The meniscus is an intra-articular fibrocartilaginous structure that serves essential biomechanical roles in the knee. With injury or arthritis, the meniscus may be exposed to significant changes in its biochemical and biomechanical environments that likely contribute to the progression of joint disease. The goal of this study was to examine the influence of mechanical stress on matrix turnover in the meniscus in the presence of interleukin-1 (IL-1) and to determine the role of nitric oxide (NO) in these processes. Explants of porcine menisci were subjected to dynamic compressive stresses at 0.1 MPa for 24 h at 0.5 Hz with 1 ng/ml IL-1, and the synthesis of total protein, proteoglycan, and NO was measured. The effects of a nitric oxide synthase 2 (NOS2) inhibitor were determined. Dynamic compression significantly increased protein and proteoglycan synthesis by 68 and 58%, respectively, compared with uncompressed explants. This stimulatory effect of mechanical stress was prevented by the presence of IL-1 but was restored by specifically inhibiting NOS2. Release of proteoglycans into the medium was increased by IL-1 or mechanical compression and further enhanced by IL-1 and compression together. Stimulation of proteoglycan release in response to compression was dependent on NOS2 regardless of the presence of IL-1. These finding suggest that IL-1 may modulate the effects of mechanical stress on extracellular matrix turnover through a pathway that is dependent on NO.
the meniscus is an intra-articular fibrocartilaginous structure that serves essential biomechanical roles in the normal function of the knee joint (50). Under physiological conditions, the menisci are subjected to significant mechanical stresses and serve to distribute loads across the tibial plateau (2, 4, 36, 57). The meniscus is maintained through the metabolic activity of cells termed “fibrochondrocytes,” cells that may exhibit phenotypic characteristics of fibroblasts as well as chondrocytes (21, 28, 39, 58).
Previous studies have shown that mechanical forces can alter the metabolic activity of chondrocytes in articular cartilage (9, 23, 25, 27, 32, 33, 46, 52, 60). However, the effects of mechanical stress on cells of the meniscus are less well understood. Exercise increases collagen and proteoglycan contents in rat menisci (54), whereas joint immobilization decreases aggrecan gene expression in intact menisci (10) and inhibits collagen accumulation in healing menisci (11). Furthermore, late-stage knee joints in experimentally immobilized chick embryos are lacking menisci (40). Importantly, injury or loss of the meniscus is known to initiate pathological changes in the articular cartilage of the knee (3, 7, 12, 15, 41, 45), which are associated with alterations in the stress-strain environment in the joint (35, 36, 47). However, little is known of the sequence of biomechanical and biochemical events linking mechanical stress to inflammatory or degenerative changes of the meniscus and its interactions with articular cartilage relative to the onset and progression of arthritis. In this respect, a better knowledge of meniscal physiology could further enhance our understanding of the pathogenesis of knee osteoarthritis.
Cartilage destruction in osteoarthritis is believed to involve the action of soluble mediators such as the cytokine interleukin-1 (IL-1) (55, 56). IL-1 exerts strong catabolic effects on cartilage extracellular matrix synthesis (29, 38, 43, 49). These effects are associated with increased production of nitric oxide (NO), a product of NO synthase (NOS) (14, 31, 37, 42, 51). IL-1 also induces production of NO from lapine and human osteoarthritic menisci (5, 34). Mechanical stress increases NO production through a NOS2-dependent mechanism in cartilage (9, 17, 18, 20, 32, 33) and meniscus (19). It is of particular interest that cyclic tensile stretch can exert a protective effect on IL-1-induced matrix degradation in fibrochondrocytes from the temporomandibular joint (1). However, the influence of physiological and pathological compressive stress in the presence of IL-1 on the NOS pathway in knee menisci is unknown.
The goal of this study was to determine the influence of dynamic mechanical compression on matrix synthesis in meniscal explants cultured in the presence of the proinflammatory cytokine IL-1. We also sought to determine whether matrix biosynthesis and turnover by meniscal cells were mediated through the endogenous NO production. Therefore, we investigated the role of NO, induced by inflammatory cytokines and/or mechanical stresses, on matrix turnover in meniscal explants.
