Directing the Morphology and Differentiation of Skeletal Muscle

Shaoqu Xie , Xiao Zhang , Michael P. Walcott , Hongfei Lin .... Zeinab Hosseinidoust , Md Nur Alam , Goeun Sim , Nathalie Tufenkji , Theo G. M. van de...
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Directing the Morphology and Differentiation of Skeletal Muscle Cells Using Oriented Cellulose Nanowhiskers James M. Dugan,† Julie E. Gough,† and Stephen J. Eichhorn*,†,‡ Materials Science Centre and Northwest Composites Centre, School of Materials, The University of Manchester, Grosvenor Street, Manchester M14 9PL, United Kingdom Received June 18, 2010; Revised Manuscript Received July 20, 2010

Radially oriented submonolayer surfaces of 10-15 nm diameter cellulose nanowhiskers (CNWs) were prepared by spin-coating. The response of myoblasts (muscle cells) to the surfaces was assessed using atomic force microscopy (AFM), immunocytochemistry, and image analysis. Despite the small size of the CNWs, the myoblasts oriented along the CNW surfaces. Upon differentiation, the myoblasts produced striking radial patterns of myotubes, following the radial pattern of the CNWs. This facile method of nanopatterning surfaces may be applied where the directed growth of tissue is required and shows for the first time the potential of CNWs for tissue engineering applications.

Introduction The interaction of living cells with both natural and synthetic materials continues to attract interest from a diverse range of scientists, engineers, and clinicians. With the capacity not only to support cell attachment and proliferation but also to direct tissue development actively, there is a great deal of excitement about the potential application of engineered material scaffolds to regenerative medicine, tissue engineering, and fundamental research in tissue development.1-3 In particular, the morphology and function of various cell types have been modulated and controlled using bioactive materials with microscale and nanoscale architectures.4-7 Here we present spin-coated surfaces of oriented CNWs that despite their small dimensions not only support the attachment and proliferation of myoblasts but also direct differentiation and fusion to form characteristic myotubes (muscle fibers). The initial response of the myoblasts was assessed using AFM to image the physical interactions between the cells and the CNWs, whereas the formation of focal adhesions and F-actin cytoskeleton was imaged using immunocytochemistry and confocal microscopy. The orientation and height of the CNWs and the effect of the CNW surfaces on myoblast morphology was determined using semiautomated morphological image analysis. Differentiation was induced in confluent monolayers of myoblasts, and the resulting myotubes were stained for myosin using immunocytochemistry and imaged using large area confocal microscopy. Skeletal muscle exhibits a high degree of order and bulk orientation, which reflects the directional nature of muscle contraction.8 On a cellular level, mature muscle consists of muscle fibers that are extremely large, highly elongated cells with multiple nuclei. Myoblasts are the proliferative precursor cells of skeletal muscle and undergo differentiation and fusion to produce muscle fibers during muscle development and repair. Upon fusion, the characteristic proteins of skeletal muscle, including those involved in muscle contraction, are produced, and proliferation is down-regulated.9 * To whom correspondence should be addressed. E-mail: s.j.eichhorn@ manchester.ac.uk. Tel: +44 (0)161 306 5982. Fax: +44 (0)161 306 3586. † Materials Science Centre. ‡ Northwest Composites Centre, School of Materials.

