Coculture of bovine cartilage with synovium and fibrous joint capsule increases aggrecanase and matrix metalloproteinase activity
Arthritis Research & Therapy volume 19, Article number: 157 (2017)
A hallmark of osteoarthritis is increased proteolytic cleavage of aggrecan. Cross talk between cartilage and the synovium + joint capsule (SJC) can drive cartilage degradation by activating proteases in both tissues. We investigated aggrecan proteolysis patterns in cartilage explants using a physiologically relevant explant model of joint injury combining cartilage mechanical compression and coincubation with SJC.
Bovine cartilage explants were untreated; coincubated with SJC; or subjected to mechanical injury and coincubated with SJC, mechanical injury alone, or mechanical injury and incubated with tumor necrosis factor-α (TNF-α). To compare the patterns of aggrecan proteolysis between 6 h and 16 days, release of sulfated glycosaminoglycans and specific proteolytic aggrecan fragments into medium or remaining in cartilage explants was measured by dimethylmethylene blue and Western blot analysis.
Aggrecanase activity toward aggrecan was observed in all conditions, but it was directed toward the TEGE↓ARGS interglobular domain (IGD) site only when cartilage was coincubated with SJC or TNF-α. Matrix metalloproteinase (MMP) activity at the aggrecan IGD site (IPES↓FFGV) was not detected when cartilage was exposed to TNF-α (up to 6 days), but it was in all other conditions. Compared with when bovine cartilage was left untreated or subjected to mechanical injury alone, additional aggrecan fragment types were released into medium and proteolysis of aggrecan started at an earlier time when SJC was present.
Indicative of different proteolytic pathways for aggrecan degradation, the SJC increases both aggrecanase and MMP activity toward aggrecan, whereas TNF-α inhibits MMP activity against the IGD of aggrecan.
Articular cartilage is a dynamic tissue in synovial joints that can withstand substantial loads within physiological levels [1, 2]. Aggrecan, the major proteoglycan of cartilage, is substituted with negatively charged sulfated glycosaminoglycans (sGAGs) and forms large aggregates by binding to hyaluronan . The negative charge density induces a high osmotic swelling pressure within cartilage, restrained by the collagen fibril network . These osmotic and electrostatic repulsive interactions provide more than 50% of cartilage’s equilibrium compressive modulus. In addition, the closely spaced sGAG chains resist fluid flow caused by dynamic compression quantified at both the molecular level  and the tissue level . The resulting intratissue pressurization caused by dynamic compression greatly increases cartilage’s dynamic modulus at high loading rates. Proteolytic cleavage of aggrecan is an important feature in osteoarthritis (OA) [6,7,8]. Aggrecanase-1 and aggrecanase-2 (a disintegrin and metalloproteinase with thrombospondin motifs 4 and 5 [ADAMTS4 and ADAMTS5, respectively]) and the matrix metalloproteinases (MMP-1, MMP-3, MMP-8, MMP-9, and MMP-13) are believed to be the most important enzymes responsible for the proteolytic degradation of aggrecan .
A severe joint injury leads to an increased risk of developing posttraumatic osteoarthritis (PTOA) . PTOA pathogenesis is multifactorial, and many risk factors associated with developing nontraumatic OA, such as age, obesity, and genetic variation, may also apply to PTOA . With regard to knee OA, the risk is related to the integrity of the menisci; to associated compressive injuries to ligaments, cartilage, and bone; and to joint synovitis [11,12,13,14]. In the acute phase of injury, impact forces applied over the knee joint in combination with hemarthrosis lead to activation and recruitment of immune cells to the knee joint, producing an inflammatory and procatabolic joint environment. Several reports have demonstrated rapid increases in pro- and anti-inflammatory cytokines, proteases, and proteolytic activity after a severe knee injury [15,16,17,18,19,20,21,22]. Cells of the synovium and fibrous joint capsule (referred to hereafter as the synovium + joint capsule [SJC]), comprising resident inflammatory cells, synovial cells, and other cells such as endothelial cells and blood leukocytes, contribute to joint inflammation by producing proinflammatory mediators and proteases such as aggrecanases and MMPs [23, 24]. Matrix molecule fragments and cell debris released into the joint after trauma lead to activation of various cells in the cartilage and the SJC, further increasing the proinflammatory response . In the long term, imbalance between pro- and anticatabolic activities may lead to progressive cartilage extracellular matrix degradation and development of OA [15, 19, 25,26,27,28,29].
In the present in vitro study, we investigated differences in the preferred enzymatic cleavage of aggrecan in bovine cartilage explants cocultured with or without SJC, cartilage exposed to mechanical trauma, or cartilage exposed to exogenous tumor necrosis factor-α (TNF-α). We hypothesized that in this coculture system, there is cross talk between traumatized cartilage and the SJC through cell- and matrix-derived factors, leading to increased aggrecanase and MMP cleavage of the cartilage aggrecan. To quantify aggrecanase and/or MMP activity toward aggrecan in these different conditions, we used a set of well-characterized antibodies directed at different cleavage sites in the aggrecan molecule.
Bovine cartilage and SJC harvesting
The methods of cartilage explant acquisition for the experiment are described elsewhere . In brief, articular cartilage discs were harvested from the femoropatellar grooves of 1- to 2-week-old calves obtained on the day of slaughter (Research 87, Boylston, MA, USA). Full-thickness cartilage cylinders were cored using a 3-mm dermal punch, and the top 1-mm disc containing an intact superficial zone was harvested with a blade. Discs were preincubated in serum-free medium (low-glucose DMEM [1 g/L]) supplemented with 10 mM HEPES buffer, 0.1 mM nonessential amino acids, 0.4 mM proline, 20 μg/ml ascorbic acid, 100 U/ml penicillin G, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B for 2–3 days (5% CO2, 37 °C). Full-thickness explant discs inclusive of the fibrous joint capsule and the synovium were harvested  from the same animals’ knees using a 5-mm dermal punch and were preincubated separately in serum-free medium for 2–3 days. The tissue consisted of the fibrous joint capsule with a single layer of synovium varying in thickness from 0.5 to 3 mm .