MATERIALS AND METHODS
Meniscal explant culture. Medial menisci were harvested from knees of 2-yr-old pigs within 4 h of death. All protocols were performed in accordance with the Duke University Institutional Animal Care and Use Committee. Six cylindrical explants (1 mm in thickness) were obtained from the femoral surface of each medial meniscus by using a 5-mm diameter biopsy punch (Miltex Instrument, Lake Success, NY). Explants were harvested from the outer half of the meniscus, and control and compressed explants originated from adjacent sites on the meniscus and were paired at harvest (Fig. 1). The explants were cultured in standard culture medium containing Dulbecco's modified Eagle medium (GIBCO, Gaithersburg, MD) with 10% heat-inactivated fetal bovine serum (Sigma Chemical, St. Louis, MO), 0.1 mM nonessential amino acids (GIBCO), 10 mM HEPES (GIBCO), 100 U/ml penicillin and streptomycin (GIBCO), and 37.5 μg/ml ascorbate-2-phosphate (Sigma Chemical). All samples were cultured in 48-well plates for 3 days before the experiment regimens were performed. For each mechanical regimen tested, 24 pairs of samples harvested from eight different pigs were used. Meniscal explants were divided into eight groups according to treatment with compression, IL-1, and the NOS2 inhibitor 1400W. Each group contained six explants, which were site matched and paired between loaded and unloaded groups.
Compression experiments. Compressive loads were applied to 24 samples simultaneously by using a modified version of the Biopress system as described previously (17–19). Compression experiments were performed for 24 h at a frequency of 0.5 Hz (square wave with 1 s on and 1 s off) at a magnitude of 0.1 MPa. Experiments were performed at 37°C in an atmosphere of 5% CO2 and 95% air. Control explants were maintained uncompressed for the same duration. For each experimental condition, 24 test explants and 24 site-matched control explants were placed into individual compression wells in 1 ml of culture medium. Each test specimen was subjected to a 10-g tare load and allowed to equilibrate for 1 h before initiation of the compression regimens. Compression experiments were performed on day 3 after harvest, at which time the rates of metabolism in culture had equilibrated (19).
Treatment with IL-1 and NOS2 inhibitor. The concentration of rpIL-1α (R & D Systems, Minneapolis, MN) was selected as 1 ng/ml after performance of a dose-response curve of proteoglycan and total protein synthesis and proteoglycan loss in response to a range (0.01–10 ng/ml) of concentrations of IL-1 for 24 h. This concentration showed ∼50% of the maximum proteoglycan loss inducible by IL-1. Adjacent explants were cultured in the presence and absence of the specific NOS2 inhibitor 1400W (2 mM) (Alexis Chemical, San Diego, CA).
Measurements of total protein and proteoglycan synthesis. Total protein synthesis was measured as the incorporation of [3H]proline (NEN Life Science Products, Boston, MA), and proteoglycan synthesis was measured as the incorporation of [35S]sulfate (Na235SO4) (NEN Life Science Products) into glycosaminoglycans. To assess the rate of total protein or proteoglycan synthesis, explants were incubated in the presence of 20 μCi/ml [3H]proline and 10 μCi/ml [35S]sulfate for 24 h while the mechanical regimen was being applied. [3H]proline and [35S]sulfate incorporation was normalized by the wet weight of each explant. In preliminary studies, [3H]proline and [35S]sulfate incorporation of the explants were measured for 7 days after harvesting to confirm the stability of the culture system. After treatment, the explants were washed four times for 15 min each in PBS containing 0.8 mM sodium sulfate and 1 mM proline to remove any unincorporated isotope. All explants were digested with papain (125 μg/ml) overnight in the 65°C oven (23), and 4 ml of biodegradable scintillation counting cocktail (Bio-Safe II, Research Products International, Mount Prospect, IL) were added to each scintillation vial. Beta counts were determined as disintegrations per minute on the Tri-Carb liquid scintillation analyzer (model 1900TR, Packard Instrument, Meriden, CT). Results were normalized to the wet weight of explant before the compression.
