During osteoarthritis (OA) chondrocytes show deviant behavior resembling terminal differentiation of growth-plate chondrocytes, characterized by elevated MMP-13 expression. The latter is also a hallmark for OA. TGF-beta is generally thought to be a protective factor for cartilage, but it has also displayed deleterious effects in some studies. Recently, it was shown that besides signaling via the ALK5 (activin-like kinase 5) receptor, TGF-beta can also signal via ALK1, thereby activating Smad1/5/8 instead of Smad2/3. The Smad1/5/8 route can induce chondrocyte terminal differentiation. Murine chondrocytes stimulated with TGF-beta activated the ALK5 receptor/Smad2/3 route as well as the ALK1/Smad1/5/8 route. In cartilage of mouse models for aging and OA, ALK5 expression decreased much more than ALK1. Thus, the ALK1/ALK5 ratio increased, which was associated with changes in the respective downstream markers: an increased Id-1 (inhibitor of DNA binding-1)/PAI-1 (plasminogen activator inhibitor-1) ratio. Transfection of chondrocytes with adenovirus overexpressing constitutive active ALK1 increased MMP-13 expression, while small interfering RNA against ALK1 decreased MMP-13 expression to nondetectable levels. Adenovirus overexpressing constitutive active ALK5 transfection increased aggrecan expression, whereas small interfering RNA against ALK5 resulted in increased MMP-13 expression. Moreover, in human OA cartilage ALK1 was highly correlated with MMP-13 expression, whereas ALK5 correlated with aggrecan and collagen type II expression, important for healthy cartilage. Collectively, we show an age-related shift in ALK1/ALK5 ratio in murine cartilage and a strong correlation between ALK1 and MMP-13 expression in human cartilage. A change in balance between ALK5 and ALK1 receptors in chondrocytes caused changes in MMP-13 expression, thereby causing an OA-like phenotype. Our data suggest that dominant ALK1 signaling results in deviant chondrocyte behavior, thereby contributing to age-related cartilage destruction and OA.

Data show that lack of TGFbeta3 is associated with cartilage damage, suggesting loss of the protective effect of TGFbeta3 during osteoarthritis progression. Additionally, our results indicate that TGFbeta3 is involved in early osteophyte development, whereas BMP might be involved in late osteophyte development.

Osteoarthritis has as main characteristics the degradation of articular cartilage and the formation of new bone at the joint edges, so-called osteophytes. In this study enhanced expression of TGF-β1 and -β3 was detected in developing osteophytes and articular cartilage during murine experimental osteoarthritis. To determine the role of endogenous TGF-β on osteophyte formation and articular cartilage, TGF-β activity was blocked via a scavenging soluble TGF-β-RII. Our results clearly show that inhibition of endogenous TGF-β nearly completely prevented osteophyte formation. In contrast, treatment with recombinant soluble TGF-β-RII markedly enhanced articular cartilage proteoglycan loss and reduced the thickness of articular cartilage. In conclusion, we show for the first time that endogenous TGF-β is a crucial factor in the process of osteophyte formation and has an important function in protection against cartilage loss.

