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Cartilage degradation is fully reversible in the presence of aggrecanase but not matrix metalloproteinase activity.

Karsdal MA, Madsen SH, Christiansen C, Henriksen K, Fosang AJ, Sondergaard BC - Arthritis Res. Ther. (2008)

Bottom Line: Highly distinct protease activities were found with aggrecanase-mediated aggrecan degradation at early stages, whereas MMP-mediated aggrecan and collagen degradation occurred during later stages.By histology, we found a complete replenishment of glycosaminoglycan at early time points and pericellular localization at an intermediate time point.In contrast, only significantly increased collagen type II formation (200%; P < 0.01) was observed at early time points.

View Article: PubMed Central - HTML - PubMed

Affiliation: Nordic Bioscience A/S, Herlev Hovedgade 207, DK-2730 Herlev, Denmark. mk@nordicbioscience.com

ABSTRACT

Introduction: Physiological and pathophysiological cartilage turnover may coexist in articular cartilage. The distinct enzymatic processes leading to irreversible cartilage damage, compared with those needed for continuous self-repair and regeneration, remain to be identified. We investigated the capacity of repair of chondrocytes by analyzing their ability to initiate an anabolic response subsequent to three different levels of catabolic stimulation.

Methods: Cartilage degradation was induced by oncostatin M and tumour necrosis factor in articular cartilage explants for 7, 11, or 17 days. The catabolic period was followed by 2 weeks of anabolic stimulation (insulin growth factor-I). Cartilage formation was assessed by collagen type II formation (PIINP). Cartilage degradation was measured by matrix metalloproteinase (MMP) mediated type II collagen degradation (CTX-II), and MMP and aggrecanase mediated aggrecan degradation by detecting the 342FFGVG and 374ARGSV neoepitopes. Proteoglycan turnover, content, and localization were assessed by Alcian blue.

Results: Catabolic stimulation resulted in increased levels of cartilage degradation, with maximal levels of 374ARGSV (20-fold induction), CTX-II (150-fold induction), and 342FFGVG (30-fold induction) (P < 0.01). Highly distinct protease activities were found with aggrecanase-mediated aggrecan degradation at early stages, whereas MMP-mediated aggrecan and collagen degradation occurred during later stages. Anabolic treatment increased proteoglycan content at all time points (maximally, 250%; P < 0.001). By histology, we found a complete replenishment of glycosaminoglycan at early time points and pericellular localization at an intermediate time point. In contrast, only significantly increased collagen type II formation (200%; P < 0.01) was observed at early time points.

Conclusion: Cartilage degradation was completely reversible in the presence of high levels of aggrecanase-mediated aggrecan degradation. After induction of MMP-mediated aggrecan and collagen type II degradation, the chondrocytes had impaired repair capacity.

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Insulin growth factor (IGF) stimulates local replenishment of cartilage. Articular cartilage explants were cultured with either oncostatin M plus tumour necrosis factor (OSM + TNF) or vehicle for 7, 11, and 17 days. Subsequently, cartilage explants were paraffin-embedded and stained for aggrecan content as described in Materials and methods. Aggrecan is completely depleted from the tissue at 7, 11, and 17 days. Other cultures were treated with either OSM + TNF or vehicle for 7, 11, and 17 days followed by stimulation with either IGF or vehicle control for 14 days. Subsequently, cartilage explants were paraffin-embedded and stained for aggrecan content as described in Materials and methods. As a control experiment, articular cartilage explants were cultured for 21 days with vehicle, OSM + TNF, IGF, or metabolically inactive (MI) control for 21 days as controls (lower panel). W/O, without stimulation.
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Figure 5: Insulin growth factor (IGF) stimulates local replenishment of cartilage. Articular cartilage explants were cultured with either oncostatin M plus tumour necrosis factor (OSM + TNF) or vehicle for 7, 11, and 17 days. Subsequently, cartilage explants were paraffin-embedded and stained for aggrecan content as described in Materials and methods. Aggrecan is completely depleted from the tissue at 7, 11, and 17 days. Other cultures were treated with either OSM + TNF or vehicle for 7, 11, and 17 days followed by stimulation with either IGF or vehicle control for 14 days. Subsequently, cartilage explants were paraffin-embedded and stained for aggrecan content as described in Materials and methods. As a control experiment, articular cartilage explants were cultured for 21 days with vehicle, OSM + TNF, IGF, or metabolically inactive (MI) control for 21 days as controls (lower panel). W/O, without stimulation.

