions on Aligned andSuspended Nanofiber Scaffolds

Kevin Sheets1� †, Ji Wang2�4� †, Sean Meehan3, Puja Sharma1, Colin Ng3,Mohammad Khan3� ‡, Brian Koons3, Bahareh Behkam1�3�4,

and Amrinder S. Nain1�2�3�4�∗1School of Biomedical Engineering and Sciences, Virginia Tech, Blacksburg, 24060, VA

2Department of Engineering Science and Mechanics, Virginia Tech, Blacksburg, 24060, VA3Department of Mechanical Engineering, Virginia Tech, Blacksburg, 24060, VA4Macromolecules and Interfaces Institute, Virginia Tech, Blacksburg, 24060, VA

Cells interact with fibrous extracellular matrix (ECM) which exhibits varying degrees of alignmentthroughout the body. In this review, we highlight cell-aligned fiber interactions using the recently-developed Spinneret-based Tunable Engineered Parameters (STEP) fiber manufacturing techniquewhich creates fibrous scaffolds with precise control on fiber diameter, spacing, orientation, and hier-archy. Through manipulation of each individual parameter, we show that multiple cell types (includingcancerous) display unique changes in cell shape, cytoskeletal arrangement, focal adhesion distribu-tion, and migration speed while interacting with the suspended STEP fibers. In addition to single-cellresponses, we present our findings on higher-level monolayer formation and wound healing mod-els, stem cell differentiation, and hepatic engineering. These single-cell and population-level studiesare conducted in the presence of aligned topographical cues that resemble native ECM. Knowl-edge gained from such studies will help create more accurate in vitro fibrous scaffolds used forthe advancement of tissue engineering, disease treatment, and the development of diagnostic anddrug testing platforms.

Keywords: Fiber Alignment, ECM, Nanofiber, Cellular Dynamics, Mechanobiology.

CONTENTS

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3552. Step Fiber Manufacturing: Role of Scaffold

Parameters on Single Cell Behavior . . . . . . . . . . . . . . . . . . 3582.1. Control of Fiber Diameter . . . . . . . . . . . . . . . . . . . . . 3592.2. Inter-Fiber Spacing . . . . . . . . . . . . . . . . . . . . . . . . . 3622.3. Fiber Orientation . . . . . . . . . . . . . . . . . . . . . . . . . . 3622.4. Multi-Layer Assemblies and Hierarchical

Structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3632.5. Role of Structural Stiffness . . . . . . . . . . . . . . . . . . . . 364

3. Population Cell Behavior and Applicationsin Tissue Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . 365

4. Concluding Remarks and Future Directions . . . . . . . . . . . . . 3665. Experimental Details . . . . . . . . . . . . . . . . . . . . . . . . . . . 366

5.1. Fiber Manufacturing and Pillar Design . . . . . . . . . . . . . 3665.2. Cell Culture and Imaging . . . . . . . . . . . . . . . . . . . . . 3665.3. Statistical Analysis . . . . . . . . . . . . . . . . . . . . . . . . . 366Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366References and Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . 366

∗Author to whom correspondence should be addressed.†These two authors equally contributed to this work.‡Mohammad Khan was a member of the STEP lab from 2010–2011.

1. INTRODUCTION

In the body, cells attach to and interact with a fibrous,mesh-like extracellular matrix (ECM) which exhibits vary-ing degrees of alignment throughout the body both spatiallyand temporally.1 Collagen in tendons, for instance, beginsas poorly aligned fibrils in early development but becomeshighly aligned in later stages.2 The fully-developed ten-don tissue, which is approximately 80% ECM mass bydry weight, relies heavily on hierarchical assembly ofhighly aligned ECM fibrils to provide functional muscle-bone interfaces.3�4 ECM facilitates cell attachment, cell–cell contacts, provides soluble growth factors and presentsgradients of elasticity to cells which directly control cellfate.5 Cells attach to the ECM through integrin-mediatedfocal adhesion complexes (FACs), which physically linkthe cell to the ECM protein networks.6 Currently, it iswidely accepted that mechanical cues and forces con-ducted through these cell-ECM junctions play a domi-nant role in cellular and sub-cellular biological functions.7

Common examples of cellular/tissue response to such

J. Biomater. Tissue Eng. 2013, Vol. 3, No. 4 2157-9083/2013/3/355/014 doi:10.1166/jbt.2013.1105 355



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Cell-Fiber Interactions on Aligned and Suspended Nanofiber Scaffolds Sheets et al.