Objective. To investigate in vivo and in vitro whether macrophages have an intermediate role in transforming growth factor ␤ (TGF␤)-induced osteophyte formation.Methods. In vivo, synovial lining macrophages were selectively depleted by injection of clodronateladen liposomes 7 days prior to injection of 20 ng or 200 ng of TGF␤ into murine knee joints 3 times, on alternate days. Total knee joint sections were obtained on day 7 after the last injection and stained with Safranin O. Production of bone morphogenetic protein 2 (BMP-2) and BMP-4 was determined by immunolocalization. The interaction between murine macrophages and mesenchymal cells (precursors with chondrogenic potential) was studied in vitro using a Transwell system in which RAW macrophages were cocultured with C3H10T1/2 mesenchymal cells. Spheroid neocartilage formation was quantified microscopically after staining with May-Grünwald-Giemsa.Results Osteoarthritis (OA) is a chronic disease characterized by severe cartilage damage and osteophyte formation. Its pathogenesis is thought to be multifactorial, and most likely, both metabolic and mechanical factors are involved (1,2).During OA, dysregulated chondrogenesis is observed along the margins of articular cartilage. It involves differentiation of chondrogenic cells that reside in the periosteum (periosteal cells). In addition, periosteal cells express cartilage-specific genes producing extracellular matrix proteins. These cells may mature from prechondrocytic cells into hypertrophic chondrocytes (3). Cartilage formation is followed by cartilage maturation, removal of cartilage, and replacement with bone, resulting in structures called osteophytes (4).Factors that regulate mesenchymal cells to differentiate along the chondrocytic lineage for new cartilage and bone formation include cytokines and growth fac-1 P.

The aim of this study was to investigate the roles of Smad2/3 and Smad1/5/8 phosphorylation in transforming growth factor-beta-induced chondrogenic differentiation of bone-marrow-derived mesenchymal stem cells (BMSCs) to assess whether specific targeting of different Smad signaling pathways offers possibilities to prevent terminal differentiation and mineralization of chondrogenically differentiated BMSCs. Terminally differentiated chondrocytes produced in vitro by chondrogenic differentiation of BMSCs or studied ex vivo during murine embryonic limb formation stained positive for both Smad2/3P and Smad1/5/8P. Hyaline-like cartilage produced in vitro by articular chondrocytes or studied in ex vivo articular cartilage samples that lacked expression for matrix metalloproteinase 13 and collagen X only expressed Smad2/3P. When either Smad2/3 or Smad1/5/8 phosphorylation was blocked in BMSC culture by addition of SB-505124 or dorsomorphin throughout culture, no collagen II expression was observed, indicating that both pathways are involved in early chondrogenesis. Distinct functions for these pathways were demonstrated when Smad signaling was blocked after the onset of chondrogenesis. Blocking Smad2/3P after the onset of chondrogenesis resulted in a halt in collagen II production. On the other hand, blocking Smad1/5/8P during this time period resulted in decreased expression of matrix metalloproteinase 13, collagen X, and alkaline phosphatase while allowing collagen II production. Moreover, blocking Smad1/5/8P prevented mineralization. This indicates that while Smad2/3P is important for continuation of collagen II deposition, Smad1/5/8 phosphorylation is associated with terminal differentiation and mineralization.

Objective. Osteoarthritis (OA) is a joint disease characterized by osteophyte development, fibrosis, and articular cartilage damage. Effects of exogenous transforming growth factor ␤ (TGF␤) isoforms and bone morphogenetic proteins (BMPs) suggest a role for these growth factors in the pathogenesis of OA. The aim of this study was to elucidate the role of endogenous TGF␤ and BMP during papain-induced OA-like changes in mice.Methods. We used adenoviral overexpression of TGF␤ and BMP antagonists to block growth factor signaling. An adenovirus expressing a secreted, panspecific TGF␤ antagonist called murine latencyassociated peptide 1 (mLAP-1) was used. In addition, we used intracellular inhibitory Smad6 as a BMP antagonist and Smad7 as a TGF␤/BMP inhibitor. Papain was injected into the knee joints of C57BL/6 mice to induce osteophyte development, synovial thickening, and articular cartilage proteoglycan (PG) loss.Results. Intraarticular injection of papain caused increased protein expression of several TGF␤ and BMP isoforms in synovium and cartilage. Adenovirus transfection into the joint resulted in a strong expression of the transgenes in the synovial lining. Overexpression of mLAP-1, Smad6, and Smad7 led to a significant reduction in osteophyte formation compared with that in controls. Smad6 and Smad7 overexpression also significantly decreased synovial thickening. Furthermore, the secreted TGF␤ inhibitor mLAP-1 increased articular cartilage PG loss.Conclusion. Our results indicate a pivotal role of endogenous TGF␤ in the development of osteophytes and synovial thickening, implicating endogenous TGF␤ in the pathogenesis of OA. In contrast, the prevention of cartilage damage by endogenous TGF␤ signifies the protective role of TGF␤ in articular cartilage. This is the first study to demonstrate that endogenous BMPs are involved in osteophyte formation and synovial thickening in experimental OA.

