P.M. van der Kraan

Chondrocyte hypertrophy and osteoarthritis: role in initiation and progression of cartilage degeneration?

OBJECTIVE To review the literature on the role and regulation of chondrocyte terminal differentiation (hypertrophy-like changes) in osteoarthritis (OA) and to integrate this in a conceptual model of primary OA development. METHODS Papers investigating chondrocyte terminal differentiation in human OA cartilage and experimental models of OA were recapitulated and discussed. Focus has been on the occurrence of hypertrophy-like changes in chondrocytes and the factors described to play a role in regulation of chondrocyte hypertrophy-like changes in OA. RESULTS Chondrocyte hypertrophy-like changes are reported in both human OA and experimental OA models by most investigators. These changes play a crucial part in the OA disease process by protease-mediated cartilage degradation. We propose that altered chondrocyte behavior and concomitant cartilage degradation result in a disease-amplifying loop, leading to a mixture of disease stages and cellular responses within an OA joint. CONCLUSION Chondrocyte hypertrophy-like changes play a role in early and late stage OA. Since not all cells in an OA joint are synchronized, inhibition of hypertrophy-like changes might be a therapeutic target to slow down further OA progression.

Culture of chondrocytes in alginate and collagen carrier gels

In this in vitro study, we compared the potential of collagen and alginate gels as carriers for chondrocyte transplantation and we studied the influence of demineralized bone matrix (DBM) on chondrocytes in the gels. Chondrocytes were assessed for cell viability, phenotype (histology), proliferation rate and sulfate incorporation. Collagen gels showed a significant increase in cell numbers, but the chondrocytes dedifferentiated into fibroblast-like cells from day 6 onwards. In alginate gels, initial cell loss was found, but the cells maintained their typical chondrocyte phenotype. Although the total quantity of proteoglycans initially synthesized per cell in collagen gel was significantly higher, expressed per cell, the quantity in alginate gel eventually surpassed collagen. No effects of culturing chondrocytes in combination with DBM could be demonstrated on cell proliferation and sulfate incorporation. The collagen and alginate gels have different advantages as carriers for chondrocyte transplantation. The high proliferation rate of chondrocytes in collagen gel may be an advantage, but the preservation of the chondrocyte phenotype and the gradually increasing proteoglycan synthesis in alginate gel is a promising method for creating a hyaline cartilage implant in vitro.

Increase in ALK1/ALK5 Ratio as a Cause for Elevated MMP-13 Expression in Osteoarthritis in Humans and Mice

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-β 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-β 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-β 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.

Crosslinked type II collagen matrices: preparation, characterization, and potential for cartilage engineering.

The limited intrinsic repair capacity of articular cartilage has stimulated continuing efforts to develop tissue engineered analogues. Matrices composed of type II collagen and chondroitin sulfate (CS), the major constituents of hyaline cartilage, may create an appropriate environment for the generation of cartilage-like tissue. In this study, we prepared, characterized, and evaluated type 11 collagen matrices with and without CS. Type II collagen matrices were prepared using purified, pepsin-treated, type II collagen. Techniques applied to prepare type I collagen matrices were found unsuitable for type II collagen. Crosslinking of collagen and covalent attachment of CS was performed using 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide. Porous matrices were prepared by freezing and lyophilization, and their physico-chemical characteristics (degree of crosslinking, denaturing temperature, collagenase-resistance, amount of CS incorporated) established. Matrices were evaluated for their capacity to sustain chondrocyte proliferation and differentiation in vitro. After 7 d of culture, chondrocytes were mainly located at the periphery of the matrices. In contrast to type I collagen, type II collagen supported the distribution of cells throughout the matrix. After 14 d of culture, matrices were surfaced with a cartilagenous-like layer, and occasionally clusters of chondrocytes were present inside the matrix. Chondrocytes proliferated and differentiated as indicated by biochemical analyses, ultrastructural observations, and reverse transcriptase PCR for collagen types I, II and X. No major differences were observed with respect to the presence or absence of CS in the matrices.

