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Porous silk scaffolds can be used for tissue engineering annulus fibrosus

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Abstract

There is no optimal treatment for symptomatic degenerative disc disease which affects millions of people worldwide. One novel approach would be to form a patch or tissue replacement to repair the annulus fibrosus (AF) through which the NP herniates. As the optimal scaffold for this has not been defined the purpose of this study was to determine if porous silk scaffolds would support AF cell attachment and extracellular matrix accumulation and whether chemically decorating the scaffold with RGD peptide, which has been shown to enhance attachment for other cell types, would further improve AF cell attachment and tissue formation. Annulus fibrosus cells were isolated from bovine caudal discs and seeded into porous silk scaffolds. The percent cell attachment was quantified and the cell morphology and distribution within the scaffold was evaluated using scanning electron microscopy. The cell-seeded scaffolds were grown for up to 8 weeks and evaluated for gene expression, histological appearance and matrix accumulation. AF cells attach to porous silk scaffolds, proliferate and synthesize and accumulate extracellular matrix as demonstrated biochemically and histologically. Coupling the silk scaffold with RGD-peptides did not enhance cell attachment nor tissue formation but did affect cell morphology. As well, the cells had higher levels of type II collagen and aggrecan gene expression when compared to cells grown on the non-modified scaffold, a feature more in keeping with cells of the inner annulus. Porous silk is an appropriate scaffold on which to grow AF cells. Coupling RGD peptide to the scaffold appears to influence AF cell phenotype suggesting that it may be possible to select an appropriate scaffold that favours inner annulus versus outer annulus differentiation which will be important for tissue engineering an intervertebral disc.

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References

  1. Adams MA, Roughley PJ (2006) What is intervertebral disc degeneration, and what causes it? Spine 31(18):2151–2161

    Article  PubMed  Google Scholar 

  2. Alini M, Li W, Markovic P, Aebi M, Spiro RC, Roughley PJ (2003) The potential and limitations of a cell-seeded collagen/hyaluronan scaffold to engineer an intervertebral disc-like matrix. Spine 28(5):446–454

    Article  PubMed  Google Scholar 

  3. Altman GH, Diaz F, Jakuba C, Calabro T, Horan RL, Chen J et al (2003) Silk-based biomaterials. Biomaterials 24(3):401–416

    Article  PubMed  CAS  Google Scholar 

  4. Anderson DG, Tannoury C (2005) Molecular pathogenic factors in symptomatic disc degeneration. Spine J 5(6 Suppl):S260–S266

    Article  Google Scholar 

  5. Baer AE, Wang JY, Kraus VB, Setton LA (2001) Collagen gene expression and mechanical properties of intervertebral disc cell-alginate cultures. J Orthop Res 19(1):2–10

    Article  PubMed  CAS  Google Scholar 

  6. Battie MC, Videman T (2006) Lumbar disc degeneration: epidemiology and genetics. J Bone Joint Surg Am 88(Suppl 2):3–9

    Article  PubMed  Google Scholar 

  7. Bogduk N (1997) The inter-body joints and the intervertebral discs. In: Bogduk N (ed) Clinical anatomy of the lumbar spine and sacrum. Churchill Livingstone, New York, pp 13–31

    Google Scholar 

  8. Broberg KB (1983) On the mechanical behaviour of intervertebral discs. Spine 8(2):151–165

    Article  PubMed  CAS  Google Scholar 

  9. Carman CV, Springer TA (2003) Integrin avidity regulation: are changes in affinity and conformation underemphasized? Curr Opin Cell Biol 15(5):547–556

    Article  PubMed  CAS  Google Scholar 

  10. Chen J, Altman GH, Karageorgiou V, Horan R, Collette A, Volloch V et al (2003) Human bone marrow stromal cell and ligament fibroblast responses on RGD-modified silk fibers. J Biomed Mater Res A 67(2):559–570

    Article  PubMed  Google Scholar 

  11. Gruber HE, Leslie K, Ingram J, Norton HJ, Hanley EN (2004) Cell-based tissue engineering for the intervertebral disc: in vitro studies of human disc cell gene expression and matrix production within selected cell carriers. Spine J 4(1):44–55

    Article  PubMed  Google Scholar 

  12. Hayes AJ, Benjamin M, Ralphs JR (1999) Role of actin stress fibres in the development of the intervertebral disc: cytoskeletal control of extracellular matrix assembly. Dev Dyn 215(3):179–189