Cartilage injury and treatment conditions
After preequilibration, six cartilage discs per condition were treated according to one of the following conditions: (A) untreated (uninjured cartilage), (B) mechanically injured (injured cartilage), (C) coincubated with SJC (uninjured cartilage + SJC), (D) mechanically injured and coincubated with SJC (injured cartilage + SJC), or (E) mechanically injured and incubated with exogenous TNF-α at 25 ng/ml (injured + TNF-α), and (F) six SJC explants were also cultured alone (SJC alone) . Tissues and medium were removed from culture at different incubation times ranging from 6 h to 16 days (Table 1). Explant and SJC samples were harvested from six joints of three animals. One cartilage explant from one joint of one animal was cultured alone (conditions A, B, and E) or in the same well as an SJC explant from the same joint (conditions C and D). In condition F, separate SJC specimens of were cultured. There were 6 cartilage explants used for each time point, giving us 36 cartilage explants for the 6 time points in each condition (A–D) plus separate specimens for SJC (conditions C and D). In conditions E and F, six explants were cultured for 6 and 16 days, respectively. Medium was changed (300 μl) after 6 h and 2 days of incubation and thereafter every 2 days until the end of incubation (Table 1).
In conditions involving mechanical injury, injurious unconfined compression to a final strain of 50% at a strain rate of 100%/second was applied to the cartilage (conditions B and D) using a custom-designed loading apparatus just prior to the start of culture [30, 33]. Purified bovine aggrecan (A1D1 fraction, see below) was incubated (0.22 μg sGAG/μl) at 37 °C in serum-free medium for 1 h or 24 h in the presence or absence of bovine SJC (67 μg wet wt/μl) and TNF-α (10 pg/ml). After incubation, the aggrecan solution and tissue were frozen separately.
Purification of proteoglycans and aggrecan
Proteoglycans (including full-length aggrecan and proteolytic fragments of aggrecan) were purified by guanidine extraction from all cartilage discs and SJCs. The discs (approximately 10 mg each) were extracted by stirring at 4 °C in a total of 17 μl of extraction buffer (50 mM Na+-acetate, 4 M guanidinium HCl, 10 mM ethylenediaminetetraacetic acid [EDTA], 100 mM 6-aminocaproic acid, 10 mM N-ethylmaleimide, 5 mM benzamidine HCl, 1 mM phenylmethanesulfonyl fluoride, pH 6) per milligram of tissue. Thereafter, the guanidine-extracted proteoglycan sample was centrifuged (20,000 × g, 20 minutes at 4 °C) to collect the supernatant, which was precipitated for 1 h at −20 °C with 5 vol of ice-cold acetone. The proteoglycan pellets were then collected by centrifugation (20,000 × g, 20 minutes at 4 °C) and dissolved in deglycosylation buffer (50 mM Tris-acetate, 50 mM Na+-acetate, 10 mM EDTA, pH 7.6). In separate tests, aggrecan was purified from freshly harvested, untreated bovine calf femoropatellar groove cartilage samples that were immediately frozen after removal from the same knee joints as the treated cartilage explants. Briefly, proteoglycans were first purified by guanidine extraction (as described above), and aggrecan was then purified by CsCl density centrifugation to collect the A1D1 fraction .
Analysis of sGAG and aggrecan
Culture medium, guanidine-extracted proteoglycan samples from cartilage, and SJCs were analyzed for sGAG content using 1,9-dimethylmethylene blue (DMMB) , with the following changes: 20-μl samples or standards (chondroitin sulfate [CS], catalogue number C4384; Sigma-Aldrich, St. Louis, MO, USA) were mixed on a 96-well microtiter plate with 200 μl of DMMB, and absorbance was measured at 520 nm with a plate reader.
For Western blot analysis, samples were deglycosylated as described elsewhere , with the exception that incubation was performed with lower keratanase II concentrations but for a longer time with chondroitinase ABC (EC 220.127.116.11, 2 h at 37 °C, 1 mU/mg sGAG; Sigma-Aldrich), keratanase (EC 18.104.22.168, 1 h at 37 °C, 1 mU/mg sGAG; Seikagaku, Tokyo, Japan), and keratanase II (Bacillus sp. Ks36, 3 h at 37 °C, 0.01 mU/mg sGAG; Seikagaku). Deglycosylated samples were precipitated with 5 vol of ice-cold acetone, and proteins were collected by centrifugation (20,000 × g, 20 minutes at 4 °C) and dissolved in 40–50 μl of 2× concentrated sample buffer (SB) (NuPAGE Novex; Life Technologies, Carlsbad, CA, USA) in the following conditions: 4 μl of medium/1 μl of SB, 10–150 μg wet wt cartilage/1 μl of SB, and 80–200 μg wet wt SJC/1 μl of SB.