Kevin Sheets is a Ph.D. candidate at the Virginia Tech-Wake Forest School of Biomed-ical Engineering and Sciences. He earned his Bachelor of Science in Materials Scienceand Engineering from Virginia Tech in 2009 and his Master of Science in 2010. Kevin isworking on understanding fundamental cell-fiber interactions to study cell migration, forcemodulation, and disease models using STEP scaffolds.

Ji Wang is a Ph.D. candidate in the Department of Engineering Science and Mechanics atVirginia Tech. Ji earned his Bachelor of Science degree in Polymer Science and Engineeringfrom the Beijing Institute of Textile Technology, China. He has worked on determiningthe spinnability of various polymers using the STEP technique and also on single fibermechanical/material characterization.

Sean Meehan earned his Master of Science in Mechanical engineering from Virginia Techin 2013. He earned a Bachelor of Science in Mechanical Engineering and a minor in Physicsfrom Rowan University in Glassboro, NJ. Sean’s research focused on the effects of structuralstiffness (N/m) on single cell migration mechanics along a single nanofiber. He is currentlyworking at Duke University Medical Center in Durham, NC.

Puja Sharma is Ph.D. student at Virginia Tech-Wake Forest School of Biomedical Engi-neering and Sciences. She received a Bachelor of Science in Biology (Honors) and minors inPhysics and Math from Hollins University, Virginia. Her research in the STEP Lab focuseson studying single cancer cell behavior (migration, cytoskeleton and blebbing dynamics) asa function of nanofiber structural stiffness, protein coatings and fiber curvature. She is alsostudying cancer cell force modulation in a tunable mechanistic environment.

Colin Ng graduated with a Bachelor’s in Mechanical Engineering from Virginia Tech in2012, and is currently a Masters student in the same department. His research focuses onthe cellular investigation of wound healing on nanofiber scaffolds, and single cell migratoryforces.

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Sheets et al. Cell-Fiber Interactions on Aligned and Suspended Nanofiber Scaffolds

Mohammad Khan was a member of STEP Lab from 2010–2011. He is currently attendingUniversity of Illinois Urbana-Champaign graduate school.

Brian Koons is a graduate student studying Mechanical Engineering with an emphasis onbiomedical applications at Virginia Tech. He earned his Bachelor of Science in mechanicalengineering from Virginia Tech, 2013. Currently, his research focus is on integrating func-tional suspended acrobatic hepatic monolayers within a bioreactor device. Brian also doesextensive work in creating customized fiber networks to determine cancer cell protrusiondynamics.

Bahareh Behkam earned her B.Sc. degree from Sharif University of Technology (Iran),and her M.Sc. and Ph.D. degrees from Carnegie Mellon University, all in Mechanical Engi-neering. She has been on the faculty in the Department of Mechanical Engineering withan affiliate appointment in School of Biomedical Engineering and Sciences and Macro-molecules and Interfaces Institute at Virginia Tech since August 2008. Dr. Behkam is thedirector of MicroN BASE Laboratory at Virginia Tech. Her research interests include bio-hybrid micro/nano systems and living machines, biological microfluidics, wound healingand physical chemistry of cell-surface interaction.

Amrinder S. Nain earned his B.E degree from Manipal Institute of Technology (MechanicalEngineering, India), and his M.Sc. (Chemical Engineering) and Ph.D. (Mechanical Engi-neering) degrees from Carnegie Mellon University. He has been on the faculty in the Depart-ment of Mechanical Engineering with an affiliate appointment in the School of BiomedicalEngineering and Sciences, the Department of Engineering Science and Mechanics and theMacromolecules and Interfaces Institute at Virginia Tech since August 2009. Dr. Nain is thedirector of the STEP Laboratory at Virginia Tech. His research interests include advancedmaterials, tissue engineering and single cell disease models.

forces include smooth muscle cell remodeling of arte-rial walls to accommodate changes in flow rate,8 softtissue remodeling (collagen fibers),9 and bone loss inmicrogravity.10–12 In addition, external forces carried bythe ECM guide the cell through a series of mechanicalcues, each triggering a cascade of biological responses.For example, mesenchymal stem cells (MSCs) differenti-ate towards osteogenic phenotypes at low strains and car-diovascular lineages at high strains.13�14 Cell migration hasalso been shown to be a product of a cell’s ability to gen-erate and align these traction forces, and cancer cells are

thought to metastasize by separating from a primary tumorand migrating along ECM towards blood vessels.15

