Bioactive Chemical Nanopatterns Impact Human Mesenchymal Stem

Letter pubs.acs.org/NanoLett

Bioactive Chemical Nanopatterns Impact Human Mesenchymal Stem Cell Fate Zhe A. Cheng,†,‡ Omar F. Zouani,*,† Karine Glinel,‡ Alain M. Jonas,‡ and Marie-Christine Durrieu*,† †

Institut Européen de la Chimie et Biologie, CBMN-UMR5248, Université de Bordeaux 1, 2 Rue Robert Escarpit, 33607 Pessac, France ‡ Institut de la Matière Condensée et des Nanosciences, Université Catholique de Louvain, 1 Croix du Sud, B-1348 Louvain-la-Neuve, Belgium S Supporting Information *

ABSTRACT: We present a method of preparing and characterizing nanostructured bioactive motifs using a combination of nanoimprint lithography and surface functionalization. Nanodots were fabricated on silicon surfaces and modified with a cell-adhesive RGD peptide for studies in human mesenchymal stem cell adhesion and differentiation. We report that bioactive nanostructures induce mature focal adhesions on human mesenchymal stem cells with an impact on their behavior and dynamics specifically in terms of cell spreading, cell-material contact, and cell differentiation. KEYWORDS: Nanoimprint lithography, surface functionalization, mesenchymal stem cell, focal adhesion, differentiation

T

in stem cell research is thus to understand the mechanisms that direct cell differentiation into a specific cell lineage within a given nanoscale environment. A typical example of a nanoscale signaling inducing a regulation of cell behavior is provided by focal adhesions (FAs). FAs lie at the convergence of integrin clustering, signal transduction, and actin cytoskeleton organization.21,22 Cells modify FAs in response to changes in the molecular composition and physical forces present in their ECM environment.23−31 The exact composition of a given FA will in turn regulate intracellular tension, effecting cellular behaviors such as adhesion, migration, proliferation, and differentiation.32−36 In this study, we combined nanoimprint lithography with surface modification techniques to prepare material surfaces that are chemically patterned with nanosized bioactive features, over a total area of about 1 cm2. These platforms allow cellular assays to be carried out for the investigation of hMSC-material nanointeraction. Various nanofabrication techniques such as ebeam lithography, colloidal lithography, and nanoimprint lithography (NIL) were explored to produce nanopatterned surfaces. Among them, NIL offers a series of advantages related to its ease-of-processing, rapidity, and versatility.37−45 As a template-based system, NIL operates by transferring a predefined pattern from a master mold to a material surface. Thus, surface features ranging from the microscale down to the nanoscale can be constructed with no limit on geometry. Motifs with varying shapes (circles, squares, lines), sizes, and

he optimization of biomaterial surface interactions with biological components is important for sustaining an artificial environment capable of directing and maintaining favorable cell and tissue growth. Essentially, biomaterial surfaces should be able to function compatibly in physiological conditions by imitating the role of the in vivo extracellular matrix (ECM) with which cells and tissues naturally come in contact. In turn, a correct type of cells, an appropriate scaffold designed at different spatial scales, and a smart choice of signaling biomolecules must all be incorporated in the design of a biomaterial.1 As in vivo cellular interactions occur on the nanoscale, surface nanopatterning has gained interest and attention as a unique means of mimicking micro and nanoenvironments in which cells and tissues thrive.2 For instance, nanoscale topographies have a noticeable effect on the behavior of stem cells, and reports have shown the effects of nanofeature dimensions on stem cell fate. 3−8 Though numerous studies have been published on the effects of physical surface nanotopography on stem cell behavior, few are concerned with the nanoorganization of signaling molecules. We demonstrate here the impact of biochemical nanostructuring on the behavior of human mesenchymal stem cells (hMSCs), in particular the formation of mature focal adhesions (FAs) and changes in cell commitment. hMSCs are a type of adult stem cells derived from bone marrow that show promise in the regeneration of damaged tissues due to their multipotent capacities, being able to differentiate into osteoblasts, adipocytes, and chondrocytes, among other mature cell types.9,10 The differentiation of hMSCs can be modulated by chemical and physical forces present in their ECM environment.1,11−20 One important goal © XXXX American Chemical Society