In vitro, myoblasts have been shown to differentiate and fuse to produce immature muscle fibers known as myotubes. Fusion has been shown to occur in an end-to-end configuration,10 so the morphology and spatial arrangement of myoblasts as well as the freedom to move in different directions have therefore been shown to have an effect on the morphology and relative orientation of the resulting myotubes.11-13 Under standard conditions in vitro, the fusion and differentiation of myoblasts yields monolayers of myotubes with no bulk orientation and thus little similarity with mature skeletal muscle tissue. It is important, therefore, to develop methods to direct the differentiation of myoblasts to produce myotubes with a high degree of relative orientation. As well as finding potential applications in regenerative medicine, such techniques are likely to prove useful for understanding the development of muscle and other anisotropic tissues. Previous studies using model topographies have shown a strong dependence of myoblast morphology, fusion, and differentiation on the height, width, pitch, and relative orientation of ridged and grooved surfaces.11,14-16 Similar responses have also been shown on more complex materials such as oriented electrospun polymer scaffolds.12,13,17,18 To date, however, such studies have only employed topographical features on the micrometer or submicrometer scales. Here we present the first study of myoblast response and differentiation on truly nanoscale materials. CNWs are rod-like nanoparticles produced by the incomplete hydrolysis of native cellulose. Their shape is a consequence of the natural structure of cellulose microfibrils, and their aspect ratio depends on the source organism and the reaction conditions of hydrolysis.19,20 As a result of the abundance and relatively low cost of native cellulose coupled to the extremely high aspect ratio, mechanical properties, surface area, and electrical and magnetic properties of the CNWs, potential applications have been proposed in advanced composite materials as well as optical and electronic devices.21,22 Although other forms of cellulose have been investigated with respect to biocompatibility and degradation,23-26 the bioactivity of CNWs has never been reported, and no potentially therapeutic applications have previously been demonstrated. We have produced surfaces of adsorbed CNWs with a high degree of bulk orientation, which

10.1021/bm100684k  2010 American Chemical Society Published on Web 08/06/2010

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not only direct the morphology of myoblasts but also control the orientation of muscle fiber formation. The tunicates, particularly the ascidians, are unusual among animals in that they produce a nanofibrillar structural polysaccharide (tunicin cellulose) that is similar in many ways to the cellulose produced by plants. Tunicin cellulose has a higher degree of crystallinity than plant cellulose, however, allowing the preparation of CNWs with particularly high aspect ratios.27 We employed spin-coating to produce surfaces of CNWs with a high degree of radial orientation on pieces of optical glass pretreated with the cationic polyelectrolyte polyallylamine hydrochloride (PAHCl). Our method was inspired by that of Cranston and Gray, who used spin-coating to produce oriented layer-by-layer composites of cotton CNWs.28 The degree of orientation in these composites was found to increase with increasing numbers of adsorbed double-layers. Here we have produced submonolayers of sparsely adsorbed tunicin CNWs. By using the higher aspect ratio tunicin CNWs and by depositing the CNW suspension onto the samples at high rotational speed, we have achieved a high degree of orientation without building up multiple layers of polyelectrolyte.

Materials and Methods Preparation of Cellulose Nanowhiskers. An aqueous suspension of tunicin CNWs was prepared according to the method of ElazzouziHafraoui et al.20 In brief, the gutted tunics of Halocynthia roretzi were deproteinized and bleached with alternating treatments of potassium hydroxide and sodium chlorite. The purified tunicin cellulose was then partially hydrolyzed with sulfuric acid (48% w/w) at 55 °C for 13 h. The crude suspension was purified by repeated centrifugation and dialyzed against deionized water. The suspension was finally stabilized by ultrasonification for 1 min. Preparation of Spin Coated Surfaces of Cellulose Nanowhiskers. The CNW surfaces were prepared using a Laurell Technologies spin processor (model ws-400b-6npp/lite). Optical glass coverslips (Fisher Scientific, Loughborough, U.K.) were cut into rectangular fragments measuring 9 × 18 mm2 and cleaned by soaking for 20 min in a 3:1 (v/v) mixture of sulfuric acid (98% w/w) and hydrogen peroxide (30% w/w aq) (“Piranha solution” - highly corrosive), followed by rinsing in copious quantities of deionized water. Each fragment was then accelerated to 3000 rpm on the spin-coater, and an aliquot of PAHCl (0.6% w/v aq, 200 µL) was dropped onto the spinning glass. After 10 s, two aliquots of deionized water (2 × 500 µL) were dropped onto the glass to rinse the surfaces. To prepare CNW surfaces with a high degree of orientation, we increased the spin speed to 6000 rpm, and the CNW suspension (200 µL, 0.02% w/v) was dropped onto the glass fragment. After 10 s, the speed was lowered to 3000 rpm, and the sample was rinsed with two aliquots of deionized water. These samples are designated “C6000”. To produce surfaces with a lower degree of radial orientation, the glass fragments were decelerated to 0 rpm following the PAHCl adsorption step. The CNW suspension (200 µL, 0.02% w/v) was then dropped onto the stationary glass fragment, and adsorption was allowed to take place for 20 s before acceleration to 500 rpm. The samples were rapidly rinsed three times at 500 rpm before finally spinning at 3000 rpm for 20 s to dry the samples. These surfaces are designated “C500”. A schematic of the surface axes is given in Figure 1. AFM Microscopy and Image Analysis. The CNW surfaces were characterized using atomic force microscopy (AFM). A Veeco MultiMode AFM with Digital Instruments Nanoscope II controller was used in tapping mode with N-doped silicon anisotropic probes with typical resonant frequency of ∼320 kHz and force constant of 42 N m-1 (TESPA, Veeco Probes, Cambridge, U.K.). All AFM images were flattened using Gwyddion SPM software (Czech Metrology Institute, Czech Republic).

