European Spine Journal

, Volume 16, Issue 12, pp 2193–2205 | Cite as

Biglycan and fibromodulin fragmentation correlates with temporal and spatial annular remodelling in experimentally injured ovine intervertebral discs

  • James Melrose
  • Susan M. Smith
  • Emily S. Fuller
  • Allan A. Young
  • Peter J. Roughley
  • Andrew Dart
  • Christopher B. Little
Original Article

Abstract

This study evaluated spatial and temporal extracellular matrix changes, induced by controlled surgical defects in the outer third of the annulus fibrosus (AF) of ovine intervertebral discs (IVDs). Thirty-two 4 year old sheep received a 4 mm deep × 10 mm wide standard annular surgical incision in the L1L2 and L3L4 IVDs (lesion group), 32 sheep were also subjected to the same surgical approach but the AF was not incised (sham-operated controls). Remodeling of the IVD matrix in the lesion and sham discs was assessed histochemically at 3, 6,12 and 26 month post operation (PO). Discs were also dissected into annular lesion site and contra-lateral AF and NP and equivalent zones in the sham sheep group, extracted with GuHCl, dialysed, freeze dried, digested with chondroitinase ABC/keratanase-I and aliquots examined for small leucine repeat proteoglycan (SLRP) core protein species by Western blotting using C-terminal antibodies to decorin, biglycan, lumican and fibromodulin and monoclonal antibody (Mab) 2B6 to unsaturated stub epitopes on chondroitin-4-sulphate generated by chondroitinase ABC. Masson Trichrome and Picrosirius red staining demonstrated re-organisation of the outermost collagenous lamellae in the incised discs 3–6 month PO. Toluidine blue staining also demonstrated a focal loss of anionic proteoglycan (PG) from the annular lesion 3–6 month PO with partial recovery of PG levels by 26 month. Specific fragments of biglycan and fibromodulin were associated with remodeling of the AF 12–26 month PO in the lesion IVDs but were absent from the NP of the lesion discs or all tissue zones in the sham animal group. Fragments of decorin were also observed in lesion zone extracts from 3 to 6 months but diminished after this. Isolation and characterization of the biglycan/fibromodulin fragments may identify them as prospective biomarkers of annular remodeling and characterization of the enzyme systems responsible for their generation may identify therapeutic target molecules.

Keywords

IVD Annular remodelling Experimental disc degeneration SLRP fragmentation 

Notes

Acknowledgments

Funding for this project was provided by the National Health and Medical Research Council of Australia (Project grants 211266 and 352562) whose support is gratefully acknowledged.

