Effect of topological cues on material-driven fibronectin fibrillogenesis and cell differentiation

  • José Ballester-Beltrán
  • Marco Cantini
  • Myriam Lebourg
  • Patricia Rico
  • David Moratal
  • Andrés J. García
  • Manuel Salmerón-Sánchez


Fibronectin (FN) assembles into fibrillar networks by cells through an integrin-dependent mechanism. We have recently shown that simple FN adsorption onto poly(ethyl acrylate) surfaces (PEA), but not control polymer (poly(methyl acrylate), PMA), also triggered FN organization into a physiological fibrillar network. FN fibrils exhibited enhanced biological activities in terms of myogenic differentiation compared to individual FN molecules. In the present study, we investigate the influence of topological cues on the material-driven FN assembly and the myogenic differentiation process. Aligned and random electrospun fibers were prepared. While FN fibrils assembled on the PEA fibers as they do on the smooth surface, the characteristic distribution of globular FN molecules observed on flat PMA transformed into non-connected FN fibrils on electrospun PMA, which significantly enhanced cell differentiation. The direct relationship between the fibrillar organization of FN at the material interface and the myogenic process was further assessed by preparing FN gradients on smooth PEA and PMA films. Isolated FN molecules observed at one edge of the substrate gradually interconnected with each other, eventually forming a fully developed network of FN fibrils on PEA. In contrast, FN adopted a globular-like conformation along the entire length of the PMA surface, and the FN gradient consisted only of increased density of adsorbed FN. Correspondingly, the percentage of differentiated cells increased monotonically along the FN gradient on PEA but not on PMA. This work demonstrates an interplay between material chemistry and topology in modulating material-driven FN fibrillogenesis and cell differentiation.



The support of the Spanish Ministry of Science and Innovation through project MAT2009-14440-C02-01 is acknowledged. CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. This work was supported by funds for research in the field of Regenerative Medicine through the collaboration agreement from the Conselleria de Sanidad (Generalitat Valenciana), and the Instituto de Salud Carlos III.


