Advertisement

Histochemistry and Cell Biology

, Volume 138, Issue 3, pp 461–475 | Cite as

Chondroitin sulphate and heparan sulphate sulphation motifs and their proteoglycans are involved in articular cartilage formation during human foetal knee joint development

  • James Melrose
  • Marc D. Isaacs
  • Susan M. Smith
  • Clare E. Hughes
  • Christopher B. Little
  • Bruce Caterson
  • Anthony J. HayesEmail author
Original Paper

Abstract

Novel sulphation motifs within the glycosaminoglycan chain structure of chondroitin sulphate (CS) containing proteoglycans (PGs) are associated with sites of growth, differentiation and repair in many biological systems and there is compelling evidence that they function as molecular recognition sites that are involved in the binding, sequestration or presentation of soluble signalling molecules (e.g. morphogens, growth factors and cytokines). Here, using monoclonal antibodies 3B3(−), 4C3 and 7D4, we examine the distribution of native CS sulphation motifs within the developing connective tissues of the human foetal knee joint, both during and after joint cavitation. We show that the CS motifs have broad, overlapping distributions within the differentiating connective tissues before the joint has fully cavitated; however, after cavitation, they all localise very specifically to the presumptive articular cartilage tissue. Comparisons with the labelling patterns of heparan sulphate (HS), HS-PGs (perlecan, syndecan-4 and glypican-6) and FGF-2, molecules with known signalling roles in development, indicate that these also become localised to the future articular cartilage tissue after joint cavitation. Furthermore, they display interesting, overlapping distributions with the CS motifs, reflective of early tissue zonation. The overlapping expression patterns of these molecules at this site suggests they are involved, or co-participate, in early morphogenetic events underlying articular cartilage formation; thus having potential clinical relevance to mechanisms involved in its repair/regeneration. We propose that these CS sulphation motifs are involved in modulating the signalling gradients responsible for the cellular behaviours (proliferation, differentiation, matrix turnover) that shape the zonal tissue architecture present in mature articular cartilage.

Keywords

Chondroitin sulphate Heparan sulphate Perlecan Syndecan Glypican FGF Articular cartilage 

Notes

Acknowledgments

Thanks to Drs. Mari Nowell (Cardiff School of Medicine), Hannah Shaw and Jim Ralphs (Cardiff School of Biosciences), for help in the anatomy of the foetal human knee. This work was funded with financial support from the Arthritis Research UK (ARUK grant numbers 18331 and 19858).

Supplementary material

418_2012_968_MOESM1_ESM.doc (7.2 mb)
Supplementary material 1 (DOC 7370 kb)