For quantitative Western blotting of aggrecan fragments, purified calf cartilage aggrecan A1D1 fraction (8 standard points per gel; 0.02–0.48 μg of sGAG loaded per well) was used as the standard for aggrecan G3 fragments . Calf cartilage aggrecan A1D1 fraction (500 μg) was digested for 24 h by ADAMTS4 (0.5 μg)  (6 standard points per gel; 0.01–0.36 μg of sGAG loaded per well) and was used as the standard for ARGS-aggrecan fragments. Calf cartilage aggrecan A1D1 fraction (100 μg) was digested for 24 h by MMP-3 (1 μg)  (7 standard points per gel [0.002–0.018 μg of sGAG loaded per well]) and was used as the standard for FFGV-aggrecan fragments. Because KEEE standards were not available, calf cartilage aggrecan A1D1 fraction (80 μg) was digested 10 minutes with ADAMTS4 (0.08 μg) for use as a control sample (1 μg of sGAG loaded per well three times) for quantification of KEEE fragments, expressing the data as arbitrary units of the control sample. All standards were deglycosylated. FFGV, ARGS, KEEE, and G3 aggrecan fragments were quantified by Western blot analysis (using the LAS-1000 imaging system and Image Gauge version 3.2 software [FUJIFILM Medical Systems, Stamford, CT, USA], with settings of regions of interest done by one experienced operator [MH]) in medium samples (i.e., six samples per time point and condition). On the basis of the film images of these experiments, an experienced operator (AS) determined the presence and absence of bands and the time point when a band was first observed.
Immunoreactions were done  using anti-ARGS (neoepitope monoclonal OA-1 antibody, 5.3–14 μg/ml; GlaxoSmithKline), anti-AGEG (neoepitope sera, 1:500; GlaxoSmithKline), anti-FFGV (neoepitope monoclonal AF-28 antibody, 0.5 μg/ml; EMD Millipore, Billerica, MA, USA), anti-G3 (polyclonal antibody, 5 μg/ml, PA1-1745; Pierce Biotechnology, Rockford, IL, USA), anti-KEEE (neoepitope sera, 1:1000), anti-LGQR (neoepitope sera, 1:500), anti-ARLE (neoepitope sera, 1:2000), anti-G1 (sera, 1:5000) against G1 sequence ATEGQVRVNSIYQDKVSL, together with secondary peroxidase-conjugated antibodies of horse antimouse immunoglobulin G (IgG) (1:10,000–1:25,000, catalogue number 7076; Cell Signaling Technology, Danvers, MA, USA) and goat antirabbit IgG (20 ng/ml, catalogue number 074-1516; Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA). The immunobands were visualized using ECL 2 Western Blotting substrate (Pierce Biotechnology) together with a luminescence image analyzer (LAS-1000) and film (Amersham Hyperfilm ECL; GE Healthcare Life Sciences, Little Chalfont, UK). Western blotting was done to verify immunoband blocking of antibody reactions . All the bovine aggrecan fragments detected by Western blot analysis in the medium and cartilage plugs in this study are described in Additional file 1 and are illustrated (Fig. 1 and Additional file 1: Figure S1).
The ratios of FFGV, ARGS, G1-KEEE, and G1–G3 fragments in medium to the total amount of aggrecan (sGAG left in the cartilage explant plus sGAG released into the medium during the 16 days of culture) in the explant system were calculated. The total amount of sGAG released into the medium was calculated as the cumulative amount of sGAG detected in the medium changed every 2 days over the 16 days of incubation. Because of the low number of animals (n = 3 cows), a normal distribution was assumed but not tested. All tests were performed using the paired-samples Student’s t test of statistical significance (IBM SPSS Statistics version 20 software; IBM, Armonk, NY, USA). A P value <0.05 was considered statistically significant. The number of animals used in the statistical analysis was 3, where n = 1 was defined as the mean amount of aggrecan fragment from two cartilage explants (one from each separate knee joint) of the same cow. This approach accounts for intra- and interanimal variability. When the quantified amount was below the limit of detection, it was set to half the lowest standard point before statistical testing.
Aggrecan left in cartilage or released into medium
We used sGAG as a measure of total aggrecan content. The amount of sGAG extracted from the SJC or released into medium from SJC explants was very low: approximately 200-fold less than cartilage explants (Fig. 2a). Hence, the contribution of aggrecan from the SJC to the explant system was considered negligible in the present study.
The total amount of sGAG (sum of what was left in cartilage and released into the medium) increased from 6 h to day 16 in conditions A (uninjured cartilage; 1.7-fold), B (injured cartilage; 1.6-fold), and C (uninjured cartilage + SJC; 1.4-fold), but not in condition D (injured cartilage + SJC; 0.95-fold) (Fig. 2a), suggesting de novo synthesis of aggrecan in conditions A–C during the culture period. However, full-length G1–G3 aggrecan present in the cartilage, quantified by Western blot analysis, decreased by between 57% and 66% from 6 h to day 16 in all conditions (data not shown). This suggests that a substantial fraction of full-length aggrecan present at the start of the experiment was either released from the cartilage or C-terminally truncated, thus missing the G3 domain.
The accumulated release of sGAG into medium over the 16 days of incubation was similar in conditions A–D (with or without injury, with or without SJC). After 6 days of culture, the release of sGAG into medium was approximately 50 μg in conditions A–D, whereas the corresponding amount for condition E (injured cartilage + TNF-α) was substantially higher at about 270 μg (P ≤ 0.022 for all) (Fig. 2b). In summary, culture of cartilage explants over 16 days was associated with de novo aggrecan production, except for conditions where cartilage was mechanically injured and cultured in the presence of SJC.
Enzymatically cleaved aggrecan fragments detected in medium
Although total amounts of bovine aggrecan released from the cartilage into the medium (approximated as total sGAG) did not differ between the culture conditions A–D (Fig. 2b), analysis of the time-dependent release of bovine aggrecan fragments revealed striking differences between these conditions (Table 2 and Additional file 2: Figure S2). Aggrecan fragments with different proteolytic cleavage sites were generated and released into the medium when the cartilage was coincubated with SJC (i.e., 18 fragments in condition C and 19 fragments in condition D compared with without SJC [A and B] with 12 fragments in each condition) (Table 2). For example, aggrecanase-generated ARGS and ARLE-G3 fragments were observed only in medium from culture conditions in which SJC was present or when exogenous TNF-α was added (E). MMP-generated FFGV fragments were observed in all culture conditions, except when exogenous TNF-α was added (E) (Table 2 and Additional file 2: Figure S2). Conditions in which injury was applied to cartilage (B and D) did not generate different types of aggrecan fragments compared with conditions without applied injury (A vs. B and C vs. D).
