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Biomaterials xxx (2009) 1–9

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Biomaterials

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

Myoblast morphology and organization on biochemically micro-patternedhydrogel coatings under cyclic mechanical strain

Wylie W. Ahmed a, Tobias Wolfram b, Alexandra M. Goldyn b,c, Kristina Bruellhoff d, Borja Aragues Rioja b,Martin Moller c, Joachim P. Spatz b,c, Taher A. Saif a, Jurgen Groll d, Ralf Kemkemer b,*

a Department of Mechanical Sciences and Engineering, University of Illinois at Urbana-Champaign, 1206 West Green Street, Urbana, IL 61801, USAb Department of New Materials and Biosystems, Max Planck Institute for Metals Research, Heisenbergstrasse 3, 70569 Stuttgart, Germanyc Biophysical Chemistry, University of Heidelberg, Heidelberg, Germanyd DWI e.V. and Institute of Technical and Macromolecular Chemistry, Pauwelstrasse 8, D-52056 Aachen, Germany

a r t i c l e i n f o

Article history:Received 17 August 2009Accepted 11 September 2009Available online xxx

Keywords:Cell adhesionPassivationCyclic strainMuscle cell differentiationPolydimethylsiloxane (PDMS)Micro-patterning

* Corresponding author. Tel.: þ49 711 689 3516.E-mail address: ralf.kemkemer@mf.mpg.de (R. Kem

0142-9612/$ – see front matter � 2009 Elsevier Ltd.doi:10.1016/j.biomaterials.2009.09.047

Please cite this article in press as: Ahmedcoatings under cyclic mechanical strain, Bio

a b s t r a c t

Mechanical forces and geometric constraints play critical roles in determining cell functionality andtissue development. Novel experimental methods are essential to explore the underlying biologicalmechanisms of cell response. We present a versatile method to culture cells on adhesive micro-patternedsubstrates while applying long-term cyclic tensile strain (CTS). A polydimethysiloxane (PDMS) mold iscoated with a cell repulsive NCO-sP(EO-stat-PO) hydrogel which in turn is covalently patterned byfibronectin using micro-contact printing. This results in two-dimensional, highly selective cell-adhesivemicro-patterns. The substrates allow application of CTS to adherent cells for more than 4 days under cellculture conditions without unspecific adhesion. The applicability of our system is demonstrated bystudying the adaptive response of C2C12 skeletal myoblasts seeded on fibronectin lines with differentorientations relative to the strain direction. After application of CTS (amplitude of 7%, frequency of0.5 Hz) we find that actin fiber organization is dominantly controlled by CTS. Nuclei shape is predomi-nantly affected by the constraint of the adhesive lines, resulting in significant elongation. Morphologi-cally, myotube formation was incomplete after 4 days of culture, but actin striations were observedexclusively on the 45� line patterns subjected to CTS, the direction of maximum shear strain.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Various tools have been developed to investigate the response ofbiological systems to mechanical forces on the molecular and cellularscale. Single cell properties and behavior can be studied by methodssuch as atomic force microscopy (AFM), magnetic twisting cytometry(MTC), and optical tweezers. Methods for investigating the responseof cell populations to mechanical stress include the application ofshear flow or substrate strain [1,2]. In the latter, cells are typicallycultured on an elastic membrane that is cyclically stretched to mimicperiodic forces in vivo. Interestingly, it has been demonstrated thatcell responses depend on the direction of the stretch with respect tothe cell orientation and internal cell structure [3,4]. For example,anisotropic mechanosensing can modulate the gene expression ofmesenchymal stem cells [5]. To further investigate such phenomenawe developed a method to functionalize non-adhesive elastic

kemer).

All rights reserved.

WW, et al., Myoblast morphmaterials (2009), doi:10.1016

substrates by micro-patterned covalently bound extracellular matrixmolecules. These patterns are stable for extended durations andprovide control of cell orientation relative to the applied straindirection and thus allow the investigation of the various stress stateson adherent cells. As a demonstration of our novel experimentalsystem we investigated the mechanoresponse of C2C12 myoblastcells.

Skeletal muscle is responsible for nearly all voluntary move-ment in the body. It is constantly subjected to stress in vivo but therole of mechanical forces in myogenesis is not completely known.Myogenesis is a process in which precursor muscle cells, known asmyoblasts, develop into mature skeletal muscle [6–9]. However, theresponse of myoblasts to different environmental cues is importantto understand their development into skeletal muscle [10–13].Specifically, their response to cyclic loading is a crucial modelsystem for exploring cell based therapies because it closely mimicsin vivo cell differentiation conditions.

