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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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Quantification of aggrecan within the articular cartilage explants. The proteins of the cultured explants were extracted by liquid N2 pulverization. (a) Cartilage was extracted immediately after isolation (t = 0) or after culture for 21 days with vehicle, insulin growth factor (IGF), oncostatin M plus tumour necrosis factor (OSM + TNF), or metabolically inactive (MI) control for assessing passive physiochemical release. (b) Cartilage was extracted after the three different levels of cytokine treatment followed by an identical 14 days with either vehicle or IGF. IGF significantly stimulated proteoglycan content within the cartilage explants at all time points. (c) Quantification of sulphated glycosaminoglycan (S-GAG) from all treatments over the entire experimental period. S-GAG released from cartilage explants to the conditioned medium was quantified by the Alcian blue-binding assay. The curves represent the release at days when the conditioned medium was fully replaced, and the values were accumulated over the entire period. MI, metabolically inactive; O + T, oncostatin M plus tumour necrosis factor; W/O, without stimulation (vehicle control). (d) Quantification of S-GAG turnover 2 weeks after the catabolic induction. The aggrecan release in the identical 14-day period, with or without IGF stimulation following three different periods of catabolic stimulation, was measured by the Alcian blue-binding assay. The results show the accumulated release of S-GAG during the 2 weeks with anabolic stimulation (IGF) and without stimulation (vehicle). *P < 0.05, **P < 0.01, ***P < 0.001.
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Figure 1: Quantification of aggrecan within the articular cartilage explants. The proteins of the cultured explants were extracted by liquid N2 pulverization. (a) Cartilage was extracted immediately after isolation (t = 0) or after culture for 21 days with vehicle, insulin growth factor (IGF), oncostatin M plus tumour necrosis factor (OSM + TNF), or metabolically inactive (MI) control for assessing passive physiochemical release. (b) Cartilage was extracted after the three different levels of cytokine treatment followed by an identical 14 days with either vehicle or IGF. IGF significantly stimulated proteoglycan content within the cartilage explants at all time points. (c) Quantification of sulphated glycosaminoglycan (S-GAG) from all treatments over the entire experimental period. S-GAG released from cartilage explants to the conditioned medium was quantified by the Alcian blue-binding assay. The curves represent the release at days when the conditioned medium was fully replaced, and the values were accumulated over the entire period. MI, metabolically inactive; O + T, oncostatin M plus tumour necrosis factor; W/O, without stimulation (vehicle control). (d) Quantification of S-GAG turnover 2 weeks after the catabolic induction. The aggrecan release in the identical 14-day period, with or without IGF stimulation following three different periods of catabolic stimulation, was measured by the Alcian blue-binding assay. The results show the accumulated release of S-GAG during the 2 weeks with anabolic stimulation (IGF) and without stimulation (vehicle). *P < 0.05, **P < 0.01, ***P < 0.001.

Mentions: All cell cultures with bovine articular cartilage explants were approved by the local ethics committee. Articular cartilage explants were stimulated for 7, 11, or 17 days with the cytokines OSM (10 ng/mL) and TNF (20 ng/mL). Each catabolic period was followed by either (a) no stimulation or (b) (100 ng/mL) IGF stimulation for 2 weeks, resulting in total culture times of 21, 25, or 31 days (Figure 1c). Between the catabolic and anabolic phases, the explants were washed three times in PBS. On the last day of culture, samples from each treatment were either formaldehyde-fixed or snap-frozen. For other controls, additional samples were cultured for either 7, 11, or 17 days and treated without stimulation (vehicle), OSM + TNF, and IGF, and these samples were also formaldehyde-fixed and snap-frozen. Control treatments were analyzed in parallel on the same plate for vehicle, MI, (100 ng/mL) IGF, and OSM (10 ng/mL) + TNF (20 ng/mL) for 21 days and, on the last day, were formaldehyde-fixed or frozen for protein extraction. All treatment conditions were refreshed three times a week with freshly prepared medium plus stimulants. The conditioned medium was collected and stored at -20°C for further analysis. The use of MI cartilage as a control serves to control for the passive physical-chemical release of proteins and other molecules into the culture medium. Thereby, the difference between MI and vehicle is the cell-mediated release.