Together, these results suggest that coincubation with SJC generates an increased number of different types of aggrecan fragments compared with conditions without SJCs and that cartilage injury does not increase the number of different aggrecan fragments.
Initiation and preferred order of enzymatic aggrecan cleavage
Compared with culture conditions without SJC (A and B), cartilage cultured in the presence of SJC (C and D) showed a more rapid aggrecan degradation, observed as an earlier release of several types of aggrecan fragments into the medium (Table 2). No clear differences in the initiation or the preferred order of cleavage of aggrecan were detected between untreated and mechanically injured cartilage (A and B). For all explant conditions, except when injured cartilage was cultured in the presence of TNF-α (E), the earliest aggrecan cleavage was found at the aggrecanase sites GELE↓GRGT and KEEE↓GLGS in the CS2 region, based on analysis of the GRGT-KEEE and G1-KEEE fragments, and at the MMP site IPES↓FFGV in the interglobular domain (IGD), based on analysis of the FFGV-G3 fragment. Cuts at these sites were observed 6 h to 1 day after the start of the incubation (Table 2). Early aggrecan digestion (6 h to 2 days) was also observed for the MMP cuts in the CS1 region (based on analysis of the CS1-KEEE fragments). The cleavage of aggrecan at the aggrecanase site TEGE↓ARGS in the IGD was detected much later, after 6–8 days of incubation (C and D). Together, these results suggest that in these explant systems, aggrecan degradation starts earlier in time in the presence of SJC, and the most preferred cleavage sites are the aggrecanase sites in the CS2 regions and the MMP site in the IGD, whereas the least preferred cleavage site is the aggrecanase site in the IGD.
Quantitative analyses of aggrecanase- and MMP-generated aggrecan fragments in medium
After 16 days of culture, the mean accumulated release of FFGV into medium was higher from injured cartilage (B, 1.4-fold) and from injured cartilage cocultured with SJC (D, 4.6-fold) than from uninjured cartilage (A) (Table 3, Fig. 3). The mean accumulated release of ARGS was 4.8-fold higher from injured cartilage cocultured with SJC than the release from uninjured cartilage (D vs. A) and 4.6-fold higher than the release from injured cartilage (D vs. B) (Table 3, Fig. 3b). Owing to large variations in ARGS concentrations between the cartilage plugs (Additional file 3: Table S1), no statistically significant differences were observed between uninjured cartilage cocultured with SJC (C) and conditions A and B (Table 3), even though visual differences were observed on the Western blots (Fig. 3b, Additional file 2: Figure S2). The mean accumulated release of G1-KEEE was higher in conditions C (9- and 10-fold) and D (28- and 25-fold) than in condition A and condition B (Table 3, Fig. 3c). There were no statistically significant differences in the mean accumulated release of G1–G3 between any of the conditions (Table 3, Fig. 3d).
These results together suggest that increased levels of aggrecanase-generated ARGS and KEEE fragments, as well as MMP-generated FFGV fragments, are released into medium when cartilage is coincubated with SJC compared with when SJC is absent. An increased release of MMP-generated FFGV fragments into medium was also observed when cartilage was mechanically injured compared with when cartilage was left untreated.
The SJC produces active aggrecanases and MMPs
Incubation of purified bovine aggrecan with bovine SJC for 1 h induced aggrecan cleavage at the MMP site IPES↓FFGV in the IGD, seen as an FFGV-G3 fragment (Fig. 4a), and cleavage at the aggrecanase site GELE↓GRGT in the CS2 region, seen as a weak GRGT-G3 fragment (Fig. 4c). Incubation for 24 h further processed the FFGV-G3 to a FFGV-CS2 fragment (Fig. 4a), induced cleavage at the aggrecanase site TEGE↓ARGS in the IGD, seen as ARGS-CS1/CS2 fragments (Fig. 4b), and further induced aggrecanase cleavage in the CS2 region, generating AGEG-G3, LGQR-G3, and ARLE-G3 fragments (Fig. 4c). Addition of TNF-α to these incubations increased the aggrecanase activity, seen either as faster generation of fragments (AGEG-G3 and LGQR-G3) (Fig. 4c) or as more intense immunobands (ARGS-CS1/CS2 and GRGT-G3) (Fig. 4b and c). This increase in protease activity was not observed for MMP activity (Fig. 4a). These results suggest that the SJC produces both active aggrecanases and MMPs and that addition of TNF-α increases the induction of aggrecanases.
Proteolysis of human aggrecan showing the fragmentology of aggrecan has been described previously [8, 9, 34, 38], but details such as those in this study, showing aggrecan fragments present in cartilage and released into medium, have not been described to date, to our knowledge. Our findings suggest that coincubation of cartilage with SJC leads to increased proteolytic activity of both MMPs and aggrecanases against aggrecan. These quantitative findings were supported by the qualitative analysis of aggrecan fragments in medium, showing that conditions where the SJC was present were associated with additional enzymatically cleaved aggrecan fragment types released into medium and that proteolytic cleavage of aggrecan started earlier in time in these conditions. Mechanical injury of cartilage alone was associated with a small increase in the amount of FFGV fragments released into the medium, suggesting increased MMP activity toward aggrecan compared with the uninjured control.