The fact that mechanical and geometric stimuli influencemyoblast differentiation is now well accepted [3,14–25]. Forexample, myoblasts respond differently to uniaxial and multiaxial

ology and organization on biochemically micro-patterned hydrogel/j.biomaterials.2009.09.047

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stresses by exhibiting altered intracellular signaling and alignment[23,26]. In the absence of mechanical strain myotube formationoccurs on substrates regardless of stiffness [14], however maturesarcomeric striations form only on substrates with elastic modulusranging from 10 to 12 kPa, which implicates that restoring forces ofthe surrounding matrix might influence myotube development[15]. In addition to mechanical forces, adhesion geometry andstiffness of the culture matrix influence the behavior of myoblasts.Griffin et al. [15] examined the effect of geometric constraints byobserving myoblast behavior in long-term culture on micro-patterned collagen lines on glass and found that nascent myotubesformed but did not differentiate further. Geometric constraints alsoplay a role in myoblast alignment and subsequent fusion intomyotubes [17] where a line width of 30 mm has been reported to beoptimal for single myotube formation [18].

Although it is thus clear that both mechanical and geometricstimuli play a key role in the development of myoblasts, the complexinteractions of these signals on the cells are not well understood.There are, for example, reports on the down-regulation [20,25,27] ofmyogenic gene expression as well as on the up-regulation [28] inresponse to cyclic tensile strain (CTS). These studies suggest thatslight variations in strain parameters such as magnitude, duration,direction, frequency, and whether the strain is uniaxial or multiaxialcan induce substantially different responses in C2C12 myoblasts.Most current stretching systems apply uniaxial or biaxial strain topopulations of cells on homogeneously functionalized substrates.Some studies employ micro-grooves [5,26,29,30] to control cellalignment, however, this also introduces a three-dimensionaltopography to the substrate which may cause a significantly differentbiological response when compared to a two-dimensional homo-geneous surface. Therefore when using micro-grooves, the effect ofthree-dimensional topography and cell alignment cannot be de-coupled.

Fig. 1. Schematic diagram of the PDMS stretchable substrate (not to scale). The entire surfacestat-PO) coating. Insert on upper right hand corner shows the layer structure of bovineorientations of 0� , 45� , and 90� relative to the strain direction (pointing arrows).

Please cite this article in press as: Ahmed WW, et al., Myoblast morphcoatings under cyclic mechanical strain, Biomaterials (2009), doi:10.1016

To control the cell orientation with regard to the direction ofdeformation by CTS, we developed an experimental system withfunctionalized micro-patterns to explore the combined effect onC2C12 myoblasts. The cells were constrained by adhesion to 30 mmwide lines of fibronectin that is covalently micro-patterned ontohydrogel coated deformable substrates. To achieve this, we modi-fied polydimethysiloxane (PDMS) substrates with an ultrathin non-adhesive NCO-sP(EO-stat-PO) hydrogel film. Covalent linking ofadhesive protein micro-patterns to the hydrogel ensures specificadhesion of the cells, thus allowing control of cell orientationwithout the use of micro-grooves. These lines have been directed invarious orientations relative to the strain direction. Thus byapplying strain in one direction the effect of tension, compression,and shear strain may be investigated on the same substrate byvarying the cell orientation. The robustness and versatility of ourmethod for use with different cell adhesion molecules and micro-patterns demonstrates its potential for long-term studies of celldifferentiation.

2. Materials and methods

2.1. PDMS substrates

PDMS substrates [31,32] with Young’s modulus, E w 1 MPa were prepared bythoroughly mixing a 10:1 ratio of Dow Corning Sylgard 184 silicone elastomer andcuring agent. The mixture was cast in a custom fabricated acrylic mold and cured at65 �C for 24 h to create stretchable substrates with a 70� 50� 5 mm culturemedium reservoir (see Fig. 1).

2.1.1. Fabrication of micro-contact printing stampsPhotolithography was used to pattern Si wafers as molds to create the PDMS

stamps for micro-contact printing. SU-8 photoresist was spin-coated on a 3 inchwafer and it was soft-baked in two stages (2 min, 65 �C) (10 min, 95 �C). UV expo-sure was applied by a SUSS Microtec MJB4 contact aligner (hard contact, 5 s expo-sure) with a mask manufactured by Masken Lithographie and Consulting GmbH. Thewafer was then post-baked in two stages (2 min, 65 �C) (4 min, 95 �C) and hard-

of the culture medium reservoir (70� 50� 5 mm) was passivated with an NCO-sP(EO-fibronectin (5 mg/mL) onto the NCO-sP(EO-stat-PO) and PDMS. Adhesive lines have

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baked (15 min, 200 �C). All lithography was performed at 20 �C and 49% humidity.PDMS was poured directly on the patterned Si wafer to create the micro-contactprinting stamps.