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Figure 1. Schematic showing a plan view of a rectangular glass fragment, upon which the CNW surfaces were spin coated. Examples of several radial axes as well as the x axis and y axes of the sample are shown (arrows).

The degree of orientation and the height distributions of the CNW surfaces were quantified using morphological image analysis. MATLAB and the MATLAB Image Processing Toolbox (Mathworks, v2009a) were used to carry out the image analysis procedures. In brief, the degree of orientation was calculated from 5 AFM topography images for each condition. Images were thresholded, binarised, and disconnected using a series of morphological operations. For each image, the objects corresponding to the CNWs were fitted to ellipses, and the angles of the major ellipse axes relative to the image x axis were calculated. The standard deviation of angles was calculated for each image, and the mean standard deviation (σCNW) was calculated for each condition. A Student’s t test was carried out on the standard deviation data. To quantify the diameters of the CNWs and therefore the height distribution of the adsorbed surfaces, samples were prepared by the same method as for the C500 surfaces with a slight modification. A CNW suspension with a concentration of 0.004% (w/w) was used to achieve a more sparsely adsorbed surface. Three AFM topography images were captured and separated into foreground (CNW) and background (glass) components by thresholding and filtering by object size. The mean background height was subtracted from the foreground topography data, and the adjusted heights of the pixels corresponding to CNWs were combined and binned to produce a histogram. Maintenance and Differentiation of Myoblasts. C2C12 is a murine myoblast cell line known to be useful in research because of its rapid differentiation, fusion, and production of characteristic muscle fiber proteins.29 Proliferative C2C12 myoblasts were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Paisley, U.K.) supplemented with 10% (v/v) fetal bovine serum (FBS, Invitrogen) and 1% penicillin/streptomycin preparation (Invitrogen) at 37 °C and 5% CO2. Cells were passaged when subconfluent with Trypsin EDTA preparation (0.25%, percentage, Invitrogen), and growth medium was replaced every 2 days. To investigate differentiation on the CNW surfaces, myoblasts were seeded at a high density of 290 mm-2 and were allowed to become confluent over 24 h. Differentiation was induced by replacing the growth medium with a formulation in which the 10% (v/v) FBS was replaced with 2% (v/v) horse serum. Differentiation and fusion were allowed to occur over 7 days. For all other experiments, the myoblasts were seeded at a lower density of 115 mm-2. Processing of Tissue Cultures for Microscopy. Myoblasts were fixed for microscopy at different time points after seeding to the CNW surfaces. For investigation of the distribution of F-actin and focal adhesions, the myoblasts were fixed after 4 and 12 h, and for microscopy of the differentiated myotubes, the cells were fixed after 7 days. In each case, the growth medium was removed, and the surfaces were rinsed twice with Dulbecco’s phosphate-buffered saline (PBS, Invitrogen). The cells were fixed with paraformaldehyde (SigmaAldrich, Poole, U.K., 3% w/v in PBS) for 10 min and were then permeabilized and blocked simultaneously for 30 min using a blocking buffer consisting of 0.1% (v/v) Triton X-100 (Sigma-Aldrich), 0.1%