References

  1. 1.
    Ameye L, Young MF (2002) Mice deficient in small leucine-rich proteoglycans: novel in vivo models for osteoporosis, osteoarthritis, Ehlers-Danlos syndrome, muscular dystrophy, and corneal diseases. Glycobiology 12(9):107R–116RPubMedCrossRefGoogle Scholar
  2. 2.
    Ameye LG, Young MF (2006) Animal models of osteoarthritis: lessons learned while seeking the “Holy Grail”. Curr Opin Rheumatol 18(5):537–547PubMedCrossRefGoogle Scholar
  3. 3.
    Bancroft JD, Cook HC (eds) (1994) Manual of histological techniques and their diagnostic application, Chapt. 3. Churchill-Livingstone, London, p 42Google Scholar
  4. 4.
    Boos N, Weissbach S, Rohrbach H, Weiler C, Spratt KF, Nerlich AG (2002) Classification of age-related changes in lumbar intervertebral discs: 2002 Volvo award in basic science. Spine 27(23):2631–2644PubMedCrossRefGoogle Scholar
  5. 5.
    Buckwalter JA (1995) Aging and degeneration of the human intervertebral disc. Spine 20(11):1307–1314PubMedGoogle Scholar
  6. 6.
    Caterson B, Christner JE, Baker JR, Couchman JR (1985) Production and characterization of monoclonal antibodies directed against connective tissue proteoglycans. Fed Proc 44(2):386–393PubMedGoogle Scholar
  7. 7.
    Chakravarti S (2002) Functions of lumican and fibromodulin: lessons from knockout mice. Glycoconj J 19(4–5):287–293PubMedCrossRefGoogle Scholar
  8. 8.
    Christner JE, Caterson B, Baker JR (1980) Immunological determinants of proteoglycans. Antibodies against the unsaturated oligosaccharide products of chondroitinase ABC-digested cartilage proteoglycans. J Biol Chem 255(15):7102–7105PubMedGoogle Scholar
  9. 9.
    Cs-Szabo G, Ragasa-San Juan D, Turumella V, Masuda K, Thonar EJ, An HS (2002) Changes in mRNA and protein levels of proteoglycans of the anulus fibrosus and nucleus pulposus during intervertebral disc degeneration. Spine 27(20):2212–2219PubMedCrossRefGoogle Scholar
  10. 10.
    Flannery CR (2006) Usurped SLRPs: novel arthritis biomarkers exposed by catabolism of small leucine-rich proteoglycans? Arthritis Res Ther 8(2):106PubMedCrossRefGoogle Scholar
  11. 11.
    Geng Y, McQuillan D, Roughley PJ (2006) SLRP interaction can protect collagen fibrils from cleavage by collagenases. Matrix Biol 25(8):484–491PubMedCrossRefGoogle Scholar
  12. 12.
    Getzy LL, Malemud CJ, Goldberg VM, Moskowitz RW (1982) Factors influencing metachromatic staining in paraffin embedded sections of rabbit and human articular cartilage: a comparison of the Safranin O and toluidine blue techniques. J Histotechnol 5:111–116Google Scholar
  13. 13.
    Guner A, Oktay G, Kerman M, Guner G (1995) Immunoglobulins and alpha-1-proteinase inhibitor in human intervertebral disc material. Biochem Soc Trans 23(2):212SPubMedGoogle Scholar
  14. 14.
    Habtemariam A, Gronblad M, Virri J, Seitsalo S, Ruuskanen M, Karaharju E (1996) Immunocytochemical localization of immunoglobulins in disc herniations. Spine 21(16):1864–1869PubMedCrossRefGoogle Scholar
  15. 15.
    Haefeli M, Kalberer F, Saegesser D, Nerlich AG, Boos N, Paesold G (2006) The course of macroscopic degeneration in the human lumbar intervertebral disc. Spine 31(14):1522–1531PubMedCrossRefGoogle Scholar
  16. 16.
    Hatano E, Fujita T, Ueda Y, Okuda T, Katsuda S, Okada Y, Matsumoto T (2006) Expression of ADAMTS-4 (aggrecanase-1) and possible involvement in regression of lumbar disc herniation. Spine 31(13):1426–1432PubMedCrossRefGoogle Scholar
  17. 17.
    Hausser H, Groning A, Hasilik A, Schonherr E, Kresse H (1994) Selective inactivity of TGF-beta/decorin complexes. FEBS Lett 353(3):243–245PubMedCrossRefGoogle Scholar
  18. 18.