Received: June 3, 2013 Revised: July 24, 2013

A

dx.doi.org/10.1021/nl4020149 | Nano Lett. XXXX, XXX, XXX−XXX

Nano Letters

Letter

interspacing can be fabricated, contributing to the versatility of the system. Furthermore, the imprinting method allows large areas to be patterned on the nanoscale without the need of a time-consuming process.46 We choose a cell adhesion-promoting peptide as the biomolecule of interest to study whether the nanosized distribution of the peptide induces a different hMSC adhesion and commitment behavior from homogeneous controls through integrin clustering. The arginine-glycine-aspartic acid (RGD) peptide sequence is a short region found in a number of ECM proteins, such as fibronectin and vitronectin, acting as a key recognition site in these proteins, principally enhancing integrin-mediated cell adhesion.47−49 The method of preparing nanopatterned bioactive surfaces using NIL and surface functionalization50−52 is described in Figure S1. Briefly, square arrays of nanodots of unit cell parameter P were transferred from a silicon mold onto silicon substrates via a polymer mask. Two types of arrays were realized, hereafter noted as D150S350 and D80S110, where D and S = P − D denote the nanodot diameter and interdot gap width, respectively. These patterns were functionalized with a 3-aminopropyldimethylethoxysilane (APDMS) layer subsequently grafted with a cysteine-modified GRGDSPC molecule through a 3-succinimidyl-3-maleimidopropionate (SMP), a heterobifunctional cross-linker. After polymer mask removal, the nonpatterned background was passivated with a cell-repellent 2-[methoxypoly(ethyleneoxy)propyl]trimethoxysilane (PEO silane) layer. Homogeneous surfaces used as references were prepared in parallel to nanopatterned samples to characterize the three-step surface modification process. To evidence the grafting of the biomolecule on the surface, tests were first performed using an RGD molecule tagged with a TAMRA fluorophore. Epifluorescence microscopy observations (Figure S2A) revealed a clear fluorescent signal on these surfaces, indicating that the RGD was well-grafted. X-ray reflectivity (XRR) and Xray photoelectron spectroscopy (XPS) were also performed on homogeneous samples after each step of the functionalization to monitor the change in molecular layer thickness, molecular density, and elemental composition. As seen in the XRR analysis (Figures S2B, S2C and Table S1), the deposition of APDMS, SMP, and RGD yielded successively increasing thicknesses, confirming successful grafting. The decreasing molecular density along the three modification steps was attributed to the increase in the size of each grafted molecule, resulting in steric hindrance that limits the grafting efficiency (i.e., not every single amino group from silane molecule reacts with an SMP molecule and not every SMP molecule reacts with a peptide molecule, resulting in a 25 μm2, 10− 25 μm2, 5−10 μm2, and 25 μm2, 33% increase for FA areas between 10 and 25 μm2, and a 15% increase for FA areas between 5 and 10 μm2. Two sample t tests were used to compare significant difference between the percentages. *** represents a p-value of less than 0.05.

between Si poli and all RGD-grafted surfaces, while the difference between the nanopatterns and homogeneous RGD was also significant (Figure 5E). A decrease in STRO-1 activity implies that hMSC population has decreased over 4 weeks on nanopatterns relative to homogeneous controls. While a part of the population is still STRO-1 positive (hence retaining its “stemness”), the decrease indicates that more cells have differentiated on nanopatterns than homogeneous controls. The study of hMSC behavior on nanopatterned surfaces offers interesting insights. Herein, unlike conventional studies involving physical nanotopography, we prepared materials with chemical nanopatterns, providing a bioactive stage for the induction of specific cell response. We first noted that projected hMSC area is significantly larger on all RGD-grafted surfaces compared to the bare silicon control, after 24 h in culture. RGD, being an adhesion-promoting motif, provides the platform for anchorage between the cells and the material. With the presence of RGD, an adherent cell is able to extend its cytoskeleton and probe into its surroundings while bare Si samples, without such mediator, result in poor adhesion in comparison. Further investigation in integrin-mediated adhesion is carried out, in particular the interaction between the cell and material through staining for vinculin, an integrin-related protein. The onset of efficient cell adhesion requires the clustering of integrins and their receptors which, when mature, D

