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GEORG SCHMORL PRIZE OF THE GERMAN SPINE SOCIETY (DWG) 2018: combined inflammatory and mechanical stress weakens the annulus fibrosus: evidences from a loaded bovine AF organ culture

  • Taryn Saggese
  • Graciosa Q. Teixeira
  • Kelly Wade
  • Lydia Moll
  • Anita Ignatius
  • Hans-Joachim Wilke
  • Raquel M. Goncalves
  • Cornelia Neidlinger-WilkeEmail author
Original Article

Abstract

Purpose

The pathomechanism of annulus fibrosus (AF) failure is still unknown. We hypothesise that mechanical overload and an inflammatory microenvironment contribute to AF structural weakening. Therefore, the objective of this study was to investigate the influence of these factors on the AF, particularly the translamellar bridging network (TLBN) which connects the AF lamellae.

Methods

A bovine AF organ culture (AF-OC) model of standardised AF rings was used to study the individual and combined effects of cyclic tensile strain (CTS) and IL-1β (1 ng/mL) culture medium supplementation. AF-OCs were analysed for PGE2 production (ELISA) and deposition of IL-6, COX-2, fibrillin, and MMP3 in the tissue (immunohistochemistry, IHC). The mechanical strength of the TLBN was evaluated using a peel test to measure the strength required to separate an AF segment along a lamellar bound.

Results

The combination of CTS + IL-1β led to a significant increase in PGE2 production compared to Control (p < 0.01). IHC evaluations showed that the CTS + IL-1β group exhibited higher production of COX-2 and MMP3 within the TLBN regions compared to the adjacent lamellae and a significant increase in IL-6 ratio compared to Control (p < 0.05). A significant decrease in the annular peel strength was observed in the CTS + IL1β group compared to Control (p < 0.05).

Conclusion

Our findings suggest that CTS and IL-1β act synergistically to increase pro-inflammatory and catabolic molecules within the AF, particularly the TLBN, leading to a weakening of the tissue. This standardised model enables the investigation of AF/TLBN structure–function relationship and is a platform to test AF-focused therapeutics.

Graphical abstract

These slides can be retrieved under Electronic Supplementary Material.

Keywords

Annulus fibrosus Organ culture Inflammation Mechanical loading Disc herniation 

Notes

Acknowledgements

The authors of the present study wish to thank the local certified abattoir Fleischmarkt Donautal for kindly providing to us the bovine tails. We wish to thank Mrs. Iris Baum, Mrs. Patrizia Horny, Mrs. Marion Tomo, Mrs. Anna Weigl, Mrs. Anastasia Raiber, Mr. Zhiyao Yong and Mr. Alexander Vogel for the excellent support. We also acknowledge the German Spine Foundation (Deutsche Wirbelsäulenstitung), the German Academic Exchange Service (DAAD), the Conselho de Reitores das Universidades Portuguesas, the Ulm University (L.SBN.0157) and the Alexander von Humboldt Foundation for their financial support. The funding agencies did not have any involvement in the study design, data collection/analysis/interpretation, manuscript preparation or in the decision to submit the manuscript for publication.

Compliance with ethical standards

Conflict of interest

All authors declare that they have no conflict of interest.

Ethical approval

This study did not involve animal experiments or any studies with human participants performed by any of the authors. The bovine tails were obtained from cattle which were slaughtered for alimentary purposes.

Supplementary material

586_2019_5901_MOESM1_ESM.pptx (450 kb)
Supplementary material 1 (PPTX 450 kb)
586_2019_5901_MOESM2_ESM.docx (660 kb)
Supplementary material 2 (DOCX 660 kb)

