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The evolutionary importance of cell ratio between notochordal and nucleus pulposus cells: an experimental 3-D co-culture study

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Abstract

Introduction

Notochordal cells and nucleus pulposus cells are co-existing in the intervertebral disc at various ratios among different mammalians. This fact rises the question about the interactions and the evolutionary relevance of this phenomenon. It has been described that these relatively large notochordal cells are mainly dominant in early lifetime of all vertebrates and then differences occur with ageing. Human, cattle, sheep, and goat lose the cells with age, whereas rodents and lagomorphs maintain these throughout their lifetime.

Materials and methods

Here, we addressed the importance of cell ratio using alginate bead 3-D co-culture of bovine nucleus pulposus cells (bNPC) and porcine notochordal cells (pNCs) for 14 days using culture inserts.

Result

We found a significant stimulation of bNPC in the presence of pNC in terms of cell activity and glycosaminoglycan production, but not for proliferation (DNA content). Relative gene expression was significantly stimulated for collagen type 2 and aggrecan.

Conclusion

The stimulating effect of NC was confirmed and the ideal ratio of NPC: NC was found to be ~50:50. This has direct implications for tissue-engineering approaches, which aim to repopulate discs with NP-like precursor cells.

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References

  1. Aguiar DJ, Johnson SL, Oegema TR (1999) Notochordal cells interact with nucleus pulposus cells: regulation of proteoglycan synthesis. Exp Cell Res 246(1):129–137. doi:10.1006/excr.1998.4287

    Google Scholar 

  2. Ahmed SA, Gogal RM, Walsh JE (1994) A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [3H] thymidine incorporation assay. J Immunol Methods 170(2):211–224

    Article  PubMed  CAS  Google Scholar 

  3. Boyd LM, Chen J, Kraus VB, Setton LA (2004) Conditioned medium differentially regulates matrix protein gene expression in cells of the intervertebral disc. Spine 29(20):2217–2222

    Article  PubMed  Google Scholar 

  4. Butler WF (1989) Comparative anatomy and development of the mammalian disc. In: Gosh P (ed) The biology of the intervertebral disc. CRC Press, Boca Raton, pp 84–108

    Google Scholar 

  5. Chen J, Yan W, Setton LA (2006) Molecular phenotypes of notochordal cells purified from immature nucleus pulposus. Eur Spine J 15(Suppl 3):S303–S311. doi:10.1007/s00586-006-0088-x

    Article  PubMed  Google Scholar 

  6. Doskocil M, Valouch P, Pazderka V (1993) On vertebral body growth. Funct Dev Morphol 3(3):149–155

    PubMed  CAS  Google Scholar 

  7. Enobakhare BO, Bader DL, Lee DA (1996) Quantification of sulfated glycosaminoglycans in chondrocyte/alginate cultures, by use of 1,9-dimethylmethylene blue. Anal Biochem 243(1):189–191. doi:10.1006/abio.1996.0502

    Article  PubMed  CAS  Google Scholar 

  8. Erwin WM (2010) The enigma that is the nucleus pulposus cell: the search goes on. Arthritis Res Ther 12(3):118. doi:10.1186/ar3001

    Article  PubMed  Google Scholar 

  9. Erwin WM, Ashman K, O’Donnel P, Inman RD (2006) Nucleus pulposus notochord cells secrete connective tissue growth factor and up-regulate proteoglycan expression by intervertebral disc chondrocytes. Arthritis Rheum 54(12):3859–3867. doi:10.1002/art.22258

    Article  PubMed  CAS  Google Scholar 

  10. Erwin WM, Las Heras F, Islam D, Fehlings MG, Inman RD (2009) The regenerative capacity of the notochordal cell: tissue constructs generated in vitro under hypoxic conditions. J Neurosurg Spine 10(6):513–521. doi:10.3171/2009.2.SPINE08578

    Article  PubMed  Google Scholar 

  11. Farndale RW, Buttle DJ, Barrett AJ (1986) Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue 1. Biochim Biophys Acta 883(2):173–177

    Article  PubMed  CAS  Google Scholar 

  12. Faul F, Erdfelder E, Lang AG, Buchner A (2007) G*Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav Res Methods 39(2):175–191

