Skip to main content
SpringerLink
Log in

Cell-Instructive Graphene-Containing Nanocomposites Induce Multinucleated Myotube Formation

  • Emerging Trends in Biomaterials Research
  • Published:
Annals of Biomedical Engineering Aims and scope Submit manuscript

Abstract

Myoblast differentiation is a key step in myogenesis and has long been considered to be controlled mainly by biochemical cues such as growth factors. However, the tissue engineering approaches based on biochemical cues demonstrate low reproducibility as a precise spatial control over their bioactivity is challenging. Recently, substrate micro/nano-structure and electro-responsive properties are recognized for their important roles in myoblast differentiation. In this study, we hypothesized that engineering biophysical features such as nano/micro-fibrous structure and conductive properties into a single biomaterial scaffold will instruct the myoblasts to differentiate into multinucleated myotubes even in the absence of differentiation media. We fabricated nanocomposite scaffolds composed of conductive graphene nanosheets and polycaprolactone (PCL), a widely used biocompatible material. The resulting graphene-PCL scaffolds possess excellent conductivity due to graphene nanosheets and great processability, biodegradability and elastic mechanical properties conferred by PCL. Additionally, physicochemical and mechanical properties of nanocomposite scaffolds can be tuned by varying graphene concentration. Further, graphene-PCL nanocomposites and their 8-week degradation products exhibited remarkable cytocompatibility and promoted adhesion and proliferation of C2C12 mouse myoblast cells. Importantly, these nanocomposite scaffolds induced graphene concentration-dependent differentiation of C2C12 cells into multinucleated myotubes even in normal growth media suggesting their cell-instructive potential. Thus, graphene-PCL nanocomposite scaffolds can serve as a strategy to promote skeletal muscle regeneration without biochemical cues.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

Similar content being viewed by others

References

  1. Ahadian, S., J. Ramon-Azcon, H. Chang, X. Liang, H. Kaji, H. Shiku, K. Nakajima, M. Ramalingam, H. Wu, T. Matsue, and A. Khademhosseini. Electrically regulated differentiation of skeletal muscle cells on ultrathin graphene-based films. Rsc Adv. 4:9534–9541, 2014.

    Article  CAS  Google Scholar 

  2. Ahadian, S., R. B. Sadeghian, S. Yaginuma, J. Ramon-Azcon, Y. Nashimoto, X. Liang, H. Bae, K. Nakajima, H. Shiku, T. Matsue, K. S. Nakayama, and A. Khademhosseini. Hydrogels containing metallic glass sub-micron wires for regulating skeletal muscle cell behaviour. Biomater. Sci. 3:1449–1458, 2015.

    Article  CAS  PubMed  Google Scholar 

  3. Ahmed, W. W., T. Wolfram, A. M. Goldyn, K. Bruellhoff, B. A. Rioja, M. Möller, J. P. Spatz, T. A. Saif, J. Groll, and R. Kemkemer. Myoblast morphology and organization on biochemically micro-patterned hydrogel coatings under cyclic mechanical strain. Biomaterials 31:250–258, 2010.

    Article  CAS  PubMed  Google Scholar 

  4. Alzari, V., D. Nuvoli, S. Scognamillo, M. Piccinini, E. Gioffredi, G. Malucelli, S. Marceddu, M. Sechi, V. Sanna, and A. Mariani. Graphene-containing thermoresponsive nanocomposite hydrogels of poly(N-isopropylacrylamide) prepared by frontal polymerization. J. Mater. Chem. 21:8727, 2011.

    Article  CAS  Google Scholar 

  5. Aryaei, A., A. H. Jayatissa, and A. C. Jayasuriya. The effect of graphene substrate on osteoblast cell adhesion and proliferation. J Biomed Mater Res A 102:3282–3290, 2014.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Bajaj, P., J. A. Rivera, D. Marchwiany, V. Solovyeva, and R. Bashir. Graphene-based patterning and differentiation of C2C12 myoblasts. Adv. Healthc. Mater 3:949, 2014.

    Article  Google Scholar 

  7. Benezra, R., R. L. Davis, D. Lockshon, D. L. Turner, and H. Weintraub. The protein Id: a negative regulator of helix-loop-helix DNA binding proteins. Cell 61:49–59, 1990.

    Article  CAS  PubMed  Google Scholar 

  8. Bentzinger, C. F., Y. X. Wang, and M. A. Rudnicki. Building muscle: molecular regulation of myogenesis. Cold Spring Harb. Perspect. Biol. 4:a008342–a008342, 2012.