References

  1. 1.
    Hoy D, Bain C, Williams G, March L, Brooks P, Blyth F, Woolf A, Vos T, Buchbinder R (2012) A systematic review of the global prevalence of low back pain. Arthritis Rheum 64:2028–2037.  https://doi.org/10.1002/art.34347 Google Scholar
  2. 2.
    Rajasekaran S, Bajaj N, Tubaki V, Kanna RM, Shetty AP (2013) ISSLS Prize winner: the anatomy of failure in lumbar disc herniation: an in vivo, multimodal, prospective study of 181 subjects. Spine 38:1491–1500.  https://doi.org/10.1097/BRS.0b013e31829a6fa6 Google Scholar
  3. 3.
    Adams MA, Dolan P (2012) Intervertebral disc degeneration: evidence for two distinct phenotypes. J Anat 221:497–506.  https://doi.org/10.1111/j.1469-7580.2012.01551.x Google Scholar
  4. 4.
    Stefanakis M, Luo J, Pollintine P, Dolan P, Adams MA (2014) ISSLS Prize winner: mechanical influences in progressive intervertebral disc degeneration. Spine 39:1365–1372.  https://doi.org/10.1097/BRS.0000000000000389 Google Scholar
  5. 5.
    Battie MC, Videman T, Levalahti E, Gill K, Kaprio J (2007) Heritability of low back pain and the role of disc degeneration. Pain 131:272–280.  https://doi.org/10.1016/j.pain.2007.01.010 Google Scholar
  6. 6.
    Adams MA, Freeman BJ, Morrison HP, Nelson IW, Dolan P (2000) Mechanical initiation of intervertebral disc degeneration. Spine 25:1625–1636.  https://doi.org/10.1097/00007632-200007010-00005 Google Scholar
  7. 7.
    Lotz JC, Chin JR (2000) Intervertebral disc cell death is dependent on the magnitude and duration of spinal loading. Spine 25:1477–1483.  https://doi.org/10.1097/00007632-200006150-00005 Google Scholar
  8. 8.
    Risbud MV, Shapiro IM (2014) Role of cytokines in intervertebral disc degeneration: pain and disc content. Nat Rev Rheumatol 10:44–56.  https://doi.org/10.1038/nrrheum.2013.160 Google Scholar
  9. 9.
    Yu J, Schollum ML, Wade KR, Broom ND, Urban JP (2015) ISSLS Prize Winner: a detailed examination of the elastic network leads to a new understanding of annulus fibrosus organization. Spine 40:1149–1157.  https://doi.org/10.1097/BRS.0000000000000943 Google Scholar
  10. 10.
    Yu J, Winlove PC, Roberts S, Urban JP (2002) Elastic fibre organization in the intervertebral discs of the bovine tail. J Anat 201:465–475.  https://doi.org/10.1046/j.1469-7580.2002.00111.x Google Scholar
  11. 11.
    Melrose J, Smith SM, Appleyard RC, Little CB (2008) Aggrecan, versican and type VI collagen are components of annular translamellar crossbridges in the intervertebral disc. Eur Spine J 17:314–324.  https://doi.org/10.1007/s00586-007-0538-0 Google Scholar
  12. 12.
    Schollum ML, Appleyard RC, Little CB, Melrose J (2010) A detailed microscopic examination of alterations in normal anular structure induced by mechanical destabilization in an ovine model of disc degeneration. Spine 35:1965–1973.  https://doi.org/10.1097/BRS.0b013e3181e0f085 Google Scholar
  13. 13.
    Mengoni M, Luxmoore BJ, Wijayathunga VN, Jones AC, Broom ND, Wilcox RK (2015) Derivation of inter-lamellar behaviour of the intervertebral disc annulus. J Mech Behav Biomed Mater 48:164–172.  https://doi.org/10.1016/j.jmbbm.2015.03.028 Google Scholar
  14. 14.
    Han SK, Chen CW, Labus KM, Puttlitz CM, Chen Y, Hsieh AH (2016) Optical coherence tomographic elastography reveals mesoscale shear strain inhomogeneities in the annulus fibrosus. Spine 41:E770–E777.  https://doi.org/10.1097/BRS.0000000000001463 Google Scholar
  15. 15.