References

  1. Arai T, Parker A, Walker B, Clemmons DR (1994) Heparan, heparan sulphate, and dermatan sulphate regulate formation of the insulin-like growth factor-I and insulin-like growth factor-binding protein complexes. J Biol Chem 269:20388–20393PubMedGoogle Scholar
  2. Archer CW, Morrison H, Pitsillides AA (1994) Cellular aspects of the development of diarthroidal joints and articular cartilage. J Anat 184:447–456PubMedGoogle Scholar
  3. Archer CW, Morrison EA, Bayliss MT, Ferguson MWJ (1996) The development of articular cartilage: II. The spatial and temporal patterns of glycosaminoglycans and small-leucine-rich proteoglycans. J Anat 189:23–35PubMedGoogle Scholar
  4. Archer CW, Dowthwaite GP, Francis-West P (2003) Development of synovial joints. Birth Defects Res (Part C) 69:144–155CrossRefGoogle Scholar
  5. Asimakopoulou AP, Thecharis AD, Tzanakakis GN, Karamanos NK (2008) The biological role of chondroitin sulfate in cancer and chondroitin-based anticancer agents. In Vivo 22:385–389PubMedGoogle Scholar
  6. Bernfield M, Gotte M, Park PW, Reizes O, Fitzgerald ML, Lincecum J, Zako M (1999) Functions of cell surface proteoglycans. Annu Rev Biochem 68:729–777PubMedCrossRefGoogle Scholar
  7. Bishop JR, Schukz M, Esko JD (2007) Heparan sulphate proteoglycans fine-tune mammalian physiology. Nature 446:1030–1037PubMedCrossRefGoogle Scholar
  8. Campos-Xavier AB, Martinet D, Bateman J, Belluoccio D, Rowley L, Tan TY, Baxová A, Borochowitz ZU, Innes AM, Unger S, Beckmann JS, Mittaz L, Ballhausen D, Superti-Furga A, Savarirayan R, Bonafé L (2009) Mutations in the heparan-sulfate proteoglycan glypican 6 (GPC6) impair endochondral ossification and cause recessive omodysplasia. Am J Hum Genet 84:760–770PubMedCrossRefGoogle Scholar
  9. Caterson B, Mahmoodian F, Sorrell JM, Hardingham TE, Mayliss MT, Carney SL, Ratcliffe A, Muir H (1990) Modulation of native chondroitin sulphate structure in tissue development and in disease. J Cell Sci 97:411–417PubMedGoogle Scholar
  10. Chen X, Macica CM, Nasiri A, Broadus AE (2008) Regulation of articular chondrocyte proliferation and differentiation by Indian hedgehog and parathyroid hormone-related protein in mice. Arthritis Rheum 58:3788–3797PubMedCrossRefGoogle Scholar
  11. Cortes M, Baria AT, Schwartz NB (2009) Sulfation of chondroitin sulphate proteoglycans is necessary for proper Indian hedgehog signalling in the developing growth plate. Development 136:1697–1706PubMedCrossRefGoogle Scholar
  12. Couchman JR, Caterson B, Christner JE, Baker JR (1984) Mapping by monoclonal antibody detection of glycosaminoglycans in connective tissues. Nature 307:650–665PubMedCrossRefGoogle Scholar
  13. David G, Bai XM, Van der Schueren B, Cassiman JJ, Van den Berge H (1992) Developmental changes in heparan sulfate expression: in situ detection with mAbs. J Cell Biol 119:961–975PubMedCrossRefGoogle Scholar
  14. Deepa SS, Yamada S, Zako M, Goldberger O, Sugahara K (2004) Chondroitin sulphate chains on syndecan-1 and syndecan-4 from normal murine mammary gland epithelial cells are structurally and functionally distinct and cooperate with heparan sulphate chains to bind growth factors. A novel function to control binding of midkine, pleiotrophin, and basic fibroblast growth factor. J Biol Chem 36:37368–37376CrossRefGoogle Scholar
  15. Dowthwaite GP, Bishop JC, Redman SN, Khan IM, Rooney P, Evans DJR, Haughton L, Bayram Z, Boyer S, Thomson B, Wolfe MS, Archer CW (2004) The surface of articular cartilage contains a progenitor cell population. J Cell Sci 117:889–897PubMedCrossRefGoogle Scholar
  16. Echtermeyer F, Bertrand J, Dreier R, Ingmar M, Neugebauer K, Fuerst M, Lee YJ, Song YW, Herzog C, Theilmeier G, Pap T (2009) Syndecan-4 regulates ADAMTS-5 activation and cartilage breakdown in osteoarthritis. Nat Med 15:1072–1076PubMedCrossRefGoogle Scholar
