Background
Joint diseases apply to approximately 15% of world’s population. In a healthy knee joint, the meniscus integrity is a prerequisite for the joints kinematics and its load balancing. In the case of a partial meniscectomy a 10% reduction in meniscal contact area was demonstrated, which produced a 65% increase in peak joint-contact stress [
1], leading to an early development of osteoarthritis (OA) [
2]. Hence, post-traumatic and degenerative damages of the meniscus are well accepted as major predisposing factors for OA and post-traumatic OA (PTOA) [
3,
4].
Degenerative meniscal tears predominantly occur in the postero-medial meniscus horn because of the highest force uptake during weight bearing in this area [
5]. Meniscus degeneration takes place as a biomechanical adaptation to elevated loads and is accompanied by fibrocartilage formation and calcification in the medial meniscus posterior horn, which in turn is associated with the degree of the tear [
6]. Both mechanisms are known to impair the resistance to tension and act as adaptation to increased mechanical overload [
7,
8]. Histopathological characteristics of fibrocartilage metaplasia include joint inflammation, matrix remodeling by matrix metalloproteinases, collagen disruption and calcification. Du et al. demonstrated that mechanical overload induces meniscus hypertrophy and therefore called for anti-hypertrophic drugs to prevent meniscus degeneration and subsequent PTOA [
9]. In addition, traumatic injuries of the meniscus lead to raised levels of proinflammatory cytokines, such as TNF-α [
10]. In vitro models have pointed out the relevance of an inflammatory reaction induced by mechanical overload, which subsequently caused matrix degeneration and induction of cell apoptosis [
11,
12]. In terms of traumatic anterior cruciate ligament rupture, meniscal tear and early OA, this inflammation was demonstrated acute after the injury [
13]. Inflammatory events occured after meniscal injury regardless of preexisting osteoarthritic changes in the joint [
14]. Proinflammatory cytokines have been shown to suppress matrix biosynthesis and increase enzymatic matrix degradation. TNF-α is known to induce cellular apoptosis by direct stimulation of death receptors. In addition, TNF-α induces matrix-metalloproteinases and aggrecanases as well as nitric oxide (NO) production, which in turn cause cell death and matrix degeneration [
15‐
17]. The intrinsic repair capacity of the meniscus tissue is significantly inhibited at high concentrations of TNF-α (10 ng/ml) [
18].
Yet, therapeutically augmentation strategies to promote the meniscus regeneration are still a field of study. Current interest is growing in the use of tissue growth factors to facilitate meniscus regeneration and restore a functional meniscus. The administration of growth factors may be a supplementary option in terms of surgical meniscus repair and usage of exogenous materials for meniscus regeneration. In this context, various studies have shown protective effects of anti-inflammatory interleukin 10 (IL-10) on a wide range of tissues including articular cartilage and synovialocytes [
19]. Interleukin 10 is a potent anti-inflammatory and immune-regulatory cytokine. Apart from its immunological properties IL-10 is involved in processes such as connective tissue extracellular matrix remodeling and matrix homeostasis [
19]. It has been shown to antagonize matrix degeneration, induced by proinflammatory cytokines such as TNF-α in experimental models of OA and rheumatic arthritis [
19,
20]. IL-10 restored inhibition of proteoglycan synthesis in cartilage explants of joints with end-stage haemophilic arthropathy [
21]. In addition, IL-10 seems to have anti-apoptotic effects since it was shown to inhibit TNF-α induced caspase activities and restored the impaired bax/bcl-2 ratio in articular chondrocytes [
22]. Meniscus degeneration and development of OA are closely interactive. The aim of this study was to test the hypothesis that IL-10 treatment will lead to a decrease of inflammation-induced loss of tissue viability and matrix degeneration in an in vitro model of bovine meniscal tissue.
Method
Isolation and culture of mature meniscal explants
This study was conducted using explants from five donor animals. Approval was obtained from the institutional ethics committee prior to performing the study. Meniscus explants were isolated from the articular cavity of knee joints from 16 to 24 months old cows procured from a local abattoir authorized by the relevant meat inspectors as described previously [
23,
24]. Using a 10 mm biopsy punch full thickness tissue cylinders were harvested from the red-white zone of the meniscus. Tissue cylinders were loaded in a sample holder and trimmed to yield 1 mm thick disks. Thereby the specimens lost the original meniscal surface. Four to five smaller explants (3 mm in diameter × 1 mm thickness) were harvested from each disk using a 3 mm biopsy punch (Hebu-medical, Tuttlingen, Germany). Weight of explants were measured. Explants with appropriate thickness were isolated, randomly distributed (for one individual experiment) and thereafter cultured separately in 250 μl medium in 96-well plates. An equilibration was performed for 24 h at 37 °C in an atmosphere of 5% CO
2 in culture medium (low-glucose Dulbecco’s modified Eagle’s medium (Biochrom, Berlin, Germany) supplemented with 10 mM HEPES buffer (Biochrom, Berlin, Germany), 0.4 mM proline (Sigma-Aldrich, Darmstadt, Germany), 50 μg vitamin c, 100 units/ml of penicillin G, 100 mg/ml of streptomycin, and 0.25 mg/ml of amphotericin B (PAA Laboratories, Pasching, Germany)).