2.1.2. Activation and NCO-sP(EO-stat-PO) coating of PDMS substratesNCO-functional star shaped poly(ethylene oxide-stat-propylene oxide) (NCO-

sP(EO-stat-PO)) was prepared as described before by Gasteier et al. [33]. Prior tocoating with NCO-sP(EO-stat-PO), the PDMS chambers were treated with ammoniaplasma as reported previously [34]. Immediately after plasma-treatment, the amine-functionalized PDMS chambers were coated with NCO-sP(EO-stat-PO) by spin-casting a solution of 10 mg/mL in a 9:1 v:v mixture of water and tetrahydrofuran(THF). The solution was prepared by pre-dissolving the polymer in THF followed bydilution with water to the desired concentration and solvent ratio. The solution wasused for coating after the solvents had been mixed for 5 min. After film preparation,the coating was allowed to partially cross-link for 1 h to develop enough mechanicalstrength to enable standard micro-contact printing techniques without topologicalpatterning defects.

2.1.3. Patterning of the substratesPDMS stamps were ‘‘inked’’ with bovine derived fibronectin (5 mg/mL), partially

dried and brought in contact with the NCO-sP(EO-stat-PO) surface as reportedbefore [33–35]. Micro-patterns consisted of 30 mm wide parallel lines with 40 mmspacing between. The unlabeled fibronectin was mixed with Alexa Fluor 568 labeledfibronectin (10:1) to visualize the micro-patterns. The stamps were used to patternthree different orientations of the line pattern (0� , 45� , and 90�) with respect to thestrain direction as shown in Fig. 1.

2.1.4. Cyclic stretching systemCyclic tensile strain (CTS) was applied to the PDMS substrates using the

stretching system developed at the Max Planck Institute for Metals Research inStuttgart, Germany [31]. Briefly, one side of the substrate was clamped in a fixedposition while the other side was coupled to a servo motor (Faulhaber Group) withan eccentric disc which applied strain as it rotated. It should be noted that thetransverse direction was not constrained and therefore experienced slightcompression. The entire stretching apparatus was placed inside an incubator tomaintain suitable cell culture conditions. Cells were subjected to cyclic strain (7%,0.5 Hz) for 4 days (24 h/day) and fixed immediately afterwards. These CTS param-eters were chosen to closely mimic in vivo muscle deformation [36]. The magnitudeof substrate strain and transverse compression was characterized by trackingparticle displacement at various randomly chosen positions on the substrate.Additionally, a finite element calculation was performed to verify uniform substratedeformation in the area of the micro-patterns (results not shown). A perforatedcover was used to minimize evaporation of the medium and to allow adequate gasexchange.

2.1.5. Cell culture and immunofluorescent stainingC2C12 skeletal myoblasts (<14 passage) from ATCC were cultured in an incu-

bator at 37 �C and 5% CO2 in growth medium consisting of Dulbecco’s Modified EagleMedium (DMEM) 41965-039 with 20% Fetal Bovine Serum (FBS) and 1% Penicillin/Streptomycin (Pen/Strep) (all from Gibco). PDMS substrates were sterilized with 70%ethanol and washed with phosphate buffered saline (PBS). Then cells were plated(confluent layer) on PDMS substrates for 24 h before cell medium was changed todifferentiation medium consisting of DMEM 41965-039 with 1% FBS and 1% Pen/Strep. CTS experiments began immediately after culture medium was changed.

Immediately after cyclic strain was stopped the cells were fixed in 3.7% para-formaldehyde (PFA) for 15 min at 37 �C. Cells were permeabilized with 0.3% Triton-X100 for 3 min and blocked in 5% Bovine Serum Albumin (BSA) for 10 min at roomtemperature. Cells were then incubated in DAPI (1:1000 from 5 mg/mL stock) (Roth)and Alexa Fluor 488 conjugated Phalloidin (1:50 from methanolic stock) (InvitrogenA12379) for 30 min at 37 �C. Cells were washed twice with PBS between each stepand were then incubated in PBS overnight.