  17. Fico A, Maina F, Dono R (2011) Fine-tuning of cell signalling by glypicans. Cell Mol Life Sci 68:923–929PubMedCrossRefGoogle Scholar
  18. Fisher MC, Li Y, Seghatoleslami MR, Dealy CN, Kosher RA (2006) Heparan sulphate proteoglycans including syndecan-3 modulate BMP activity during limb cartilage differentiation. Matrix Biol 25:27–39PubMedCrossRefGoogle Scholar
  19. Francis-West PH, Parish J, Lee K, Archer CW (1999) BMP/GDF-signalling interactions during synovial joint development. Cell Tissue Res 296:111–119PubMedCrossRefGoogle Scholar
  20. Gama CI, Tully SE, Sotogaku N, Clark PM, Rawat M, Vaidehi N, Goddard WA, Nishi A, Hsieh-Wilson LC (2006) Sulfation patterns of glycosaminoglycans encode molecular recognition and activity. Nat Chem Biol 2:467–473PubMedCrossRefGoogle Scholar
  21. Gardner E, O’Rahilly R (1968) The early development of the knee joint in staged human embryos. J Anat 102:289–299PubMedGoogle Scholar
  22. Govindraj P, West L, Koob TJ, Neame P, Doege K, Hassell JR (2002) Isolation and identification of the major heparan sulphate proteoglycans in the developing bovine rib growth plate. J Biol Chem 277:19461–19469PubMedCrossRefGoogle Scholar
  23. Govindraj P, West L, Smith S, Hassell JR (2006) Modulation of FGF-2 binding to chondrocytes from the developing growth plate by perlecan. Matrix Biol 25:232–239PubMedCrossRefGoogle Scholar
  24. Guerne PA, Sublet A, Lotz M (1994) Growth factor responsiveness of human articular chondrocytes: distinct profiles in primary chondrocytes, subcultured chondrocytes, and fibroblasts. J Cell Physiol 158:476–484PubMedCrossRefGoogle Scholar
  25. Hardingham T (1994) The sulphation pattern in chondroitin sulphate chains investigated by chondroitinase ABC and ACII digestion and reactivity with monoclonal antibodies. Carbohydrate Res 255:241–254CrossRefGoogle Scholar
  26. Hayes AJ, MacPherson S, Morrison H, Dowthwaite G, Archer CW (2001) The development of articular cartilage: evidence for an appositional growth mechanism. Anat Embryol (Berl) 203:469–479CrossRefGoogle Scholar
  27. Hayes AJ, Dowthwaite GP, Webster SV, Archer CW (2003) The distribution of Notch receptors and their ligands during articular cartilage development. J Anat 202:495–502PubMedCrossRefGoogle Scholar
  28. Hayes AJ, Tudor D, Nowell MA, Caterson B, Hughes CE (2008) Chondroitin sulphate sulfation motifs as putative biomarkers for isolation of articular cartilage progenitor cells. J Histochem Cytochem 56:125–138PubMedCrossRefGoogle Scholar
  29. Hayes AJ, Hughes CE, Ralphs JR, Caterson B (2011) Chondroitin sulphate sulphation motif expression in the ontogeny of the intervertebral disc. Eur Cell Mater 21:1–14PubMedGoogle Scholar
  30. Hill DJ, Logan A (1992) Peptide growth factors and their interactions during chondrogenesis. Prog Growth Factor Res 4:45–68PubMedCrossRefGoogle Scholar
  31. Horowitz A, Tkachenko E, Simons M (2002) Fibroblast growth factor-specific modulation of cellular response by syndecan-4. J Cell Biol 157:715–725PubMedCrossRefGoogle Scholar
  32. Karp SJ, Schipani E, St-Jacques B, Hunzelman J, Kronenberg H, McMahon AP (2000) Indian Hedgehog coordinates endochondral bone growth and morphogenesis via parathyroid hormone related-protein-dependent and -independent pathways. Development 127:543–548PubMedGoogle Scholar
  33. Kavanagh E, Church VL, Osbourne AC, Lamb KJ, Archer CW, Francis-West PH, Pitsillides AA (2006) Differential regulation of GDF-5 and FGF-2/4 by immobilisation in ovo exposes distinct roles in joint formation. Dev Dyn 235:826–834PubMedCrossRefGoogle Scholar
  34. Khan IM, Evans SL, Young RD, Blain EJ, Quantock AJ, Avery N, Archer CW (2011) Fibroblast growth factor 2 and transforming growth factor β1 induce precocious maturation of articular cartilage. Arthritis Rheum 63:3417–3427PubMedCrossRefGoogle Scholar
  35. Kirsch T, Koyama E, Liu M, Golub EE, Pacifici M (2002) Syndecan-3 is a selective regulator of chondrocyte proliferation. J Biol Chem 277:42171–42177PubMedCrossRefGoogle Scholar