dx.doi.org/10.1021/nl4020149 | Nano Lett. XXXX, XXX, XXX−XXX

Nano Letters

Letter

It may be ambiguous and misleading to analyze certain parameters, such as projected cell area and total FA area, individually without making a connection between the two. Since FAs are indicative of direct cell contact with the material, we looked at the degree of attachment between the cell and substrate by relating it to cell area. Normalizing the two parameters by taking the ratio of the total FA area to total cell area, we obtained the percentage of the cell that is in contact with the underlying material through FAs (Figure S4C). A statistically significant increase in cell-material contact area was seen on homogeneously grafted RGD (RGD H) compared with bare silicon. Similarly, a statistically significant increase was seen on both nanopatterns compared with RGD H. The difference in adhesive behavior between the homogeneous and nanopatterned samples is attributed to the dynamics of FA formation, where efficient integrin clustering leads to the maturation of FA points (Figure 4A−D). To probe deeper into the effects of integrin clustering, we measured the area of each individual FA, classifying them into three groups according to area. Large FAs were more abundant in nanopatterns compared with homogeneous surfaces, with a 48% increase for FA areas >25 μm2, a 33% increase for FA areas between 10 and 25 μm2, and a 15% increase for FA areas between 5 and 10 μm2. This result complements the results of total FA area and cell-material contact area. Since nanopatterned surfaces have a higher percentage of FAs with large areas, it is reasonable to say that the total FA area should also be higher (and is indeed the case as shown) and in parallel that the cell-material contact area is higher on nanopatterned surfaces. These results also point to a general trend that integrins and their receptors are able to cluster more efficiently on nanopatterned surfaces, readily enabling stable FAs to form. As previously discussed, nanopatterns have a positive effect on cell-material contact area, and the data obtained from measuring individual FA area reaffirm the role of mature FAs in cell adhesion. As FAs have a direct effect on cell mechanotransduction and signaling pathways, we hypothesized that since FAs behave differently on homogeneous and nanopatterned surfaces, the commitment of hMSCs should be also affected. We attempted to establish a direct link between FA activity and hMSC commitment and performed differentiation-specific immunofluorescence staining on hMSCs cultured on various surfaces for 4 weeks to draw a preliminary conclusion. STRO-1 is a marker of hMSCs and is expressed when stemness is present. The decrease in STRO-1 expression on D150S350 and D80S110 relative to RGD H (Figure 5E) is a sign that cells are less “stem” on nanopatterns than homogeneously grafted RGD surfaces after 4 weeks in culture, thus indicating that they have differentiated into a mature cell lineage. As it is known that cell differentiation and proliferation follow an inverse relationship,17,57 we evaluated hMSC proliferation during the 4 weeks in culture (Figure S6). hMSCs on nanopatterned RGD-grafted surfaces showed a lower proliferation profile compared with the homogeneous RGD control, which is expected with increased differentiation. We can attribute the change in behavior to the way the FAs form stable contacts between the cells and the substrate, causing changes in the cytoskeletal contractility and thereby altering the cell mechanism and chemical signal pathways that follow. Nanoscale surface cues, such as the RGD nanopatterns presented in this study, might have important indications in tissue-specific hMSC differentiation. Previous reports have proven that nanotopographical cues,3 chemical patterning,16

Figure 5. Preliminary commitment studies of hMSCs after 4 weeks in culture on various types of material surfaces. (A−D) STRO-1, a hMSC marker, is immunofluorescently stained and shown in red, with F-actin stained in green and cell nucleus in blue; the scale bar represents 20 μm. (E) The amount of STRO-1 present in the cell is expressed as average fluorescent density, normalized with the number of cells (50 per condition). Compared to bare silicon controls, STRO-1 activity is lower on homogeneous RGD-grafted surfaces and still lower on patterned RGD-grafted surfaces, both D150S350 and D80S110, indicating that the cells have lost some “stemness” and differentiated into mature lineages. *** represents a p-value of less than 0.05.

