Journal of Medical and Biological Engineering

, Volume 38, Issue 4, pp 596–606 | Cite as

How Deep Might Myoblasts Sense: The Effect of Substrate Stiffness and Thickness on the Behavior of Myoblasts

  • Shan Li
  • Feng Zhao
  • Yuewei Zhan
  • Xiaoyi Liu
  • Tingting Hun
  • Haokang Zhang
  • Changjun Qiu
  • Jingwen He
  • Zongchun Yi
  • Yan SunEmail author
  • Yubo FanEmail author
Original Article


Mechanical characters of extracellular matrix, such as the stiffness and thickness, have been shown to impact an abundance of cellular processes, including cell spreading, adhesion, proliferation and differentiation. In this study, we used polydimethylsiloxane (PDMS) films of variable thickness and stiffness to investigate the impact on skeletal muscles cell (C2C12 cells) behavior, in more detail. Furthermore, we utilized, for the first time, a heat sensitive material, poly-[N-isopropylacrylamide], in the process of film thickness measurement to obtain more complete films. Results confirmed that C2C12 cells grow better on stiff substrates. Also, our research demonstrated that film thickness has an influence on C2C12 cells attachment and growth. Specifically, when the elastic modulus of the substrate was 5 kPa, cells seeded on thin gels (h < 38 μm) were found to establish large, well-organized and well-spread focal adhesions. In addition, an increase in proliferation can be observed when the gels were 18 μm or thinner. The differentiation of C2C12 cells was also influenced by gel thickness. Myotubes formed on thick PDMS films (h > 38 μm) were generally differentiated by single myoblasts. When growing on thinner gels, myotubes appeared more elongated and multinuclear. Moreover, sarcomeres began to form when cells were seeded on substrates of 38 μm (or less). However, when the elastic modulus was 1.72 MPa, altering the thickness of the PDMS films had no significant impact on spreading, adhesion or proliferation. In short, we conclude that C2C12 cells are able to sense the underlay when growing on a stiff or a thin (h < 38 μm) substrate, which is reflected in their development.


Polydimethylsiloxane Stiffness Thickness Skeletal muscles cell Proliferation Differentiation Adhesion Spreading 



This work was supported by the National Natural Science Foundation of China [31470942, 11072021, 31670982], International Joint Research Center of Aerospace Biotechnology and 344 Medical Engineering from Ministry of Science and Technology of China, 111 Project 345 [B13003].