  36. Kosher RA (1998) Syndecan-3 in limb skeletal development. Microsc Res Tech 43:123–130PubMedCrossRefGoogle Scholar
  37. Kronenberg HM (2006) PTHrP and skeletal development. Ann NY Acad Sci 1068:1–13PubMedCrossRefGoogle Scholar
  38. Lamanna WC, Kalus I, Padva M, Baldwin RJ, Merry CLR, Dierks T (2007) The heparanome—the enigma of encoding and decoding heparan sulphate sulfation. J Biotech 129:290–307CrossRefGoogle Scholar
  39. Lanske B, Karaplis AC, Lee K, Luz A, Vortkamp A, Pirro A, Karperien M, Defize LH, Ho C, Mulligan RC, Abou-Samra AB, Juppner H, Degre GV, Kronenberg HM (1996) PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth. Science 273:663–666PubMedCrossRefGoogle Scholar
  40. LeClair EE, Mui SR, Huang A, Topczewska JM, Topczewski J (2009) Craniofacial skeletal defects of adult zebrafish glypican 4 (knypek) mutants. Dev Dyn 238:2550–2563PubMedCrossRefGoogle Scholar
  41. Luan Y, Praul CA, Gay CV, Leach RM (1996) Basic fibroblast growth factor: an autocrine growth factor for epiphyseal growth plate chondrocytes. J Cell Biochem 62:372–382PubMedCrossRefGoogle Scholar
  42. Melrose J, Roughley P, Knox S, Smith S, Lord M, Whitelock J (2006) The structure, location, and function of perlecan, a prominent pericellular proteoglycan of fetal, postnatal, and mature hyaline cartilages. J Biol Chem 281:36905–36914PubMedCrossRefGoogle Scholar
  43. Melrose J, Hayes AJ, Whitelock JM, Little CB (2008a) Perlecan, the “jack of all trades” proteoglycan of cartilaginous weight-bearing connective tissues. BioEssays 30:457–469PubMedCrossRefGoogle Scholar
  44. Melrose J, Smith SM, Smith MM, Little CB (2008b) The use of Histochoice for histological examination of articular and growth plate cartilages, intervertebral disc and meniscus. Biotech Histochem 83:47–53PubMedCrossRefGoogle Scholar
  45. Mérida-Velasco JA, Sánchez-Montesinos I, Espín-Ferra J, Mérida-Velasco JR, Rodríguez-Vázquez JF, Jiménez-Collado J (1997a) Development of the human knee joint ligaments. Anat Rec 248:259–268PubMedCrossRefGoogle Scholar
  46. Mérida-Velasco JA, Sánchez-Montesinos I, Espín-Ferra J, Rodríguez-Vázquez JF, Mérida-Velasco JR, Jiménez-Collado J (1997b) Development of the human knee joint. Anat Rec 248:269–278PubMedCrossRefGoogle Scholar
  47. Mundy C, Yasuda T, Kinumatsu T, Yamaguchi Y, Iwamoto M, Enomoto-Iwamoto M, Koyama E, Pacifici M (2011) Synovial joint formation requires local EXT1 expression and heparan sulfate production in developing mouse embryo limbs and spine. Dev Biol 351:70–81PubMedCrossRefGoogle Scholar
  48. O’Rahilly R (1951) The early prenatal development of the human knee joint. J Anat 85:166–170PubMedGoogle Scholar
  49. O’Rahilly R, Müller F (1987) Developmental stages in human embryos. Carnegie Institution of Washington, Washington, DCGoogle Scholar
  50. Ornitz DM (2000) FGFs, heparan sulphate and FGFRs: complex interactions essential for development. BioEssays 22:108–112PubMedCrossRefGoogle Scholar
  51. Otsuki S, Taniguchi N, Grogan SP, D’Lima D, Kinoshita M, Lotz M (2008) Expression of novel extracellular sulfatases Sulf-1 and Sulf-2 in normal and osteoarthritic cartilage. Arthritis Res Therapy 10:R61CrossRefGoogle Scholar
  52. Pacifici M, Koyama E, Iwamoto M, Gentili C (2000) Development of articular cartilage: what do we know about it and how may it occur? Connect Tissue Res 41:175–184PubMedCrossRefGoogle Scholar
  53. Pacifici M, Koyama E, Iwamoto M (2005a) Mechanisms of synovial joint and articular cartilage formation: recent advances, but many lingering mysteries. Birth Defects Res (Part C) 75:237–248CrossRefGoogle Scholar
  54. Pacifici M, Shimo T, Gentili C, Kirsch T, Freeman TA, Enomoto-Iwamoto M, Iwamoto M, Koyama E (2005b) Syndecan-3: a cell surface heparan sulphate proteoglycan important for chondrocyte proliferation and function during limb skeletogenesis. J Bone Miner Metab 23:191–199PubMedCrossRefGoogle Scholar