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Figure 2. (a) AFM topography image of a C500 surface. Arrow indicates the approximate local radial axis. Scale bar ) 5 µm. (b) AFM topography image of a C6000 surface. Arrow indicates the approximate local radial axis. Scale bar ) 5 µm. (c) Bar chart of the standard deviation of CNW orientation on C500 and C6000 surfaces. Error bars were calculated as 95% confidence intervals (*** denotes p < 0.001). (d) Histogram of CNW pixel heights from AFM topography images.

(w/v) bovine serum albumin (Merck, Darmstadt, Germany), and 1% (v/v) goat serum (Sigma-Aldrich) in PBS. For AFM imaging of myoblasts on the CNW surfaces, the cells were fixed 12 h after seeding. The growth medium was removed, and the surfaces were rinsed twice with PBS. The cells were then fixed with glutaraldehyde (TAAB, Aldermaston, U.K., 1.5% v/v in phosphate buffer) for 30 min at 4 °C. The samples were washed three times with double-distilled water before being allowed to dry in air at room temperature. Immunocytochemistry. Immunocytochemistry was carried out using well-documented methods.30,31 We investigated focal adhesions by staining for vinculin using a primary monoclonal mouse antibody raised against human vinculin (Sigma-Aldrich, product number v9131, 0.001% v/v in blocking buffer) and a secondary AlexaFluor-546-conjugated goat antibody raised against mouse IgG (Invitrogen, product number a-11003, 0.001% v/v in blocking buffer). Filamentous-actin was stained with FITC-conjugated phalloidin (Sigma-Aldrich), and differentiated myotubes were stained with a monoclonal mouse antibody raised against rabbit heavy-chain fast skeletal myosin (Abcam, Cambridge, U.K., product number ab7784, 0.001% v/v in blocking buffer), followed by a secondary Alexa Fluor 488 conjugated goat antibody raised against mouse IgG (Invitrogen, product number a11001, 0.001% v/v in blocking buffer). All samples were mounted with ProLong Gold mounting reagent containing DAPI nuclear counterstain (Invitrogen). Fluorescence microscopy was carried out using a Leica SP5 confocal microscope equipped with motorized stage. Determination of Myoblast Orientation by Microscopy and Image Analysis. To quantify the orientation of the myoblasts on the CNW surfaces, a morphological image analysis technique was used. Myoblasts were seeded to the surfaces at a density of 115 mm-2 with glass coverslips as controls. After 12 h, the growth medium was removed, and the surfaces were rinsed twice with PBS. The live cells were then stained with carboxyfluorescein diacetate succinimidyl ester (1 µM in Hank’s balanced salt solution, Sigma-Aldrich) for 10 min at 37 °C before being fixed with paraformaldehyde (Sigma-Aldrich, 3% w/v in PBS) for 10 min and permeabilized with blocking buffer for 5 min. The samples were then mounted with ProLong Gold mounting reagent containing DAPI nuclear counterstain (Invitrogen). For each

Figure 3. (a) Fluorescence micrograph of myoblasts growing on a C500 surface 12 h after seeding, stained with CFDA SE. Arrow indicates the approximate local radial axis. Scale bar ) 75 µm. (b) Fluorescence micrograph of myoblasts growing on a C6000 surface 12 h after seeding, stained with CFDA SE. Arrow indicates the approximate local radial axis. Scale bar ) 75 µm. (c) Histogram of myoblast orientation angles relative to the image x axis on a glass coverslip control surface. (d) Histogram of myoblast orientation angles relative to the x axis of the image on C500 surfaces. (e) Histogram of myoblast orientation angles relative to the x axis of the image on C6000 surfaces. (f) Mean standard deviation of myoblast orientation angles on glass coverslip control surfaces, C500 and C6000 surfaces (*** denotes p < 0.001).