  1. 1.
    Buckingham, M. (2006). Myogenic progenitor cells and skeletal myogenesis in vertebrates. Current Opinion in Genetics & Development., 16(5), 525–532.CrossRefGoogle Scholar
  2. 2.
    Brown, X. Q., Bartolak-Suki, E., Williams, C., Walker, M. L., Weaver, V. M., & Wong, J. Y. (2010). Effect of substrate stiffness and PDGF on the behavior of vascular smooth muscle cells: implications for atherosclerosis. Journal of cellular Physiology., 225(1), 115–122.CrossRefGoogle Scholar
  3. 3.
    Boontheekul, T., Hill, E. E., Kong, H. J., & Mooney, D. J. (2007). Regulating myoblast phenotype through controlled gel stiffness and degradation. Tissue Engineering., 13(7), 1431–1442.CrossRefGoogle Scholar
  4. 4.
    Pelham, R. J., Jr., & Wang, Y. (1997). Cell locomotion and focal adhesions are regulated by substrate flexibility. Proceedings of the National Academy of Sciences of the United States of America., 94(25), 13661–13665.CrossRefGoogle Scholar
  5. 5.
    Discher, D. E., Janmey, P., & Wang, Y. L. (2005). Tissue cells feel and respond to the stiffness of their substrate. Science., 310(5751), 1139–1143.CrossRefGoogle Scholar
  6. 6.
    Engler, A. J., Griffin, M. A., Sen, S., Bonnemann, C. G., Sweeney, H. L., & Discher, D. E. (2004). Myotubes differentiate optimally on substrates with tissue-like stiffness: pathological implications for soft or stiff microenvironments. The Journal of Cell Biology., 166(6), 877–887.CrossRefGoogle Scholar
  7. 7.
    Engler, A. J., Sen, S., Sweeney, H. L., & Discher, D. E. (2006). Matrix elasticity directs stem cell lineage specification. Cell., 126(4), 677–689.CrossRefGoogle Scholar
  8. 8.
    Levymishali, M., Zoldan, J., & Levenberg, S. (2009). Effect of Scaffold stiffness on myoblast differentiation. Tissue Engineering: Part A., 15(4), 935–944.CrossRefGoogle Scholar
  9. 9.
    Palchesko, R. N., Zhang, L., Sun, Y., & Feinberg, A. W. (2012). Development of polydimethylsiloxane substrates with tunable elastic modulus to study cell mechanobiology in muscle and nerve. PLoS ONE., 7(12), e51499.CrossRefGoogle Scholar
  10. 10.
    Feng, C. H., Cheng, Y. C., & Chao, P. H. (2013). The influence and interactions of substrate thickness, organization and dimensionality on cell morphology and migration. Acta Biomaterialia., 9(3), 5502–5510.CrossRefGoogle Scholar
  11. 11.
    Lin, Y. C., Tambe, D. T., & Park, C. Y. (2010). Mechanosensing of substrate thickness. Physical Review E, Statistical, Nonlinear, and Soft Matter Physics., 82(4 Pt 1), 041918.CrossRefGoogle Scholar
  12. 12.
    Leong, W. S., Tay, C. Y., Yu, H., Li, A., Wu, S. C., Duc, D.-H., et al. (2010). Thickness sensing of hMSCs on collagen gel directs stem cell fate. Biochemical and Biophysical Research Communications., 401(2), 287–292.CrossRefGoogle Scholar
  13. 13.
    Chowdhury, F., Li, Y., Poh, Y. C., Yokohama-Tamaki, T., Wang, N., & Tanaka, T. S. (2010). Soft substrates promote homogeneous self-renewal of embryonic stem cells via downregulating cell-matrix tractions. PLoS ONE., 5(12), e15655.CrossRefGoogle Scholar
  14. 14.
    Wang, Y. L., & Pelham, R. J., Jr. (1998). Preparation of a flexible, porous polyacrylamide substrate for mechanical studies of cultured cells. Methods in Enzymology., 298, 489–496.CrossRefGoogle Scholar
  15. 15.
    Fioretta, E. S., Fledderus, J. O., Baaijens, F. P., & Bouten, C. V. (2012). Influence of substrate stiffness on circulating progenitor cell fate. Journal of Biomechanics, 45(5), 736–744.CrossRefGoogle Scholar
  16. 16.
    Charest, J. M., Califano, J. P., Carey, S. P., & Reinhart-King, C. A. (2012). Fabrication of substrates with defined mechanical properties and topographical features for the study of cell migration. Macromolecular Bioscience., 12(1), 12–20.CrossRefGoogle Scholar
  17. 17.
    Jiang, F. X., Yurke, B., Firestein, B. L., & Langrana, N. A. (2008). (2012)Neurite outgrowth on a DNA crosslinked hydrogel with tunable stiffnesses. Annals of Biomedical Engineering., 36(9), 1565–1579.CrossRefGoogle Scholar
  18. 18.