Mentions: To visualize the repair enhanced by IGF treatment, cultured cartilage was harvested at different time points. Proteoglycans in the cartilage were visualized using Alcian blue staining, the same dye used in the S-GAG assay. The control articular cartilage explants (shown in the bottom row of Figure 5) were cultured for 21 days with vehicle, OSM + TNF, IGF, or MI control for 21 days. In complete agreement with the S-GAG quantifications in Figure 2, IGF increased whereas OSM + TNF decreased GAG content compared with vehicle. MI control contained more GAG compared with vehicle as the cell-mediated loss of proteoglycan content was abrogated. With regard to the dynamics in the reversibility experiments presented in the upper panels, the vehicle control explants gradually lost S-GAG content over time, whereas the explants treated with IGF maintained the S-GAGs, even after 17 days in culture. OSM + TNF treatment depleted proteoglycans from the matrix maximally by day 7, consistent with the results in Figure 1c which show that S-GAG release into the medium is also maximal by day 7. Treatment with IGF stimulated GAG synthesis in the explants that were treated with cytokines for 7 and 11 days, but not for 17 days. IGF treatment of explants after 7 days of catabolic stimuli restored proteoglycan content throughout the entire cartilage matrix. IGF treatment after 11 days of catabolic treatment showed new proteoglycan synthesis around chondrocytes, indicative of repair. There was also evidence of repair in the absence of IGF treatment after 11 days of catabolic stimuli; however, the repair was substantially improved in the presence of IGF. Chondrocytes treated with catabolic cytokines for 17 days reinitiated, only to a very minor extent, proteoglycan synthesis in the presence of IGF compared with that of vehicle. These data support the idea that cartilage degradation may be more reversible before induction of MMP activity.


Cartilage degradation is fully reversible in the presence of aggrecanase but not matrix metalloproteinase activity.

Karsdal MA, Madsen SH, Christiansen C, Henriksen K, Fosang AJ, Sondergaard BC - Arthritis Res. Ther. (2008)

Insulin growth factor (IGF) stimulates local replenishment of cartilage. Articular cartilage explants were cultured with either oncostatin M plus tumour necrosis factor (OSM + TNF) or vehicle for 7, 11, and 17 days. Subsequently, cartilage explants were paraffin-embedded and stained for aggrecan content as described in Materials and methods. Aggrecan is completely depleted from the tissue at 7, 11, and 17 days. Other cultures were treated with either OSM + TNF or vehicle for 7, 11, and 17 days followed by stimulation with either IGF or vehicle control for 14 days. Subsequently, cartilage explants were paraffin-embedded and stained for aggrecan content as described in Materials and methods. As a control experiment, articular cartilage explants were cultured for 21 days with vehicle, OSM + TNF, IGF, or metabolically inactive (MI) control for 21 days as controls (lower panel). W/O, without stimulation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2483454&req=5