establish FAs and enhance adhesive strength, in turn initiating a host of related signal transductions.21,22 Mature FAs have been shown to stretch along the direction of actin stress fiber elongation.22,55,56 Morphologically, on D150S350 and D80S110 surfaces, FAs are formed in a fibrillar shape and concentrated around the edge of the cell. They are well-aligned with the stress fibers found in the cytoskeleton, serving as anchors that maintain fiber tension. On the other hand, FAs on homogeneous RGD orient in a random and disordered way, without the fibrillar configuration and profound cytoskeletal alignment observed on nanopatterned surfaces. Since cytoskeletal contractility is implicated in cellular signal transduction, the alignment of fibril-shaped contacts with stress fibers may upregulate this contractility through tension caused by the pulling action of FAs, in turn affecting cell phenotype. Quantification of vinculin staining (Figure S4B) revealed a trend similar to that observed for cell area. On RGD-grafted surfaces, total FA area, represented by vinculin expression, is greater than on the polished Si control, yet the difference between the three RGD substrates cannot be considered statistically significant. E

dx.doi.org/10.1021/nl4020149 | Nano Lett. XXXX, XXX, XXX−XXX

Nano Letters

Letter

and material stiffness18 all play roles in hMSC differentiation. The ensemble of these studies indicates that even slight changes in the hMSC environment is sufficient to promote a change in cell behavior, further extending the need to study the function of the stem cell niche. The induction of hMSC differentiation through contact with a material occurs through the formation of mature FAs, which in turn impacts cytoskeletal tension as FAs act as anchoring points between the cytoskeleton and a material surface. Consequently, changes in cytoskeletal tension indirectly affect a cell’s mechanotransductive pathways, as demonstrated by cell response to material stiffness or external stress,18,58 for example. In our case, the presence of nanopatterned chemical cues also contributes to this change in cytoskeletal tension. Additionally, it has been shown that changes in cytoskeletal tension in response to surface nanocues could influence interphase nucleus organization, thus affecting cellular gene expression.59 The changes in cytoskeletal tension result in changes in cell morphology, as was seen in our study. We observe that specific maturation of FAs and the interaction of FAs with the cytoskeleton lead to different conformations of FA-based proteins, such as vinculin. Recruitment and activation of FAs induce these effects, as shown by the activation of vinculin binding via the unfolding of single talin rods,60,61 a process which forms part of the cellular mechanotransduction mechanism. The patterning technique developed in this study allows the deposition of biomolecules on nanoregions on material surfaces, at the scale of cell-surface interactions. Hence, instead of observing cell behavior through conventional modifications in physical nanotopography, we are able to study hMSCs on a spatially organized surface bioactivity. As we have demonstrated here that nanopatterned RGD peptides can induce a noticeable effect on the specific mature FA and differentiation behavior of hMSCs, the same approach will be applied in future studies on other types of biomolecules to examine the lineage commitment and differentiation of hMSCs.

■

work was performed within the frame of the International Doctoral School in Functional Materials funded by the ERASMUS MUNDUS Programme of the European Union. KG is a Research Associate of the Belgium F.R.S.-FNRS.

■

ABBREVIATIONS AFM, atomic force microscopy; APDMS, 3-aminopropyldimethylethoxysilane; ECM, extracellular matrix; FA, focal adhesion; hMSC, human mesenchymal stem cell; NIL, nanoimprint lithography; RGD, arginine-glycine-aspartic acid; SMP, 3-succinimidyl-3-maleimidopropionate; XPS, X-ray photoelectron spectroscopy; XRR, X-ray reflectivity

■

ASSOCIATED CONTENT

S Supporting Information *

Detailed materials and methods are provided: figures for NIL and functionalization schemes, homogeneous surface characterization, and cell-material contact quantification; tables for XPS and XRR. This material is available free of charge via the Internet at http://pubs.acs.org.