2.1.6. Microscopy and image analysisImages were acquired with a Zeiss AxioImager Z1 using a 40� (0.8 NA) water

immersion objective and an AxioCam MRm CCD Camera. Z-Stacks were collapsed withthe Zeiss Extended Focus Image (EFI) algorithm. Each nucleus was treated as a singleparticle and an ellipse was fit to determine the aspect ratio and orientation angle(n> 500 cells for each line orientation). Actin orientation was determined by manuallytracing actin fibers and measuring the angle relative to the strain direction (n> 100cells for each line orientation). Actin orientation angle is reported as symmetric aboutthe axis of strain (0�) unless otherwise stated. An ANOVA test was performed toevaluate statistical significance. ImageJ was used for all image analysis [37].

3. Results

The PDMS chambers were coated with a cell repulsive NCO-sP(EO-stat-PO) polymer film on which fibronectin was covalentlylinked using micro-contact printing techniques. The patterned

Please cite this article in press as: Ahmed WW, et al., Myoblast morphcoatings under cyclic mechanical strain, Biomaterials (2009), doi:10.1016

substrates maintained high integrity of the fibronectin functional-ization and NCO-sP(EO-stat-PO) passivation showing no sign ofdegradation with time. There were no obvious defects in thehydrogel surface passivation observable by light microscopy. Thisensured specific adhesion of the cells exclusively onto the fibro-nectin lines even after 4 days of continuous CTS with an amplitudeof 7% and frequency of 0.5 Hz under typical cell culture conditions.

As characterized by tracking the particle displacement of thesubstrate we found a transverse compression of 3.5% for 7% tensilestrain. That matches the expected values for PDMS that has a Pois-son’s ratio of nearly 0.5 [32]. The strain was observed to be uniformin the region of interest (3 cm� 1 cm area of micro-patterning) atdifferent randomly chosen positions on the substrate. Additionally,the finite element calculation verified uniform substrate deforma-tion in the area of the micro-patterns (results not shown).

3.1. Actin stress fiber orientation

CTS and micro-patterned lines both affected actin stress fiberorientation. In the absence of applied strain, on the homogeneouslyfunctionalized substrates, the myoblasts exhibited no specificorientation and formed actin fibers extending in all directionsradially outward from the cell center (Fig. 2a). The resulting randomdistribution for the measured actin fiber orientation is given inFig. 3a. On 0� micro-patterned lines, the cells elongated along thepattern and developed actin fibers aligned along the line axis(Fig. 2c). The measured average orientation angle is 0.1� (�10.8�)with a symmetric distribution around 0� (Fig. 3c).

When CTS was applied to the homogeneously functionalizedsubstrates the cells reoriented their stress fibers and cell bodies toa mean value of 71.5� (�17.2�) relative to the strain direction (Figs.2b and 3b). Similar cell reorientation of approximately 70� due toCTS has been observed in endothelial cells [38]. Stress fiber orien-tation due to applied CTS was observed to be dependent on the lineorientation relative to the strain direction. On the 0� micro-patternsthe myoblasts did not exhibit elongated morphology and the actinstress fibers were not well organized (Fig. 2d). The fibers appearedto orient along an oblique direction with large standard deviation(47.9� �19.5�) (Fig. 3d). Furthermore, cross-bridging of cellsbetween micro-patterned lines occurred frequently. The myoblastsformed localized agglomerations and bridged the 40 mm passivatedregion between the lines (Fig. 4), with actin fibers at oblique anglesin the bridges. On the 45� lines, the cells became elongated anddeveloped well defined stress fibers oriented nearly along the lineat an average angle of 51.8� (�16.8�) relative to the strain direction(Fig. 2e). The angle distribution is not symmetric to the direction ofthe micro-patterned lines but is bias toward larger angles (Fig. 3e).On the 90� lines, the cells became highly elongated and showedstress fibers oriented at 91.0� (�14.0�) (Figs. 2f and 3f).