  55. Pacifici M, Koyama E, Shibukawa Y, Wu C, Tamamura Y, Enomoto-Iwamoto M, Iwamoto M (2006) Cellular and molecular mechanisms of synovial joint and articular cartilage formation. Ann N Y Acad Sci 1068:74–86PubMedCrossRefGoogle Scholar
  56. Pain-Saunders S, Viviano BL, Zupicich J, Skarnes WC, Saunders S (2000) Glypican-3 controls cellular responses to BMP-4 in limb patterning and skeletal development. Dev Biol 225:179–187CrossRefGoogle Scholar
  57. Poole AR, Kojima T, Yasuda T, Mwale F, Kobayashi M, Laverty S (2001) Composition and structure of articular cartilage: a template for tissue repair. Clin Orthop Relat Res 391:S26–S33PubMedCrossRefGoogle Scholar
  58. Powell AK, Yates EA, Fernig DG, Turnbull JE (2004) Interactions of heparin/heparan sulfate with proteins: appraisal of structural features and experimental approaches. Glycobiology 14:17–30CrossRefGoogle Scholar
  59. Purushothaman A, Sugahara K, Faissner A (2011) Chondroitin sulfate wobble motifs modulate the maintenance and differentiation of neural stem cells and their progeny. J Biol Chem [Epub ahead of print]Google Scholar
  60. Ratajczak W (2000) Early development of the cruciate ligaments in staged human embryos. Folia Morphol 59:285–290Google Scholar
  61. Reddi AH (2011) Cartilage morphogenetic proteins: role in joint development, homeostasis, and regeneration. Ann Rheum Dis 62:73–78Google Scholar
  62. Rider CC (2006) Heparin/heparan sulphate binding in the TGF-beta cytokine family. Biochem Soc Trans 34:458–460PubMedCrossRefGoogle Scholar
  63. Rountree RB, Schoor M, Chen H, Marks ME, Harley V, Mishina Y, Kingsley DM (2004) BMP receptor signalling is required for postnatal maintenance of articular cartilage. PLoS Biol 2:e355PubMedCrossRefGoogle Scholar
  64. Sassi N, Laadhar L, Driss M, Kallel-Sellami M, Sellami S, Makni S (2011) The role of the Notch pathway in healthy and osteoarthritic articular cartilage: from experimental models to ex vivo studies. Arthritis Res Therapy 13:208CrossRefGoogle Scholar
  65. Shimazu A, Nah HD, Kirsch T, Koyama E, Leatherman JL, Golden EB, Kosher RA, Pacifici M (1996) Syndecan-3 and the control of chondrocyte proliferation during endochondral ossification. Exp Cell Res 229:126–136PubMedCrossRefGoogle Scholar
  66. Shimo T, Gentili C, Iwamoto M, Wu C, Koyama E, Pacifici M (2004) Indian hedgehog and syndecan-3 coregulate chondrocyte proliferation and function during chick limb skeletogenesis. Dev Dyn 229:607–617PubMedCrossRefGoogle Scholar
  67. Sirko S, von Holst A, Weber A, Wizenmann A, Theocharidis U, Götz M, Faissner A (2010) Chondroitin sulfates are required for fibroblast growth factor-2-dependent proliferation and maintenance in neural stem cells and for epidermal growth factor-dependent migration of their progeny. Stem Cells 28:775–787PubMedCrossRefGoogle Scholar
  68. Smith SML, West LA, Govindraj P, Zhang X, Ornitz DM, Hassell JR (2007) Heparan and chondroitin sulfate on growth plate perlecan mediate biding and delivery of FGF-2 to FGF receptors. Matrix Biol 26:175–184PubMedCrossRefGoogle Scholar
  69. Smith SM, Shu C, Melrose J (2010) Comparative immunolocalisation of perlecan with collagen II and aggrecan in human foetal, newborn and adult ovine joint tissues demonstrates perlecan as an early developmental chondrogenic marker. Histochem Cell Biol 134:251–263PubMedCrossRefGoogle Scholar
  70. Sorrell JM, Caterson B (1989) Detection of age-related changes in the distribution of keratan sulfates and chondroitin sulfates in developing chick limbs: an immunocytochemical study. Development 106:657–663PubMedGoogle Scholar
  71. Sorrell JM, Lintala AM, Mahmoodian F, Caterson B (1988) Epitope-specific changes in chondroitin sulphate/dermatan sulphate proteoglycans as markers in the lymphopoietic and granulopoietic compartments of developing bursae of Fabricius. J Immunol 140:4263–4270PubMedGoogle Scholar