    Heathfield TF, Onnerfjord P, Dahlberg L, Heinegard D (2004) Cleavage of fibromodulin in cartilage explants involves removal of the N-terminal tyrosine sulfate-rich region by proteolysis at a site that is sensitive to matrix metalloproteinase-13. J Biol Chem 279(8):6286–6295PubMedCrossRefGoogle Scholar
  19. 19.
    Hedbom E, Heinegard D (1993) Binding of fibromodulin and decorin to separate sites on fibrillar collagens. J Biol Chem 268(36):27307–27312PubMedGoogle Scholar
  20. 20.
    Hildebrand A, Romaris M, Rasmussen LM, Heinegard D, Twardzik DR, Border WA, Ruoslahti E (1994) Interaction of the small interstitial proteoglycans biglycan, decorin and fibromodulin with transforming growth factor beta. Biochem J 302(Pt 2):527–534PubMedGoogle Scholar
  21. 21.
    Hilton RC, Ball J (1984) Vertebral rim lesions in the dorsolumbar spine. Ann Rheum Dis 43(2):302–307PubMedCrossRefGoogle Scholar
  22. 22.
    Iozzo RV (1999) The biology of the small leucine-rich proteoglycans. Functional network of interactive proteins. J Biol Chem 274(27):18843–18846PubMedCrossRefGoogle Scholar
  23. 23.
    Johnstone B, Markopoulos M, Neame P, Caterson B (1993) Identification and characterization of glycanated and non-glycanated forms of biglycan and decorin in the human intervertebral disc. Biochem J 292(Pt 3):661–666PubMedGoogle Scholar
  24. 24.
    Junqueira LC, Bignolas G, Brentani RR (1979) Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections. Histochem J 11(4):447–455PubMedCrossRefGoogle Scholar
  25. 25.
    Kaapa E, Holm S, Inkinen R, Lammi MJ, Tammi M, Vanharanta H (1994) Proteoglycan chemistry in experimentally injured porcine intervertebral disk. J Spinal Disord 7(4):296–306PubMedGoogle Scholar
  26. 26.
    Kaapa E, Han X, Holm S, Peltonen J, Takala T, Vanharanta H (1995) Collagen synthesis and types I, III, IV, and VI collagens in an animal model of disc degeneration. Spine 20(1):59–66; discussion 66–67PubMedCrossRefGoogle Scholar
  27. 27.
    Katz JN (2006) Lumbar disc disorders and low-back pain: socioeconomic factors and consequences. J Bone Joint Surg Am 88(Suppl 2):21–24PubMedCrossRefGoogle Scholar
  28. 28.
    Lipson SJ, Muir H (1981) 1980 Volvo award in basic science. Proteoglycans in experimental intervertebral disc degeneration. Spine 6(3):194–210PubMedCrossRefGoogle Scholar
  29. 29.
    Lotz JC (2004) Animal models of intervertebral disc degeneration: lessons learned. Spine 29(23):2742–2750PubMedCrossRefGoogle Scholar
  30. 30.
    Masson P (1929) Some histological methods. Trichrome stainings and their preliminary technique. Bulletin of The International Association of Medicine. J Tech Methods 12:75Google Scholar
  31. 31.
    Masuda K, Aota Y, Muehleman C, Imai Y, Okuma M, Thonar EJ, Andersson GB, An HS (2005) A novel rabbit model of mild, reproducible disc degeneration by an anulus needle puncture: correlation between the degree of disc injury and radiological and histological appearances of disc degeneration. Spine 30(1):5–14PubMedGoogle Scholar
  32. 32.
    Melching LI, Fisher WD, Lee ER, Mort JS, Roughley PJ (2006) The cleavage of biglycan by aggrecanases. Osteoarthr Cartil 14(11):1147–1154PubMedCrossRefGoogle Scholar
  33. 33.
    Melrose J, Ghosh P, Taylor TK, Hall A, Osti OL, Vernon-Roberts B, Fraser RD (1992) A longitudinal study of the matrix changes induced in the intervertebral disc by surgical damage to the annulus fibrosus. J Orthop Res 10(5):665–676PubMedCrossRefGoogle Scholar
  34. 34.
    Melrose J, Ghosh P, Taylor TK, Latham J, Moore R (1997) Topographical variation in the catabolism of aggrecan in an ovine annular lesion model of experimental disc degeneration. J Spinal Disord 10(1):55–67PubMedCrossRefGoogle Scholar