3.1.1. Nuclei shape and orientationCTS and micro-patterned lines both affected nuclei orientation

and their aspect ratio. Representative examples of DAPI stainednuclei are given in Fig. 5 for the different conditions. On homo-geneously functionalized surfaces the nuclei were randomlyoriented with no preferred direction (Figs. 5a and 6a). Afterapplication of CTS, nuclei were slightly oriented in a moreperpendicular direction (Figs. 5b and 6b). For cells seeded onmicro-patterned lines, a drastic orientation of the nuclei along theline direction can be observed (Fig. 5c) which results in a meanorientation of 0� (�8.3�). The corresponding frequency distribu-tion of the nuclei orientation is given in Fig. 6c demonstrating thehigh degree of alignment along the lines. Nuclei orientation wasnot drastically changed for cells cultured on lines parallel to thestretching direction (0� CTS, Fig. 5d). The mean orientation angle

ology and organization on biochemically micro-patterned hydrogel/j.biomaterials.2009.09.047

Fig. 2. Images of actin stress fiber orientation of C2C12 myoblasts subjected to cyclic tensile strain (CTS) (7%, 0.5 Hz, horizontal direction): (a) On the control (unstretched) substratewith a homogeneously fibronectin functionalized surface the cells had no specific orientation with phalloidin stained actin stress fibers extending radially outwards in all directions.(b) CTS was applied to a homogeneously functionalized surface and caused cells to develop actin stress fibers oriented at an average angle of 71.5� (�17.2�). (c) On the controlsubstrate with micro-patterned lines the cells exhibited an elongated morphology with actin fibers oriented along the direction of the micro-patterned line at 0.1� (�10.8�). (d) CTSapplied to 0� micro-patterned lines caused cells to orient irregularly and form actin stress fibers oblique to the strain direction. (e, f) CTS applied to 45� and 90� micro-patternsresulted in an average actin stress fiber orientation angle of 51.8� (�16.8�) and 91� (�14.0�) respectively. (scale bar¼ 50 mm) (HS¼ homogeneous surface).

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is still close to 0� (1.1� �22.5�), however, the distribution becamebroader (Fig. 6d) compared to the no CTS condition. Similarly, themean orientation of the nuclei followed the orientation ofthe micro-patterned lines for the conditions 45� CTS (Figs. 5e and6e) and 90 �CTS (Figs. 5f and 6f).

Fig. 3. Histograms of actin stress fiber orientation: (a) Homogeneously fibronectin functionabsence of CTS, actin stress fibers align preferentially along the micro-pattern axis. (d) Howestrain direction at an average of 47.9� (�19.5�) (b, d, e, f) In all cases where CTS is applied,

Please cite this article in press as: Ahmed WW, et al., Myoblast morphcoatings under cyclic mechanical strain, Biomaterials (2009), doi:10.1016

On homogeneously functionalized surfaces the nuclei wererather round shaped (Fig. 5a) with an average aspect ratio of 1.3.Nuclei shape was slightly more elongated after the application ofCTS (Fig. 5b) which resulted in a small but significant increase of theaverage nuclei aspect ratio by 7%. In the absence of strain, when

alized control substrates show large scatter in actin fiber orientation angle. (c) In thever when CTS is applied to the 0� orientation the actin stress fibers orient oblique to thethere is a significant effect on actin stress fiber orientation.

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Fig. 4. Image of cross-bridging phenomenon observed where cells would form localagglomerations and bridge the 40 mm passivated distance between micro-patternedlines. Cross-bridging was observed 13 times on the 0� lines, 4 times on the 45� lines,and once on the 90� lines. (scale bar¼ 50 mm).

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cells were seeded on micro-patterned lines, a drastic elongation ofthe nuclei can be observed (Fig. 5c). The mean values for the aspectratio of nuclei elongation are summarized for all conditions in Fig. 7.The nuclei aspect ratio increased by over 40% relative to thehomogeneous surface to a value of 1.9 (Fig. 7). When CTS wasapplied to the micro-patterned lines, a similar nuclei elongationwas observed for all line orientations relative to applied straindirection (Fig. 5d–f). The mean aspect ratio increased from 1.9(0 �CTS), to 2.0 (45 �CTS), to 2.1 (90 �CTS), respectively which meansby less than 10% from 0� to 90� line (Fig. 7). The direction of thepatterned lines appears to determine the nuclei orientation; CTSmight have a negligible influence.

Fig. 5. Images of nuclei of C2C12 myoblasts subjected to CTS (7%, 0.5 Hz, horizontal directsubstrates exhibited aspect ratios of 1.3 (�0.2) and were oriented randomly. (b) The applicatratio to 1.4 (�0.2). (c) When micro-patterned lines were used (in the absence of CTS) the nmicro-pattern. (d–f) The application of CTS to micro-patterned lines showed an increase oorientations, respectively. (scale bar¼ 50 mm).

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3.1.2. Actin striationActin striations were observed for the 45� orientation lines

when subjected to CTS. A representative example for the actinstriation under the mentioned condition is given in Fig. 8. Therewere no signs of actin striations for any other combination ofparameters including the control non-stretched substrates. Toverify the actin striations, ImageJ was used to plot the intensityprofile from the images of phalloidin staining showing the periodicpattern of actin striations (Fig. 8) with a periodicity of about1.0–1.8 mm.