    Vergroesen PP, Kingma I, Emanuel KS, Hoogendoorn RJ, Welting TJ, van Royen BJ, van Dieen JH, Smit TH (2015) Mechanics and biology in intervertebral disc degeneration: a vicious circle. Osteoarthr Cartil 23:1057–1070.  https://doi.org/10.1016/j.joca.2015.03.028 Google Scholar
  16. 16.
    Molinos M, Almeida CR, Caldeira J, Cunha C, Goncalves RM, Barbosa MA (2015) Inflammation in intervertebral disc degeneration and regeneration. J R Soc Interface 12:20141191.  https://doi.org/10.1098/rsif.2014.1191 Google Scholar
  17. 17.
    Le Maitre CL, Hoyland JA, Freemont AJ (2007) Catabolic cytokine expression in degenerate and herniated human intervertebral discs: IL-1beta and TNFalpha expression profile. Arthritis Res Ther 9:R77.  https://doi.org/10.1186/ar2275 Google Scholar
  18. 18.
    Le Maitre CL, Freemont AJ, Hoyland JA (2005) The role of interleukin-1 in the pathogenesis of human intervertebral disc degeneration. Arthritis Res Ther 7:R732–R745.  https://doi.org/10.1186/ar1732 Google Scholar
  19. 19.
    Teixeira GQ, Boldt A, Nagl I, Pereira CL, Benz K, Wilke HJ, Ignatius A, Barbosa MA, Goncalves RM, Neidlinger-Wilke C (2016) A degenerative/proinflammatory intervertebral disc organ culture: an ex vivo model for anti-inflammatory drug and cell therapy. Tissue Eng C Methods 22:8–19.  https://doi.org/10.1089/ten.tec.2015.0195 Google Scholar
  20. 20.
    Neidlinger-Wilke C, Mietsch A, Rinkler C, Wilke HJ, Ignatius A, Urban J (2012) Interactions of environmental conditions and mechanical loads have influence on matrix turnover by nucleus pulposus cells. J Orthop Res 30:112–121.  https://doi.org/10.1002/jor.21481 Google Scholar
  21. 21.
    Neidlinger-Wilke C, Wilke HJ, Claes L (1994) Cyclic stretching of human osteoblasts affects proliferation and metabolism: a new experimental method and its application. J Orthop Res 12:70–78.  https://doi.org/10.1002/jor.1100120109 Google Scholar
  22. 22.
    Stokes IA (1987) Surface strain on human intervertebral discs. J Orthop Res 5:348–355.  https://doi.org/10.1002/jor.1100050306 Google Scholar
  23. 23.
    Gregory DE, Bae WC, Sah RL, Masuda K (2012) Anular delamination strength of human lumbar intervertebral disc. Eur Spine J 21:1716–1723.  https://doi.org/10.1007/s00586-012-2308-x Google Scholar
  24. 24.
    Shen B, Melrose J, Ghosh P, Taylor F (2003) Induction of matrix metalloproteinase-2 and -3 activity in ovine nucleus pulposus cells grown in three-dimensional agarose gel culture by interleukin-1beta: a potential pathway of disc degeneration. Eur Spine J 12:66–75.  https://doi.org/10.1007/s00586-002-0454-2 Google Scholar
  25. 25.
    Cho H, Lee S, Park SH, Huang J, Hasty KA, Kim SJ (2013) Synergistic effect of combined growth factors in porcine intervertebral disc degeneration. Connect Tissue Res 54:181–186.  https://doi.org/10.3109/03008207.2013.775258 Google Scholar
  26. 26.
    Sakai D, Schol J (2017) Cell therapy for intervertebral disc repair: clinical perspective. J Orthop Translat 9:8–18.  https://doi.org/10.1016/j.jot.2017.02.002 Google Scholar
  27. 27.
    Vadalà G, Russo F, Di Martino A, Denaro V (2015) Intervertebral disc regeneration: from the degenerative cascade to molecular therapy and tissue engineering. J Tissue Eng Regen Med 9:679–690.  https://doi.org/10.1002/term.1719 Google Scholar
  28. 28.
    Masuda K (2008) Biological repair of the degenerated intervertebral disc by the injection of growth factors. Eur Spine J 17(Suppl 4):441–451.  https://doi.org/10.1007/s00586-008-0749-z Google Scholar
  29. 29.