TGF-beta signaling in chondrocyte terminal differentiation and osteoarthritis: Modulation and integration of signaling pathways through receptor-Smads

OBJECTIVE Chondrocytes and alteration in chondrocyte differentiation play a central role in osteoarthritis. Chondrocyte differentiation is amongst others regulated by members of the transforming growth factor-beta (TGF-beta) superfamily. The major intracellular signaling routes of this family are via the receptor-Smads. This review is focused on the modulation of receptor-Smad signaling and how this modulation can affect chondrocyte differentiation and potentially osteoarthritis development. METHODS Peer reviewed publications published prior to April 2009 were searched in the Pubmed database. Articles that were relevant for the role of TGF-beta superfamily/Smad signaling in chondrocyte differentiation and for differential modulation of receptor-Smads were selected. RESULTS Chondrocyte terminal differentiation is stimulated by Smad1/5/8 activation and inhibited the by Smad2/3 pathway, most likely by modulation of Runx2 function. Several proteins and signaling pathways differentially affect Smad1/5/8 and Smad2/3 signaling. This will result in an altered Smad1/5/8 and Smad2/3 balance and subsequently have an effect on chondrocyte differentiation and osteoarthritis development. CONCLUSION Modulation of receptor-Smads signaling can be expect to play an essential role in both the regulation of chondrocyte differentiation and osteoarthritis development and progression.

Reduced transforming growth factor-beta signaling in cartilage of old mice: role in impaired repair capacity

Osteoarthritis (OA) is a common joint disease, mainly effecting the elderly population. The cause of OA seems to be an imbalance in catabolic and anabolic factors that develops with age. IL-1 is a catabolic factor known to induce cartilage damage, and transforming growth factor (TGF)-beta is an anabolic factor that can counteract many IL-1-induced effects. In old mice, we observed reduced responsiveness to TGF-beta-induced IL-1 counteraction. We investigated whether expression of TGF-beta and its signaling molecules altered with age. To mimic the TGF-beta deprived conditions in aged mice, we assessed the functional consequence of TGF-beta blocking. We isolated knee joints of mice aged 5 months or 2 years, half of which were exposed to IL-1 by intra-articular injection 24 h prior to knee joint isolation. Immunohistochemistry was performed, staining for TGF-beta1, -2 or -3, TGF-betaRI or -RII, Smad2, -3, -4, -6 and -7 and Smad-2P. The percentage of cells staining positive was determined in tibial cartilage. To mimic the lack of TGF-beta signaling in old mice, young mice were injected with IL-1 and after 2 days Ad-LAP (TGF-beta inhibitor) or a control virus were injected. Proteoglycan (PG) synthesis (35S-sulfate incorporation) and PG content of the cartilage were determined. Our experiments revealed that TGF-beta2 and -3 expression decreased with age, as did the TGF-beta receptors. Although the number of cells positive for the Smad proteins was not altered, the number of cells expressing Smad2P strongly dropped in old mice. IL-1 did not alter the expression patterns. We mimicked the lack of TGF-beta signaling in old mice by TGF-beta inhibition with LAP. This resulted in a reduced level of PG synthesis and aggravation of PG depletion. The limited response of old mice to TGF-beta induced-IL-1 counteraction is not due to a diminished level of intracellular signaling molecules or an upregulation of intracellular inhibitors, but is likely due to an intrinsic absence of sufficient TGF-beta receptor expression. Blocking TGF-beta distorted the natural repair response after IL-1 injection. In conclusion, TGF-beta appears to play an important role in repair of cartilage and a lack of TGF-beta responsiveness in old mice might be at the root of OA development.

Pralnacasan, an inhibitor of interleukin-1β converting enzyme, reduces joint damage in two murine models of osteoarthritis

OBJECTIVE To study the effect of pralnacasan, the orally bioavailable pro-drug of a potent, non-peptide inhibitor of interleukin-1beta converting enzyme (ICE), RU 36384/VRT-18858, on joint damage in two mouse models of knee osteoarthritis (OA). DESIGN In a collagenase-induced OA model, pralnacasan was given orally by gavage to female Balb/c mice at 0, 12.5, 25 and 50 mg/kg twice a day. In the second study, pralnacasan was tested in male STR/1N mice, which develop OA spontaneously, by administering food-drug mixtures ad libitum at concentrations of 0, 700 and 4200 ppm (mg/kg food). OA joint damage was assessed by a semi-quantitative histopathological score in both studies. In the STR/1N mouse study, urinary levels of collagen cross-links hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) were determined by high-pressure liquid chromatography at baseline, after 3 and 6 weeks of treatment and RU 36384/VRT-18858 plasma concentrations was measured after 6 weeks. RESULTS In both studies, the mice developed moderate to severe knee joint OA in the medial joint compartments (tibial plateau and femoral condyle), the non-treated control groups showing median histopathological scores from 18 to 21 of a maximal score of 32. Pralnacasan was well tolerated. At the doses of 12.5 and 50 mg/kg in collagenase-induced OA and at the high dose of 4200 ppm in STR/1N mice pralnacasan treatment significantly reduced OA by 13-22%. In the STR/1N mice, urinary levels of HP cross-links and the ratio of HP/LP, which are indicators of joint damage in OA, were significantly reduced in the high dose group by 59 and 84%, respectively. CONCLUSIONS The ICE inhibitor pralnacasan reduced joint damage in two experimental models of OA and has the potential to become a disease-modifying drug for the treatment of OA.