In summary, CTS and the micro-patterned lines in variousorientations relative to the strain direction had a significant effecton actin fiber orientation, nuclei aspect ratio, and nuclei orienta-tion. Strikingly, actin striations were observed exclusively on the45� line orientation when subjected to CTS, the direction ofmaximum shear strain. Morphologically, myotube formation wasincomplete after 4 days in both the CTS and control specimens.

4. Discussion

Functionalized PDMS membranes are frequently used to stretchadherent cells to mimic mechanical forces occurring in vivo. To applythe mechanical strain with specific direction to the cells’ initialorganization requires the combination of robust immobilizedadhesive micro-patterns and passivation of the PDMS. There areseveral approaches to modify PDMS to locally immobilize celladhesion molecules on the low-adhesive PDMS [39]. However, manymethods to functionalize PDMS with micro-patterns are not welltested for their efficacy to endure long-term mechanical stretching.For example, Liu et al reported a method to passivate PDMS surfacesfor more than 28 days by functionalizing it with Pluronic F108 andPEO but applied no CTS [40]. A similar approach for the modificationof PDMS was followed by Gopalan et al. in order to apply anisotropicstrain to cardio myocytes adherent on micro-patterned collagen for24 h [3]. We demonstrated that our experimental system with the

ion): (a) DAPI stained nuclei on the homogeneously fibronectin functionalized controlion of CTS to the homogeneously functionalized substrates increased the nuclei aspectuclei aspect ratio increased to 1.9 (�0.4) and the nuclei oriented along the axis of thef nuclei aspect ratio to 1.9 (�0.4), 2.0 (�0.4), and 2.1 (�0.4) for the 0� , 45� , and 90�

ology and organization on biochemically micro-patterned hydrogel/j.biomaterials.2009.09.047

Fig. 6. Histograms of nuclei orientation: (a) Homogeneously functionalized control substrates show large scatter in nuclei orientation angle. (b) When CTS is applied to thehomogeneously functionalized surface the nuclei orient preferentially but with large scatter at 81.4� (�46.3�). (c) In the absence of CTS, nuclei orientation angle strongly corre-sponds with micro-pattern axis at 0.0� (�8.3�). (d–f) Nuclei orient preferentially along the axis of the micro-pattern in all cases. It should be noted that less scatter in nucleiorientation angle is observed in the absence of CTS (c).

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protein repulsive NCO-sP(EO-stat-PO) polymer film on PDMS isstable enough to endure several days of cyclic tensile strain undercell culture conditions. Even after nearly 180,000 cycles of CTS undercell culture conditions we did not observe non-specific cell adhesionin areas adjacent to the micro-patterned fibronectin lines. Since the

Fig. 7. Nuclei aspect ratio: The small square in the center of the shaded box representsthe mean nuclei aspect ratio. The horizontal line through the shaded box indicates themedian nuclei aspect ratio and the lower and upper edges represent the 25th and 75thpercentile, respectively. The standard deviation is shown by the whiskers extendingvertically from each box. The (*) indicates that values differ significantly (ANOVAp< 0.05). (angles indicate orientation of micro-patterned lines with respect to thestrain direction).

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fibronectin is covalently linked to the NCO-sP(EO-stat-PO) polymerfilm the micro-pattern also did not show degradation. Therefore, webelieve that our system is well suited for applications requiring long-term stability of adhesive patterns under mechanical manipulationssuch as tissue engineering.

Using our model system we show that both geometricconstraints and cyclic tensile strain significantly affect the structureand function of C2C12 myoblasts. Our experiments with un-patterned adhesive substrata confirmed previous reports that formany cell types cyclic uniaxial strain lead to alignment of the actincytoskeleton and cell away from the axis of strain [5,38,41–44]. Wefound a significant alignment of the actin stress fibers in C2C12 cellswith an angle of approximately 70� relative to the strain direction(Figs. 2b and 3b). The deviation from a perpendicular alignment(90�) may be due to the lateral compression the cells experience inthe transverse direction [38,44]. The magnitude of compressiondepends on the Poisson’s ratio of the material (n z 0.5 for PDMS)and was measured to be 3.5%. From these calculations one wouldexpect an actin or cell orientation of approximately 55� if the cellsaligned in the direction of minimal strain. A recent model supportsthis prediction in the case that cells sense mechanical strain [45].However, if cells sense stress rather than strain, this model predictsa more perpendicular orientation of the cells with respect to thestretch direction. It remains speculative though, if the stresssensing effect can explain our observed deviation of stress fiberorientation from the direction of minimum strain.