Due to the varied alignments, compositions, and physicalproperties ECM exhibits throughout the body, mimickingcell-ECM interactions in vitro using state-of-the-art fibermanufacturing platforms can be challenging.1�16 Never-theless, a number of recent works have established keyscaffold parameters to directly influence cell behaviorincluding fiber diameter, spacing (or porosity), align-ment, and hierarchy.17–20 Cellular interaction with fibersoccurs on various lengthscales affecting fundamental cell

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Fig. 1. Fibrous scaffold hierarchy and mechanobiological relevance(shaded region depicts current manufacturing challenges).

behavior including migration, proliferation, differentiation,and eventual tissue remodeling.21�22 The lengthscales canbe separated into four characteristic domains: macroscale(> 100�m), microscale (1 �m–100 �m), sub-microscale(100 nm–1 �m), and nanoscale (< 100 nm), with eachdomain detailing biological aspects critical for the scaffoldto capture (Fig. 1).23�24

At the macroscale, the scaffold should allow cell–cellfusion and monolayer formation necessary for tissue gen-eration. Control at the microscale (∼ cellular dimensions)should allow individual cell dynamics and cell–cell inter-actions to be studied where fiber spacing and orienta-tion control single-cell decision making.18�19�23�25 Whilethe macro and microscales have been extensively stud-ied in the past, the sub-micro and nanoscales are activelybeing investigated for understanding fundamental single-cell behavior (biophysical and biochemical) using a num-ber of different fiber manufacturing techniques.26�27 Thesestudies aimed at elucidating single-cell response to alter-ations in fibrous environments smaller than cellular dimen-sions have the potential to unveil previously undescribedmodes of cell attachment, force production, and migra-tion mechanisms, thereby providing fresh insights into tis-sue engineering and also advancing our understanding ofmechanisms of disease onset, progression, and eventualtreatment.Fiber fabrication technologies have advanced tremen-

dously over the past decade to the point where macroscaleand microscale resolution is rather easily achieved.28 How-ever, studies aimed at the single cell-fiber interaction levelare still in their infancy and common fiber manufactur-ing technologies require either new development or refine-ment to achieve such resolution (shaded region of Fig. 1).Processes such as electrospinning are excellent platformsfor depositing fibers of a very wide variety of mate-rials but require specialized techniques to achieve fiberalignment in single and double layers (Supplementary

Information).28–31 The manufacturing challenges in con-trolling diameter, spacing, and alignment restrict the scopeto which cell-fiber interactions can be investigated usingelectrospinning methods.32 In this review, we highlightthe use of the STEP manufacturing platform to createfibrous scaffolds of well-defined diameters, spacing, ori-entation, and hierarchical assembly and its flexibility ininvestigating single-cell behavior at different length scales.We then present our results in extending STEP technol-ogy to develop cell population level applications in tissueengineering.

2. STEP FIBER MANUFACTURING: ROLEOF SCAFFOLD PARAMETERS ONSINGLE CELL BEHAVIOR

STEP is a pseudo-dry spinning nanofiber fabrication tech-nique which does not rely on an electric field to stretchthe solution filament. This method is fundamentally differ-ent from electrospinning in that the absence of an electricfield eliminates random fiber deposition associated with acharged Taylor cone and distant target,33 allowing arraysof highly aligned fibers to be created. STEP consists ofboth continuous (high density fiber array) and sequen-tial (single fiber) fiber deposition approaches.34–36 In thecontinuous approach outlined in this review, polymer solu-tion is pumped through a nozzle and pulled into a sin-gle filament by a rotating substrate (Fig. 2(A)). As therotating substrate translates at a user-defined speed (�),well-aligned fibers with desired scaffold porosity (inter-fiber spacing) are obtained. In addition to depending onpolymer molecular weight and concentration, fiber diam-eter is observed to scale with rotational speed of the sub-strate (�) (Fig. 2(B)).34 Multi-layer fiber networks areobtained by depositing fiber arrays over the previous layer,as demonstrated in Figure 2(C). By varying fiber diam-eters and the angles between different fiber layers, fiberassemblies with desired patterns can be fabricated in mul-tiple layers (Figs. 2(D, E)). Fibers of different surface andinterior features are obtained by controlling fiber solidifica-tion and phase separation processes during fabrication. Asshown in Figure 2(F), porous fiber surfaces are obtainedat high relative humidity (RH), which is attributed to acombination of vapor induced phase separation (VIPS) andtemperature induced phase separation (TIPS) processes.37