    Article  PubMed  Google Scholar 

  13. Gilson A, Dreger M, Urban JP (2010) Differential expression levels of cytokeratin 8 in cells of the bovine nucleus pulposus complicates the search for specific intervertebral disc cell markers. Arthritis Res Ther 12(1):R24. doi:10.1186/ar2931

    Article  PubMed  Google Scholar 

  14. Guehring T, Nerlich A, Kroeber M, Richter W, Omlor GW (2010) Sensitivity of notochordal disc cells to mechanical loading: an experimental animal study. Eur Spine J 19(1):113–121. doi:10.1007/s00586-009-1217-0

    Article  PubMed  Google Scholar 

  15. Guehring T, Urban JP, Cui Z, Tirlapur UK (2008) Noninvasive 3D vital imaging and characterization of notochordal cells of the intervertebral disc by femtosecond near-infrared two-photon laser scanning microscopy and spatial-volume rendering. Microsc Res Tech 71(4):298–304. doi:10.1002/jemt.20557

    Article  PubMed  Google Scholar 

  16. Guehring T, Wilde G, Sumner M, Grünhagen T, Karney GB, Tirlapur UK, Urban JP (2009) Notochordal intervertebral disc cells: sensitivity to nutrient deprivation. Arthritis Rheum 60(4):1026–1034. doi:10.1002/art.24407

    Article  PubMed  Google Scholar 

  17. Horwitz T (1977) The human notochord: a study of its development and regression, variations, and pathologic derivative, chordoma. Horwitz: Indianapolis

  18. Hunter CJ, Bianchi S, Cheng P, Muldrew K (2007) Osmoregulatory function of large vacuoles found in notochordal cells of the intervertebral disc running title: an osmoregulatory vacuole. Mol Cell Biomech 4(4):227–237

    PubMed  Google Scholar 

  19. Hunter CJ, Matyas JR, Duncan NA (2003) The notochordal cell in the nucleus pulposus: a review in the context of tissue engineering. Tissue Eng 9(4):667–677. doi:10.1089/107632703768247368

    Article  PubMed  CAS  Google Scholar 

  20. Hunter CJ, Matyas JR, Duncan NA (2004) Cytomorphology of notochordal and chondrocytic cells from the nucleus pulposus: a species comparison. J Anat 205(5):357–362. doi:10.1111/j.0021-8782.2004.00352.x

    Article  PubMed  Google Scholar 

  21. Kim KW, Ha KY, Lee JS, Nam SW, Woo YK, Lim TH, An HS (2009) Notochordal cells stimulate migration of cartilage end plate chondrocytes of the intervertebral disc in in vitro cell migration assays. Spine J 9(4):323–329. doi:10.1016/j.spinee.2008.05.003

    Article  PubMed  Google Scholar 

  22. Korecki CL, Taboas JM, Tuan RS, Iatridis JC (2010) Notochordal cell conditioned medium stimulates mesenchymal stem cell differentiation toward a young nucleus pulposus phenotype. Stem Cell Res Ther 1(2):18. doi:10.1186/scrt18

    Article  PubMed  Google Scholar 

  23. Lee CR, Grad S, Maclean JJ, Iatridis JC, Alini M (2005) Effect of mechanical loading on mRNA levels of common endogenous controls in articular chondrocytes and intervertebral disk. Anal Biochem 341(2):372–375. doi:10.1016/j.ab.2004.10.005

    Article  PubMed  CAS  Google Scholar 

  24. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25(4):402–408. doi:10.1006/meth.2001.1262

    Article  PubMed  CAS  Google Scholar 

  25. Maldonado BA, Oegema TR (1992) Initial characterization of the metabolism of intervertebral disc cells encapsulated in microspheres. J Orthop Res 10(5):677–690

    Article  PubMed  CAS  Google Scholar 

  26. Marino JH, Cook P, Miller KS (2003) Accurate and statistically verified quantification of relative mRNA abundances using SYBR Green I and real-time RT-PCR. J Immunol Methods 283(1–2):291–306

    Article  PubMed  CAS  Google Scholar 

  27. Minogue BM, Richardson SM, Zeef LA, Freemont AJ, Hoyland JA (2010) Transcriptional profiling of bovine intervertebral disc cells: implications for identification of normal and degenerate human intervertebral disc cell phenotypes. Arthritis Res Ther 12(1):R22. doi:10.1186/ar2929

