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. 2021 Feb 20;3(2):100145.
doi: 10.1016/j.ocarto.2021.100145. eCollection 2021 Jun.

Iron triggers the early stages of cartilage degeneration in vitro: The role of articular chondrocytes

A V Ferreira  1   2 , T L Duarte  1   2 , S Marques  1   2 , P Costa  1   2 , S C Neves  2   3 , T Dos Santos  2   3 , P L Granja  2   3 , G Porto  1   2   4   5
Affiliations

Iron triggers the early stages of cartilage degeneration in vitro: The role of articular chondrocytes

A V Ferreira et al. Osteoarthr Cartil Open. .

Abstract

Objective: Arthropathy is a major clinical problem in patients with hemochromatosis, the most common genetic disorder of iron overload. The pathological features of hemochromatosis arthropathy (HA) are heterogeneous and its specific nature remains unknown. One important drawback is the lack of proper in vitro models. The aim of the present study was to set up a model to investigate the biological response of cartilage to iron exposure.

Design: Bovine articular cartilage explants were incubated with ferric citrate for up to 9 days. We evaluated chondrocyte viability, iron deposition, and biomarkers of cartilage degradation in the conditioned medium.

Results: Iron accumulated within chondrocytes, which was associated with programmed cell death through chondroptosis. Iron treatment increased the release of sulfated glycosaminoglycans (sGAG), a component of the extracellular matrix, into the medium (p=0.0189). This was dependent on the presence of viable chondrocytes and was associated with increased activity of matrix-degrading metalloproteinases (MMP) (pro/active MMP-9, p=0.0317; pro MMP-2, p=0.0092; active MMP-2, p=0.0288). Co-treatment with the broad MMP/aggrecanase inhibitor prinomastat reduced iron-mediated sGAG release (0.02 ​μM, p=0.0425; 2 ​μM, p=0.0014), confirming that iron induces sGAG release via the activation of catabolic enzymes. Notably, iron-treated cartilage continued to release an increased amount of sGAG into the medium for 6 days after termination of the ferric citrate treatment (p=0.0259).

Conclusions: Iron triggers the early stages of cartilage degeneration. Removal of iron exposure does not prevent further damage to the cartilage, thus providing a possible explanation why HA is not prevented after iron depletion by phlebotomy treatment.