    Article  PubMed  CAS  Google Scholar 

  13. Hayes AJ, Benjamin M, Ralphs JR (2001) Extracellular matrix in development of the intervertebral disc. Matrix Biol 20(2):107–121

    Article  PubMed  CAS  Google Scholar 

  14. Horan RL, Antle K, Collette AL, Wang Y, Huang J, Moreau JE et al (2005) In vitro degradation of silk fibroin. Biomaterials 26(17):3385–3393

    Article  PubMed  CAS  Google Scholar 

  15. Hynes RO (2002) Integrins: bidirectional, allosteric signaling machines. Cell 110(6):673–687

    Article  PubMed  CAS  Google Scholar 

  16. Iatridis JC, Maclean JJ, Roughley PJ, Alini M (2006) Effects of mechanical loading on intervertebral disc metabolism in vivo. J Bone Joint Surg Am 88(Suppl 2):41–46

    Article  PubMed  Google Scholar 

  17. Iatridis JC, MaClean JJ, Ryan DA (2005) Mechanical damage to the intervertebral disc annulus fibrosus subjected to tensile loading. J Biomech 38(3):557–565

    Article  PubMed  Google Scholar 

  18. Kim UJ, Park J, Li C, Jin HJ, Valluzzi R, Kaplan DL (2004) Structure and properties of silk hydrogels. Biomacromolecules 5(3):786–792

    Article  PubMed  CAS  Google Scholar 

  19. Klein JA, Hickey DS, Hukins DW (1983) Radial bulging of the annulus fibrosus during compression of the intervertebral disc. J Biomech 16(3):211–217

    Article  PubMed  CAS  Google Scholar 

  20. Kluba T, Niemeyer T, Gaissmaier C, Grunder T (2005) Human anulus fibrosis and nucleus pulposus cells of the intervertebral disc: effect of degeneration and culture system on cell phenotype. Spine 30(24):2743–2748

    Article  PubMed  Google Scholar 

  21. Martin G (1980) Recurrent disc prolapse as a cause of recurrent pain after laminectomy for lumbar disc lesions. NZ Med J 91(656):206–208

    CAS  Google Scholar 

  22. Meinel L, Hofmann S, Karageorgiou V, Kirker-Head C, McCool J, Gronowicz G et al (2005) The inflammatory responses to silk films in vitro and in vivo. Biomaterials 26(2):147–155

    Article  PubMed  CAS  Google Scholar 

  23. Meinel L, Hofmann S, Karageorgiou V, Zichner L, Langer R, Kaplan D et al (2004) Engineering cartilage-like tissue using human mesenchymal stem cells and silk protein scaffolds. Biotechnol Bioeng 88(3):379–391

    Article  PubMed  CAS  Google Scholar 

  24. Meinel L, Karageorgiou V, Hofmann S, Fajardo R, Snyder B, Li C et al (2004) Engineering bone-like tissue in vitro using human bone marrow stem cells and silk scaffolds. J Biomed Mater Res A 71(1):25–34

    Article  PubMed  Google Scholar 

  25. Mizuno H, Roy AK, Vacanti CA, Kojima K, Ueda M, Bonassar LJ (2004) Tissue-engineered composites of anulus fibrosus and nucleus pulposus for intervertebral disc replacement. Spine 29(12):1290–1297

    Article  PubMed  Google Scholar 

  26. Mizuno H, Roy AK, Zaporojan V, Vacanti CA, Ueda M, Bonassar LJ (2006) Biomechanical and biochemical characterization of composite tissue-engineered intervertebral discs. Biomaterials 27(3):362–370

    Article  PubMed  CAS  Google Scholar 

  27. Nazarov R, Jin HJ, Kaplan DL (2004) Porous 3-D scaffolds from regenerated silk fibroin. Biomacromolecules 5(3):718–726

    Article  PubMed  CAS  Google Scholar 

  28. Nettles DL, Richardson WJ, Setton LA (2004) Integrin expression in cells of the intervertebral disc. J Anat 204(6):515–520

    Article  PubMed  CAS  Google Scholar 

  29. Ochsenhirt SE, Kokkoli E, McCarthy JB, Tirrell M (2006) Effect of RGD secondary structure and the synergy site PHSRN on cell adhesion, spreading and specific integrin engagement. Biomaterials 27(20):3863–3874