  1. 1.
    Singh P, Carraher C, Schwarzbauer JE. Assembly of fibronectin extracellular matrix. Ann Rev Cell Dev Biol. 2010;26:397–419.CrossRefGoogle Scholar
  2. 2.
    Hynes RO. Fibronectins springer series in molecular biology. New York: Springer; 1990.Google Scholar
  3. 3.
    Mao Y, Schwarzbauer JE. FN fibrillogenesis, a cell-mediated matrix assembly process. Matrix Biol. 2005;24:389–99.CrossRefGoogle Scholar
  4. 4.
    Geiger B, Bershadsky A, Pankov R, Yamada KM. Transmembrane extracellular matrix–cytoskeleton crosstalk. Natl Rev Mol Cell Biol. 2001;2:793–805.CrossRefGoogle Scholar
  5. 5.
    Sakai K, Fujii T, Hayashi T. Cell-free formation of disulfide-bonded multimer from isolated plasma fibronectin in the presence of a low concentration of SH reagent under a physiological condition. J Biochem. 1994;115:415–21.Google Scholar
  6. 6.
    Vartio T. Disulfide-bonded polymerization of plasma fibronectin in the presence of metal ions. J Biol Chem. 1986;261:9433–7.Google Scholar
  7. 7.
    Mosher DF, Johnson RB. In vitro formation of disulfide-bonded fibronectin multimers. J Biol Chem. 1983;258:6595–601.Google Scholar
  8. 8.
    Vuento M, Vartio T, Saraste M, von Bonsdorff CH, Vaheri A. Spontaneous and polyamine-induced formation of filamentous polymers from soluble fibronectin. Eur J Biochem. 1980;105:33–42.CrossRefGoogle Scholar
  9. 9.
    Richter H, Wendt C, Hörmann H. Aggregation and fibril formation of plasma fibronectin by heparin. Biol Chem Hoppe-Seyler. 1985;366:509–14.CrossRefGoogle Scholar
  10. 10.
    Morla A, Zhang Z, Ruoslahti E. Superfibronectin is a functionally distinct form of fibronectin. Nature. 1994;367:193–6.CrossRefGoogle Scholar
  11. 11.
    Baneyx G, Vogel V. Self-assembly of fibronectin into fibrillar networks underneath dipalmitoylphosphatidylcholine monolayers: role of lipid matrix and tensile forces. Proc Natl Acad Sci USA. 1999;96:12518–23.CrossRefGoogle Scholar
  12. 12.
    Ulmer J, Geiger B, Spatz JP. Force-induced fibronectin fibrillogenesis in vitro. Soft Matter. 2008;4:1998–2007.CrossRefGoogle Scholar
  13. 13.
    Brown RA, Blunn GW, Ejim OS. Preparation of orientated fibrous mats from fibronectin: composition and stability. Biomaterials. 1994;15:457–64.CrossRefGoogle Scholar
  14. 14.
    Little WC, Smith ML, Ebneter U, Vogel V. Assay to mechanically tune and optically probe fibrillar fibronectin conformations from fully relaxed to breakage. Matrix Biol. 2008;27:451–61.CrossRefGoogle Scholar
  15. 15.
    Klotzsch E, Smith ML, Kubow KE, Muntwyler S, Little WC, Beyeler F, Gourdon D, Nelson BJ, Vogel V. Fibronectin forms the most extensible biological fibers displaying switchable force-exposed cryptic binding sites. Proc Natl Acad Sci USA. 2009;106:18267–72.CrossRefGoogle Scholar
  16. 16.
    Rico P, Rodríguez Hernández JC, Moratal D, Altankov G, Monleón Pradas M, Salmerón-Sánchez M. Substrate-induced assembly of fibronectin into networks: influence of surface chemistry and effect on osteoblast adhesion. Tissue Eng Part A. 2009;15:3271–81.CrossRefGoogle Scholar
  17. 17.
    Gugutkov D, González-García C, Rodríguez Hernández JC, Altankov G, Salmerón-Sánchez M. Biological activity of the substrate-induced FN network: insight into the third dimension through electrospun fibers. Langmuir. 2009;25:10893–900.CrossRefGoogle Scholar
  18. 18.
    Salmerón-Sánchez M, Rico P, Moratal D, Lee T, Schwarzbauer J, García AJ. Role of material-driven fibronectin fibrillogenesis in cell differentiation. Biomaterials. 2011;32:2099–115.CrossRefGoogle Scholar
  19. 19.
    Sabourin LA, Rudnicki MA. The molecular regulation of myogenesis. Clin Genet. 2000;57:16–25.CrossRefGoogle Scholar
  20. 20.
    Agarwal S, Wendorff J, Greiner A. Use of electrospinning technique for biomedical applications. Polymer. 2008;49:5603–21.CrossRefGoogle Scholar
  21. 21.
    Sill TJS, von Recum HA. Electrospinning: applications in drug delivery and tissue engineering. Biomaterials. 2008;29:1989–2006.CrossRefGoogle Scholar
  22. 22.
    Huber A, Pickett A, Shakesheff KM. Reconstruction of spatially orientated myotubes in vitro using electrospun, parallel microfibre arrays. Eur Cells Mater. 2007;14:56–63.Google Scholar
  23. 23.
    Jun I, Jeong S, Shin H. The stimulation of myoblast differentiation by electrically conductive sub-micron fibers. Biomaterials. 2009;30:2038–47.CrossRefGoogle Scholar
  24. 24.
    Clark P, Dunn GA, Knibbs A, Peckham M. Alignment of myoblasts on ultrafine gratings inhibits fusion in vitro. Int J Biochem Cell Biol. 2002;34:816–25.CrossRefGoogle Scholar
  25. 25.
    Lam MT, Sim S, Zhu X, Takayama S. The effect of continuous wavy micropatterns on silicone substrates on the alignment of skeletal muscle myoblasts and myotubes. Biomaterials. 2007;27:4340–7.CrossRefGoogle Scholar
  26. 26.
    Altomare L, Gadegaard N, Visai L, Tanzi MC, Farè S. Biodegradable microgrooved polymeric surfaces obtained by photolithography for skeletal muscle cell orientation and myotube development. Acta Biomater. 2010;6:1948–57.CrossRefGoogle Scholar
  27. 27.
