Effect of a synthetic link N peptide nanofiber scaffold on the matrix deposition of aggrecan and type II collagen in rabbit notochordal cells

  • Kaige Ma
  • Yongchao Wu
  • Baichuan Wang
  • Shuhua Yang
  • Yulong Wei
  • Zengwu Shao


Self-assembling peptide nanofiber scaffolds have been studied extensively as biological materials for 3-dimensional cell culture and repairing tissue defects in animals. However, few studies have applied peptide nanofiber scaffolds in the tissue engineering of intervertebral discs (IVDs). In this study, a novel functionalized peptide scaffold was specifically designed for IVD tissue engineering, and notochordal cells (NCs) as an alternative cell source for IVD degeneration were selected to investigate the bioactive scaffold material. The novel RADA16-Link N self-assembling peptide scaffold material was designed by direct coupling to a bioactive motif link N. The link N nanofiber scaffold (LN-NS) material was obtained by mixing pure RADA16-I and RADA16-Link N (1:1) designer peptide solutions. Although live/dead cell assays showed that LN-NS and RADA16-I scaffold materials were both biocompatible with NCs, the LN-NS material significantly promoted NC adhesion compared with that of the pure RADA16-I SAP scaffold material. The depositions of aggrecan and type II collagen, which are significant markers for IVD cells, were remarkably increased. Furthermore, the results indicated that the link N motif, the matrix analog of the nucleus pulposus, significantly promoted the accumulation of other extracellular matrices in vitro. We conclude that the novel LN-NS material is a promising biological scaffold material, and may have a broad range of applications in IVD tissue engineering.


  1. 1.
    Ganey T, Libera J, Moos V, Alasevic O, Fritsch KG, Meisel HJ, Hutton WC. Disc chondrocyte transplantationin a canine model: a treatment for degenerated or damaged intervertebral disc. Spine. 2003;28(23):2609–20.CrossRefGoogle Scholar
  2. 2.
    Cheung KM, Karppinen J, Chan D, Ho DW, Song YQ, Sham P, Cheah KS, Leong JC, Luk KD. Prevalence and pattern of lumbar magnetic resonance imaging changes in a population study of one thousand forty-three individuals. Spine. 2009;34(9):934–40.CrossRefGoogle Scholar
  3. 3.
    Clouet J, Pot-Vaucel M, Grimandi G, Masson M, Lesoeur J, Fellah BH, Gauthier O, Fusellier M, Cherel Y, Maugars Y, Guicheux J, Vinatier C. Characterization of the age-dependent intervertebral disc changes in rabbit by correlation between MRI, histology and gene expression. BMC Musculoskelet Disord. 2011;12:147.CrossRefGoogle Scholar
  4. 4.
    Gilson A, Dreger M, Urban JP. Differential expression level of cytokeratin 8 in cells of the bovine nucleus pulposus complicates the search for specific intervertebral disc cell markers. Arthritis Res Ther. 2010;12(1):R24.CrossRefGoogle Scholar
  5. 5.
    Hohaus C, Ganey TM, Minkus Y, Meisel HJ. Cell transplantation in lumbar spine disc degeneration disease. Eur Spine J. 2008;17(Suppl 4):492–503.CrossRefGoogle Scholar
  6. 6.
    Kepler CK, Anderson DG, Tannoury C, Ponnappan RK. Intervertebral disk degeneration and emerging biologic treatments. J Am Acad Orthop Surg. 2011;19(9):543–53.Google Scholar
  7. 7.
    Kandel R, Roberts S, Urban JP. Tissue engineering and the intervertebral disc: the challenges. Eur Spine J. 2008;17(Suppl 4):480–91.CrossRefGoogle Scholar
  8. 8.
    Seguin CA, Grynpas MD, Pilliar RM, Waldman SD, Kandel RA. Tissue engineered nucleus pulposus tissue formed on aporous calcium polyphosphate substrate. Spine. 2004;29(12):1299–306.CrossRefGoogle Scholar
  9. 9.
    Gaetani P, Torre ML, Klinger M, Faustini M, Crovato F, Bucco M, Marazzi M, Chlapanidas T, Levi D, Tancioni F, Vigo D, Rodriguez y Baena R. Adipose-derived stem cell therapy for intervertebral disc regeneration: an in vitro reconstructed tissue in alginate capsules. Tissue Eng Part A. 2008;14(8):1415–23.CrossRefGoogle Scholar
  10. 10.
    Sakai D. Future perspectives of cell-based therapy for intervertebral disc disease. Eur Spine J. 2008;17(Suppl 4):452–8.CrossRefGoogle Scholar
  11. 11.