Whereas high aggrecanase activity against the IGD cleavage site, as seen by Western blotting, was observed in cartilage stimulated by mechanical injury + TNF-α, no MMP activity against the IGD was observed in this condition during the 6 days of culture. This novel finding suggests that the activated proteolytic pathways are different between the TNF-α-stimulated cartilage and when cartilage is cultured alone, mechanically traumatized, or coincubated with SJC.
Activation of inflammatory pathways may be involved in the pathogenesis of OA and has been shown to associate with pain and to be prognostic of OA progression [14, 39]. As this study and others have shown, the synovial cells and/or other cells of the fibrous joint capsule may induce aggrecan cleavage by producing proteases themselves and could, by producing inflammatory mediators, induce chondrocytes to produce aggrecanases and MMPs [23, 26]. In the sequelae of severe joint injury, the activation of Toll-like receptors (TLRs) by matrix fragments may induce a vicious cycle by downstream activation of inflammation pathways [40, 41]. Aggrecan fragments may also contribute to continued activation of inflammatory pathways, and a 32-mer aggrecan peptide was recently shown to have antianabolic, procatabolic, and proinflammatory bioactivity mediated through TLR2 and nuclear factor-κB . Importantly, with regard to the findings of the present study, cells other than synoviocytes (leukocytes, macrophages, fibroblasts, and endothelial cells) reside in the matrix underlying the synovial lining and could contribute to increased protease activity in the cartilage explant in vitro system [43, 44].
Our results extend previous findings suggesting that cross talk between cartilage and SJC could increase aggrecanase activity in the cartilage [23, 45] by showing that coincubation of mechanically injured cartilage with SJC increases both MMP and aggrecanase activity. We also found that the SJC produces active aggrecanases and MMPs. Our study further extends previous investigations by showing that the coincubation of cartilage with SJC induces not only aggrecan cleavage in the IGD (generating ARGS-CS2 fragments) [45, 46] but also proteolytic cleavage between CS2 and G3 (generating ARLE-G3 fragments), and leads to a higher degree of cleavage in the CS2 region (generating G1-KEEE fragments). This is important, considering that aggrecan degradation in this explant study, and in other in vitro studies [38, 47], starts with cleavage in the CS region followed by cleavage in the IGD.
Interestingly, the findings of the present study indicate that mechanical injury to cartilage alone, previously shown to induce increased MMP and aggrecanase gene expression (250-fold and 40-fold, respectively) , did not increase aggrecanase-mediated digestion of aggrecan. This novel observation suggests that additional soluble factors from the SJC are essential to increasing aggrecanase degradative activity toward aggrecan in the cartilage. We also found evidence of more MMP activity in mechanically injured cartilage with or without coincubation with SJC compared with cartilage cultured alone. These findings are consistent with previous studies which have shown that the combination of mechanical injury of cartilage plus incubation with inflammatory cytokines led to the release of significantly greater amounts of sGAG and specific mass spectrometry-detected matrix fragments (e.g., aggrecan, collagen III, cartilage oligomeric matrix protein) from the cartilage than cytokine treatment alone [27, 48]. Taken together, the findings of the present study are consistent with the hypothesis that mechanical injury alone does not induce or alter the mechanisms underlying aggrecan proteolysis but can affect the rate of release of the resulting fragments from the tissue via altered transport/diffusion. Also, the increased MMP activity observed in the mechanically injured cartilage coincubated with SJC could be related to increased access to the cartilage for proinflammatory molecules and proteases released from the SJC .
In contrast, when mechanically injured cartilage was cultured in the presence of TNF-α, only aggrecanase and no MMP activity was detected. These findings are interesting and could imply that different intracellular pathways for inducing aggrecan degradation are activated, depending on if cartilage is stimulated by SJC or by TNF-α. Such differences may be crucial when interpreting results from in vitro studies and when assessing the physiological relevance of any given model.
These findings are furthermore consistent with previous in vitro and in vivo investigations showing that high concentrations of catabolic cytokines such as TNF-α lead to early (8 h to 2 days) aggrecanase activity [49, 50]. In agreement with Madsen et al. , who used a similar bovine explant system, we did not detect MMP-generated FFGV fragments in the medium or in the cartilage during the 6 days of cartilage explant culture in the presence of TNF-α. In all other conditions, FFGV-aggrecan was detected as early as 1–2 days after incubation started. More complex systems, such as in vivo lipopolysaccharide-induced joint inflammation or acute knee injury, have implicated increased MMP activity toward type II collagen within 1 day of insult [15, 20, 51, 52]. However, whether MMP activity toward aggrecan is increased in these conditions is not well understood . Studies designed to identify which factors released from the SJC increase aggrecanase and MMP activity are warranted. If these factors could be singled out, then they could represent novel targets for treatment of PTOA or joint injury.
There are several limitations to the present investigation. Owing to the small study sample size (three animals), our findings need to be repeated. Of note, the excision of SJC before culture traumatizes the SJC tissue, and resident cells may respond to this injury with an inflammatory phenotype. This is also applicable to the cartilage. We must also keep in mind that very young bovine cartilage was used in the experiment, and we do not know if the pattern of proteolysis is similar in older bovine cartilage or in adult human cartilage, even though the aggrecan fragment pattern observed in the bovine medium resembles the pattern seen in synovial fluid from patients with OA (Additional file 1: Figure S1). However, using this young bovine system of very tightly controlled age, we have previously shown that animal-to-animal variations in cartilage behavior are typically no larger than specimen-to-specimen variations within a single animal, giving us confidence that the results presented are meaningful. A strength of the present study is that we were able to follow a course of events using different experimental conditions, using well-characterized bovine aggrecan fragments rather than observing momentary time frames of the joint milieu, as is the case in analysis of joint fluid after, for example, a joint injury.