    Article  PubMed  PubMed Central  Google Scholar 

  9. Bonderer, L. J., A. R. Studart, and L. J. Gauckler. Bioinspired design and assembly of platelet reinforced polymer films. Science 319:1069–1073, 2008.

    Article  CAS  PubMed  Google Scholar 

  10. Bridges, A. W., and A. J. Garcia. Anti-inflammatory polymeric coatings for implantable biomaterials and devices. J Diabetes Sci Technol 2:984–994, 2008.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Cezar, C. A., E. T. Roche, H. H. Vandenburgh, G. N. Duda, C. J. Walsh, and D. J. Mooney. Biologic-free mechanically induced muscle regeneration. Proc. Natl. Acad. Sci. 113:1534–1539, 2016.

    Article  CAS  PubMed  Google Scholar 

  12. Chaudhuri, B., D. Bhadra, L. Moroni, and K. Pramanik. Myoblast differentiation of human mesenchymal stem cells on graphene oxide and electrospun graphene oxide-polymer composite fibrous meshes: importance of graphene oxide conductivity and dielectric constant on their biocompatibility. Biofabrication 7:015009, 2015.

    Article  PubMed  Google Scholar 

  13. Depan, D., and R. D. K. Misra. The development, characterization, and cellular response of a novel electroactive nanostructured composite for electrical stimulation of neural cells. Biomater. Sci. 2:1727–1739, 2014.

    Article  CAS  Google Scholar 

  14. Ding X., H. Liu, and Y. Fan. Graphene-based materials in regenerative medicine. Adv. Healthc. Mater. 2015.

  15. Ferri, P., E. Barbieri, S. Burattini, M. Guescini, A. D’Emilio, L. Biagiotti, P. Del Grande, A. De Luca, V. Stocchi, and E. Falcieri. Expression and subcellular localization of myogenic regulatory factors during the differentiation of skeletal muscle C2C12 myoblasts. J. Cell. Biochem. 108:1302–1317, 2009.

    Article  CAS  PubMed  Google Scholar 

  16. Gaharwar, A. K., M. Nikkhah, S. Sant, and A. Khademhosseini. Anisotropic poly (glycerol sebacate)-poly (ϵ-caprolactone) electrospun fibers promote endothelial cell guidance. Biofabrication 7:015001, 2015.

    Article  PubMed Central  Google Scholar 

  17. Gawlitta, D., K. J. M. Boonen, C. W. J. Oomens, F. P. T. Baaijens, and C. V. C. Bouten. The influence of serum-free culture conditions on skeletal muscle differentiation in a tissue-engineered model. Tissue Eng. Part A 14:161–171, 2008.

    Article  CAS  PubMed  Google Scholar 

  18. Goenka, S., V. Sant, and S. Sant. Graphene-based nanomaterials for drug delivery and tissue engineering. J Control Release 173:75–88, 2014.

    Article  CAS  PubMed  Google Scholar 

  19. Gutierrez, J., and E. Brandan. A novel mechanism of sequestering fibroblast growth factor 2 by glypican in lipid rafts, allowing skeletal muscle differentiation. Mol. Cell. Biol. 30:1634–1649, 2010.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Han, Y., Q. Zeng, H. Li, and J. Chang. The calcium silicate/alginate composite: preparation and evaluation of its behavior as bioactive injectable hydrogels. Acta Biomater. 9:9107–9117, 2013.

    Article  CAS  PubMed  Google Scholar 

  21. Hill, E., T. Boontheekul, and D. J. Mooney. Regulating activation of transplanted cells controls tissue regeneration. Proc. Natl. Acad. Sci. 103:2494–2499, 2006.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Holloway, J. L., A. M. Lowman, M. R. VanLandingham, and G. R. Palmese. Interfacial optimization of fiber-reinforced hydrogel composites for soft fibrous tissue applications. Acta Biomater. 10:3581–3589, 2014.

    Article  CAS  PubMed  Google Scholar 

  23. Horsley, V., and G. K. Pavlath. Forming a multinucleated cell: molecules that regulate myoblast fusion. Cells Tissues Organs 176:67–78, 2004.

    Article  PubMed  Google Scholar 

  24. Juhas, M., and N. Bursac. Engineering skeletal muscle repair. Curr. Opin. Biotechnol. 24:880–886, 2013.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Jun, I., S. Jeong, and H. Shin. The stimulation of myoblast differentiation by electrically conductive sub-micron fibers. Biomaterials 30:2038–2047, 2009.