Objective. Synovial fibrosis is a major contributor to joint stiffness in osteoarthritis (OA). Transforming growth factor ␤ (TGF␤), which is elevated in OA, plays a key role in the onset and persistence of synovial fibrosis. However, blocking of TGF␤ in OA as a therapeutic intervention for fibrosis is not an option since TGF␤ is crucial for cartilage maintenance and repair. Therefore, we undertook the present study to seek targets downstream of TGF␤ for preventing OA-related fibrosis without interfering with joint homeostasis.Methods. Experiments were performed to determine whether genes involved in extracellular matrix turnover were responsive to TGF␤ and were elevated in OA-related fibrosis. We analyzed gene expression in TGF␤-stimulated human OA synovial fibroblasts and in the synovium of mice with TGF␤-induced fibrosis, mice with experimental OA, and humans with end-stage OA. Gene expression was determined by microarray, lowdensity array, or quantitative polymerase chain reaction analysis.Results. We observed an increase in expression of procollagen genes and genes encoding collagen crosslinking enzymes under all of the OA-related fibrotic conditions investigated. Comparison of gene expression in TGF␤-stimulated human OA synovial fibroblasts, synovium from mice with experimental OA, and synovium from humans with end-stage OA revealed that the genes PLOD2, LOX, COL1A1, COL5A1, and TIMP1 were up-regulated in all of these conditions. Additionally, we confirmed that these genes were up-regulated by TGF␤ in vivo in mice with TGF␤-induced synovial fibrosis.Conclusion. Most of the up-regulated genes identified in this study would be poor targets for therapy development, due to their crucial functions in the joint. However, the highly up-regulated gene PLOD2, responsible for the formation of collagen crosslinks that make collagen less susceptible to enzymatic degradation, is an attractive and promising target for interference in OArelated synovial fibrosis.

Articular cartilage has a very limited intrinsic repair capacity leading to progressive joint damage. Therapies involving tissue engineering depend on chondrogenic differentiation of progenitor cells. This chondrogenic differentiation will have to survive in a diseased joint. We postulate that catabolic factors in this environment inhibit chondrogenesis of progenitor cells. We investigated the effect of a catabolic environment on chondrogenesis in pellet cultures of human mesenchymal stem cells (hMSCs). We exposed chondrogenically differentiated hMSC pellets, to interleukin (IL)-1α, tumor necrosis factor (TNF)-α or conditioned medium derived from osteoarthritic synovium (CM-OAS). IL-1α and TNF-α in CM-OAS were blocked with IL-1Ra or Enbrel, respectively. Chondrogenesis was determined by chondrogenic markers collagen type II, aggrecan, and the hypertrophy marker collagen type X on mRNA. Proteoglycan deposition was analyzed by safranin o staining on histology. IL-1α and TNF-α dose-dependently inhibited chondrogenesis when added at onset or during progression of differentiation, IL-1α being more potent than TNF-α. CM-OAS inhibited chondrogenesis on mRNA and protein level but varied in extent between patients. Inhibition of IL-1α partially overcame the inhibitory effect of the CM-OAS on chondrogenesis whereas the TNF-α contribution was negligible. We show that hMSC chondrogenesis is blocked by either IL-1α or TNF-α alone, but that there are additional factors present in CM-OAS that contribute to inhibition of chondrogenesis, demonstrating that catabolic factors present in OA joints inhibit chondrogenesis, thereby impairing successful tissue engineering.