  35. 35.
    Melrose J, Ghosh P, Taylor TK, Vernon-Roberts B, Latham J, Moore R (1997) Elevated synthesis of biglycan and decorin in an ovine annular lesion model of experimental disc degeneration. Eur Spine J 6(6):376–384PubMedCrossRefGoogle Scholar
  36. 36.
    Melrose J, Roberts S, Smith S, Menage J, Ghosh P (2002) Increased nerve and blood vessel ingrowth associated with proteoglycan depletion in an ovine anular lesion model of experimental disc degeneration. Spine 27(12):1278–1285PubMedCrossRefGoogle Scholar
  37. 37.
    Melrose J, Smith S, Little CB, Kitson J, Hwa SY, Ghosh P (2002) Spatial and temporal localization of transforming growth factor-beta, fibroblast growth factor-2, and osteonectin, and identification of cells expressing alpha-smooth muscle actin in the injured anulus fibrosus: implications for extracellular matrix repair. Spine 27(16):1756–1764PubMedCrossRefGoogle Scholar
  38. 38.
    Monfort J, Tardif G, Reboul P, Mineau F, Roughley P, Pelletier JP, Martel-Pelletier J (2006) Degradation of small leucine-rich repeat proteoglycans by matrix metalloprotease-13: identification of a new biglycan cleavage site. Arthritis Res Ther 8(1):R26PubMedCrossRefGoogle Scholar
  39. 39.
    Moore RJ, Osti OL, Vernon-Roberts B, Fraser RD (1992) Changes in endplate vascularity after an outer anulus tear in the sheep. Spine 17(8):874–878PubMedCrossRefGoogle Scholar
  40. 40.
    Moore RJ, Vernon-Roberts B, Osti OL, Fraser RD (1996) Remodeling of vertebral bone after outer anular injury in sheep. Spine 21(8):936–940PubMedCrossRefGoogle Scholar
  41. 41.
    Moore RJ, Crotti TN, Osti OL, Fraser RD, Vernon-Roberts B (1999) Osteoarthrosis of the facet joints resulting from anular rim lesions in sheep lumbar discs. Spine 24(6):519–525PubMedCrossRefGoogle Scholar
  42. 42.
    Nerlich AG, Schleicher ED, Boos N (1997) 1997 Volvo award winner in basic science studies. Immunohistologic markers for age-related changes of human lumbar intervertebral discs. Spine 22(24):2781–2795PubMedCrossRefGoogle Scholar
  43. 43.
    Osti OL, Vernon-Roberts B, Fraser RD (1990) 1990 Volvo award in experimental studies. Anulus tears and intervertebral disc degeneration. An experimental study using an animal model. Spine 15(8):762–767PubMedCrossRefGoogle Scholar
  44. 44.
    Pennington JB, McCarron RF, Laros GS (1988) Identification of IgG in the canine intervertebral disc. Spine 13(8):909–912PubMedCrossRefGoogle Scholar
  45. 45.
    Roberts S, Caterson B, Menage J, Evans EH, Jaffray DC, Eisenstein SM (2000) Matrix metalloproteinases and aggrecanase: their role in disorders of the human intervertebral disc. Spine 25(23):3005–3013PubMedCrossRefGoogle Scholar
  46. 46.
    Rousseau MA, Ulrich JA, Bass EC, Rodriguez AG, Liu JJ, Lotz JC (2007) Stab incision for inducing intervertebral disc degeneration in the rat. Spine 32(1):17–24PubMedCrossRefGoogle Scholar
  47. 47.
    Scott JE (2003) Elasticity in extracellular matrix ‘shape modules’ of tendon, cartilage, etc. A sliding proteoglycan-filament model. J Physiol 553(Pt 2):335–343PubMedCrossRefGoogle Scholar
  48. 48.
    Scott JE, Stockwell RA (2006) Cartilage elasticity resides in shape module decoran and aggrecan sumps of damping fluid: implications in osteoarthrosis. J Physiol 574(Pt 3):643–650PubMedCrossRefGoogle Scholar
  49. 49.
    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(1):66–75PubMedGoogle Scholar
  50. 50.
    Smit TH (2002) The use of a quadruped as an in vivo model for the study of the spine—biomechanical considerations. Eur Spine J 11(2):137–144PubMedCrossRefGoogle Scholar
  51. 51.
    Smith JW, Walmsley R (1951) Experimental incision of the intervertebral disc. J Bone Joint Surg Br 33-B(4):612–625PubMedGoogle Scholar