The time-dependent release of bovine aggrecan fragments from cartilage explants during different culture conditions revealed striking differences related to activation of different proteolytic pathways by the presence of SJC or TNF-α. On the whole, our findings are consistent with the important involvement of the SJC in joint cartilage aggrecan cleavage. The SJC may induce aggrecan cleavage in the mechanically injured cartilage by producing both aggrecanases and MMPs, whereas exogenous TNF-α leads to aggrecan cleavage by activating aggrecanase and inhibiting MMP activity. The cells of the synovium and fibrous joint capsule may also secrete active molecules (e.g., cytokines) that can stimulate cartilage-originated aggrecanase and MMP activity, implicating that the cartilage coculture system including SJC may reveal additional physiologically relevant matrix degradation mechanisms associated with joint injury.
A disintegrin and metalloproteinase with thrombospondin motifs
Chondroitin sulfate region
Keratan sulfate region
Synovium + joint capsule
Tumor necrosis factor-α
Sah RL, Kim YJ, Doong JY, Grodzinsky AJ, Plaas AH, Sandy JD. Biosynthetic response of cartilage explants to dynamic compression. J Orthop Res. 1989;7:619–36.
Bingham JT, Papannagari R, Van de Velde SK, Gross C, Gill TJ, Felson DT, et al. In vivo cartilage contact deformation in the healthy human tibiofemoral joint. Rheumatology (Oxford). 2008;47:1622–7.
Heinegård D. Fell-Muir Lecture: proteoglycans and more – from molecules to biology. Int J Exp Pathol. 2009;90:575–86.
Nia HT, Han L, Bozchalooi IS, Roughley P, Youcef-Toumi K, Grodzinsky AJ, et al. Aggrecan nanoscale solid–fluid interactions are a primary determinant of cartilage dynamic mechanical properties. ACS Nano. 2015;9:2614–25.
Soltz MA, Ateshian GA. Interstitial fluid pressurization during confined compression cyclical loading of articular cartilage. Ann Biomed Eng. 2000;28:150–9.
Sandy JD, Flannery CR, Neame PJ, Lohmander LS. The structure of aggrecan fragments in human synovial fluid: evidence for the involvement in osteoarthritis of a novel proteinase which cleaves the Glu 373-Ala 374 bond of the interglobular domain. J Clin Invest. 1992;89:1512–6.
Lohmander LS, Neame PJ, Sandy JD. The structure of aggrecan fragments in human synovial fluid: evidence that aggrecanase mediates cartilage degradation in inflammatory joint disease, joint injury, and osteoarthritis. Arthritis Rheum. 1993;36:1214–22.
Struglics A, Larsson S, Pratta MA, Kumar S, Lark MW, Lohmander LS. Human osteoarthritis synovial fluid and joint cartilage contain both aggrecanase- and matrix metalloproteinase-generated aggrecan fragments. Osteoarthritis Cartilage. 2006;14:101–13.
Sandy JD, Verscharen C. Analysis of aggrecan in human knee cartilage and synovial fluid indicates that aggrecanase (ADAMTS) activity is responsible for the catabolic turnover and loss of whole aggrecan whereas other protease activity is required for C-terminal processing in vivo. Biochem J. 2001;358:615–26.
Lohmander LS, Englund PM, Dahl LL, Roos EM. The long-term consequence of anterior cruciate ligament and meniscus injuries: osteoarthritis. Am J Sports Med. 2007;35:1756–69.
Johnson DL, Urban Jr WP, Caborn DN, Vanarthos WJ, Carlson CS. Articular cartilage changes seen with magnetic resonance imaging-detected bone bruises associated with acute anterior cruciate ligament rupture. Am J Sports Med. 1998;26:409–14.
Oiestad BE, Engebretsen L, Storheim K, Risberg MA. Knee osteoarthritis after anterior cruciate ligament injury: a systematic review. Am J Sports Med. 2009;37:1434–43.
Potter HG, Jain SK, Ma Y, Black BR, Fung S, Lyman S. Cartilage injury after acute, isolated anterior cruciate ligament tear: immediate and longitudinal effect with clinical/MRI follow-up. Am J Sports Med. 2012;40:276–85.
Berenbaum F. Osteoarthritis as an inflammatory disease (osteoarthritis is not osteoarthrosis!). Osteoarthritis Cartilage. 2013;21:16–21.
Lohmander LS, Hoerrner LA, Lark MW. Metalloproteinases, tissue inhibitor, and proteoglycan fragments in knee synovial fluid in human osteoarthritis. Arthritis Rheum. 1993;36:181–9.
Lohmander LS, Atley LM, Pietka TA, Eyre DR. The release of crosslinked peptides from type II collagen into human synovial fluid is increased soon after joint injury and in osteoarthritis. Arthritis Rheum. 2003;48:3130–9.
Higuchi H, Shirakura K, Kimura M, Terauchi M, Shinozaki T, Watanabe H, et al. Changes in biochemical parameters after anterior cruciate ligament injury. Int Orthop. 2006;30:43–7.
Elsaid KA, Fleming BC, Oksendahl HL, Machan JT, Fadale PD, Hulstyn MJ, et al. Decreased lubricin concentrations and markers of joint inflammation in the synovial fluid of patients with anterior cruciate ligament injury. Arthritis Rheum. 2008;58:1707–15.
Swärd P, Frobell R, Englund M, Roos H, Struglics A. Cartilage and bone markers and inflammatory cytokines are increased in synovial fluid in the acute phase of knee injury (hemarthrosis) – a cross-sectional analysis. Osteoarthritis Cartilage. 2012;20:1302–8.
Kumahashi N, Swärd P, Larsson S, Lohmander LS, Frobell R, Struglics A. Type II collagen C2C epitope in human synovial fluid and serum after knee injury – associations with molecular and structural markers of injury. Osteoarthritis Cartilage. 2015;23:1506–12.