    Article  PubMed  Google Scholar 

  28. Miyazaki T, Kobayashi S, Takeno K, Meir A, Urban J, Baba H (2009) A phenotypic comparison of proteoglycan production of intervertebral disc cells isolated from rats, rabbits, and bovine tails; which animal model is most suitable to study tissue engineering and biological repair of human disc disorders? Tissue Eng Part A 15(12):3835–3846. doi:10.1089/ten.tea.2009.0250

    Article  PubMed  CAS  Google Scholar 

  29. Oguz E, Tsai TT, Di Martino A, Guttapalli A, Albert TJ, Shapiro IM, Risbud MV (2007) Galectin-3 expression in the intervertebral disc: a useful marker of the notochord phenotype? Spine 32(1):9–16. doi:10.1097/01.brs.0000250302.74574.98

    Article  PubMed  Google Scholar 

  30. Risbud MV, Schaer TP, Shapiro IM (2010) Toward an understanding of the role of notochordal cells in the adult intervertebral disc: from discord to accord. Dev Dyn 239:2141–2148. doi:10.1002/dvdy.22350

    Article  PubMed  CAS  Google Scholar 

  31. Rufai A, Benjamin M, Ralphs JR (1995) The development of fibrocartilage in the rat intervertebral disc. Anat Embryol (Berl) 192(1):53–62

    Article  CAS  Google Scholar 

  32. Sakai D, Nakai T, Mochida J, Alini M, Grad S (2009) Differential phenotype of intervertebral disc cells: microarray and immunohistochemical analysis of canine nucleus pulposus and anulus fibrosus. Spine 34(14):1448–1456. doi:10.1097/BRS.0b013e3181a55705

    Article  PubMed  Google Scholar 

  33. Sarkar D, Shields B, Davies ML, Müller J, Wakeman JA (2011) BRACHYURY confers cancer stem cell characteristics on colorectal cancer cells. Int J Cancer. doi:10.1002/ijc.26029

  34. Schmittgen TD, Zakrajsek BA (2000) Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR. J Biochem Biophys Methods 46(1–2):69–81

    Article  PubMed  CAS  Google Scholar 

  35. Shapiro IM, Risbud MV (2010) Transcriptional profiling of the nucleus pulposus: say yes to notochord. Arthritis Res Ther 12(3):117. doi:10.1186/ar3003

    Article  PubMed  Google Scholar 

  36. Vujovic S, Henderson S, Presneau N, Odell E, Jacques TS, Tirabosco R, Boshoff C, Flanagan AM (2006) Brachyury, a crucial regulator of notochordal development, is a novel biomarker for chordomas. J Pathol 209(2):157–165. doi:10.1002/path.1969

    Article  PubMed  CAS  Google Scholar 

  37. Walmsley R (1953) The development and growth of the intervertebral disc. Edinburgh Med J 60:341–365

    Google Scholar 

  38. Weiler C, Nerlich AG, Schaaf R, Bachmeier BE, Wuertz K, Boos N (2010) Immunohistochemical identification of notochordal markers in cells in the aging human lumbar intervertebral disc. Eur Spine J. doi:10.1007/s00586-010-1392-z

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Acknowledgments

This work was supported by the Swiss National Science Foundation (SNF #310030-127586/1) and the Department for Orthopaedic Surgery, Insel University Hospital, Bern.

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Authors and Affiliations

  1. ARTORG Center for Biomedical Engineering Research, Institute for Surgical Technology and Biomechanics, Medical Faculty, University of Bern, Stauffacherstrasse 78, 3014, Bern, Switzerland

    Benjamin Gantenbein-Ritter & Samantha C. W. Chan

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  1. Benjamin Gantenbein-Ritter
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  2. Samantha C. W. Chan
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Correspondence to Benjamin Gantenbein-Ritter.

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Gantenbein-Ritter, B., Chan, S.C.W. The evolutionary importance of cell ratio between notochordal and nucleus pulposus cells: an experimental 3-D co-culture study. Eur Spine J 21 (Suppl 6), 819–825 (2012). https://doi.org/10.1007/s00586-011-2026-9

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  • DOI: https://doi.org/10.1007/s00586-011-2026-9

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