Prolonged solidification leads to the creation of wrin-kled fiber surfaces as a result of shrinkage mismatchbetween the shell and the core (Fig. 2(G)).38�39 Further-more, controlling the solvent volatility in the manufactur-ing process can lead to hollow tube structures as shownin Figure 2(H). STEP manufacturing has been extendedto deposit highly aligned fiber assemblies of differentpolymers including poly(lactic-co-glycolic acid) (PLGA),poly(methyl methacrylate)(PMMA), poly(ethylene oxide)




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Fig. 2. (A) Schematic illustration of continuous STEP manufacturing process, (B) typical fiber diameter profile as a function of substrate rotationalspeed (�), in this case normalized to 400 nm for PS 1.8 M g ·mol−1 at 10% by wt. (C) SEM images of a crisscross fiber network with controlled fiberspacing, (D) fiber assembly incorporating different diameters: d1 = 800 nm, d2 = 400 nm, d3 = 80 nm, d4 = 160 nm, (E) three-dimensional scaffoldswith controlled pore sizes (1 �m × 2 �m) and (2 �m × 2 �m), with the scale bar in insert representing 1 �m, (F) porous, (G) wrinkled, and (H)hollow fibers, unidirectional and cross-hatch scaffolds of (I) PLGA, (J) PMMA, (K) PEO fiber assemblies, (L) PU, (M) collagen and (N).

(PEO), polyurethane (PU), collagen (type I), and fib-rinogen in single and multiple layers of varying diame-ters with control of porosity, morphology, and orientation(Figs. 2(I)–(N)). Recently, we have also demonstrated wellaligned metal oxide such as TiO2 and BaTiO3 by combin-ing STEP with sol gel process.40�41

The suspended nanofibers cause cells to react to surfacecurvature and dimensionality that flat substrates inherentlymask, thus providing a unique platform for investigat-ing cell-fiber interactions (Supplementary Videos 1–4).

Fig. 3. Suspended fiber design space, which accounts for the substrate’selastic modulus (E), structural stiffness (k), diameter, inter-fiber spacing,orientation, and hierarchical assembly, compared to traditionally-studiedflat substrates.

As shown in the schematic of Figure 3, cells on fiberssense and respond to changes in curvature (or diameter),form spindle or parallel shapes due to differences in fiberspacing, spread and attach differently on oriented fibers,and finally form kite-like polygonal shapes on hierarchicalassemblies. We speculate that these parameters along withthe combination of material and structural stiffness in sus-pended fiber configurations leads to differences in forcegeneration (F ) compared to flat substrates, which elicitsdiverse behavior as measured by a large number of metricsincluding cytoskeletal arrangement, nuclear shape index,FAC cluster length and distribution, migration speed, pro-liferation, and differentiation.

2.1. Control of Fiber Diameter

2.1.1. Molecular Chain Entanglement

Solvent-based STEP fiber spinning requires sufficientmolecular chain entanglements in order to maintainsmooth, continuous fiber formation. Taking polystyrene




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Fig. 4. Isodiametric design space of PS fibers, Insert 1: beaded fibers formed below Ce, Insert 2–3: uniform fibers formed from semi-dilute toconcentrated regions, Insert 4: macro fibers formed from highly concentrated solutions, reproduced with permission.32

(PS) as a model spinning system, the solution entangle-ment number (ne�soln is defined as:42

�ne�soln =�pMW

M∗e

(1)

where �p is the polymer volume fraction and M∗e is the

corrected PS entanglement molecular weight (125 Me =16�600 g · mol−1). For electrospinning, fiber initiation(beaded fibers) starts at (ne�soln ∼ 20 and uniform fibers(defect free fiber) form at �ne�soln ∼ 35.42�43 In contrast,using the STEP technique, smooth, uniform diameter fiberwith lengths of several millimeters begin forming with(ne�soln ranging from 2.0 to 3.3 for all molecular weights,which is lower than those of electrospinning. Since thesame amount of molecular entanglements can be achievedby either high molecular weight species in low concentra-tions or low molecular weight species in high concentra-tions, isodiametric lines can be generated beyond a criticalchain entanglement concentration, Ce (Fig. 4). Polymersolutions prepared in the semi-dilute, unentangled regionlead to beaded fibers or droplets (Fig. 4, Insert 1), and con-centrated solutions with high entanglement numbers pro-duce macro-scale fibers (Fig. 4, Insert 4). Long, smooth,and uniform diameter fibers are obtained in the semi-diluteentangled and low-moderate concentrated domains (Fig. 4,Inserts 2 and 3). Fiber diameter is found to scale withpolymer concentration at the same molecular weight, andmolecular weight at the same concentration.34�36

2.1.2. Effect of Solvent Volatility

In addition to polymer molecular weight and solution con-centration, solvent volatility is another factor influenc-ing fiber diameters. Solvents with low boiling points lead

to rapid solvent loss and a shortened fiber solidificationprocess, thus limiting the stretching of the solution fila-ment and producing larger diameter fibers. As shown inFigure 5, spinning PEO solutions of the same molecularweight and concentration results in increased fiber diame-ters with increased ethanol content of the solvent mixture.