condition, 3 samples were used, and 5 images were captured for each sample, giving a total of 15 images for each condition. Fluorescence micrographs were captured using a Leica SP5 confocal microscope, sampling images at arbitrary points along the x axis of the samples to ensure that the approximate local radial axis was the same for each image. These images were thresholded and disconnected using CellProfiler software (Broad Institute of MIT and Harvard University). The orientation of the individual cells relative to the x axis of the images was quantified by a method similar to that used for the CNWs. The standard deviation for each image was calculated as a descriptor for the relative degree of orientation, and the mean standard deviation (σM) was calculated for each condition. A one-way ANOVA with Tukey’s post hoc comparison was carried out to test for a significant difference in standard deviation between conditions.

Results and Discussion Characterization of Surfaces of Cellulose Nanowhiskers. AFM topography images of the C500 and C6000 surfaces are shown in Figure 2a,b respectively. It is clear that the adsorption of CNWs on both of the surfaces was homogeneous and that

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Figure 4. (a-f) Myoblasts stained for vinculin (red), F-actin (green), and nuclei (blue), scale bar ) 50 µm. (a) 4 h after seeding on glass coverslip control, (b) 4 h after seeding on C500 surface, (c) 4 h after seeding on C6000 surface, (d) 12 h after seeding on glass coverslip control, (e) 12 h after seeding on C500 surface, and (f) 12 h after seeding on C6000 surface. (g-i) Close-up images of myoblasts stained for vinculin, scale bar )10 µm. (g) 12 h after seeding on glass coverslip control, (h) 12 h after seeding on C500 surface, and (i) 12 h after seeding on C6000 surface.

the degree of adsorption was similar in both cases. The degree of orientation of the long axes of the CNWs relative to each other was clearly much greater on the C6000 surface, however. Most of the CNWs on the C6000 surface were oriented with their long axes roughly in line with the approximate local radial axis, indicating that the CNWs were oriented radially as a consequence of the higher spin speed used in the preparation of the surfaces. The standard deviation of the CNW orientation angles (σCNW) was significantly lower for the C6000 surfaces (Figure 2c), indicating a narrower distribution and therefore a greater degree of orientation (p < 0.001). A histogram of CNW pixel heights from the analysis of AFM topography images is shown in Figure 2d. The mean CNW pixel height was 17.6 nm, and the modal pixel heights lay in the range of 10-15 nm. The distribution shown in Figure 2d will include contributions from the true diameters of the CNWs as well as the effect of overlapping CNWs and areas where several CNWs are stacked on top of each other. The modal range is therefore likely to be the best estimate of the average CNW diameter, whereas the mean may be a good estimate of the average height experienced by the myoblasts growing on the surfaces.

Determination of Myoblast Orientation. The relative orientation of the myoblasts on the C500 surfaces was significantly lower than that on the C6000 surfaces, which is evident from the micrographs in Figure 3a,b. Although not all cells are oriented in the same direction as the approximate local radial axis, the most elongated of the cells in Figure 3b are oriented radially, indicating that the greater radial orientation of CNWs on the C6000 surfaces does indeed increase orientation of the myoblasts growing on the surfaces. Indeed, significantly less orientation on the glass control and C500 surfaces is evident from the orientation angle distributions shown in Figure 3c-e; the distributions are much broader for the glass control and C500 surfaces, indicating a lower degree of orientation. The orientation distribution of the cells on the C6000 surfaces, as shown in Figure 3e, is, however, much narrower, indicating a greater degree of alignment. The mean standard deviations of orientation angles are shown in Figure 3f. The standard deviation of orientation angles was significantly lower for cells growing on the C6000 surfaces compared with the C500