    Stabenfeldt, S. E., & LaPlaca, M. C. (2011). Variations in rigidity and ligand density influence neuronal response in methylcellulose-laminin hydrogels. Acta Biomaterialia, 7(12), 4102–4108.CrossRefGoogle Scholar
  19. 19.
    Candiello, J., Balasubramani, M., Schreiber, E. M., Cole, G. J., Mayer, U., Halfter, W., et al. (2007). Biomechanical properties of native basement membranes. The FEBS Journal., 274(11), 2897–2908.CrossRefGoogle Scholar
  20. 20.
    Fisher, R. F., & Wakely, J. (1976). The elastic constants and ultrastructural organization of a basement membrane (lens capsule). Proceedings of the Royal Society of London Series B, Biological Sciences., 193(1113), 335–358.CrossRefGoogle Scholar
  21. 21.
    Brown, X. Q., Ookawa, K., & Wong, J. Y. (2005). Evaluation of polydimethylsiloxane scaffolds with physiologically-relevant elastic moduli: interplay of substrate mechanics and surface chemistry effects on vascular smooth muscle cell response. Biomaterials., 26(16), 3123–3129.CrossRefGoogle Scholar
  22. 22.
    Gutierrez, E., & Groisman, A. (2011). Measurements of elastic moduli of silicone gel substrates with a microfluidic device. PLoS ONE., 6(9), e25534.CrossRefGoogle Scholar
  23. 23.
    Wang, P. Y., Tsai, W. B., & Voelcker, N. H. (2012). Screening of rat mesenchymal stem cell behaviour on polydimethylsiloxane stiffness gradients. Acta Biomaterialia., 8(2), 519–530.CrossRefGoogle Scholar
  24. 24.
    Rivcline, D., Zamir, E., Balaban, M. Q., Schwarz, U. S., Ishizaki, T., Narumiya, S., et al. (2001). Focal contacts as mechanosensors: externally applied local mechanical force induces growth of focal contacts by an mDia1-dependent and ROCK-independent mechanism. The Journal of Cell Biology., 153(6), 1175–1186.CrossRefGoogle Scholar
  25. 25.
    Hemphill, M. A., Dabiri, B. E., Gabriele, S., Kerscher, L., Franck, C., Goss, J. A., et al. (2011). A possible role for integrin signaling in diffuse axonal injury. PLoS ONE., 6(7), e22899.CrossRefGoogle Scholar
  26. 26.
    Zhang WY, Ferguson GS, Tatic-Lucic S (2004) Elastomer-supported cold welding for room temperature wafer-level bonding. In: IEEE (pp. 741–744).Google Scholar
  27. 27.
    Zhang, Y. H., Zhao, C. Q., Jiang, L. S., & Dai, L. Y. (2010). Substrate stiffness regulates apoptosis and the mRNA expression of extracellular matrix regulatory genes in the rat annular cells. Matrix Biology, 30(2), 135–144.CrossRefGoogle Scholar
  28. 28.
    Merkel, R., Kirchgessner, N., Cesa, C. M., & Hoffmann, B. (2007). Cell force microscopy on elastic layers of finite thickness. Biophysical Journal., 93(9), 3314–3323.CrossRefGoogle Scholar
  29. 29.
    Sen, S., Engler, A. J., & Discher, D. E. (2009). Matrix strains induced by cells: Computing how far cells can feel. Cellular and Molecular Bioengineering., 2(1), 39–48.CrossRefGoogle Scholar
  30. 30.
    Buxboim, A., Rajagopal, K., Brown, A. E., & Discher, D. E. (2010). How deeply cells feel: methods for thin gels. Journal of Physics Condensed Matter: An Institute of Physics Journal, 22(19), 194116.CrossRefGoogle Scholar
  31. 31.
    Gribova, V., Gauthier-Rouviere, C., Albiges-Rizo, C., et al. (2013). Effect of RGD functionalization and stiffness modulation of polyelectrolyte multilayer films on muscle cell differentiation(J). Acta Biomaterialia., 9(5), 6468–6480.CrossRefGoogle Scholar

Copyright information

© Taiwanese Society of Biomedical Engineering 2017

Authors and Affiliations

  • Shan Li
    • 1
    • 3
  • Feng Zhao
    • 1
  • Yuewei Zhan
    • 1
  • Xiaoyi Liu
    • 1
  • Tingting Hun
    • 1
  • Haokang Zhang
    • 1
  • Changjun Qiu
    • 1
  • Jingwen He
    • 1
  • Zongchun Yi
    • 1
  • Yan Sun
    • 1
    • 3
    Email author
  • Yubo Fan
    • 1
    • 2
    Email author
  1. 1.Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical EngineeringBeihang UniversityBeijingPeople’s Republic of China
  2. 2.National Research Center for Rehabilitation Technical AidsBeijingPeople’s Republic of China
  3. 3.State Key Laboratory of Transducer TechnologyChinese Academy of SciencesShanghaiPeople’s Republic of China

Personalised recommendations