2.1.3. Fibers of Different Polymer Systems

By adjusting these parameters, a wide range of fiber diam-eters can be obtained for multiple polymer systems, includ-ing PS, PU, PEO and PMMA systems (Table I). Note thatthe deviation of fiber diameter is well-controlled (within20%) due to the absence of filament branching in the

Fig. 5. PEO design space: fiber diameter change as a function of solu-tion concentration, ethanol ratio and molecular weight.




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Table I. Fiber diameter spectrum for various polymer species spun using STEP at room temperature.

Polymer Parameter Small diameter Large diameter

Polystyrene Molecular weight (g ·mol−1� 0.86 M 2 M(PS) Solution concentration (%wt) 6 14

Solvent p-Xylene THFDiameter (nm) 34±4 8067±881

Polyurethane Molecular weight (g ·mol−1� 0.12 M 0.12 M(PU) Solution concentration (%wt) 20 30

Solvent THF/DMF= 3/7, V:V THF/DMF= 8/2, V:VDiameter (nm) 382±52 2610±231

Polyethylene Molecular weight (g ·mol−1� 0.4 M 0.9 M(PEO) Solution concentration (%wt) 3 9

Solvent Ethanol/Water= 2:8, V:V Ethanol/Water= 8:2, V:VDiameter (nm) 188±35 961±180

Poly methyl Molecular weight (g ·mol−1� 0.075 M 1 Mmethacrylate Solution concentration (%wt) 20 16(PMMA) Solvent Chlorobenzene Nitromethane

Diameter (nm) 643±147 1921±111

Note: M =million in molecular weight description.

fiber formation process. All fibers reported in Table I havelengths of at least several millimeters.

2.1.4. Cell Response to Fiber Diameter

Cells attach to and interact with fibers differently thanwith flat substrates. When attached to single fibers,

Fig. 6. A fluorescently labeled cell on a flat substrate, (B) a fluorescently labeled cell attached to a single STEP fiber in the spindle morphology,(C) SEM image of a cell on a thick bundle of fibers slightly elongated along the fiber axes, (D) SEM image of a spindle cell on a thin bundle offibers, (E) nuclear circularity measurements (circularity= 4A/P 2, A= nucleus area and P = nucleus perimeter) show increased elongation on fibers(N = 479), and (F) migration speed is found to be a function of fiber diameter (N = 30). Cell speeds were measured for cells migrating near thescaffold center, and scale bars represent 10 �m. ∗indicates statistical significance (p < 005).

cells spread along the fiber axes and take on a highlyelongated, spindle-like morphology characterized by quan-tifiable changes in nucleus circularity (Figs. 6(A)–(E)).Cells attached to STEP fibers are topographically con-fined, and the degree of this confinement and subse-quent migratory response are dependent on fiber diameter(Fig. 6(F)).




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2.2. Inter-Fiber Spacing

STEP manufacturing provides control of spacing betweenadjacent fibers by adjusting the rotational velocity of thesubstrate and the translational speed of the manipulatorholding the substrate (Fig. 2(A)). Biologically, controllinginter-fiber spacing and alignment enables cells to eitherspread between neighboring parallel fibers or remain on asingle fiber if the gap is too large (Fig. 7).On suspended fibers, FAC cluster lengths are approxi-

mately four times longer than cells on flat PS (Fig. 7(D)).This phenomenon likely occurs due to comparably lim-ited available cell attachment locations compared to flatsurfaces. The cell likely compensates by forming multi-ple adhesions within the same spatial confinement, whichwhen fluoresced appear as a longer adhesion cluster.With the addition of common cytoskeletal knockdowndrugs (blebbistatin-myosin II, nocodazole-microtubules,cytochalasin-D-actin), cells still display increased FACcluster lengths on fibers compared to flat (with the excep-tion of cytochalasin-D in which no appreciable adhesionsare observed for either substrate). This demonstrates thateven in the absence of several key cytoskeletal compo-nents, surface topography still influences cell attachment.Recognizing that substrate design influences cell spread-

ing, a direct application of aligned fiber networks is in thedesign and development of monolayer-based wound heal-ing assays. Traditionally, wound healing assays or scratch