    Article  CAS  PubMed  Google Scholar 

  26. Kreider, B., R. Benezra, G. Rovera, and T. Kadesch. Inhibition of myeloid differentiation by the helix-loop-helix protein Id. Science 255:1700–1702, 1992.

    Article  CAS  PubMed  Google Scholar 

  27. Langer, R., and J. Vacanti. Tissue engineering. Science 260:920–926, 1993.

    Article  CAS  PubMed  Google Scholar 

  28. Lawson, M. A., and P. P. Purslow. Differentiation of myoblasts in serum-free media: effects of modified media are cell line-specific. Cells Tissues Organs 167:130–137, 2000.

    Article  CAS  PubMed  Google Scholar 

  29. Lee, J., H.-R. Chae, Y. J. Won, K. Lee, C.-H. Lee, H. H. Lee, I.-C. Kim, and J.-M. Lee. Graphene oxide nanoplatelets composite membrane with hydrophilic and antifouling properties for wastewater treatment. J. Membr. Sci. 448:223–230, 2013.

    Article  CAS  Google Scholar 

  30. Li, L., J. C. Chambard, M. Karin, and E. N. Olson. Fos and Jun repress transcriptional activation by myogenin and MyoD: the amino terminus of Jun can mediate repression. Genes Dev. 6:676–689, 1992.

    Article  CAS  PubMed  Google Scholar 

  31. Li, Y., H. Yuan, A. von dem Bussche, M. Creighton, R. H. Hurt, A. B. Kane, and H. Gao. Graphene microsheets enter cells through spontaneous membrane penetration at edge asperities and corner sites. Proc. Natl. Acad. Sci. 110:12295–12300, 2013.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Liu, D. TGF-beta inhibits muscle differentiation through functional repression of myogenic transcription factors by Smad3. Genes Dev. 15:2950–2966, 2001.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Liu, W., D. Zhai, Z. Huan, C. Wu, and J. Chang. Novel tricalcium silicate/magnesium phosphate composite bone cement having high compressive strength, in vitro bioactivity and cytocompatibility. Acta Biomater. 21:217–227, 2015.

    Article  CAS  PubMed  Google Scholar 

  34. Luo, Y., H. Shen, Y. Fang, Y. Cao, J. Huang, M. Zhang, J. Dai, X. Shi, and Z. Zhang. Enhanced proliferation and osteogenic differentiation of mesenchymal stem cells on graphene oxide-incorporated electrospun poly(lactic-co-glycolic acid) nanofibrous mats. ACS Appl Mater Interfaces 7:6331–6339, 2015.

    Article  CAS  PubMed  Google Scholar 

  35. Mukundan, S., V. Sant, S. Goenka, J. Franks, L. C. Rohan, and S. Sant. Nanofibrous composite scaffolds of poly(ester amides) with tunable physicochemical and degradation properties. Eur. Polymer J. 68:21–35, 2015.

    Article  CAS  Google Scholar 

  36. Nair, L. S., and C. T. Laurencin. Biodegradable polymers as biomaterials. Prog. Polym. Sci. 32:762–798, 2007.

    Article  CAS  Google Scholar 

  37. Nayak, T. R., H. Andersen, V. S. Makam, C. Khaw, S. Bae, X. Xu, P.-L. R. Ee, J.-H. Ahn, B. H. Hong, G. Pastorin, and B. Özyilmaz. Graphene for controlled and accelerated osteogenic differentiation of human mesenchymal stem cells. ACS Nano 5:4670–4678, 2011.

    Article  CAS  PubMed  Google Scholar 

  38. Olson, E. N. Interplay between proliferation and differentiation within the myogenic lineage. Dev Biol 154:261–272, 1992.

    Article  CAS  PubMed  Google Scholar 

  39. Ostrovidov, S., V. Hosseini, S. Ahadian, T. Fujie, S. P. Parthiban, M. Ramalingam, H. Bae, H. Kaji, and A. Khademhosseini. Skeletal muscle tissue engineering: methods to form skeletal myotubes and their applications. Tissue Eng Part B-Rev 20:403–436, 2014.

    Article  PubMed  PubMed Central  Google Scholar 

  40. Patel A., S. Mukundan, W. Wang, A. Karumuri, V. Sant, S. M. Mukhopadhyay and S. Sant. Carbon-based hierarchical scaffolds for myoblast differentiation: Synergy between nano-functionalization and alignment. Acta Biomater. 2016.