    Mwale F, Masuda K, Pichika R, Epure LM, Yoshikawa T, Hemmad A, Roughley PJ. The efficacy of Link N as a mediator of repair in a rabbit model of intervertebral disc degeneration. Antoniou J Arthritis Res Ther. 2011;13(4):R120.CrossRefGoogle Scholar
  12. 12.
    Pearce RH, Mathieson JM, Mort JS, Roughley PJ. Effect of age on the abundance and fragmentation of link protein of the human intervertebral disc. J Orthop Res. 1989;7(6):861–7.CrossRefGoogle Scholar
  13. 13.
    Mwale F, Demers CN, Petit A, Roughley P, Poole AR, Steffen T, Aebi M, Antoniou J. A synthetic peptide of link protein stimulates the biosynthesis of collagens II, IX and proteoglycan by cells of the intervertebral disc. J Cell Biochem. 2003;88(6):1202–13.CrossRefGoogle Scholar
  14. 14.
    Roberts JJ, Nicodemus GD, Giunta S, Bryant SJ. Incorporation of biomimetic matrix molecules in PEG hydrogels enhances matrix deposition and reduces load-induced loss of chondrocyte-secreted matrix. J Biomed Mater Res A. 2011;97(3):281–91.Google Scholar
  15. 15.
    Rodriguez E, Roughley P. Link protein can retard the degradation of hyaluronan in proteoglycan aggregates. Osteoarthritis Cartilage. 2006;14(8):823–9.CrossRefGoogle Scholar
  16. 16.
    Yokoi H, Kinoshita T, Zhang S. Dynamic reassembly of peptide RADA16 nanofiber scaffold. Proc Natl Acad Sci USA. 2005;102(24):8414–9.CrossRefGoogle Scholar
  17. 17.
    Wang B, Wu Y, Shao Z, Yang S, Che B, Sun C, Ma Z, Zhang Y. Functionalized self-assembling peptide nanofiber hydrogel as a scaffold for rabbit nucleus pulposus cells. J Biomed Mater Res A. 2012;100(3):646–53.Google Scholar
  18. 18.
    Le Maitre CL, Freemont AJ, Hoyland JA. Expression of cartilage-derived morphogenetic protein in human intervertebral discs and its effect on matrix synthesis in degenerate human nucleus pulposus cells. Arthritis Res Ther. 2009;11(5):R137.CrossRefGoogle Scholar
  19. 19.
    Kim KW, Lim TH, Kim JG, Jeong ST, Masuda K, An HS. The origin of chondrocytes in the nucleus pulposus and histologic findings associated with the transition of a notochordal nucleus pulposus to a fibrocartilaginous nucleus pulposus in intact rabbit intervertebral discs. Spine. 2003;28(10):982–90.Google Scholar
  20. 20.
    Hunter CJ, Matyas JR, Duncan NA. The functional significance of cell clusters in the notochordal nucleus pulposus: survival and signaling in the canine intervertebral disc. Spine. 2004;29(10):1099–104.CrossRefGoogle Scholar
  21. 21.
    Oegema TR Jr. The role of disc cell heterogeneity in determining disc biochemistry: a speculation. Biochem Soc Trans 2002;30(Pt6):839–44.Google Scholar
  22. 22.
    Weiler C, Nerlich AG, Schaaf R, Bachmeier BE, Wuertz K, Boos N. Immunohistochemical identification of notochordal markers in cells in the aging human lumbar intervertebral disc. Eur Spine J. 2010;19(10):1761–70.CrossRefGoogle Scholar
  23. 23.
    Erwin WM, Ashman K, O’Donnel P, Inman RD. Nucleus pulposus notochord cells secrete connective tissue growth factor and up-regulate proteoglycan expression by intervertebral disc chondrocytes. Arthritis Rheum. 2006;54(12):3859–67.CrossRefGoogle Scholar
  24. 24.
    Hayes AJ, Benjamin M, Ralphs JR. Extracellular matrix in development of the intervertebral disc. Matrix Biol. 2001;20(2):107–21.CrossRefGoogle Scholar
  25. 25.
    Erwin WM, Inman RD. Notochord cells regulate intervertebral disc chondrocyte proteoglycan production and cell proliferation. Spine. 2006;31(10):1094–9.CrossRefGoogle Scholar
  26. 26.
    Hunter CJ, Matyas JR, Duncan NA. The notochordal cell in the nucleus pulposus: a review in the context of tissue engineering. Tissue Eng. 2003;9(4):667–77.CrossRefGoogle Scholar
  27. 27.