Fig. 7. Inter-fiber spacing effects, (A) at 10 um, C2C12s spread among multiple fibers (parallel cells). They maintain this shape up to about 15–20 um gap sizes. At higher spacing, cells are unable to spread between fibers and attach to only one instead (spindle cells) (Supplementary Video 5),(B) schematic representation of parallel and spindle cell shapes as well as their FAC length measurements showing typical locations, (C) FAC clusterlengths for cells treated with pharmacological agents affecting cytoskeletal components, which show ∼ 4× increase on fibers compared to flat, and(D) differences in migration speed.44

tests probe population-based migratory potential of a cellline under a given physiochemical state. Such tests are usu-ally conducted by making an incision to a cell monolayeron a flat substrate and observing the closure dynamics.45

In contrast, STEP suspended parallel fibers enable theinvestigation of closure dynamics, yet cells are interact-ing along topographic cues which they would more likelyencounter in vivo.46

Closure in our wound healing assay occurs in two prin-cipal steps: axial migration (where NIH-3T3 fibroblastsemerge and migrate along the fiber), and gap closure(where cells fill in the space between the fibers). Over aweek, the cells will migrate and eventually cover the fibers(Fig. 8). We envision that this STEP based fundamentalstudy of single cells spreading and migrating along alignedand suspended fibers of different diameters deposited atcontrolled spacing would aid in the development of woundhealing sutures as a means of facilitating the migration ofhealthy cells into wounded damaged tissue.

2.3. Fiber Orientation

In addition to creating substrates with a single layer offibers parallel to one another, the angle between subse-quent layers of fibers can be controlled. Here, we illustratethat increments of 30� have an effect on cell spreadingbehavior (Fig. 9).




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Fig. 8. (A) STEP based wound healing assay which demonstrates gap closure over time, (B) cell migration along the aligned fibers after one day,and (C) wound closure after one week. The gaps in the closure model are dependent upon inter-fiber spacing. Scale bar represents 500 �m.

C2C12 cells seeded on these scaffolds typicallyform stable configurations at the fiber intersections(Supplementary Video 6). Those that do not form stableconfigurations start migrating along diverging fibers. Theincreasing gap size that the cell encounters as it attemptsto remain attached to both diverging fibers causes thecell to eventually detach from one and assume a spin-dle shape, where presumably less energy is required tomaintain this cell configuration (Supplementary Video 7).This system enables us to visualize F-actin stress fiberlocation and relate it to nucleus shape and FAC forma-tion, potentially allowing us to determine if cells mod-ulate their elasticity in accordance with the angle ofthe fibers. Increased stress fiber presence is commonlyassociated with increased cell contractility and thereforedecreased migration speed.47�48 Interestingly, we find that

Fig. 9. (A)–(D) Angular control of STEP fibers, (E)–(H) corresponding changes in cytoskeletal arrangement of C2C12 cells, and (I)–(L) Schematicsof cytoskeletal arrangements on the angled substrates highlighting orientation of stress fibers (dark red) and typical cell spread area measurements.Scale bars represent 20 �m.

cell migration speed is dependent on fiber orientation, withspindle cells able to migrate the fastest (∼ 50 �m/hr) andpolygonals migrating the slowest (∼ 20 �m/hr). Spindlecells only form two main clusters which are oriented alongthe same axis, whereas parallel cells contain four alongthe same axis and polygonal cells have four oppositely-oriented adhesion clusters on two different axes. Eventhough FAC cluster lengths are comparable on fibers, it istheir orientation relative to one another which influencesmaximum migration speed.

2.4. Multi-Layer Assemblies andHierarchical Structures

Arrays of fibers arranged in hierarchical structures beginto resemble the complex ECM fibrous arrangement. Such




