title: Assessment of the matrix degenerative effects of MMP-3, ADAMTS-4, and HTRA1, injected into a bovine intervertebral disc organ culture model.
abstract: METHODS
  In vitro study to develop an intervertebral disc degeneration organ culture model, using coccygeal bovine intervertebral discs (IVDs) and injection of proteolytic enzymes MMP-3, ADAMTS-4, and HTRA1.
  
  
  OBJECTIVE
  This study aimed to develop an in vitro model of enzyme-mediated intervertebral disc degeneration to mimic the clinical outcome in humans for investigation of therapeutic treatment options.
  
  
  BACKGROUND
  Bovine IVDs are comparable with human IVDs in terms of cell composition and biomechanical behavior. Researchers injected papain and trypsin into them to create an intervertebral disc degeneration model with a degenerated nucleus pulposus (NP) area. They achieved macroscopic cavities as well as a loss of glycosaminoglycans (GAGs). However, none of these enzymes are clinically relevant.
  
  
  METHODS
  Bovine IVDs were harvested maintaining the endplates. Active forms of MMP-3, ADAMTS-4, and HTRA1 were injected at a dose of 10 μg/mL each. Phosphate-buffered saline was injected as a control. Discs were cultured for 8 days and loaded diurnally (days 1-4 with ≈0.4 MPa for 16 hr) and left under free swelling condition from days 4 to 8 to avoid expected artifacts because of dehydration of the NP. Outcome parameters included disc height, metabolic cell activity, DNA content, GAG content, total collagen content, relative gene expression, and histological investigation.
  
  
  RESULTS
  The mean metabolic cell activity was significantly lower in the NP area of discs injected with ADAMTS-4 than the day 0 control discs. Disc height was decreased after injection with HTRA1 and was significantly correlated with changes in GAG/DNA of the NP tissue. Total collagen content tended to be lower in groups injected with ADAMTS4 and MMP-3.
  
  
  CONCLUSIONS
  MMP-3, ADAMTS-4, and HTRA1 provoked neither visible matrix degradation nor major shifts in gene expression. However, cell activity was significantly reduced and HTRA1 induced loss of disc height that positively correlated with changes in GAG/DNA content. The use of higher doses of these enzymes or a combination thereof may, therefore, be necessary to induce disc degeneration.
authors.affiliation: *Institute for Surgical Technology and Biomechanics, University of Bern, Berne, Switzerland; †Bone and Stem Cell Research Group, Center for Applied Biotechnology and Molecular Medicine, University of Zurich, Zurich, Switzerland; and ‡Zurich Center for Integrative Human Physiology, University of Zurich, Zurich, Switzerland.
authors.forename: Tina
authors.initials: T
authors.lastname: Furtwängler
authors.forename: Samantha C W
authors.initials: SC
authors.lastname: Chan
authors.forename: Gregor
authors.initials: G
authors.lastname: Bahrenberg
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Richards
authors.forename: Benjamin
authors.initials: B
authors.lastname: Gantenbein-Ritter
mesh_terms: D051722:ADAM Proteins / Q000378:metabolism / Q000494:pharmacology*; D000071121:ADAMTS4 Protein; D000818:Animals; D002417:Cattle; D003094:Collagen / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004195:Disease Models, Animal; D015870:Gene Expression / Q000187:drug effects; D006025:Glycosaminoglycans / Q000378:metabolism; D006801:Humans; D007403:Intervertebral Disc / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D055959:Intervertebral Disc Degeneration / Q000235:genetics / Q000378:metabolism; D053509:Matrix Metalloproteinase 13 / Q000235:genetics; D019278:Matrix Metalloproteinase 3 / Q000378:metabolism / Q000494:pharmacology*; D009924:Organ Culture Techniques; D011348:Procollagen N-Endopeptidase / Q000378:metabolism / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012697:Serine Endopeptidases / Q000378:metabolism / Q000494:pharmacology*; D013997:Time Factors; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000235:genetics
chemical_list: D006025:Glycosaminoglycans; D019715:Tissue Inhibitor of Metalloproteinase-1; D019717:Tissue Inhibitor of Metalloproteinase-3; D003094:Collagen; D004247:DNA; D012697:Serine Endopeptidases; D051722:ADAM Proteins; D053509:Matrix Metalloproteinase 13; D011348:Procollagen N-Endopeptidase; D019278:Matrix Metalloproteinase 3; D000071121:ADAMTS4 Protein; C000603918:ADAMTS4 protein, human