Incubation with TNF-α and stimulation with IL-10
Treatment was initiated after 24 h of equilibration by renewing the culture medium containing bovine TNF-α (R&D systems, Wiesbaden-Nordenstadt Germany; 10 and 100 ng/ml) with or without bovine IL-10 (Kingfisher Biotech, Saint-Paul, MN, USA) in different concentrations (10 pg/ml – 20 ng/ml). An alignment analysis of the protein sequences of bovine and human IL-10 revealed a homology of 77.52%. Treatment was performed for 72 h at 37 °C in an atmosphere of 5% CO2. After that supernatants were frozen and explants were collected for real time qPCR or processed to histological analysis.
Histological detection of cell death and immunohistochemistry
In order to visualize cells with nuclear bleebing indicating apoptotic cell death [
25], explants were fixed overnight using 4% paraformaldehyde (in PBS), embedded in Paraplast, sectioned (7 μm) and stained with Mayer’s haematoxylin as described previously [
24]. Images were taken using a Zeiss Axiophot microscope (Zeiss, Wetzlar, Germany) and the number of apoptotic cells was quantified [in %] in relation to the total number of cells per optical field by a blinded investigator. While margins of the sections (150 μm depth) were excluded, the value for each explant was calculated from three different areas.
For immunohistochemical visualisation of aggrecanase-specific aggrecan neoepitope NITEGE formation, immobilized explants were incubated for 2.5 min in a digester at 100 °C (in 0.01 M citric acid, pH 6.0) and thereafter incubated overnight at 4 °C with the primary antibody (Rabbit-IgG-Aggrecan Neo Polyclonal Antibody PA-1746; 1:50 dilution in 1% BSA; Thermofisher Scientific, Waltham, MA, USA) as described previously [
24]. Sections were rinsed in Tris-NaCl and incubated with the secondary antibody AlexaFluor 488 goat anti-rabbit IgG (1:700; Invitrogen, Carlsbad, CA, USA) for one hour at room temperature. Additional nuclear staining was performed with bisbenzimide (Sigma-Aldrich, St. Louis, MO, USA). Images were taken using the Apotome (ZEISS, Jena, Germany) fluorescence microscope.
Measurement of glycosaminoglycans (GAG) and nitric oxide (NO)
Release of GAGs in the supernatant was quantified by dimethlmethylene blue dye (DMMB) assay at a wavelength of 525 nm (Photometer Ultraspec II, Biochrom, Cambridge, UK) using shark chondroitin-sulfate as standard [
26]. Values are given as μg GAG/mg explant wet weight.
Release of NO in the supernatant was quantified by Griess reagent (1% sulphanilamide and 0.1% N-(1-naphtyl)-ethylene diamine-dihydro-chloride in 5% phosphoric acid (H3O4P), Sigma-Aldrich, St. Louis, MO, USA) using sodium nitrite (NaNO2, Merck, Darmstadt, Germany) as standard. Absorption was measured in an automated plate reader (SLT Reader 340 ATTC, SLT-Labinstruments, Achterwehr, Germany) at 540 nm. Values are given as μmol nitrite/mg explant wet weight.
Quantitative real-time PCR (qPCR)
Relative quantification of different gene transcription levels was performed via real-time PCR. After 72 h of treatment eight explants of one experimental subgroup were pooled, immediately frozen in liquid nitrogen and pulverized. RNA extraction was performed using TRIZOL reagent (1 ml/100 mg wet weight tissue; Invitrogen, Carlsbad, CA, USA) and RNA quality was controlled by measuring OD260/OD280 ratio. Remaining DNA contamination was removed by digestion with DNase (65 °C for 10 min; Promega, Madison, WI, USA). qPCR was performed in a 7500 Fast Real-Time PCR System (Applied Biosystems, Darmstadt, Germany) using the Qiagen QuantiTect SYBR® Green PCR Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions: reverse transcription 30 min at 50 °C; PCR initial activation step 15 min at 95 °C; denaturation 15 s at 94 °C; annealing 30 s at 60 °C; extension 30 s at 72 °C; optional: data acquisition 30 s at melting temperature 70 to 78 °C.