Punzi L, Galozzi P, Luisetto R, Favero M, Ramonda R, Oliviero F, et al. Post-traumatic arthritis: overview on pathogenic mechanisms and role of inflammation. RMD Open. 2016;2:e000279.
Struglics A, Larsson S, Kumahashi N, Frobell R, Lohmander LS. Changes in cytokines and aggrecan ARGS neoepitope in synovial fluid and serum and in C-terminal crosslinking telopeptide of type II collagen and N-terminal crosslinking telopeptide of type I collagen in urine over five years after anterior cruciate ligament rupture: an exploratory analysis in the knee anterior cruciate ligament, nonsurgical versus surgical treatment trial. Arthritis Rheumatol. 2015;67:1816–25.
Ilic MZ, Vankemmelbeke MN, Holen I, Buttle DJ, Clem Robinson H, Handley CJ. Bovine joint capsule and fibroblasts derived from joint capsule express aggrecanase activity. Matrix Biol. 2000;19:257–65.
Goldring MB, Otero M. Inflammation in osteoarthritis. Curr Opin Rheumatol. 2011;23:471–8.
Lee JH, Fitzgerald JB, DiMicco MA, Grodzinsky AJ. Mechanical injury of cartilage explants causes specific time-dependent changes in chondrocyte gene expression. Arthritis Rheum. 2005;52:2386–95.
Lee JH, Fitzgerald JB, DiMicco MA, Cheng DM, Flannery CR, Sandy JD, et al. Co-culture of mechanically injured cartilage with joint capsule tissue alters chondrocyte expression patterns and increases ADAMTS5 production. Arch Biochem Biophys. 2009;489:118–26.
Sui Y, Lee JH, DiMicco MA, Vanderploeg EJ, Blake SM, Hung HH, et al. Mechanical injury potentiates proteoglycan catabolism induced by interleukin-6 with soluble interleukin-6 receptor and tumor necrosis factor α in immature bovine and adult human articular cartilage. Arthritis Rheum. 2009;60:2985–96.
Larsson S, Lohmander LS, Struglics A. Synovial fluid level of aggrecan ARGS fragments is a more sensitive marker of joint disease than glycosaminoglycan or aggrecan levels: a cross-sectional study. Arthritis Res Ther. 2009;11:R92.
Swärd P, Struglics A, Englund M, Roos HP, Frobell RB. Soft tissue knee injury with concomitant osteochondral fracture is associated with higher degree of acute joint inflammation. Am J Sports Med. 2014;42:1096–102.
Li Y, Frank EH, Wang Y, Chubinskaya S, Huang HH, Grodzinsky AJ. Moderate dynamic compression inhibits pro-catabolic response of cartilage to mechanical injury, tumor necrosis factor-α and interleukin-6, but accentuates degradation above a strain threshold. Osteoarthritis Cartilage. 2013;21:1933–41.
Patwari P, Lin SN, Kurz B, Cole AA, Kumar S, Grodzinsky AJ. Potent inhibition of cartilage biosynthesis by coincubation with joint capsule through an IL-1-independent pathway. Scand J Med Sci Sports. 2009;19:528–35.
Patwari P, Cook MN, DiMicco MA, Blake SM, James IE, Kumar S, et al. Proteoglycan degradation after injurious compression of bovine and human articular cartilage in vitro: interaction with exogenous cytokines. Arthritis Rheum. 2003;48:1292–301.
Frank EH, Jin M, Loening AM, Levenston ME, Grodzinsky AJ. A versatile shear and compression apparatus for mechanical stimulation of tissue culture explants. J Biomech. 2000;33:1523–7.
Struglics A, Larsson S. A comparison of different purification methods of aggrecan fragments from human articular cartilage and synovial fluid. Matrix Biol. 2010;29:74–83.
Farndale RW, Buttle DJ, Barrett AJ. Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochim Biophys Acta. 1986;883:173–7.
Struglics A, Hansson M, Lohmander LS. Human aggrecanase generated synovial fluid fragment levels are elevated directly after knee injuries due to proteolysis both in the inter globular and chondroitin sulfate domains. Osteoarthritis Cartilage. 2011;19:1047–57.
Struglics A, Larsson S, Hansson M, Lohmander LS. Western blot quantification of aggrecan fragments in human synovial fluid indicates differences in fragment patterns between joint diseases. Osteoarthritis Cartilage. 2009;17:497–506.
Struglics A, Hansson M. MMP proteolysis of the human extracellular matrix protein aggrecan is mainly a process of normal turnover. Biochem J. 2012;446:213–23.
Lieberthal J, Sambamurthy N, Scanzello CR. Inflammation in joint injury and post-traumatic osteoarthritis. Osteoarthritis Cartilage. 2015;23:1825–34.
Piccinini AM, Midwood KS. DAMPening inflammation by modulating TLR signalling. Mediators Inflamm. 2010;2010:672395.
de Lange-Brokaar BJ, Ioan-Facsinay A, van Osch GJ, Zuurmond AM, Schoones J, Toes RE, et al. Synovial inflammation, immune cells and their cytokines in osteoarthritis: a review. Osteoarthritis Cartilage. 2012;20:1484–99.
Lees S, Golub SB, Last K, Zeng W, Jackson DC, Sutton P, et al. Bioactivity in an aggrecan 32-mer fragment is mediated via Toll-like receptor 2. Arthritis Rheumatol. 2015;67:1240–9.
Bondeson J, Wainwright SD, Lauder S, Amos N, Hughes CE. The role of synovial macrophages and macrophage-produced cytokines in driving aggrecanases, matrix metalloproteinases, and other destructive and inflammatory responses in osteoarthritis. Arthritis Res Ther. 2006;8:R187.
