Henk M. van Beuningen

Inhibition of endogenous TGF-beta during experimental osteoarthritis prevents osteophyte formation and impairs cartilage repair.

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.

Linkage of chondroitin-sulfate to type I collagen scaffolds stimulates the bioactivity of seeded chondrocytes in vitro

An increasing amount of interest is focused on the potential use of tissue-engineered articular cartilage implants, for repair of defects in the joint surface. In this perspective, various biodegradable scaffolds have been evaluated as a vehicle to deliver chondrocytes into a cartilage defect. This cell-matrix implant should eventually promote regeneration of the traumatized articular joint surface with hyaline cartilage. Successful regeneration can only be achieved with such a tissue-engineered cartilage implant if the seeded cells reveal an appropriate proliferation rate in the biodegradable scaffold together with the production of a new cartilage-specific extracellular matrix. These metabolic parameters can be influenced by the biochemical composition of a cell-delivery scaffold. Further elucidation of specific cell-matrix interactions is important to define the optimal biochemical composition of a cell-delivery vehicle for cartilage repair. In this in vitro study, we investigated the effect of the presence of cartilage-specific glycosaminoglycans in a type I collagen scaffold on the metabolic activity of seeded chondrocytes. Isolated bovine chondrocytes were cultured in porous type I collagen matrices in the presence and absence of covalently attached chondroitin sulfate (CS) up to 14 days. CS did indeed influence the bioactivity of the seeded chondrocytes. Cell proliferation and the total amount of proteoglycans retained in the matrix, were significantly higher (p < 0.001) in type I collagen scaffolds with CS. Light microscopy showed the formation of a more dense cartilaginous layer at the matrix periphery. Scanning electron microscopy revealed an almost complete surfacing of the initially porous surface of both matrices. Histology and reverse transcriptase PCR for various proteoglycan subtypes suggested a good preservation of the chondrocytic phenotype of the seeded cells during culture. The stimulatory potential of CS on both the cell-proliferation and matrix retention, turns this GAG into an interesting biochemical component of a cell-delivery scaffold for use in tissue-engineering articular cartilage.

Cartilage destruction and osteophytes in instability-induced murine osteoarthritis: Role of TGFβ in osteophyte formation?

Osteoarthritis is characterized by focal cartilage destruction and marked formation of osteophytes. We have investigated the possible relationship between site specific occurrence of cartilage damage and osteophytes in the collagenase induced murine osteoarthritis model. The degree of instability of the joint correlated with the amount of cartilage loss. Moreover, cartilage damage in the medial tibial plateau correlated only strongly with the osteophyte at the medial plateau, whereas a similar, site directed trend was noted for lateral damage and lateral osteophytes. A separate study with intraarticular injection of TGFβ1 in normal murine knee joints revealed that this factor can induce osteophytes at characteristic sites, suggesting a role of endogenous TGFβ in this phenomenon.

Analysis of changes in proteoglycan content in murine articular cartilage using image analysis

The extracellular matrix of articular cartilage consists mainly of type II collagen and large aggregating proteoglycan (aggrecan). During arthritis and other joint diseases, the proteoglycan (PG) level of cartilage matrix is diminished, leading to impairment of normal joint function. A new method is described for measuring the changes in PG content of murine articular cartilage. The method is based on the automated densitometric analysis of patellar cartilage of standard, safranin O-stained sections of whole murine knee joints. It appeared to be possible to measure optical density in parallel layers of articular cartilage with high reproducibility. Approximately 25 sections can be evaluated within 1 h. Measuring a single section 10 times resulted in a coefficient of variation (CV) of 0.1-1.4%. A mean CV of 5-14% was calculated when a group of 18 sections was analyzed in quintuplicate. To validate the procedure, changes in PG content induced by arthritis or by intra-articular injection of TGFbeta-1 were analyzed by the image analysis method, the dimethylmethylene blue (DMB) assay and by visual grading. Although not a quantitive method, the newly developed image analysis method appeared to be more sensitive in detecting significant change in PG content of murine articular cartilage than the DMB method or visual grading. The image analysis method makes it possible to measure changes in PG content of specific areas of articular cartilage with higher sensitivity than the DMB method and eliminating the bias inherent to visual grading by human observers.

Enhanced suppressor of cytokine signaling 3 in arthritic cartilage dysregulates human chondrocyte function

OBJECTIVE To determine the expression of suppressor of cytokine signaling 3 (SOCS-3) in human articular chondrocytes and its functional consequences. METHODS Chondrocytes were isolated from the cartilage of patients with osteoarthritis (OA), patients with rheumatoid arthritis (RA), and trauma patients and from the healthy cartilage of patients with a femoral neck fracture. The human chondrocyte cell line G6 and primary bovine chondrocytes were used in validation experiments. SOCS-3 messenger RNA (mRNA) expression was measured by quantitative polymerase chain reaction, and SOCS-3 protein levels were determined by Western blotting and immunohistochemical analysis. To ascertain the role of SOCS-3 in the chondrocyte response to interleukin-1β (IL-1β) or lipopolysaccharide (LPS), the expression of SOCS3 was either reduced by small interfering RNA or enhanced by viral transduction. RESULTS The expression of SOCS-3 mRNA (but not that of SOCS-1 mRNA) was significantly enhanced in chondrocytes obtained from OA cartilage (mean ± SD ΔC(t) 3.4 ± 1.0) and RA cartilage (ΔC(t) 3.4 ± 1.4) compared with cartilage obtained from patients with femoral neck fracture (ΔC(t) 5.3 ± 1.2). The expression of SOCS3 correlated significantly with that of other genes known to be expressed in arthritic chondrocytes, such as MMP13 (r = 0.743), ADAMTS4 (r = 0.779), and ADAMTS5 (r = 0.647), and an inverse relationship was observed with COL2A1 (r = -0.561). Up-regulation of SOCS-3 by IL-1 in G6 chondrocytes and its spontaneous expression in OA chondrocytes were reduced by mithramycin, a specific inhibitor of transcription factor Sp-1. Overexpression of SOCS-3 in bovine chondrocytes reduced IL-1- and LPS-induced nitric oxide production and insulin-like growth factor 1-induced proteoglycan synthesis. Interestingly, a similar impairment of function was observed in OA chondrocytes, which was partially restored by SOCS-3 gene knockdown. CONCLUSION This study demonstrated that both SOCS-3 mRNA and SOCS-3 protein are expressed in human arthritic chondrocytes and affect cellular responses involved in cartilage pathology.

Dualistic role of TGFβ in osteoarthritis cartilage destruction and osteophyte formation

Osteoarthritis is a condition of focal cartilage lesions, combined with alterations in the subchondral bone and new hone formation (osteophytes) at the joint margins.