    Altomare L, Riehle M, Gadegaard N, Tanzi MC, Farè S. Microcontact printing of fibronectin on a biodegradable polymeric surface for skeletal muscle cell orientation. Int J Artif Organs. 2010;33:535–43.Google Scholar
  28. 28.
    Neumann T, Hauschka SD, Sanders JE. Tissue engineering of skeletal muscle using polymer fiber arrays. Tissue Eng. 2003;9:995–1003.CrossRefGoogle Scholar
  29. 29.
    Tse JR, Engler A. Stiffness gradients mimicking in vivo tissue variation regulate mesenchymal stem cell fate. PLoS One. 2011;6:e15978.CrossRefGoogle Scholar
  30. 30.
    Gómez-Tejedor JA, Van Overberghe N, Rico P, Gómez Ribelles JL. Assessment of the parameters influencing the fiber characteristics of electrospun poly(ethyl methacrylate) membranes. Eur Polym J. 2011;47:119–29.CrossRefGoogle Scholar
  31. 31.
    O’Connell B. Oval Profile Plot. Research Services Branch, National Institute of Mental Health, National Institute of Neurological Disorders and Stroke. Available from: http://rsbweb.nih.gov/ij/plugins/oval-profile.html. Accessed 30 November 2011.
  32. 32.
    Gugutkov D, Altankov G, Rodríguez Hernández JC, Monleón Pradas M, Salmerón Sánchez M. Fibronectin activity on substrates with controlled–OH density. J Biomed Mater Res. 2010;A92:322–31.CrossRefGoogle Scholar
  33. 33.
    Schwarzbauer JE. Identification of FN sequences required for assembly of a fibrillar matrix. J Cell Biol. 1991;113:1463–73.CrossRefGoogle Scholar
  34. 34.
    Mukhatyar VJ, Salmerón-Sánchez M, Rudra S, Mukhopadaya S, Barker TH, García AJ, Bellamkonda RV. Role of fibronectin in topographical guidance of neurite extension on electrospun fibers. Biomaterials. 2011;32:3958–68.CrossRefGoogle Scholar
  35. 35.
    Wakelam MJ. The fusion of myoblasts. Biochem J. 1985;228:1–12.Google Scholar
  36. 36.
    Quach NL, Rando TA. Focal adhesion kinase is essential for costamerogenesis in cultured skeletal muscle cells. Dev Biol. 2006;293:38–52.CrossRefGoogle Scholar
  37. 37.
    Charest JL, García AJ, King WP. Myoblast alignment and differentiation on cell culture substrates with microscale topography and model chemistries. Biomaterials. 2007;28:2202–10.CrossRefGoogle Scholar
  38. 38.
    Berendse M, Grounds MD, Lloyd CM. Myoblast structure affects subsequent skeletal myotube morphology and sarcomere assembly. Exp Cell Res. 2003;291:435–50.CrossRefGoogle Scholar
  39. 39.
    Li B, Lin M, Tang Y, Wang B, Wang JHC. Novel functional assessment of the differentiation of micropatterned muscle cells. J Biomech. 2008;41:3349–53.CrossRefGoogle Scholar
  40. 40.
    Blunn GW, Brown RA. Production of artificial-oriented mats and strands from plasma fibronectin: a morphological study. Biomaterials. 1993;14:743–8.CrossRefGoogle Scholar
  41. 41.
    Smith JT, Tomfohr JK, Wells MC, Beebe TP, Kepler TB, Reichert WM. Measurement of cell migration on surface-bound fibronectin gradients. Langmuir. 2004;20:8279–86.CrossRefGoogle Scholar
  42. 42.
    Smith JT, Elkin JT, Reichert WM. Directed cell migration on fibronectin gradients: effect of gradient slope. Exp Cell Res. 2006;312:2424–32.CrossRefGoogle Scholar
  43. 43.
    Rhoads DS, Guan JL. Analysis of directional cell migration on defined FN gradients: role of intracellular signaling molecules. Exp Cell Res. 2007;313:3859–67.CrossRefGoogle Scholar
  44. 44.
    Liu L, Ratner BD, Sage EH, Jiang S. Endothelial cell migration on surface-density gradients of fibronectin, VEGF, or both proteins. Langmuir. 2007;23:11168–73.CrossRefGoogle Scholar
  45. 45.
    Shi J, Wang L, Zhang F, Li H, Lei L, Liu L, Chen Y. Incorporating protein gradient into electrospun nanofibres as scaffolds for tissue engineering. ACS Appl Mater Interfaces. 2010;2:1025–30.CrossRefGoogle Scholar
  46. 46.
    Goetsch KP, Kallmeyer K, Niesler CU. Decorin modulates collagen I-stimulated, but not fibronectin-stimulated, migration of C2C12 myoblasts. Matrix Biol. 2011;30:109–17.CrossRefGoogle Scholar
  47. 47.
    Bondesen BA, Jones KA, Glasgow WC, Pavlath GK. Inhibition of myoblast migration by prostacyclin is associated with enhanced cell fusion. FASEB J. 2007;21:3338–45.CrossRefGoogle Scholar
  48. 48.
    Olguin HC, Santander C, Brandan E. Inhibition of myoblast migration via decorin expression is critical for normal skeletal muscle differentiation. Dev Biol. 2003;259:209–24.CrossRefGoogle Scholar
  49. 49.
    Konigsberg IR. Diffusion-mediated control of myoblast fusion. Dev Biol. 1971;26:133–52.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • José Ballester-Beltrán
    • 1
  • Marco Cantini
    • 1
  • Myriam Lebourg
    • 1
    • 2
  • Patricia Rico
    • 1
    • 2
  • David Moratal
    • 1
  • Andrés J. García
    • 3
  • Manuel Salmerón-Sánchez
    • 1
    • 2
  1. 1.Center for Biomaterials and Tissue EngineeringUniversitat Politècnica de ValènciaValenciaSpain
  2. 2.CIBER de Bioingeniería, Biomateriales y NanomedicinaValenciaSpain
  3. 3.Woodruff School of Mechanical Engineering and Petit Institute for Bioengineering and BioscienceGeorgia Institute of TechnologyAtlantaUSA

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