    Cappello R, Bird JL, Pfeiffer D, Bayliss MT, Dudhia J. Notochordal cell produce and assemble extracellular matrix in a distinct manner, which may be responsible for the maintenance of healthy nucleus pulposus. Spine. 2006;31(8):873–82; discussion 883.Google Scholar
  28. 28.
    Aguiar DJ, Johnson SL, Oegema TR. Notochordal cells interact with nucleus pulposus cells: regulation of proteoglycan synthesis. Exp Cell Res. 1999;246(1):129–37.CrossRefGoogle Scholar
  29. 29.
    Tiwari M, Lopez-Cruzan M, Morgan WW, Herman B. Loss of caspase-2-dependent apoptosis induces autophagy after mitochondrial oxidative stress in primary cultures of young adult cortical neurons. J Biol Chem. 2011;286(10):8493–506.CrossRefGoogle Scholar
  30. 30.
    Khandwekar AP, Patil DP, Shouche YS, Doble M. The biocompatibility of sulfobetaine engineered polymethylmethacrylate by surface entrapment technique. J Mater Sci Mater Med. 2010;21(2):635–46.CrossRefGoogle Scholar
  31. 31.
    Gelain F, Lomander A, Vescovi AL, Zhang S. Systematic studies of a self-assembling peptide nanofiber scaffold with other scaffolds. J Nanosci Nanotechnol. 2007;7(2):424–34.CrossRefGoogle Scholar
  32. 32.
    Horii A, Wang X, Gelain F, Zhang S. Biological designer self-assembling peptide nanofiber scaffolds significantly enhance osteoblast proliferation, differentiation and 3-D migration. PLoS ONE. 2007;2(2):e190.CrossRefGoogle Scholar
  33. 33.
    Knight CG, Morton LF, Peachey AR, Tuckwell DS, Farndale RW, Barnes MJ. The collagen-binding A-domains of integrins alpha (1) beta (1) and alpha (2) beta (1) recognize the same specific amino acid sequence, GFOGER, in native (triple-helical) collagens. J Biol Chem. 2000;275(1):35–40.CrossRefGoogle Scholar
  34. 34.
    Wang X, Horii A, Zhang S. Designer functionalized self-assembling peptide nanofiber scaffolds for growth, migration, and tubulogenesis of human umbilical vein endothelial cells. Soft Matter. 2008;4(12):2388–95.CrossRefGoogle Scholar
  35. 35.
    Kim JH, Deasy BM, Seo HY, Studer RK, Vo NV, Georgescu HI, Sowa GA, Kang JD. Differentiation of intervertebral notochordal cells through live automated cell imaging system in vitro. Spine. 2009;34(23):2486–93.CrossRefGoogle Scholar
  36. 36.
    Okuma M, Mochida J, Nishimura K, Sakabe K, Seiki K. Reinsertion of stimulated nucleus pulposus cells retards intervertebral disc degeneration: an in vitro and in vivo experimental study. J Orthop Res. 2000;18(6):988–97.CrossRefGoogle Scholar
  37. 37.
    Maldonado BA, Oegema TR Jr. Initial characterization of the metabolism of intervertebral disc cells encapsulated in microspheres. J Orthop Res. 1992;10(5):677–90.CrossRefGoogle Scholar
  38. 38.
    Poiraudeau S, Monteiro I, Anract P, Blanchard O, Revel M, Corvol MT. Phenotypic characteristics of rabbit intervertebral disc cells. Comparison with cartilage cells from the same animals. Spine. 1999;24(9):837–44.CrossRefGoogle Scholar
  39. 39.
    Ellis-Behnke RG, Liang YX, You SW, Tay DK, Zhang S, So KF, Schneider GE. Nano neuro knitting: peptide nanofiber scaffold for brain repair and axon regeneration with functional return of vision. Proc Natl Acad Sci USA. 2006;103(13):5054–9.CrossRefGoogle Scholar
  40. 40.
    Gelain F, Bottai D, Vescovi A, Zhang S. Designer self-assembling peptide nanofiber scaffolds for adult mouse neural stem cell 3-dimensional cultures. PLoS ONE. 2006;1:e119.CrossRefGoogle Scholar
  41. 41.
    Zhao X, Nagai Y, Reeves PJ, Kiley P, Khorana HG, Zhang S. Designer short peptide surfactants stabilize G protein-coupled receptor bovine rhodopsin. Proc Natl Acad Sci USA. 2006;103(47):17707–12.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Kaige Ma
    • 1
  • Yongchao Wu
    • 1
  • Baichuan Wang
    • 1
  • Shuhua Yang
    • 1
  • Yulong Wei
    • 1
  • Zengwu Shao
    • 1
  1. 1.Department of Orthopedics, Union Hospital, Tongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina

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