    Wuertz K, Urban JP, Klasen J, Ignatius A, Wilke HJ, Claes L, Neidlinger-Wilke C (2007) Influence of extracellular osmolarity and mechanical stimulation on gene expression of intervertebral disc cells. J Orthop Res 25:1513–1522.  https://doi.org/10.1002/jor.20436 Google Scholar
  30. 30.
    Alini M, Eisenstein SM, Ito K, Little C, Kettler AA, Masuda K, Melrose J, Ralphs J, Stokes I, Wilke HJ (2008) Are animal models useful for studying human disc disorders/degeneration? Eur Spine J 17:2–19.  https://doi.org/10.1007/s00586-007-0414-y Google Scholar
  31. 31.
    Horner HA, Roberts S, Bielby RC, Menage J, Evans H, Urban JP (2002) Cells from different regions of the intervertebral disc: effect of culture system on matrix expression and cell phenotype. Spine 27:1018–1028.  https://doi.org/10.1097/00007632-200205150-00004 Google Scholar
  32. 32.
    Sowa G, Agarwal S (2008) Cyclic tensile stress exerts a protective effect on intervertebral disc cells. Am J Phys Med Rehabil 87:537–544.  https://doi.org/10.1097/PHM.0b013e31816197ee Google Scholar
  33. 33.
    Sowa G, Coelho P, Vo N, Bedison R, Chiao A, Davies C, Studer R, Kang J (2011) Determination of annulus fibrosus cell response to tensile strain as a function of duration, magnitude, and frequency. J Orthop Res 29:1275–1283.  https://doi.org/10.1002/jor.21388 Google Scholar
  34. 34.
    Gilbert HT, Hoyland JA, Freemont AJ, Millward-Sadler SJ (2011) The involvement of interleukin-1 and interleukin-4 in the response of human annulus fibrosus cells to cyclic tensile strain: an altered mechanotransduction pathway with degeneration. Arthritis Res Ther 13:R8.  https://doi.org/10.1186/ar3229 Google Scholar
  35. 35.
    Miyamoto H, Doita M, Nishida K, Yamamoto T, Sumi M, Kurosaka M (2006) Effects of cyclic mechanical stress on the production of inflammatory agents by nucleus pulposus and anulus fibrosus derived cells in vitro. Spine 31:4–9.  https://doi.org/10.1097/01.brs.0000192682.87267.2a Google Scholar
  36. 36.
    Mastbergen SC, Jansen NW, Bijlsma JW, Lafeber FP (2006) Differential direct effects of cyclo-oxygenase-1/2 inhibition on proteoglycan turnover of human osteoarthritic cartilage: an in vitro study. Arthritis Res Ther 8:R2.  https://doi.org/10.1186/ar1846 Google Scholar
  37. 37.
    Chan MM, Moore AR (2010) Resolution of inflammation in murine autoimmune arthritis is disrupted by cyclooxygenase-2 inhibition and restored by prostaglandin E2-mediated lipoxin A4 production. J Immunol 184:6418–6426.  https://doi.org/10.4049/jimmunol.0903816 Google Scholar
  38. 38.
    Lang G, Liu Y, Geries J, Zhou Z, Kubosch D, Sudkamp N, Richards RG, Alini M, Grad S, Li Z (2018) An intervertebral disc whole organ culture system to investigate proinflammatory and degenerative disc disease condition. J Tissue Eng Regen Med 12:e2051–e2061.  https://doi.org/10.1002/term.2636 Google Scholar
  39. 39.
    Gregory DE, Bae WC, Sah RL, Masuda K (2014) Disc degeneration reduces the delamination strength of the annulus fibrosus in the rabbit annular disc puncture model. Spine J 14:1265–1271.  https://doi.org/10.1016/j.spinee.2013.07.489 Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Institute of Orthopaedic Research and Biomechanics, Trauma Research CentreUlm UniversityUlmGermany
  2. 2.Instituto de Investigação e Inovação em Saúde (i3S)Universidade do PortoPortoPortugal
  3. 3.Instituto de Engenharia Biomédica (INEB)Universidade do PortoPortoPortugal
  4. 4.Instituto de Ciências Biomédicas Abel Salazar (ICBAS)Universidade do PortoPortoPortugal

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