In addition, cells as well as actin stress fibers align alonggeometric constraints either provided by adhesive patterns ofsurfaces or by micro-grooved substrates which has also beenobserved in myoblasts [29]. We show that geometric constraints ofthe adhesive lines with 30 mm width have a significant effect onstress fiber orientation. On a homogeneously functionalized

ology and organization on biochemically micro-patterned hydrogel/j.biomaterials.2009.09.047

Fig. 8. Image of actin striations (phalloidin staining) observed in myoblasts subjected to CTS. Actin striations were observed in many different cells but strikingly only on the 45�

micro-patterns. The red arrows in (a) and (b) indicate the region from which the intensity profile was measured. Both show a periodicity of approximately 1.5 mm. Actin striationssuggest the induction of differentiation pathways to functional skeletal muscle. (scale bar¼ 10 mm).

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substrate the cells spread out randomly resulting in a wide range ofactin stress fiber angles (Figs. 2a and 3a). In the strain-free state, themyoblasts sense the geometric constraint imposed by the micro-pattern and develop stress fibers highly organized along the axis ofthe pattern (Figs. 2c and 3c). A similar alignment of myoblasts canbe achieved by micro-grooved substrates but such alignment didnot have influence on proliferation or differentiation [29].

The combination of CTS and substrate patterning allowmimicking of in vivo conditions where cells are aligned along thestrain direction as in muscle tissue. The physiological significance ofalignment and loading [3,5] led to increasing efforts in the devel-opment of appropriate experimental systems [22,46]. Severalpossible mechanisms have been proposed to explain cell responseto two types of mechanotransduction which include applied forcesand response to topography [47]. For example, topographic cuesmay directly transduce signals from the plasma membrane to thenucleus through the microtubule and intermediate filamentnetwork resulting in changes in gene expression. Another mecha-notransduction pathway could be induced by applied forces such assubstrate strain, transmitting tensile signals from focal adhesions tothe nucleus via actin stress fibers. In our study, the applied force isthe substrate CTS and the topographic stimuli is the geometricconstraint created by the micro-pattern. By using covalently boundfibronectin micro-patterns we are able to decouple the effect ofimposed cell orientation from three-dimensional topography. Thestretching system utilized here provides a simple method toexplore the synergistic effect of these two types of mechano-transduction on the organization of cell shape, internal cellularstructures, and changes in function. Important for such investiga-tions is the applicability of micro-patterned stretchable PDMSsubstrates for long-term studies. Our substrates fully meet thisrequirement since no defects were observed after 4 days ofcontinuous cyclic stretching (7%, 0.5 Hz).

Please cite this article in press as: Ahmed WW, et al., Myoblast morphcoatings under cyclic mechanical strain, Biomaterials (2009), doi:10.1016

When CTS is applied to the micro-patterned substrates the cellsreorganize their actin stress fibers while geometrically constrainedto a 30 mm line width. Thus, the CTS and geometric constraints arecompeting forms of stimuli where the applied strain drives actinstress fiber organization orthogonal to the strain direction, but theadhesive micro-patterns promote stress fiber orientation alongtheir axis. This may explain the observation that stress fiberorientation was 47.9� (�19.5�) on the 0� patterns subjected to CTSbecause the geometric constraint promotes 0� stress fiber organi-zation but the applied strain drives stress fibers perpendicular tothe strain direction. Actin stress fiber angle was 91.0� (�14.0�) onthe 90� micro-pattern because the geometric constraint andapplied stress may both promote stress fiber orientation along 90�.The mean actin stress fiber orientation of 51.8� (�16.8�) measuredfor cells on the 45� micro-pattern and the corresponding aniso-tropic distribution (bias to higher angles) can also be explained bythe competition between the two competing stimuli and shift tohigher orientation angles by the periodic strain.

