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A8.13 Syndecan-4 Function is Essential for Matrix Remodelling Under Inflammatory Conditions, But Dispensable During Embryogenesis
  1. Jessica Bertrand1,
  2. Richard Stange2,
  3. Heriburg Hidding1,
  4. Frank Echtermeyer3,
  5. Giovanna Nalesso4,
  6. Lars Godmann1,
  7. Melanie Timmen1,
  8. Peter Bruckner5,
  9. Francesco Dell’Accio4,
  10. Michael J Raschke2,
  11. Thomas Pap1,
  12. Rita Dreier5
  1. 1Institute for Experimental Musculoskeletal Medicine, University Hospital Muenster, Germany
  2. 2Dept. of Trauma, Hand and Reconstructive Surgery; University Hospital Muenster, Germany
  3. 3Dept. of Anaesthesiology and Intensive Care Medicine, Medical School Hanover, Germany
  4. 4Centre for Experimental Medicine and Rheumatology Queen Mary London, UK
  5. 5Institute for Physiological Chemistry and Pathobiochemistry, University Hospital Muenster, Germany

Abstract

Objective The heparan sulphate proteoglycan syndecan-4 (Sdc4) has been associated strongly with osteoarthritis, a disease that mimics key aspects of early cartilage remodelling during endochondral ossification, but its role in embryonic and adult bone formation remains unclear. Therefore, we used Sdc4 -/- mice to analyse the distribution and functional role of Scd4 in endochondral ossification of mouse embryos and in adult fracture repair, which recapitulates endochondral ossification, but like osteoarthritis, involves an inflammatory component.

Methods Sdc4 promoter activity was analysed in Sdc4 -/- /LacZ knock-in animals using β-galactosidase stainings. E16.5 embyros were used for histological (alcian blue/alizarin red) and immunohistological (PCNA, Col10a1, ADAMTS-4, BC-3, Sdc2) staining and the calcified bone area was quantified using whole mount staining of these embryos. Histological (Masson-Goldner, alcian blue) and immunohistological (Col10a1, Sdc2, PCNA) staining at day 7, 14 and 28 fracture calli were performed. These experiments were repeated with anti-TNF treatment during fracture healing. Callus size and cartilage area were quantified using image J Chondrocytes were isolated from neonatal knee joints and embyronal cartilage. Proliferation was investigated using MTT assay. Gene expression analysis for Sdc-2, Sdc-4 with and without stimulation using TNFα and WNT3a was performed using quantitative RT-PCR.

Results In Sdc4 -/- /LacZ knock-in animals, Sdc4 promoter activity was detectable in all stages of chondrocyte differentiation during embryogenesis. Sdc4 deficiency inhibited chondrocyte proliferation both in vivo and in vitro, but this did not lead to a growth phenotype at birth. In contrast to embryogenesis, fracture healing in adult mice was markedly delayed in Sdc4 -/- animals and accompanied by increased callus formation. Analysing the discrepancy between the mild embryonic and the severe adult phenotype, we found a compensatory up-regulation of Sdc2 in the developing cartilage of Sdc4 -/- mice that was absent in adult tissue. Stimulation of chondrocytes with Wnt3a in vitro, led to an increased expression of Sdc2, while stimulation with TNFα resulted in an up-regulation of Sdc4 but a decreased expression of Sdc2. In consequence treatment with a blocking anti-TNF antibody during fracture healing abolished the difference in callus size between wildtype and sdc4 -/- mice.

Conclusions We conclude that Sdc4 is functionally involved in endochondral ossification and that the loss of Sdc4 impairs adult fracture healing due to the inhibition of compensatory mechanisms under inflammatory conditions.

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