Moradi B, Rosshirt N, Tripel E, Kirsch J, Barie A, Zeifang F, et al. Unicompartmental and bicompartmental knee osteoarthritis show different patterns of mononuclear cell infiltration and cytokine release in the affected joints. Clin Exp Immunol. 2015;180:143–54.
Vankemmelbeke MN, Ilic MZ, Handley CJ, Knight CG, Buttle DJ. Coincubation of bovine synovial or capsular tissue with cartilage generates a soluble “aggrecanase” activity. Biochem Biophys Res Commun. 1999;255:686–91.
Ilic MZ, Martinac B, Handley CJ. Effects of long-term exposure to glucosamine and mannosamine on aggrecan degradation in articular cartilage. Osteoarthritis Cartilage. 2003;11:613–22.
Tortorella MD, Liu RQ, Burn T, Newton RC, Arner E. Characterization of human aggrecanase 2 (ADAM-TS5): substrate specificity studies and comparison with aggrecanase 1 (ADAM-TS4). Matrix Biol. 2002;21:499–511.
Wang Y, Li Y, Khabut A, Chubinskaya S, Grodzinsky AJ, Önnerfjord P. Quantitative proteomics analysis of cartilage response to mechanical injury and cytokine treatment. Matrix Biol. doi:10.1016/j.matbio.2016.12.004.
Malfait AM, Tortorella M, Thompson J, Hills R, Meyer DM, Jaffee BD, et al. Intra-articular injection of tumor necrosis factor-α in the rat: an acute and reversible in vivo model of cartilage proteoglycan degradation. Osteoarthritis Cartilage. 2009;17:627–35.
Madsen SH, Sumer EU, Bay-Jensen AC, Sondergaard BC, Qvist P, Karsdal MA. Aggrecanase- and matrix metalloproteinase-mediated aggrecan degradation is associated with different molecular characteristics of aggrecan and separated in time ex vivo. Biomarkers. 2009;15:266–76.
de Grauw JC, van de Lest CH, van Weeren PR. Inflammatory mediators and cartilage biomarkers in synovial fluid after a single inflammatory insult: a longitudinal experimental study. Arthritis Res Ther. 2009;11:R35.
Tchetverikov I, Lohmander LS, Verzijl N, Huizinga TW, TeKoppele JM, Hanemaaijer R, et al. MMP protein and activity levels in synovial fluid from patients with joint injury, inflammatory arthritis, and osteoarthritis. Ann Rheum Dis. 2005;64:694–8.
Lark MW, Bayne EK, Flanagan J, Harper CF, Hoerrner LA, Hutchinson NI, et al. Aggrecan degradation in human cartilage: evidence for both aggrecanase and matrix metalloproteinase activity in normal, osteoarthritic and rheumatoid joints. J Clin Invest. 1997;100:93–106.
Bonassar LJ, Frank EH, Murray JC, Paguio CG, Moore VL, Lark MW, et al. Changes in cartilage composition and physical properties due to stromelysin degradation. Arthritis Rheum. 1995;38:173–83.
Struglics A, Larsson S, Lohmander LS. Estimation of the identity of proteolytic aggrecan fragments using PAGE migration and Western immunoblot. Osteoarthritis Cartilage. 2006;14:898–905.
Durigova M, Nagase H, Mort JS, Roughley PJ. MMPs are less efficient than ADAMTS5 in cleaving aggrecan core protein. Matrix Biol. 2011;30:145–53.
We thank Jonas Ranstam (Lund University) for help with the statistical analysis, Michael W. Lark (Trevena, King of Prussia, PA, USA) for the kind gift of the LGQR and KEEE antibodies and MMP-3, Takehiko Nakamura (Seikagaku, Tokyo, Japan) for the kind gift of keratanase and keratanase II, Sanjay Kumar and Michael Pratta (GlaxoSmithKline, Collegeville, PA, USA) for the kind gift of ADAMTS-4 and ARGS (OA-1) and AGEG antibodies, Peter Roughly (Shriners Hospital, Montreal, QC, Canada) for the kind gift of ARLE antibody, and John Sandy (Rush University, Chicago, IL, USA) for the kind gift of G1 antibody.
Funding for this work was provided as project grants from the Swedish Research Council (to LSL); the Crafoord Foundation (to AS); the Faculty of Medicine Lund University (to AS); the King Gustaf V 80-year Birthday Fund (to AS); the Kock Foundation (to AS); the Swedish Rheumatism Association (to AS); the Alfred Österlunds Foundation (to AS); the Swedish National Centre for Research in Sports (to PS); the Agency for Science, Technology, and Research, Singapore (to YW); and National Institutes of Health grant AR060331 (to AJG).
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AJG, LSL, and AS designed the study. MH, PS, and YW performed the experiments. MH, PS, AS, and YW analyzed the results. PS takes responsibility for the integrity of the work. All authors drafted the manuscript, and all authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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Descriptive information on bovine aggrecan fragments detected by Western blot analysis. Figure S1 Bovine aggrecan fragments detected by Western blot analysis. (PDF 353 kb)
Anti-FFGV (a), anti-ARGS (b), anti-KEEE (c), and anti-G3 (d) Western blotting of medium samples. (PDF 647 kb)
The amount of aggrecan fragments released into medium. (PDF 79 kb)
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Swärd, P., Wang, Y., Hansson, M. et al. Coculture of bovine cartilage with synovium and fibrous joint capsule increases aggrecanase and matrix metalloproteinase activity. Arthritis Res Ther 19, 157 (2017). https://doi.org/10.1186/s13075-017-1318-9
- Cartilage injury
- Fibrous joint capsule
- Knee injury