    Article  PubMed  CAS  Google Scholar 

  30. Oharazawa H, Ibaraki N, Ohara K, Reddy VN (2005) Inhibitory effects of Arg–Gly–Asp (RGD) peptide on cell attachment and migration in a human lens epithelial cell line. Ophthalmic Res 37(4):191–196

    Article  PubMed  CAS  Google Scholar 

  31. Pulai JI, Del Carlo M Jr, Loeser RF (2002) The alpha5beta1 integrin provides matrix survival signals for normal and osteoarthritic human articular chondrocytes in vitro. Arthritis Rheum 46(6):1528–1535

    Article  PubMed  CAS  Google Scholar 

  32. Roberts S, Evans H, Trivedi J, Menage J (2006) Histology and pathology of the human intervertebral disc. J Bone Joint Surg Am 88(Suppl 2):10–14

    Article  PubMed  Google Scholar 

  33. Rong Y, Sugumaran G, Silbert JE, Spector M (2002) Proteoglycans synthesized by canine intervertebral disc cells grown in a type I collagen-glycosaminoglycan matrix. Tissue Eng 8(6):1037–1047

    Article  PubMed  CAS  Google Scholar 

  34. Ruoslahti E (1996) RGD and other recognition sequences for integrins. Annu Rev Cell Dev Biol 12:697–715

    Article  PubMed  CAS  Google Scholar 

  35. Sato M, Kikuchi M, Ishihara M, Ishihara M, Asazuma T, Kikuchi T et al (2003) Tissue engineering of the intervertebral disc with cultured annulus fibrosus cells using atelocollagen honeycomb-shaped scaffold with a membrane seal (ACHMS scaffold). Med Biol Eng Comput 41(3):365–371

    Article  PubMed  CAS  Google Scholar 

  36. Seguin CA, Pilliar RM, Roughley PJ, Kandel RA (2005) Tumor necrosis factor-alpha modulates matrix production and catabolism in nucleus pulposus tissue. Spine 30(17):1940–1948

    Article  PubMed  Google Scholar 

  37. Setton LA, Chen J (2006) Mechanobiology of the intervertebral disc and relevance to disc degeneration. J Bone Joint Surg Am 88:(Suppl 2):52–57

    Article  PubMed  Google Scholar 

  38. Thonar EJ, An H, Masuda K (2002) Compartmentalization of the matrix formed by nucleus pulposus and annulus fibrosus cells in alginate gel. Biochem Soc Trans 30:874–878

    Article  PubMed  CAS  Google Scholar 

  39. Waldman SD, Grynpas M, Pilliar RM, Kandel RA (2002) Characterization of cartilagenous tissue formed on calcium polyphosphate substrates in vitro. J Biomed Mater Res 62(3):323–330

    Article  PubMed  CAS  Google Scholar 

  40. Woessner JF Jr (1961) The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. Arch Biochem Biophys 93:440–447

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We thank Mr. Harry Bojarski and Ryding-Regency Meat Packers, Toronto, Canada for providing the tissues and Marie Maguire for secretarial support. This work was supported by NIH grant R21AR052801-02.

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Authors and Affiliations

  1. CIHR BioEngineering of Skeletal Tissues Team, Mount Sinai Hospital, University of Toronto, Toronto, Canada

    G. Chang & R. A. Kandel

  2. Department of Biomedical Engineering, Tufts University, Medford, MA, USA

    H.-J. Kim & D. Kaplan

  3. Department of Biomedical Engineering, Columbia University, New York, NY, USA

    G. Vunjak-Novakovic

  4. Mt. Sinai Hospital, 600 University Avenue, Room 6-500, M5G 1X5, Toronto, ON, Canada

    R. A. Kandel

Authors
  1. G. Chang
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  2. H.-J. Kim
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  3. D. Kaplan
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  4. G. Vunjak-Novakovic
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  5. R. A. Kandel
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Correspondence to R. A. Kandel.

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Chang, G., Kim, HJ., Kaplan, D. et al. Porous silk scaffolds can be used for tissue engineering annulus fibrosus. Eur Spine J 16, 1848–1857 (2007). https://doi.org/10.1007/s00586-007-0364-4

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  • DOI: https://doi.org/10.1007/s00586-007-0364-4

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