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surfaces and the glass coverslip controls (p < 0.001). There was no significant difference between the C500 surfaces and the control surfaces. Investigation of Distribution of Cytoskeleton and Focal Adhesions. The arrangement of the F-actin component of the cytoskeleton and focal adhesion formation on the CNW surfaces was investigated using immunocytochemistry and confocal microscopy. At 4 h after seeding, a difference in the morphology of the myoblasts was observed on the different surfaces. On the glass coverslip control surfaces, shown in Figure 4a, the cells appeared generally smaller and less well spread than on the C500 and C6000 surfaces (cf. Figure 4b,c, respectively). On both types of CNW surfaces, more vinculin appeared to be expressed, and the vinculin was more organized around the edges of the cells into discrete focal adhesions. No significant stress fibres were observed on any of the surfaces at the early time point of 4 h. The difference in morphology may arise from differences in roughness between the surfaces because the glass control is thought to be significantly smoother than the CNW surfaces. It has been previously reported that the roughness of biomaterials can affect the response of cells growing on surfaces.32 At 12 h after seeding, the myoblasts were well spread on all types of surface, as shown in Figure 4d-f, and stress fibers were observed on all types of surface. Many well-developed, brightly stained focal adhesions were observed on all of the surface types, although there was some differences in the organization of the vinculin on the CNW surfaces relative to the glass controls. Figure 4g-i shows high magnification micrographs of stained vinculin 12 h after seeding. On the glass control, in Figure 4g, the vinculin was predominantly organized into large, “dash-shaped” focal adhesions. On the CNW surfaces, however, there were many more small “dot-shaped” adhesions spread across the entire cell, as shown in Figure 4h,i, in addition to the mature focal adhesions. This possibly indicates that the cells may be more motile on the CNW surfaces compared with the glass control, and as such, these cells form smaller transitory adhesions to the surfaces. Alternatively, this may indicate that there may be integrin-mediated specific interactions between the CNW surfaces and the myoblasts, resulting in the formation of many more small adhesions. AFM of Myoblasts on Surfaces of Cellulose Nanowhiskers. In Figure 5, an AFM topography image of a myoblast lamellipodium is shown at 4 h. Several extremely fine filopodia were observed extending from the edge of the cell. It has been proposed that filopodia play a key role in the exploration of a cell’s surrounding area and topography.33 Therefore, it is likely that the myoblasts “explore” the shape and orientation of the CNWs in this way. Most filopodia appear to be extended in the direction of the bulk CNW orientation, suggesting that the morphology of the cells followed from the extension of filopodia to explore the surrounding topography. Interestingly, the filopodia that were not oriented in line with the approximate local radial axis were most likely to be oriented at right angles to this axis, as shown in Figure 5. It is unclear what the implications of this may be, although there have been some reports of cells elongating at right angles to the axis of ridges and grooves on surfaces, contrary to expectations.34 Differentiation and Fusion of Myoblasts on Surfaces of Cellulose Nanowhiskers. Figure 6 shows myotubes formed by the differentiation and fusion of myoblasts. The large scale micrographs in Figure 6a-c clearly show the effect of the radial alignment of the underlying CNWs on the arrangement of the myotubes. On the glass control and the C500 surface, as shown

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Figure 5. AFM topography image of a myoblast on a C6000 surface, 12 h after seeding. Inset image shows a large scale AFM topography image of the whole cell. Inset scale bar ) 20 µm. Main image shows a close up scan of the area bounded by the dashed box in the inset image. Arrows indicate filopodia. Scale bar ) 5 µm.