  72. Sorrell JM, Mahmoodian F, Schafer IA, Davis B, Caterson B (1990) Identification of monoclonal antibodies that recognize novel epitopes in native chondroitin/dermatan sulphate glycosaminoglycan chains: their use in mapping functionally distinct domains of human skin. J Histochem Cytochem 38:393–402PubMedCrossRefGoogle Scholar
  73. Sorrell JM, Carrino DA, Caplan AI (1996) Regulated expression of chondroitin sulfates at sites of epithelial–mesenchymal interaction: spatio-temporal patterning identified with anti-chondroitin sulphate monoclonal antibodies. Int J Dev Neurosci 14:233–248CrossRefGoogle Scholar
  74. St-Jacques B, Hammerschmidt M, McMahon AP (1999) Indian hedgehog signalling regulates proliferation and differentiation of chondrocytes and is essential for bone formation. Genes Dev 13:2072–2086PubMedCrossRefGoogle Scholar
  75. Tkachenko E, Rhodes JM, Simons M (2005) Syndecans. New kids on the signalling block. Circ Res 96:488–500PubMedCrossRefGoogle Scholar
  76. Trippel SB, Wroblewski J, Makower AM, Whelan MC, Schoenfeld D, Doctrow SR (1993) Regulation of growth-plate chondrocytes by insulin-like growth factor and basic fibroblast growth factor. J Bone Jt Surg Am 75:177–189Google Scholar
  77. Tumova S, Woods A, Couchman JR (2000) Heparan sulfate proteoglycans on the cell surface: versatile coordinators of cellular functions. Int J Biochem Cell Biol 32:269–288PubMedCrossRefGoogle Scholar
  78. van den Born J, Salmivirta K, Henttinen T, Östman N, Ishimaru T, Miyaura S, Yoshida K, Salmivirta M (2005) Novel heparan sulphate structures revealed by monoclonal antibodies. J Biol Chem 280:20516–20523PubMedCrossRefGoogle Scholar
  79. Veugelers M, De Cat B, Ceulemans H, Bruystens A-M, Coomans C, Durr J, Vermeesch J, Marynen P, David G (1999) Glypican-6, a new member of the glypican family of cell surface heparin sulphate proteoglycans. J Biol Chem 274:26968–26977PubMedCrossRefGoogle Scholar
  80. Vincent TL, McLean CJ, Full LE, Peston D, Saklatvala J (2007) FGF-2 is bound to perlecan in the pericellular matrix of articular cartilage, where it acts as a chondrocyte mechanotransducer. Osteoarthritis Cartilage 15:752–763PubMedCrossRefGoogle Scholar
  81. Visco DM, Johnstone B, Hill MA, Jolly GA, Caterson B (1993) Immunohistochemical analysis of 3B3(-) and 7D4 epitope expression in canine osteoarthritis. Arthritis Rheum 36:1718–1725PubMedCrossRefGoogle Scholar
  82. Wang Q, Green RP, Zhao G, Ornitz DM (2001) Differential regulation of endochondral bone growth and joint development by FGFR1 and FGFR3 tyrosine kinase domains. Development 128:3867–3876PubMedGoogle Scholar
  83. Whitelock J, Melrose J (2011) Heparan sulfate proteoglycans in healthy and diseased systems. Wiley Interdiscip Rev Syst Biol Med 3:739–751PubMedCrossRefGoogle Scholar
  84. Williams R, Khan IM, Richardson K, Nelson L, McCarthy HE, Analbelsi T, Singhrao SK, Dowthwaite GP, Jones RE, Baird DM, Lewis H, Roberts S, Shaw HM, Dudhia J, Fairclough J, Briggs T, Archer CW (2010) Identification and clonal characteristics of a progenitor cell sub-population in normal human articular cartilage. PLoS ONE 5:e13246PubMedCrossRefGoogle Scholar
  85. Yan D, Lin X (2009) Shaping morphogen gradients by proteoglycans. Cold Spring Harb Perspect Biol 1:a002493PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • James Melrose
    • 1
  • Marc D. Isaacs
    • 2
  • Susan M. Smith
    • 1
  • Clare E. Hughes
    • 2
  • Christopher B. Little
    • 1
  • Bruce Caterson
    • 2
  • Anthony J. Hayes
    • 2
    • 3
    Email author
  1. 1.Raymond Purves Laboratory, Institute of Bone and Joint Research, Kolling Institute of Medical ResearchUniversity of Sydney at The Royal North Shore HospitalSt. LeonardsAustralia
  2. 2.Connective Tissue Biology Laboratories, School of BiosciencesCardiff UniversityCardiffWales, UK
  3. 3.Bio-imaging Unit, Cardiff School of BiosciencesCardiff UniversityCardiffUK

Personalised recommendations