title: Altered mineralization of human osteoarthritic osteoblasts is attributable to abnormal type I collagen production.
abstract: OBJECTIVE
  Bone tissue in osteoarthritis (OA) is composed of abundant undermineralized osteoid matrix. The aim of this study was to investigate the mechanisms responsible for this abnormal matrix, using in vitro OA subchondral osteoblasts.
  
  
  METHODS
  Primary normal and OA osteoblasts were prepared from tibial plateaus. Phenotype was determined by alkaline phosphatase activity, and osteocalcin, osteopontin, prostaglandin E2 (PGE2), and transforming growth factor beta1 (TGFbeta1) were assessed by enzyme-linked immunosorbent assay. Expression of COL1A1 and COL1A2 was determined by real-time polymerase chain reaction. The production of type I collagen was determined by the release of its C-terminal propeptide and Western blot analysis. In vitro mineralization was evaluated by alizarin red staining. Inhibition of TGFbeta1 expression was performed using a small interfering RNA technique.
  
  
  RESULTS
  Mineralization of OA osteoblasts was reduced compared with mineralization of normal osteoblasts, even in the presence of bone morphogenetic protein 2 (BMP-2). Alkaline phosphatase and osteocalcin levels were elevated in OA osteoblasts compared with normal osteoblasts, whereas osteopontin levels were similar. The COL1A1-to-COL1A2 messenger RNA ratio was 3-fold higher in OA osteoblasts compared with normal osteoblasts, and the production of collagen by OA osteoblasts was increased. Because TGFbeta1 inhibits BMP-2-dependent mineralization, and because TGFbeta1 levels are approximately 4-fold higher in OA osteoblasts than in normal osteoblasts, inhibiting TGFbeta1 levels in OA osteoblasts corrected the abnormal COL1A1-to-COL1A2 ratio and increased alizarin red staining.
  
  
  CONCLUSIONS
  Elevated TGFbeta1 levels in OA osteoblasts are responsible, in part, for the abnormal ratio of COL1A1 to COL1A2 and for the abnormal production of mature type I collagen. This abnormal COL1A1-to-COL1A2 ratio generates a matrix that blunts mineralization in OA osteoblasts.
authors.affiliation: Unité de recherche en arthrose, Centre de Recherche du Centre Hospitalier de l'Université de Montréal, Hôpital Notre-Dame, Montréal, Québec, Canada.
authors.forename: Denis
authors.initials: D
authors.lastname: Couchourel
authors.forename: Isabelle
authors.initials: I
authors.lastname: Aubry
authors.forename: Aline
authors.initials: A
authors.lastname: Delalandre
authors.forename: Martin
authors.initials: M
authors.lastname: Lavigne
authors.forename: Johanne
authors.initials: J
authors.lastname: Martel-Pelletier
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Pelletier
authors.forename: Daniel
authors.initials: D
authors.lastname: Lajeunesse
mesh_terms: D000368:Aged; D000469:Alkaline Phosphatase / Q000032:analysis; D000880:Anthraquinones; D055396:Bone Morphogenetic Protein 2 / Q000032:analysis; D002113:Calcification, Physiologic / Q000502:physiology*; D002478:Cells, Cultured; D003094:Collagen / Q000032:analysis*; D024042:Collagen Type I / Q000032:analysis*; D000090602:Collagen Type I, alpha 1 Chain; D004396:Coloring Agents; D015232:Dinoprostone / Q000032:analysis; D016326:Extracellular Matrix Proteins / Q000032:analysis; D005260:Female; D006801:Humans; D008297:Male; D010003:Osteoarthritis / Q000378:metabolism*; D010006:Osteoblasts / Q000378:metabolism*; D015675:Osteocalcin / Q000032:analysis; D053495:Osteopontin / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D016212:Transforming Growth Factor beta / Q000032:analysis; D000099222:betaIG-H3 Protein
chemical_list: D000469:Alkaline Phosphatase; D000880:Anthraquinones; D055396:Bone Morphogenetic Protein 2; D003094:Collagen; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D004396:Coloring Agents; D015232:Dinoprostone; D016326:Extracellular Matrix Proteins; D015675:Osteocalcin; D053495:Osteopontin; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D000099222:betaIG-H3 Protein; C525718:BMP2 protein, human; C010078:alizarin