Figure 5: Insulin growth factor (IGF) stimulates local replenishment of cartilage. Articular cartilage explants were cultured with either oncostatin M plus tumour necrosis factor (OSM + TNF) or vehicle for 7, 11, and 17 days. Subsequently, cartilage explants were paraffin-embedded and stained for aggrecan content as described in Materials and methods. Aggrecan is completely depleted from the tissue at 7, 11, and 17 days. Other cultures were treated with either OSM + TNF or vehicle for 7, 11, and 17 days followed by stimulation with either IGF or vehicle control for 14 days. Subsequently, cartilage explants were paraffin-embedded and stained for aggrecan content as described in Materials and methods. As a control experiment, articular cartilage explants were cultured for 21 days with vehicle, OSM + TNF, IGF, or metabolically inactive (MI) control for 21 days as controls (lower panel). W/O, without stimulation.
Mentions: To visualize the repair enhanced by IGF treatment, cultured cartilage was harvested at different time points. Proteoglycans in the cartilage were visualized using Alcian blue staining, the same dye used in the S-GAG assay. The control articular cartilage explants (shown in the bottom row of Figure 5) were cultured for 21 days with vehicle, OSM + TNF, IGF, or MI control for 21 days. In complete agreement with the S-GAG quantifications in Figure 2, IGF increased whereas OSM + TNF decreased GAG content compared with vehicle. MI control contained more GAG compared with vehicle as the cell-mediated loss of proteoglycan content was abrogated. With regard to the dynamics in the reversibility experiments presented in the upper panels, the vehicle control explants gradually lost S-GAG content over time, whereas the explants treated with IGF maintained the S-GAGs, even after 17 days in culture. OSM + TNF treatment depleted proteoglycans from the matrix maximally by day 7, consistent with the results in Figure 1c which show that S-GAG release into the medium is also maximal by day 7. Treatment with IGF stimulated GAG synthesis in the explants that were treated with cytokines for 7 and 11 days, but not for 17 days. IGF treatment of explants after 7 days of catabolic stimuli restored proteoglycan content throughout the entire cartilage matrix. IGF treatment after 11 days of catabolic treatment showed new proteoglycan synthesis around chondrocytes, indicative of repair. There was also evidence of repair in the absence of IGF treatment after 11 days of catabolic stimuli; however, the repair was substantially improved in the presence of IGF. Chondrocytes treated with catabolic cytokines for 17 days reinitiated, only to a very minor extent, proteoglycan synthesis in the presence of IGF compared with that of vehicle. These data support the idea that cartilage degradation may be more reversible before induction of MMP activity.

Bottom Line: Highly distinct protease activities were found with aggrecanase-mediated aggrecan degradation at early stages, whereas MMP-mediated aggrecan and collagen degradation occurred during later stages.By histology, we found a complete replenishment of glycosaminoglycan at early time points and pericellular localization at an intermediate time point.In contrast, only significantly increased collagen type II formation (200%; P < 0.01) was observed at early time points.

View Article: PubMed Central - HTML - PubMed

Affiliation: Nordic Bioscience A/S, Herlev Hovedgade 207, DK-2730 Herlev, Denmark. mk@nordicbioscience.com

ABSTRACT

Introduction: Physiological and pathophysiological cartilage turnover may coexist in articular cartilage. The distinct enzymatic processes leading to irreversible cartilage damage, compared with those needed for continuous self-repair and regeneration, remain to be identified. We investigated the capacity of repair of chondrocytes by analyzing their ability to initiate an anabolic response subsequent to three different levels of catabolic stimulation.

Methods: Cartilage degradation was induced by oncostatin M and tumour necrosis factor in articular cartilage explants for 7, 11, or 17 days. The catabolic period was followed by 2 weeks of anabolic stimulation (insulin growth factor-I). Cartilage formation was assessed by collagen type II formation (PIINP). Cartilage degradation was measured by matrix metalloproteinase (MMP) mediated type II collagen degradation (CTX-II), and MMP and aggrecanase mediated aggrecan degradation by detecting the 342FFGVG and 374ARGSV neoepitopes. Proteoglycan turnover, content, and localization were assessed by Alcian blue.

Results: Catabolic stimulation resulted in increased levels of cartilage degradation, with maximal levels of 374ARGSV (20-fold induction), CTX-II (150-fold induction), and 342FFGVG (30-fold induction) (P < 0.01). Highly distinct protease activities were found with aggrecanase-mediated aggrecan degradation at early stages, whereas MMP-mediated aggrecan and collagen degradation occurred during later stages. Anabolic treatment increased proteoglycan content at all time points (maximally, 250%; P < 0.001). By histology, we found a complete replenishment of glycosaminoglycan at early time points and pericellular localization at an intermediate time point. In contrast, only significantly increased collagen type II formation (200%; P < 0.01) was observed at early time points.

Conclusion: Cartilage degradation was completely reversible in the presence of high levels of aggrecanase-mediated aggrecan degradation. After induction of MMP-mediated aggrecan and collagen type II degradation, the chondrocytes had impaired repair capacity.

Show MeSH
Related in: MedlinePlus