■

REFERENCES

(1) Liao, S.; Chan, C. K.; Ramakrishna, S. Mater. Sci. Eng.: C 2008, 28 (8), 1189−1202. (2) Anselme, K.; Davidson, P.; Popa, A. M.; Giazzon, M.; Liley, M.; Ploux, L. Acta Biomater. 2010, 6 (10), 3824−46. (3) Dalby, M. J.; Gadegaard, N.; Tare, R.; Andar, A.; Riehle, M. O.; Herzyk, P.; Wilkinson, C. D.; Oreffo, R. O. Nat. Mater. 2007, 6 (12), 997−1003. (4) Zouani, O. F.; Chanseau, C.; Brouillaud, B.; Bareille, R.; Deliane, F.; Foulc, M. P.; Mehdi, A.; Durrieu, M. C. J. Cell Sci. 2012, 125 (Pt 5), 1217−24. (5) Oh, S.; Brammer, K. S.; Li, Y. S.; Teng, D.; Engler, A. J.; Chien, S.; Jin, S. Proc. Natl. Acad. Sci. U.S.A. 2009, 106 (7), 2130−5. (6) Park, J.; Bauer, S.; Von der Mark, K.; Schmuki, P. Nano Lett. 2007, 7 (6), 1686−1691. (7) Dalby, M. J.; McCloy, D.; Robertson, M.; Wilkinson, C. D.; Oreffo, R. O. Biomaterials 2006, 27 (8), 1306−15. (8) Dalby, M. J.; McCloy, D.; Robertson, M.; Agheli, H.; Sutherland, D.; Affrossman, S.; Oreffo, R. O. Biomaterials 2006, 27 (15), 2980−7. (9) Caplan, A. I. J. Cell Physiol. 2007, 213, 341−347. (10) Pittenger, M. F. Science 1999, 284 (5411), 143−147. (11) Kilian, K. A.; Bugarija, B.; Lahn, B. T.; Mrksich, M. Proc. Natl. Acad. Sci. U.S.A. 2010, 107 (11), 4872−7. (12) Guilak, F.; Cohen, D. M.; Estes, B. T.; Gimble, J. M.; Liedtke, W.; Chen, C. S. Cell Stem Cell 2009, 5 (1), 17−26. (13) Nava, M. M.; Raimondi, M. T.; Pietrabissa, R. J. Biomed. Biotechnol. 2012, 2012, 797410. (14) Huang, N. F.; Li, S. Ann. Biomed. Eng. 2011, 39 (4), 1201−14. (15) McMurray, R. J.; Gadegaard, N.; Tsimbouri, P. M.; Burgess, R. W.; McNamara, L. E.; Tare, R.; Murawski, K.; Kingham, E.; Oreffo, R. O.; Dalby, M. J. Nat. Mater. 2011, 10, 637−644. (16) McBeath, R.; Pirone, D. M.; Nelson, C. M.; Bhadriraju, K.; Chen, C. S. Dev. Cell 2004, 6, 483−495. (17) Discher, D. E.; Mooney, D. J.; Zandstra, P. W. Science 2009, 324 (5935), 1673−7. (18) Engler, A. J.; Sen, S.; Sweeney, H. L.; Discher, D. E. Cell 2006, 126 (4), 677−89. (19) Huebsch, N.; Arany, P. R.; Mao, A. S.; Shvartsman, D.; Ali, O. A.; Bencherif, S. A.; Rivera-Feliciano, J.; Mooney, D. J. Nat. Mater. 2010, 9 (6), 518−26. (20) Legant, W. R.; Miller, J. S.; Blakely, B. L.; Cohen, D. M.; Genin, G. M.; Chen, C. S. Nat. Methods 2010, 7 (12), 969−71. (21) Geiger, B.; Spatz, J. P.; Bershadsky, A. D. Nat. Rev. Mol. Cell Biol. 2009, 10 (1), 21−33. (22) Adams, J. C. Cell. Mol. Life Sci. 2001, 58, 371−392. (23) Arnold, M.; Cavalcanti-Adam, E. A.; Glass, R.; Blummel, J.; Eck, W.; Kantlehner, M.; Kessler, H.; Spatz, J. P. ChemPhysChem 2004, 5 (3), 383−8. (24) Cavalcanti-Adam, E. A.; Micoulet, A.; Blummel, J.; Auernheimer, J.; Kessler, H.; Spatz, J. P. Eur. J. Cell Biol. 2006, 85 (3−4), 219−24. (25) Cavalcanti-Adam, E. A.; Volberg, T.; Micoulet, A.; Kessler, H.; Geiger, B.; Spatz, J. P. Biophys. J. 2007, 92 (8), 2964−74. (26) Chen, C. S. Science 1997, 276 (5317), 1425−1428.

