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 E 2 (PGE 2 ), and transforming growth factor ␤1 (TGF␤1) 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 TGF␤1 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 TGF␤1 inhibits BMP-2-dependent mineralization, and because TGF␤1 levels are ϳ4-fold higher in OA osteoblasts than in normal osteoblasts, inhibiting TGF␤1 levels in OA osteoblasts corrected the abnormal COL1A1-to-COL1A2 ratio and increased alizarin red staining.Conclusion. Elevated TGF␤1 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.

Our results show that glucocorticoids at clinically relevant concentrations exert specific actions on expression of adhesion molecules on activated neutrophils, which are mediated through ligation of glucocorticoid receptors and induction of protein synthesis, and suggest a novel mechanism by which anti-inflammatory corticosteroids may inhibit leukocyte accumulation.

The Wnt signaling pathway is crucial for osteogenesis and regulates terminal osteoblast differentiation. Although osteoarthritic (OA) osteoblasts show an abnormal phenotype and poor in vitro mineralization, the mechanism leading to this situation still remains unknow. Recent evidence indicates that Wnt signaling may be altered in OA osteoblasts. In this study we determined whether an alteration of the Wnt/b-catenin signaling pathway is responsible for the abnormal phenotype of OA osteoblasts. Expression of the Wnt signaling antagonist Dickkopf-1 (DKK1) was similar in normal and OA osteoblasts, whereas DKK2 expression was higher in OA osteoblasts than in normal osteoblasts. OA osteoblasts showed a decrease of Wnt3a-dependent Wnt/b-catenin signaling, measured by the TOPflash reporter assay and by Western blot analysis, compared with normal osteoblasts. Correcting DKK2 levels in OA osteoblasts by siRNA techniques enhanced Wnt/b-catenin signaling. Elevated DKK2 levels could be explained by elevated transforming growth factor b1 (TGF-b1) in OA osteoblasts, and exogenous TGF-b1 increased DKK2 expression in normal osteoblasts, whereas ablating TGF-b1 expression in OA osteoblasts reduced DKK2 expression. Inhibiting TGF-b1 or DKK2 expression corrected the abnormal phenotype of OA osteoblasts. In vitro mineralization of OA osteoblasts also was increased by DKK2 siRNA. We conclude that elevated TGF-b1 levels in OA osteoblasts can stimulate DKK2 expression, which, in turn, is responsible, at least in part, for their abnormal phenotype. ß