All primers were designed using primer express software 3.0.1 (Applied Biosystems, Darmstadt, Germany), ordered at Biomers (Ulm, Germany) and used at a concentration of 0.5 μM (Table
1). Values were taken as duplicates and a comparative quantification (ΔΔCT-method) was performed to analyze the data. Using this method the n-fold expression for the gene of interest is initially adjusted in relation to a reference gene (GAPDH; ΔCT) and secondly normalized to an untreated control group (ΔΔCT). The n-fold expression of the target gene was calculated by the equation 2
- ΔΔCT [
27].
Table 1
List of primers used for real-time qPCR primer
NOS2 sense | CCC-GCA-TGC-AAC-TCC-AA |
NOS2 antisense | TCG-TAA-GTC-ATG-AAC-TGC-CAC-TTC |
MMP3 sense | CAC-TCA-ACC-GAA-CGT-GAA-GCT |
MMP3 antisense | CGT-ACA-GGA-ACT-GAA-TGC-CGT |
MMP13 sense | TCT-TGT-TGC-TGC-CCA-TGA-GT |
MMP13 antisense | GGC-TTT-TGC-CAG-TGT-AGG-TGT-A |
ADAMTS4 sense | GCG-CCC-GCT-TCA-TCA-CTG |
ADAMTS4 antisense | TTG-CCG-GGG-AAG-GTC-ACG |
COL10A1 sense | CCT-GCC-CGA-GGA-CTT-TGT-AAA |
COL10A1 antisense | GAA-AGC-AGA-CAC-AGG-CAT-TCC |
GAPDH sense | ATC-AAG-AAG-GTG-GTG-AAG-CAG-G |
GAPDH antisense | TGA-GTG-TCG-CTG-TTG-AAG-TCG |
Statistics
Minimum experimental repetition number was three times (detailed n number is given in results section). For each test group different numbers of separate explants were cultured per experiment. In each case explants were harvested from both menisci of both knee joints of one individual animal (n = number of independent experiments with individual animals), randomly distributed and separately isolated and measured. All data were tested for normality using the Kolmogorov-Smirnov test. Statistical analysis was performed using Graph Pad prism 5 program (Graph Pad Software Inc., San Diego, CA, USA). One way ANOVA analysis with Bonferroni’s multiple correction was used to compare means among the independent experimental groups. Differences were considered significant if P ≤ 0.05. Different letters in the graphs identify experimental groups, which are statistically different. Quantitative data in the text are presented as mean and 95% confidence interval (95% CI).
Discussion
The present study evaluated effects of anti-inflammatory IL-10 in an in vitro model of TNF-α-related meniscus degeneration. Our results demonstrate menisco-protective effects of IL-10 in terms of cell death, cellular de-differentiation and matrix degeneration. IL-10 prevented early cell death events, which were mainly initiated by TNF-α. It could be shown that addition of IL-10 decreased the TNF-α-induced expression of matrix degrading enzymes, release of GAGs and formation on NITEGE fragments, which suggests that IL-10 prevented crucial mechanisms of meniscus degeneration. In addition, IL-10 reduced the release of NO by decreased expression of NOS2 and therefore interfered with oxidative stress mechanism.
Larsson et al. recently demonstrated a distinct association between elevated joint concentration of TNF- α after meniscectomy and increased risk for progression of radiographic OA [
28]. We revealed anti-apoptotic effects of IL-10 in vitro by distinct reduction of cells with NB. Histomorphometric analysis of NB using light microscopy has been confirmed to identify apoptotic cells by electron microscopy previously [
25]. Given the low cell density in meniscus tissue, apoptosis is considered as a major pathogenic factor in meniscus tears [
29]. Kim et al. reported a particularly significant role of chondrocyte apoptosis in the development of post-traumatic arthritis [
30]. Apart from a receptor mediated cell death, the reduction of NO production would be a mechanism in which IL-10 interferes with TNF-α induced cell death.
In addition, IL-10 interacted with inflammation-related matrix degradation and cellular phenotype differentiation. Our results suggest that IL-10 restored the integrity of the meniscal extracellular matrix as it reduced the expression of matrix-degrading enzymes, release of GAG fragments and NITEGE formation. In addition, IL-10 attenuated a hypertrophic phenotype de-differentiation by decreased expression of hypertrophy markers such as collagen type X and MMP13 in autochthonous meniscal cells. Cellular hypertrophy is associated with calcification and fibrocartilage formation, which in turn leads to meniscus and cartilage degeneration [
9].