AUTHOR INFORMATION

Corresponding Author

*M.-C.D.: e-mail: [email protected], phone: +33(0)540003037. O.F.Z.: e-mail: [email protected], phone: +33(0)540002228. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors sincerely thank Philippe Legros (Bordeaux Imaging Center, Bordeaux) for assistance with confocal imaging, André Crahay (WinFab, Belgium) for performance of oxygen plasma descum, Cédric Burhin, Delphine Magnin, Sylvie Derclaye, Bernard Nysten (IMCN, Belgium), and Yifeng Lei (IECB, Bordeaux) for technical assistance, and the Région Aquitaine, the Belgian Federal Science Policy (IAP P7/05), and Wallonie-Bruxelles International for financial support. This F

dx.doi.org/10.1021/nl4020149 | Nano Lett. XXXX, XXX, XXX−XXX

Nano Letters

Letter

(27) Deeg, J. A.; Louban, I.; Aydin, D.; Selhuber-Unkel, C.; Kessler, H.; Spatz, J. P. Nano Lett. 2011, 11 (4), 1469−76. (28) Pelham, R. J.; Wang, Y. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 13661−13665. (29) Malmstrom, J.; Lovmand, J.; Kristensen, S.; Sundh, M.; Duch, M.; Sutherland, D. S. Nano Lett. 2011, 11 (6), 2264−71. (30) Zouani, O. F.; Kalisky, J.; Ibarboure, E.; Durrieu, M. C. Biomaterials 2013, 34 (9), 2157−66. (31) Das, R. K.; Zouani, O. F.; Labrugere, C.; Oda, R.; Durrieu, M. C. ACS Nano 2013, 7, 3351−3361. (32) Chen, W.; Villa-Diaz, L. G.; Sun, Y.; Weng, S.; Kim, J. K.; Lam, R. H. W.; Han, L.; Fan, R.; Krebsbach, P. H.; Fu, J. ACS Nano 2012, 6 (5), 4094−4103. (33) Yim, E. K.; Darling, E. M.; Kulangara, K.; Guilak, F.; Leong, K. W. Biomaterials 2010, 31 (6), 1299−306. (34) Frith, J. E.; Mills, R. J.; Cooper-White, J. J. J. Cell Sci. 2012, 125 (Pt 2), 317−27. (35) Frith, J. E.; Mills, R. J.; Hudson, J. E.; Cooper-White, J. J. Stem Cells Dev. 2012, 21 (13), 2442−56. (36) Tsimbouri, P. M.; McMurray, R. J.; Burgess, K. V.; Alakpa, E. V.; Reynolds, P. M.; Murawski, K.; Kingham, E.; Oreffo, R. O. C.; Gadegaard, N.; Dalby, M. J. ACS Nano 2012, 6 (11), 10239−10249. (37) Guo, L. J. Adv. Mater. 