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hierarchical assemblies can then be employed to study andunderstand how cellular migration in the presence of topo-graphical constraints might be occurring in vivo. We haveextended the STEP platform to study migration dynam-ics and blebbing phenomena of glioblastoma multiforme(GBM) on aligned multi-layer fiber networks. GBM is themost invasive form of brain cancer, causing over 15,000annual deaths.49 It is believed that these cancerous gliomacells prefer to migrate along aligned structures called whitematter tracts within the central nervous system, whichrange from sub-500 nm to 3 �m in diameter.50 AlignedSTEP fibers therefore provide a unique environment tostudy glioma cell behavior. Using parallel, aligned, and sus-pended 400 nm diameter PS STEP fibers, we observed thatthe glioma cells (Denver Based Tumor Research Group,DBTRG-05MG line) migrate almost three times faster(range: 15–200 �m/hr, average: 70 �m/hr) on fibers whencompared to flat substrates (range: 5–50 �m/hr, average:25 �m/hr). Furthermore, blebbing, which is associated withcell migration and resistance to drugs/lysis, is observedto be affected by suspended STEP fiber networks. Weobserved a reversible blebbing non-blebbing phenomenonin DBTRG cells where the bleb size and number producedby the cells depended on the cell spread area. As the cellsacquired a spread configuration on suspended fibers, bothbleb size and number decreased. A linear regression analy-sis showed that blebs almost completely disappeared whencells spread beyond an area of 1400 �m2 (Fig. 10).51

Given the known relationships of blebbing with amoe-boid form of cancer migration and resistance to lysisand drugs, our results suggest that glioma cells underspread configurations could be relatively more vulnera-ble to drugs, and would be migrating using non-amoeboidmigration modes. This platform may also be used to max-imize the migration of individual cancer cells to present a‘worst-case scenario’ that can ultimately be used as a drugtesting platform.

Fig. 10. (A) Increased cell spreading causes a decrease in bleb occurrence (count). (B) Increased cell spreading reduces bleb size, insert (i) demon-strates a blebbing, rounded cell, and (ii) later, the same cell stretches and no longer blebs. Linear analysis of the data shows a disappearance of blebsas the cells spread beyond an area of 1400 �m2. Scale bar represents 20 �m.

2.5. Role of Structural Stiffness

It has been shown on many different substrate typesincluding micropillars, gels, and glass, that material-dependent substrate stiffness (E, measured as N/m2�directly alters cellular behaviors of migration, differenti-ation, and apoptosis.52–54 In comparison, suspended fibersare essentially one-dimensional beams of uniform mate-rial stiffness with varying structural stiffness (k, mea-sured as N/m) along the length. Cells on a flat surfaceof constant material stiffness and protein coating behavesimilarly at all locations, but cells on a suspended fiberare found to respond differently along the length of thefiber due to changes in structural stiffness along its axis.In such a system, the structural stiffness scales with fiberdiameter and length (∼ diameter4/length3� and we specu-late that cells attached to these fibers in spindle shapesapply migratory forces through FACs located at the poles.By interacting mechanically with a single fiber, the cellprobes substrate structural stiffness and FACs are found tomature accordingly, which in turn affects behaviors suchas cell spreading, migration, and cytoskeletal arrangement(Fig. 11).Cells attached to STEP suspended fibers with simply

supported ends clearly respond to the mechanical gra-dient of changes in structural stiffness by conformingtheir cytoskeletons and adjusting migration speed. C2C12mouse myoblasts tend to migrate quicker at the center ofsuspended nanofibers and decrease their migration speedas they reach the higher stiffness fiber ends. Additionally,as cells reach areas of lower structural stiffness at the mid-dle of the fiber span length (lowest k) compared to higherstructural stiffness at the edges (highest k), they demon-strate shorter cell length, shorter FAC clusters, and lowernuclear shape index (Fig. 11). The inverse relationshipbetween fiber diameter and fiber length corresponding tothe parameters which play dominant roles in the structuralstiffness can be extended to add to our understanding ofcancer cell metastasis mechanisms.55




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Fig. 11. (A) Schematic representation of cell positioning on a single fiber suspended across two micropillars, (B) SEM image demonstrating cellspreading’s dependence on fiber positioning, and (C) gradient structural stiffness along the length of a single 1 mm length PS nanofiber (700 nmdiameter) causes a significant difference in migration speed (normalized to 44 �m/hr) (N = 293), nuclear shape index (normalized to 0.7) (N = 69),and cell length (normalized to 100 �m) (N = 62). ∗indicates statistical significance (p < 005).

3. POPULATION CELL BEHAVIOR ANDAPPLICATIONS IN TISSUEENGINEERING

In addition to demonstrating their use in single-cell stud-ies, STEP fibers have shown promise in multiple tissueengineering applications, highlighting the usefulness ofproviding cells with physical environments that more

Fig. 12. (A) Effect of growth factor concentration on tendon differentiation,55 (B) sheet engineering (white dotted lines show underlying fiberdirections), (C) long-term culture of hepatocyte monolayers, and (D) early neural differentiation demonstrated by MAP2 fluorescence on single anddouble layer scaffolds shown by dotted lines.57

closely represent the ECM (Fig. 12). For instance, withthe use of STEP scaffolds we have shown monolayersof C2C12 cells subject to picogram/mm2 concentrationsof fibroblast growth factor 2 (FGF-2) to differentiate intomyotubes (Fig. 12 (A)).56 SEM images of monolayerformation reveals that cell populations remain in align-ment with their underlying fibers (Fig. 12(B)), suggestingapplications in sheet engineering.57 In addition, the fibers