  52. 52.
    Sobajima S, Kompel JF, Kim JS, Wallach CJ, Robertson DD, Vogt MT, Kang JD, Gilbertson LG (2005) A slowly progressive and reproducible animal model of intervertebral disc degeneration characterized by MRI, X-ray, and histology. Spine 30(1):15–24PubMedGoogle Scholar
  53. 53.
    Alini M, Eisenstein S, Ito K, Little C, Kettler AA, Masuda K, Melrose J, Ralphs J, Stokes I, Wilke HJ (2007) Are animal models useful for studying human disc disorders/degeneration? Eur Spine J, 14 Jul 2007; [Epub ahead of print]Google Scholar
  54. 54.
    Svensson L, Aszodi A, Reinholt FP, Fassler R, Heinegard D, Oldberg A (1999) Fibromodulin-null mice have abnormal collagen fibrils, tissue organization, and altered lumican deposition in tendon. J Biol Chem 274(14):9636–9647PubMedCrossRefGoogle Scholar
  55. 55.
    Svensson L, Narlid I, Oldberg A (2000) Fibromodulin and lumican bind to the same region on collagen type I fibrils. FEBS Lett 470(2):178–182PubMedCrossRefGoogle Scholar
  56. 56.
    Sweat F, Puchtler H, Rosenthal SI (1964) Sirius Red F3ba as a stain for connective tissue. Arch Pathol 78:69–72PubMedGoogle Scholar
  57. 57.
    Sztrolovics R, Alini M, Roughley PJ, Mort JS (1997) Aggrecan degradation in human intervertebral disc and articular cartilage. Biochem J 326(Pt 1):235–241PubMedGoogle Scholar
  58. 58.
    Sztrolovics R, Alini M, Mort JS, Roughley PJ (1999) Age-related changes in fibromodulin and lumican in human intervertebral discs. Spine 24(17):1765–1771PubMedCrossRefGoogle Scholar
  59. 59.
    Thompson JP, Oegema TR Jr, Bradford DS (1991) Stimulation of mature canine intervertebral disc by growth factors. Spine 16(3):253–260PubMedCrossRefGoogle Scholar
  60. 60.
    Vernon-Roberts B (1988) Pathology of the intervertebral disc. In: Ghosh P (ed) Biology of the intervertebral disc, Vol II. CRC, Boca Raton, pp 73–120Google Scholar
  61. 61.
    Vogel KG, Paulsson M, Heinegard D (1984) Specific inhibition of type I and type II collagen fibrillogenesis by the small proteoglycan of tendon. Biochem J 223(3):587–597PubMedGoogle Scholar
  62. 62.
    Wiberg C, Klatt AR, Wagener R, Paulsson M, Bateman JF, Heinegard D, Morgelin M (2003) Complexes of matrilin-1 and biglycan or decorin connect collagen VI microfibrils to both collagen II and aggrecan. J Biol Chem 278(39):37698–37704PubMedCrossRefGoogle Scholar
  63. 63.
    Wilke HJ, Kettler A, Claes LE (1997) Are sheep spines a valid biomechanical model for human spines? Spine 22(20):2365–2374PubMedCrossRefGoogle Scholar
  64. 64.
    Wilke HJ, Kettler A, Wenger KH, Claes LE (1997) Anatomy of the sheep spine and its comparison to the human spine. Anat Rec 247(4):542–555PubMedCrossRefGoogle Scholar
  65. 65.
    Young AA, Smith MM, Smith SM, Cake MA, Ghosh P, Read RA, Melrose J, Sonnabend DH, Roughley PJ, Little CB (2005) Regional assessment of articular cartilage gene expression and small proteoglycan metabolism in an animal model of osteoarthritis. Arthritis Res Ther 7(4):R852–R861PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • James Melrose
    • 1
  • Susan M. Smith
    • 1
  • Emily S. Fuller
    • 1
  • Allan A. Young
    • 1
  • Peter J. Roughley
    • 2
  • Andrew Dart
    • 1
    • 3
  • Christopher B. Little
    • 1
  1. 1.Raymond Purves Laboratory, Institute of Bone and Joint Research, Kolling Institute of Medical ResearchUniversity of Sydney at Royal North Shore HospitalSt. LeonardsAustralia
  2. 2.Shriners Hospital for Children, Genetics UnitMontrealCanada
  3. 3.Department of Veterinary ScienceUniversity of SydneyCamdenAustralia

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