Similarly, strain direction and geometric constraints arecompeting types of stimuli on the myoblast nucleus although itappears that the geometric constraint is the dominant factor indetermining nucleus shape and orientation. CTS increases theaspect ratio, and may have a weak influence on alignment. Nucleusorientation does not follow actin stress fiber orientation understretching conditions. This may be attributed to the fact that unlikeactin stress fibers which are linked to the substrate via focaladhesions complexes, the nucleus is not directly anchored to thesubstrate and therefore the influence of the substrate strain isdecreased. However, it has been shown that there is a mechanicallink between the nucleus and the actin cytoskeleton and that actinplays an important role in nuclear positioning [48–50]. In regards tonuclear shape and orientation, we note from past studies [51–54]that the spatial positioning of chromosomes in the nucleus is not

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random and mechanical deformation causes centromere rear-rangement and altered gene expression. Thus the observed effect ofnuclei elongation may alter gene regulation and ultimately cellfunction.

Cross-bridging was observed more often on the 0� orientationwhen subjected to CTS relative to the 45� and 90� orientations. Thismay be caused by the competing stimuli from the applied strainand the geometric constraint which may drive the cell to orientalong different directions. As a result, cells form agglomerations tominimize contact area with the substrate and eventually are able tobridge the entire 40 mm span over the passivated region betweenthe adhesive lines. It has been reported that single REF52wt cellscannot bridge passivated regions 12 mm or greater [55].

Actin striations were observed in myoblasts on the 45� micro-pattern when subjected to CTS. The formation of actin striationssuggests the beginning of development of acto-myosin sarcomeresfound in mature myotubes. Earlier studies have suggested that, inthe absence of CTS, mature sarcomeric striations form on softsubstrates with stiffness comparable to myoblasts (w12 kPa) [14].However sarcomere formation was not observed on substrates withstiffness above 40 kPa. Here, we observed sarcomere formation ona w1 MPa (two orders of magnitude stiffer) substrate when CTS isapplied to myoblasts on the 45� micro-patterns. Coincidentally, theaverage actin stress fiber orientation of the myoblasts exhibitingactin striations (51.8�) is quite close to the angle of zero normalstrain (54.7�), and the direction of maximum shear strain (45�), aspredicted by linear elasticity (n¼ 0.5). Thus in this orientation theindividual actin fibers experience a small change in length due toaxial strain but a large shear stress may be transduced to the nucleusthrough the cytoskeletal network. This highlights the role of specificstrain states on myogenesis that may overcome the inhibiting role ofstiff substrates.

In summary, our observations highlight the importance of thenew technique presented that allows simultaneous control of bothcell alignment and strain direction. Past studies of myoblastmechanosensing have addressed either geometric or mechanicalstimuli independently. Our system utilizes CTS, NCO-sP(EO-stat-PO) passivation, and micro-contact printing to provide a simplemethod to study the role of applied stress on the development ofmyoblasts and other adherent cells.

5. Conclusions

We have introduced a method to prepare deformable cell culturesubstrates that are passivated by a thin hydrogel coating eliminatingunspecific cell adhesion to apply long-term CTS on cells. Our NCO-sP(EO-stat-PO) hydrogel films are micro-patterned with cell adhesionmediating proteins and the substrate maintains this precisebiochemical functionalization and effective passivation undercontinuous cyclic loading for extended durations of several daysunder standard cell culture conditions. Our method provides controlof cell orientation relative to the applied strain direction and thusallows the investigation of the various stress states on adherent cellsfor extended durations. We used this system to study the effect ofstrain direction and geometric constraints on the development ofstructure and organization of C2C12 myoblasts. It was found that thedirection of applied strain with respect to the cell axis inducessignificantly different morphological and cytoskeletal responses: the0� micro-pattern exhibited the most irregular actin stress fiberorientation, whereas actin was highly organized along the 90�

pattern. The nuclei elongation appeared to be relatively insensitive toactin stress fiber orientation and CTS, and the geometric constraintseemed to determine nuclei aspect ratio and orientation. Sarcomericactin striations were observed in cells along 45� line patterns – thedirection of maximum shear strain and negligible axial strain.

Please cite this article in press as: Ahmed WW, et al., Myoblast morphcoatings under cyclic mechanical strain, Biomaterials (2009), doi:10.1016

Acknowledgments

The authors would like to thank Christine Mollenhauer and Hen-riette Ries for their technical assistance in the biology and chemistrylaboratories at MPI-MF. This work was supported by the NationalScience Foundation (NSF ECCS 0524675), the VW-foundation (projectself-assembled bioactive hydrogels), the DFG (graduate school 1035biointerface, TR-SFB 37), and the Max Planck Society (FHG-MPGInitiative).

Appendix

Figures with essential colour discrimination. Certain figures inthis article are difficult to interpret in black and white. The fullcolour version can be found in the on-line version at doi:10.1016/j.biomaterials.2009.09.047.

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