Expression of transforming growth factor-β (TGFβ) and the TGFβ signalling molecule SMAD-2P in spontaneous and instability-induced osteoarthritis: role in cartilage degradation, chondrogenesis and osteophyte formation

Background: The primary feature of osteoarthritis is cartilage loss. In addition, osteophytes can frequently be observed. Transforming growth factor-β (TGFβ) has been suggested to be associated with protection against cartilage damage and new cartilage formation as seen in osteophytes. Objective: To study TGFβ and TGFβ signalling in experimental osteoarthritis to gain insight into the role of TGFβ in cartilage degradation and osteophyte formation during osteoarthritis progression. Methods: Histological sections of murine knee joints were stained immunohistochemically for TGFβ3 and phosphorylated SMAD-2 (SMAD-2P). Expression patterns were studied in two murine osteoarthritis models, representing spontaneous (STR/ort model) and instability-associated osteoarthritis (collagenase-induced instability model). Results: TGFβ3 and SMAD-2P staining was increasingly reduced in cartilage during osteoarthritis progression in both models. Severely damaged cartilage was negative for TGFβ3. In contrast, bone morphogenetic protein-2 (BMP-2) expression was increased. In chondrocyte clusters, preceding osteophyte formation, TGFβ3 and SMAD-2P were strongly expressed. In early osteophytes, TGFβ3 was found in the outer fibrous layer, in the peripheral chondroblasts and in the core. Late osteophytes expressed TGFβ3 only in the fibrous layer. SMAD-2P was found throughout the osteophyte at all stages. In the late-stage osteophytes, BMP-2 was strongly expressed. Conclusion: Data show that lack of TGFβ3 is associated with cartilage damage, suggesting loss of the protective effect of TGFβ3 during osteoarthritis progression. Additionally, our results indicate that TGFβ3 is involved in early osteophyte development, whereas BMP might be involved in late osteophyte development.

Anabolic and destructive mediators in osteoarthritis

Osteoarthritis is a joint disease that is characterized by focal degradation of articular cartilage. In addition to the degeneration of articular cartilage, attempts at repair are found in the affected tissue. Cartilage cells (chondrocytes) play a key role, not only in the destructive process, but a

Protection from interleukin 1 induced destruction of articular cartilage by transforming growth factor beta: studies in anatomically intact cartilage in vitro and in vivo.

The modulation of interleukin 1 (IL-1) effects on proteoglycan metabolism in intact murine patellar cartilage by transforming growth factor beta (TGF-beta) was investigated in vitro and in vivo. In vitro TGF-beta (400 pmol/l) had no effect on basal proteoglycan degradation. Proteoglycan degradation induced by IL-1, however, was suppressed by TGF-beta in serum free medium alone and in medium supplemented with 0.5 micrograms/ml insulin-like growth factor 1. This suggests a specific regulatory role for TGF-beta under pathological conditions. In contrast with the suppression of breakdown, synthesis of proteoglycans was stimulated by TGF-beta for both basal and IL-1 suppressed proteoglycan synthesis in cultures without insulin-like growth factor. In the presence of insulin-like growth factor no extra effect of TGF-beta on proteoglycan synthesis was observed. With insulin-like growth factor, however, TGF-beta potentiated the ex vivo recovery of IL-1 induced suppression of proteoglycan synthesis. Analogous to the in vitro effects, TGF-beta injected intraarticularly suppressed IL-1 induced proteoglycan degradation. Furthermore, TGF-beta injected into the joint counteracted IL-1 induced suppression of proteoglycan synthesis. This indicates that in vivo also TGF-beta can ameliorate the deleterious effects of IL-1 on the cartilage matrix.