in Figure 6a,b, respectively, there is very little relative orientation, and the myotubes formed whorled patterns with no orientational order, as would be expected on isotropic topographies.11 On the C6000 surface, however, as shown in Figure 6c, a striking radial pattern of myotubes is clearly visible. It is interesting to note that the degree of orientation appears to be significantly greater for these differentiated myotubes than for the proliferating myoblasts. Figure 6d-f show high magnification micrographs of myotubes on the glass control and CNW surfaces. On this length scale, it is clear to see that differentiation had occurred more successfully on the CNW surfaces than on the glass control. Figure 6d shows myotubes on the glass control. The morphology on the glass control is less elongated than that on the CNW surfaces shown in Figure 6e,f, and there are more regions of myosin expression with poorly defined morphology. On both the C500 and C6000 surfaces, however, the morphology of the myotubes was well-defined and elongated with strong myosin expression. The myotubes on the C6000 surface, as shown in Figure 6f, are more highly oriented relative to the glass control and C500 surface. Although nanoscale materials of similar dimensions to CNWs have previously been shown to be bioactive,35 mammalian cells have never been shown to exhibit contact guidance on such small features as CNWs. The striking effect from merely changing the spatial arrangement of the rod-like CNWs indicates that the cells interact with the material by some mechanism that operates on a similar size scale to the CNWs and that the bioactivity of the material does not simply stem from bulk properties. Of course, the cells may interact with the sulfonate groups present on the surface of the CNWs, which are negatively charged. The nature of this interaction is left as a topic for future research. In previous studies, to determine the dependence of contact guidance on topographical features, a lower size limit of 35 nm was identified to induce contact guidance in fibroblasts.36 Indeed, in studies that employ substrates with model anisotropic topographies, such as ridges and grooves, fibroblasts have been shown to be the most sensitive cell type to

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Figure 6. (a-c) Large scale tile scans of differentiated myotubes stained for myosin 7 days after induction of differentiation: Myotubes on (a) a glass control surface, (b) a C500 surface, and (c) a C6000 surface. Arrows indicate the radial axes. Scale bars ) 1 mm. (d-f) Higher magnification micrographs of differentiated myotubes stained for myosin: Myotubes on (a) a glass control surface, (b) a C500 surface, and (c) a C6000 surface. Scale bars ) 250 µm.

topographical cues. We have shown in this work, however, that features of only ∼10-20 nm are sufficient to induce contact guidance in myoblasts. Although our CNW surfaces are not model topographies, we suggest that this work demonstrates that myoblasts are a particularly receptive cell type to topographical control. To our knowledge, no studies to determine the effects of nanoscale topography on myoblasts have been published, although some work using submicrometer- and micrometer-scale model topographies have been carried out.10,11,14,15 It has been suggested that nanotopography may prove to be a powerful tool for engineering bioactive surfaces for medical devices.37 We propose that ordered nanotopography could also prove to be useful for tissue engineering skeletal muscle, providing a means for generating tissue with a bulk order similar to mature tissue in vivo. Classical scaffolds with submicrometerscale fibrous- or sponge-like architectures may be of less use in the regeneration of this tissue because the scaffolds themselves would mechanically compete with the contractile muscle fibers.17 A more subtle approach, using truly nanoscale materials to guide the genesis of skeletal muscle in vitro, may therefore prove to be of greater utility both in regenerative medicine and for the understanding of skeletal muscle development. CNWs provide a relatively low cost and scalable method for producing nanopatterned surfaces for biological control. Moreover, there are several methods by which CNWs may be oriented, other than spin-coating, including the use of magnetic38 and electric fields39 and by nematic phase ordering into the dried state.40 In addition to possible therapeutic applications, we believe that this work could help contribute to the understanding of the mechanisms by which cells interact with nanotopography. By identifying myoblasts as a particularly sensitive cell type to

topographical control, we hope to facilitate the identification of specific differences in phenotype and signaling that modulate cell response to topography. Such anisotropic surfaces may also prove useful as biomimetic substrates and research tools for cell biology.

Conclusions We have shown that submonolayer surfaces of tunicin CNWs may be produced with a high degree of orientation using a simple spin-coating method. Myoblasts have been shown to sense the topography of these surfaces effectively and to orientate relative to the bulk direction of CNW orientation. Moreover, after induction of differentiation, the resulting skeletal muscle myotubes are almost entirely radially oriented, in line with the CNWs. With future work, we believe that this novel use of cellulose will find applications in regenerative medicine and in improving the understanding of tissue development. Acknowledgment. We thank Dr. Laurent Heux, CERMAV, Grenoble, France for providing the sample of tunicin CNWs used in this work.

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