  41. Pelipenko, J., P. Kocbek, and J. Kristl. Critical attributes of nanofibers: preparation, drug loading, and tissue regeneration. Int. J. Pharm. 484:57–74, 2015.

    Article  CAS  PubMed  Google Scholar 

  42. Rossi, C. A., M. Flaibani, B. Blaauw, M. Pozzobon, E. Figallo, C. Reggiani, L. Vitiello, N. Elvassore, and P. De Coppi. In vivo tissue engineering of functional skeletal muscle by freshly isolated satellite cells embedded in a photopolymerizable hydrogel. FASEB J. 25:2296–2304, 2011.

    Article  CAS  PubMed  Google Scholar 

  43. Sant, S., D. F. Coutinho, N. Sadr, R. L. Reis, and A. Khademhosseini. Tissue Analogs by the Assembly of Engineered Hydrogel Blocks. In: Biomimetic Approaches for Biomaterials Development, Wiley, KGaA, pp. 471–493, 2012.

  44. Sant, S., D. Iyer, A. K. Gaharwar, A. Patel, and A. Khademhosseini. Effect of biodegradation and de novo matrix synthesis on the mechanical properties of valvular interstitial cell-seeded polyglycerol sebacate-polycaprolactone scaffolds. Acta Biomater. 9:5963–5973, 2013.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Santin, M. Synthetic morphogens and pro-morphogens for aided tissue regeneration. Biol. Respons. Biomater. Tissue 1:43–64, 2013.

    CAS  Google Scholar 

  46. Sanz, A., M. Ruppel, J. F. Douglas, and J. T. Cabral. Plasticization effect of C60 on the fast dynamics of polystyrene and related polymers: an incoherent neutron scattering study. J. Phys.: Condens. Matter. 20:104209, 2008.

    Google Scholar 

  47. Sfeir C., P. A. Fang, J. Thottala, A. Raman, Z. Xiaoyuan, and E. Beniash. Synthesis of bone-like nanocomposites using multiphosphorylated peptides. Acta Biomater. 2014.

  48. Soliman, S., S. Sant, J. W. Nichol, M. Khabiry, E. Traversa, and A. Khademhosseini. Controlling the porosity of fibrous scaffolds by modulating the fiber diameter and packing density. J. Biomed. Mater. Res. Part A 96A:566–574, 2011.

    Article  CAS  Google Scholar 

  49. Stone, V., H. Johnston, and R. P. F. Schins. Development ofin vitrosystems for nanotoxicology: methodological considerations. Crit. Rev. Toxicol. 39:613–626, 2009.

    Article  CAS  PubMed  Google Scholar 

  50. Suk, J. W., R. D. Piner, J. An, and R. S. Ruoff. Mechanical properties of monolayer graphene oxide. ACS Nano 4:6557–6564, 2010.

    Article  CAS  PubMed  Google Scholar 

  51. Tomblyn S., E. L. Pettit Kneller, S. J. Walker, M. D. Ellenburg, C. J. Kowalczewski, M. Van Dyke, L. Burnett, and J. M. Saul. Keratin hydrogel carrier system for simultaneous delivery of exogenous growth factors and muscle progenitor cells. J. Biomed. Mater. Res. B Appl. Biomater. 2015.

  52. Tong, Z., S. Sant, A. Khademhosseini, and X. Jia. Controlling the fibroblastic differentiation of mesenchymal stem cells via the combination of fibrous scaffolds and connective tissue growth factor. Tissue Eng. Part A 17:2773–2785, 2011.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Ventre M., and P. A. Netti. Engineering cell instructive materials to control cell fate and functions through material cues and surface patterning. ACS Appl. Mater. Interfaces 2016.

  54. Warren, H., R. D. Gately, P. O’Brien, and R. Gorkin. Electrical conductivity, impedance, and percolation behavior of carbon nanofiber and carbon nanotube containing gellan gum hydrogels. J. Polym. Sci. Part B: Polym. Phys. 52:864–871, 2014.

    Article  CAS  Google Scholar 

  55. Wu, N., X. She, D. Yang, X. Wu, F. Su, and Y. Chen. Synthesis of network reduced graphene oxide in polystyrene matrix by a two-step reduction method for superior conductivity of the composite. J. Mater. Chem. 22:17254, 2012.

    Article  CAS  Google Scholar 

  56. Xavier, J. R., T. Thakur, P. Desai, M. K. Jaiswal, N. Sears, E. Cosgriff-Hernandez, R. Kaunas, and A. K. Gaharwar. Bioactive nanoengineered hydrogels for bone tissue engineering: a growth-factor-free approach. ACS Nano 9:3109–3118, 2015.