Keywords: Arthropathy; Chondrocyte; Glycosaminoglycan; Hemochromatosis; Iron; Metalloproteinase.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Fig. 1
Fig. 1
Experimental design overview. Bovine articular cartilage explants were prepared, immediately transferred to culture medium and incubated at 37 ​°C in a 5% CO2 incubator. At the end of 24h, the culture medium was replaced by fresh medium containing 50 ​μM ferric citrate or sodium citrate (control), and explants were further incubated for 9 days. Medium was refreshed every third day and conditioned medium was collected for the measurement of biomarkers of cartilage degradation and MMP activity. At the end of the culture, explants were collected for analyses of cell viability and tissue iron accumulation.
Fig. 2
Fig. 2
Iron treatment leads to intracellular iron deposition and increased death of superficial chondrocytes. Cartilage explants were treated for 9 days with 50 ​μM ferric citrate or sodium citrate (control). A) Cartilage coronal sections were stained with Perls’ Prussian blue stain for ferric iron. Intracellular iron deposits were found exclusively in ferric citrate-treated cartilages, especially in chondrocytes located in the superficial zone. The image is representative of 3 independent experiments. Bar ​= ​200 ​μm ​B) Chondrocyte viability as assessed by the TUNEL assay. A significant increase in the percentage of dead (TUNEL-positive) cells was observed in iron-treated cartilage explants incubated with ferric citrate, when compared to control. The graph shows the change within the same experiment, and the magnitude of this change was assessed with the paired Student’s t-test (n=4 independent experiments). Bar=100 ​μm.
Fig. 3
Fig. 3
Iron loading induces morphological alterations in chondrocytes. Transmission electron microscopy images illustrate the ultrastructure of chondrocytes in freshly collected cartilage (A) or in cartilage cultured for 9 days with sodium citrate (control) (B) or ferric citrate (iron loading condition) (C–F). Whilst the morphology of control chondrocytes was indistinct from that of cells from freshly collected cartilage, iron-treated chondrocytes presented siderosomes (white arrows) and a number of morphological changes consistent with cell death by chondroptosis. (D) is a magnification of the area within the square in (C), showing a siderosome and abundant dilated cisternae of rough ER. A chondroptotic cell with convoluted nucleus with condensed chromatin, abundant Golgi and vacuoles (arrow heads), and extrusion of cellular material into the extracellular space (black arrows) is represented in (E). An empty lacuna is depicted in (F), where the remnants of a dead chondrocyte are identified by a dotted line. Samples were contrasted with uranile acetate and lead citrate. Original magnification: 15000 ​× ​. Bar=1 ​μm.
Fig. 4
Fig. 4
Iron differently affects the release of cartilage ECM constituents into the conditioned medium. Cartilage explants were treated for 9 days with 50 ​μM ferric citrate or sodium citrate (control). Hydroxyproline and sGAG were measured every 3 days and results were expressed as the cumulative fold change compared to day 0 (A–B) or as the release rate between days 0–9 (C). A) Hydroxyproline release into the culture medium was not significantly altered by iron treatment. Statistical significance was assessed by two-way repeated measures ANOVA (n=4 independent experiments). B) Iron treatment significantly increased the release of sGAG into the medium compared to control from day 6. Statistical significance was assessed by two-way repeated measures ANOVA (p=0.0189) with Sidak’s multiple comparisons test (respective p-values depicted) (n=5 independent experiments). C) Ferric citrate-induced sGAG release was abrogated upon co-incubation with the iron chelator desferrioxamine (DFO, 100 ​μM). Statistical significance was assessed by one-way repeated measures ANOVA with Sidak’s multiple comparisons test (n=5 independent experiments).
Fig. 5
Fig. 5
Iron-mediated s-GAG release requires a metabolically active cartilage and metalloproteinase activity. A) Cartilage explants were rendered metabolically inactive (by repetitive freeze-thawing) prior to incubation with 50 ​μM ferric citrate or sodium citrate (control) for 9 days. sGAG release was decreased when compared with the active cartilage, and iron increased sGAG release exclusively in metabolically active explants. Statistical significance was assessed by one-way repeated measures ANOVA with Sidak’s multiple comparisons test (n=3 independent experiments). B) Cartilage explants were incubated with 50 ​μM ferric citrate (FC) or sodium citrate (SC, control) for 9 days. MMP-2 and MMP-9 activity in the 9-day conditioned medium was measured by gelatine zymography (top panel). Iron significantly increased the gelatinolytic activity levels of both MMPs (bottom panels). The graphs show the change within the same experiment, and the magnitude of this change was assessed with the paired Student’s t-test (n=3 independent experiments). C) Cartilage explants were incubated with ferric citrate (FC) (50 ​μM) in the presence or absence of the metalloproteinase inhibitor prinomastat (AG-3340) (0, 0.02 or 2 ​μM) for 9 days. Prinomastat significantly reduced sGAG release. Statistical significance was assessed by one-way repeated measures ANOVA with Sidak’s multiple comparisons test (n=5 independent experiments).
Fig. 6
Fig. 6
Increased sGAG release persists after iron withdrawal from the culture medium. A) Cartilage explants were incubated with 50 ​μM ferric citrate or sodium citrate (SC, control) for 9 days, after which the iron-treated explants have either received 50 ​μM ferric citrate (FC, sustained iron loading condition) or sodium citrate (iron withdrawal condition) for an additional 6-day period. B) Cartilage coronal sections were stained with Perls’ Prussian blue stain for ferric iron. Sustained incubation with ferric citrate (FC→FC) led to iron deposition within chondrocytes throughout the whole cartilage, including the transitional and radial zones; chondrocytes submitted to iron withdrawal condition (FC→SC) remained iron loaded, especially in the superficial zone. Bar=200 ​μm (insets denoting iron deposits in superficial chondrocytes, bar=50 ​μm). C) Despite the removal of iron excess from the medium at day 9 in culture, the cartilage samples cultured under iron withdrawal conditions displayed increased sGAG release between days 9–15, when compared to control. Statistical significance was assessed by one-way ANOVA with Dunnett’s multiple comparisons test (n=3 independent experiments). D) Chondrocyte death was assessed with the TUNEL assay. Sustained iron exposure for 15 days increased the percentage of dead (TUNEL-positive) chondrocytes when compared to control, which did not appear to be prevented by iron withdrawal from day 9 (n=2 independent experiments).
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