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have the ability to maintain long term functional behav-ior of hepatocytes without de-differentiating (Fig. 12(C)and unpublished data), and the same fibers have the abilityto achieve neuronal differentiation (80% neurons) from apopulation of neural stem cells (NSC’s) (Fig. 12(D)).58

4. CONCLUDING REMARKS ANDFUTURE DIRECTIONS

Given the vast design space available for manufacturingin vitro scaffolds, the ability to control key parameters infibrous scaffold production has opened up new possibilitiesin studying cell-fiber interactions. Since they originate andoperate in different physiochemical niches, every cell typebrings with it a unique set of biophysical and biochemicalchallenges which must be accounted for during substratedesign. Here, we have shown through the manipulationof key components in fibrous substrate design that cellsexhibit diverse behavior on suspended fibers compared tomore traditionally-studied flat substrates, and that thesedifferences carry great potential in uncovering mechanismsof cell adhesion and eventual force modulation. Throughthe ability to control fiber diameter and length, we haveshown that cells sense and respond to changes in struc-tural stiffness by increasing spreading on regions of higherstiffness leading to lower migration. Fiber spacing controlallows scaffold optimization for the development of a new,fully suspended wound healing assay. Oriented fiber arraysin single and multiple layers at specific angles forcescells to spread into different shapes, from highly elon-gated spindles to more evenly-spread polygonals, whichhelps us understand stress fiber organization and eventualelasticity of cells. Fiber curvature and geometry impartbehaviors into cells which flat, two dimensional substrateswith and without topographic features find challenging torecapitulate.The knowledge gained from these experiments coupled

with the ability to build more accurate in vitro scaffoldswill allow the measurement of migratory cell force changesin real-time to be related to scaffold mechanical properties.In the future, this will allow coupling of chemical growthfactors with mechanical cues to develop a unique, dual-cue gradient environment to advance diagnostic and drugtesting platforms and scaled up scaffold designs to accom-modate tissue engineering on a larger scale.

5. EXPERIMENTAL DETAILS

5.1. Fiber Manufacturing and Pillar Design

Fibers were deposited according to STEP manufacturingprinciples outlined above and reported previously.34 Pil-lars for structural stiffness measurements were punchedout of a block of polydimethylsiloxane (PDMS) of 0.635cm thickness using a 1.0 mm diameter Harris Uni-Core

Sample punch. Pairs of these pillars were then placed sev-eral mm apart on a glass slide and fixed using an epoxyadhesive. Fibers were spun over the pillars using the STEPmethod, with a drop of uncured PDMS applied to the tipof each pillar before spinning to ensure proper anchorage.

5.2. Cell Culture and Imaging

C2C12, NIH-3T3, and DBTRG cell lines were culturedfollowing suggested protocols from ATCC. SEM imageswere taken with the FEI Quanta 600 FEG ESEM in highvacuum mode. Phase contrast microscopy images weretaken with the Zeiss AxioObserver Z1 with incubationand digital stage. Fluorescence imaging was conducted viadirect and antibody staining. To visualize FACs, cells werefixed in 4% paraformaldehyde and permeabilized in TritonX100. Polyclonal rabbit anti-paxillin (Invitrogen) primaryantibodies diluted 1:250 were used in conjunction withAlexa Fluor 488 (Invitrogen) secondary antibodies. F-actinwas stained using 1:100 dilution of rhodamine phalloidin(Santa Cruz). Nuclei were counterstained with 300 nM4′,6-diamidino-2-phenylindole (DAPI).

5.3. Statistical Analysis

Statistical comparisons were made using student’s t-test inJMP-9 software. Populations were considered statisticallysignificant when p < 005 unless otherwise noted. Errorbars in figures denote one standard deviation.

Acknowledgments: The authors would like to acknow-ledge support received from the Institute for Critical Tech-nology and Applied Science (ICTAS) at Virginia Tech.This research was funded in part by Jeffress MemorialTrust Fund and the Bill and Andrea Waide Research Fund.

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Received: 10 March 2013. Accepted: 24 April 2013.


Cell-Fiber Interactions on Aligned and Suspended Nanofiber Scaffolds

Engineering

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