    Article  CAS  PubMed  Google Scholar 

  57. Xu, A., X. Liu, X. Gao, F. Deng, Y. Deng, and S. Wei. Enhancement of osteogenesis on micro/nano-topographical carbon fiber-reinforced polyetheretherketone-nanohydroxyapatite biocomposite. Mater. Sci. Eng. C Mater. Biol. Appl. 48:592–598, 2015.

    Article  CAS  PubMed  Google Scholar 

  58. Xue, Y., A. Patel, V. Sant, and S. Sant. PEGylated poly(ester amide) elastomers with tunable physico-chemical, mechanical and degradation properties. Eur. Polym. J. 72:163–179, 2015.

    Article  CAS  Google Scholar 

  59. Yan, X., and M. Gevelber. Investigation of electrospun fiber diameter distribution and process variations. J. Electrostat. 68:458–464, 2010.

    Article  CAS  Google Scholar 

  60. Yang, H. S., B. Lee, J. H. Tsui, J. Macadangdang, S.-Y. Jang, S. G. Im, and D.-H. Kim. Electroconductive nanopatterned substrates for enhanced myogenic differentiation and maturation. Adv. Healthc. Mater. 5:137–145, 2016.

    Article  CAS  PubMed  Google Scholar 

  61. Zhang, Y., S. F. Ali, E. Dervishi, Y. Xu, Z. Li, D. Casciano, and A. S. Biris. Cytotoxicity effects of graphene and single-wall carbon nanotubes in neural phaeochromocytoma-derived PC12 cells. ACS Nano 4:3181–3186, 2010.

    Article  CAS  PubMed  Google Scholar 

  62. Zhou, T. N. The preparation of the poly(vinyl alcohol)/graphene nanocomposites with low percolation threshold and high electrical conductivity by using the large-area reduced graphene oxide sheets. Exp. Polym. Lett. 7:747–755, 2013.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors acknowledge Material Characterization Lab, University of Pittsburgh for access to contact angle goniometer and Dr. Tracy Cui lab for access to conductivity measurement. SS acknowledges financial support (start-up funds) from the Department of Pharmaceutical Sciences at University of Pittsburgh.

Author information

Author notes

    Authors and Affiliations

    1. Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA, 15261, USA

      Akhil Patel, Yingfei Xue, Shilpaa Mukundan, Lisa C. Rohan, Vinayak Sant & Shilpa Sant

    2. Center for Biologic Imaging, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15261, USA

      Donna B. Stolz

    3. Departments of Cell Biology and Pathology, University of Pittsburgh, Pittsburgh, PA, 15261, USA

      Donna B. Stolz

    4. Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, 15219, USA

      Shilpa Sant

    5. McGowan Institute for Regenerative Medicine, Pittsburgh, PA, 15260, USA

      Shilpa Sant

    Authors
    1. Akhil Patel
      View author publications

      You can also search for this author in PubMed Google Scholar

    2. Yingfei Xue
      View author publications

      You can also search for this author in PubMed Google Scholar

    3. Shilpaa Mukundan
      View author publications

      You can also search for this author in PubMed Google Scholar

    4. Lisa C. Rohan
      View author publications

      You can also search for this author in PubMed Google Scholar

    5. Vinayak Sant
      View author publications

      You can also search for this author in PubMed Google Scholar

    6. Donna B. Stolz
      View author publications

      You can also search for this author in PubMed Google Scholar

    7. Shilpa Sant
      View author publications

      You can also search for this author in PubMed Google Scholar

    Corresponding author

    Correspondence to Shilpa Sant.

    Additional information

    Associate Editor Akhilesh K. Gaharwar oversaw the review of this article.

    Akhil Patel and Yingfei Xue have contributed equally to the work.

    Rights and permissions

    Reprints and permissions

    About this article

    Check for updates. Verify currency and authenticity via CrossMark

    Cite this article

    Patel, A., Xue, Y., Mukundan, S. et al. Cell-Instructive Graphene-Containing Nanocomposites Induce Multinucleated Myotube Formation. Ann Biomed Eng 44, 2036–2048 (2016). https://doi.org/10.1007/s10439-016-1586-6

    Download citation

    • Received:

    • Accepted:

    • Published:

    • Issue Date:

    • DOI: https://doi.org/10.1007/s10439-016-1586-6

    Keywords

    Search

    Navigation