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)

Quantification of aggrecan within the articular cartilage explants. The proteins of the cultured explants were extracted by liquid N2 pulverization. (a) Cartilage was extracted immediately after isolation (t = 0) or after culture for 21 days with vehicle, insulin growth factor (IGF), oncostatin M plus tumour necrosis factor (OSM + TNF), or metabolically inactive (MI) control for assessing passive physiochemical release. (b) Cartilage was extracted after the three different levels of cytokine treatment followed by an identical 14 days with either vehicle or IGF. IGF significantly stimulated proteoglycan content within the cartilage explants at all time points. (c) Quantification of sulphated glycosaminoglycan (S-GAG) from all treatments over the entire experimental period. S-GAG released from cartilage explants to the conditioned medium was quantified by the Alcian blue-binding assay. The curves represent the release at days when the conditioned medium was fully replaced, and the values were accumulated over the entire period. MI, metabolically inactive; O + T, oncostatin M plus tumour necrosis factor; W/O, without stimulation (vehicle control). (d) Quantification of S-GAG turnover 2 weeks after the catabolic induction. The aggrecan release in the identical 14-day period, with or without IGF stimulation following three different periods of catabolic stimulation, was measured by the Alcian blue-binding assay. The results show the accumulated release of S-GAG during the 2 weeks with anabolic stimulation (IGF) and without stimulation (vehicle). *P < 0.05, **P < 0.01, ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Quantification of aggrecan within the articular cartilage explants. The proteins of the cultured explants were extracted by liquid N2 pulverization. (a) Cartilage was extracted immediately after isolation (t = 0) or after culture for 21 days with vehicle, insulin growth factor (IGF), oncostatin M plus tumour necrosis factor (OSM + TNF), or metabolically inactive (MI) control for assessing passive physiochemical release. (b) Cartilage was extracted after the three different levels of cytokine treatment followed by an identical 14 days with either vehicle or IGF. IGF significantly stimulated proteoglycan content within the cartilage explants at all time points. (c) Quantification of sulphated glycosaminoglycan (S-GAG) from all treatments over the entire experimental period. S-GAG released from cartilage explants to the conditioned medium was quantified by the Alcian blue-binding assay. The curves represent the release at days when the conditioned medium was fully replaced, and the values were accumulated over the entire period. MI, metabolically inactive; O + T, oncostatin M plus tumour necrosis factor; W/O, without stimulation (vehicle control). (d) Quantification of S-GAG turnover 2 weeks after the catabolic induction. The aggrecan release in the identical 14-day period, with or without IGF stimulation following three different periods of catabolic stimulation, was measured by the Alcian blue-binding assay. The results show the accumulated release of S-GAG during the 2 weeks with anabolic stimulation (IGF) and without stimulation (vehicle). *P < 0.05, **P < 0.01, ***P < 0.001.
Mentions: All cell cultures with bovine articular cartilage explants were approved by the local ethics committee. Articular cartilage explants were stimulated for 7, 11, or 17 days with the cytokines OSM (10 ng/mL) and TNF (20 ng/mL). Each catabolic period was followed by either (a) no stimulation or (b) (100 ng/mL) IGF stimulation for 2 weeks, resulting in total culture times of 21, 25, or 31 days (Figure 1c). Between the catabolic and anabolic phases, the explants were washed three times in PBS. On the last day of culture, samples from each treatment were either formaldehyde-fixed or snap-frozen. For other controls, additional samples were cultured for either 7, 11, or 17 days and treated without stimulation (vehicle), OSM + TNF, and IGF, and these samples were also formaldehyde-fixed and snap-frozen. Control treatments were analyzed in parallel on the same plate for vehicle, MI, (100 ng/mL) IGF, and OSM (10 ng/mL) + TNF (20 ng/mL) for 21 days and, on the last day, were formaldehyde-fixed or frozen for protein extraction. All treatment conditions were refreshed three times a week with freshly prepared medium plus stimulants. The conditioned medium was collected and stored at -20°C for further analysis. The use of MI cartilage as a control serves to control for the passive physical-chemical release of proteins and other molecules into the culture medium. Thereby, the difference between MI and vehicle is the cell-mediated release.

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