2007, 19 (4), 495−513. (38) Hart, A.; Gadegaard, N.; Wilkinson, C. D.; Oreffo, R. O.; Dalby, M. J. J. Mater. Sci. Mater. Med. 2007, 18 (6), 1211−8. (39) Tan, J.; Saltzman, W. M. Biomaterials 2004, 25 (17), 3593−601. (40) Shekaran, A.; Garcia, A. J. Biochim. Biophys. Acta 2011, 1810 (3), 350−60. (41) Lei, Y.; Zouani, O. F.; Remy, M.; Ayela, C.; Durrieu, M. C. PLoS One 2012, 7 (7), e41163. (42) Dalby, M. J.; Gadegaard, N.; Wilkinson, C. D. J. Biomed. Mater. Res. A 2008, 84 (4), 973−9. (43) Yan, C.; Sun, J.; Ding, J. Biomaterials 2011, 32 (16), 3931−8. (44) Yu, B.; Chou, P.; Sun, Y.; Lee, Y.; Young, T. J. Membr. Sci. 2006, 273 (1−2), 31−37. (45) Truskett, V. N.; Watts, M. P. Trends Biotechnol. 2006, 24 (7), 312−7. (46) Christman, K. L.; Enriquez-Rios, V. D.; Maynard, H. D. Soft Matter 2006, 2 (11), 928. (47) Ruoslahti, E.; Pierschbacher, M. D. Science 1987, 238, 491−497. (48) Williams, D. F. Biomaterials 2011, 32 (18), 4195−7. (49) Collier, J. H.; Segura, T. Biomaterials 2011, 32 (18), 4198−204. (50) Pallandre, A.; Glinel, K.; Jonas, A. M.; Nysten, B. Nano Lett. 2004, 4 (2), 365−371. (51) Porte-Durrieu, M. C.; Guillemot, F.; Pallu, S.; Labrugere, C.; Brouillaud, B.; Bareille, R.; Amedee, J.; Barthe, N.; Dard, M.; Baquey, C. Biomaterials 2004, 25 (19), 4837−46. (52) Jonas, A. M.; Hu, Z.; Glinel, K.; Huck, W. T. Nano Lett. 2008, 8 (11), 3819−24. (53) Porte-Durrieu, M. C.; Labrugere, C.; Villars, F.; Lefebvre, F.; Dutoya, S.; Guette, A.; Bordenave, L.; Baguey, C. J. Biomed. Mater. Res. 1999, 46 (3), 368−375. (54) Van Noort, S. J. T.; Van der Werf, K. O.; De Grooth, B. G.; Van Hulst, N. F.; Greve, J. Ultramicroscopy 1997, 69, 117−127. (55) Balaban, N. Q.; Schwarz, U.; Riveline, D.; Goichberg, P.; Tzur, G.; Sabanay, I.; Mahalu, D.; Safran, S.; Bershadsky, A.; Addadi, L.; Geiger, B. Nat. Cell Biol. 2001, 3 (5), 466−472. (56) Chen, C. S.; Alonso, J. L.; Ostuni, E.; Whitesides, G. M.; Ingber, D. E. Biochem. Biophys. Res. Commun. 2003, 307 (2), 355−361. (57) Zhu, L.; Skoultchi, A. I. Curr. Opin. Genet. Dev. 2001, 11 (1), 91−97. (58) Chowdhury, F.; Na, S.; Li, D.; Poh, Y. C.; Tanaka, T. S.; Wang, F.; Wang, N. Nat. Mater. 2010, 9 (1), 82−8. (59) Dalby, M. J.; Biggs, M. J.; Gadegaard, N.; Kalna, G.; Wilkinson, C. D.; Curtis, A. S. J. Cell Biochem. 2007, 100 (2), 326−38. (60) Grashoff, C.; Hoffman, B. D.; Brenner, M. D.; Zhou, R.; Parsons, M.; Yang, M. T.; McLean, M. A.; Sligar, S. G.; Chen, C. S.; Ha, T.; Schwartz, M. A. Nature 2010, 466 (7303), 263−6.

(61) del Rio, A.; Perez-Jimenez, R.; Liu, R.; Roca-Cusachs, P.; Fernandez, J. M.; Sheetz, M. P. Science 2009, 323, 638−641.

G

dx.doi.org/10.1021/nl4020149 | Nano Lett. XXXX, XXX, XXX−XXX