The importance of MMP activity in TNF-α depended proteoglycan degradation in bovine meniscal tissue has been emphasized previously [
24]. We focused on ADAMTS4 since Voigt et al. [
24] demonstrated increased expression of ADAMTS4 with TNF-α-treatment, whereas ADAMTS5 mRNA expression was low or not detectable. Reduction of ADAMTS4 mRNA by IL-10 was paralleled by decreased detection of NITEGE fragments. Reduction of GAG release and expression of matrix degrading enzymes MMP3, MMP13 and ADAMTS4 indicated protective effects of IL-10. Although extracellular matrix in meniscus tissue only consists of 1% GAG, it has been demonstrated to essentially contribute to compressive and tensile properties in human meniscus [
31]. Recently, similar findings have been demonstrated in an in vitro model of mechanically injured articular cartilage. In this model IL-10 was able to inhibit the post-injurious apoptosis and matrix degeneration [
32].
Interestingly, the question about the effective dosage for IL-10 under different joint pathologies remains unanswered yet. Our results present first evidence for a distinct dose-response showing more pronounced effects with lower IL-10 dosages. This is in line with a previous study in mechanically injured cartilage, which revealed a similar dosage ranging from 10 to 100 pg/ml IL-10 to be most effective in terms of chondroprotection [
32]. The weaker effect of IL-10 at higher doses may have important implications. In this context, the IL-10 signaling pathway may provide a possible explanation. There is evidence that IL-10 auto-regulates its biological effects via a negative feed-back loop involving autocrine stimulation of the IL-10 receptor [
33,
34]. In addition, in various immune cells, including synovial macrophages, this feedback-loop has been identified to cause a cellular phenotype switch from classically activated (proinflammatory) to an alternatively activated (anti-inflammatory) phenotype [
35,
36]. One could speculate that a self-adjusting mechanism for IL-10 by an autocrine inhibition takes place in fibro-chondrocytes as well and that this is responsible for less pronounced effects with higher IL-10 dosages. Anyway, the concentration of IL-10 in a healthy joint or even after injury has been found to be lot lower than 10 ng/ml [
37,
38]. However, at this moment in time a wide range of thematically related studies, that directly applied IL-10, used dosages ranging prom 10 - 300 ng/ml [
22,
39‐
41]. The decrease of efficacy with higher dosages remains interesting and noticeable and should be taken into account by future investigations about IL-10. It remains noticeable that there were no increased effects with higher TNF-α dosages, which could be indicative for a saturation effect since TNF-α effects are mostly receptor-mediated.
A recent study found that fibrocartilage formation and calcification in meniscus tissue is positively correlated with meniscus degeneration and is a predisposing factor for cartilage lesions [
6]. This lines up with investigations by Du et al., who demonstrated that abnormal mechanical loading induced cartilage degeneration by accelerating meniscus hypertrophy and mineralization [
9]. The results of our study provide evidence that IL-10 inhibits TNF-α-induced hypertrophic de-differentiation, as it reduced the hypertrophy markers collagen type X and MMP13 [
42]. We therefore propose that IL-10, especially in lower dosages, has the ability to stabilize the physiological cellular phenotype in inflammatory-challenged meniscus tissue. In addition, Du et al. found post-traumatic cell death and GAG release to be significant pathogenetic mechanisms in their model, which were counteracted by IL-10 under inflammatory as well as in post-injurious conditions [
32,
40].
Due to its tissue source and in vitro design our research study has several limitations. Firstly, our study was performed with bovine meniscal explants and it remains questionable if these effects are the same in human OA and PTOA. Secondly, the highly inhomogeneous meniscal tissue, exhibiting regional differences in composition, structure and cellular phenotype, caused a methodical limitation of our results. Our explants were largely punched out of the middle radial third, but there are however explants that have to be attributed rather to the inner or outer radial third, respectively. Since there are zonal differences in matrix turnover and cytokine response [
43], we cannot exclude this as a confounding factor in our study. In addition, pretreatment with IL-10 could have induced more pronounced effects, but to our opinion the simultaneous treatment with and without TNF-α and IL-10 reflects the therapeutical approach more closely.
From a translational point of view, the administration of IL-10 and even more important its biologically effective dose in vivo remain challenging questions. IL-10 is a short lasting biological molecule and its effective dosage seems to vary depending on different joint pathologies. Targeted gene therapy might offer an effective approach, which allows a disease-regulated overexpression of IL-10 within the damaged joint [
44,
45]. Another approach could be the injection into the rupture margins during arthroscopic meniscus suture or interposition of an IL-10-soaked scaffold. To the best of our knowledge there are no studies that examined the ability of IL-10 to penetrate meniscal tissue by diffusion yet. Since we used no monoculture, but a tissue culture, IL-10 must have had the ability to penetrate the explant tissue and reach the fibro-chondrocytes, since we can present significant effects by administration of IL-10.
Further studies should examine the long-term effects of IL-10 in terms of tissue remodeling and cell differentiation of fibro-chondrocytes. In this regard the effective IL-10 dose in chronically inflamed meniscus tissue seems to be of particular importance.
Acknowledgements
We like to thank Rita Kirsch and Frank Lichte for their excellent technical assistance.