NO assay. NO production was determined by measuring the concentration of nitrate and nitrite in the media by techniques previously described (59). Briefly, nitrate was enzymatically reduced to nitrite by the addition of nitrate reductase (Boehringer Mannheim) to 50 μl of standard or sample. After a 30-min incubation at 37°C, 100 μl of Griess I (1% sulfanilamide) (Sigma Chemical) and 100 μl Griess II (0.1% naphthylethylenediamine) (Sigma Chemical) were added, followed by 10 min of incubation at room temperature. Nitrite was the determined spectrophotometrically with absorbance read at 540 nm and interpolated with a sodium nitrate (Sigma Chemical) standard curve. Results were expressed as micromoles per gram wet weight per 24 h.
Dimethylmethylene blue assay. The release of glycosaminoglycans from the meniscal explants, as a measure of proteoglycan degradation, was determined using the dimethylmethylene blue (DMMB) method as described previously (13, 16). Media samples were added to prepared DMMB solution, and sulfated glycosaminoglycan levels were determined spectrophotometrically with absorbance read at 570 nm.
Statistical analysis. Statistical analysis between control and compressed explants with and without IL-1 treatment was performed by using a two-way ANOVA with repeated measures and a Duncan's post hoc test. Significance was determined at the 95% confidence level.
Total protein synthesis. Dynamic mechanical compression significantly increased total protein by 68% compared with that of uncompressed explants (Fig. 2A). IL-1 increased protein synthesis relative to controls, whereas dynamic compression in the presence of IL-1 did not lead to a further increase in protein synthesis. To examine the role of NO in this response, explants were compressed and/or treated with IL-1 in the presence of 1400W, a NOS2 inhibitor (Fig. 2B). In this situation, mechanical compression increased protein synthesis in the presence or absence of IL-1.
Proteoglycan synthesis. To examine the role of mechanical stress and IL-1 on proteoglycan synthesis, we measured the incorporation of 35SO4 in meniscal explants. Dynamic compression significantly increased 35SO4 incorporation compared with uncompressed control explants (Fig. 3A). IL-1 did not affect 35SO4 incorporation in uncompressed specimens, but it abolished the stimulatory effect of mechanical compression. In the presence of 1400W, the stimulatory effect of mechanical compression was restored (Fig. 3B).
Proteoglycan release. To examine proteoglycan loss from meniscal explants, we measured the total DMMB reactivity of the media. Dynamic compression increased proteoglycan release from meniscal explants. Furthermore, IL-1 significantly increased proteoglycan release in both uncompressed and compressed explants (Fig. 4A). The presence of 1400W prevented the increases in proteoglycan release that were induced by mechanical stress or by IL-1 (Fig. 4B).
NO production. Total NO production was significantly increased by dynamic compression and by IL-1 (Fig. 5A). The greatest increase in NO production was induced by IL-1 alone. The application of compression in the presence of IL-1 reduced total NO production compared with IL-1 alone. The induction of NO by dynamic compression or IL-1 was significantly inhibited by 1400W (Fig. 5B).
The findings of this study support the hypotheses that dynamic compression significantly increases matrix biosynthesis in the meniscus and that this simulation is disrupted by IL-1. However, inhibition of NOS2 restored the biosynthetic response to dynamic compression, suggesting that the inhibitory influence of IL-1 on mechanically induced biosynthesis requires NOS2 expression and NO. Dynamic compression also increased proteoglycan release rates, which were further increased by IL-1. This stimulation of proteoglycan release was also dependent on NO production. These studies show that mechanical stress and IL-1 have interacting effects on the metabolic activity of meniscal cells, potentially through NOS2-mediated production of NO. Taken together, these findings suggest that mechanical stress may play a role in the regulation of the metabolic activity of the meniscus in vivo.
Our results are generally consistent with previous studies showing increased total protein synthesis in bovine meniscal explants exposed to oscillatory compression (30). However, the same study showed no influence of dynamic stress on proteoglycan synthesis (30), whereas our data demonstrated that dynamic compression stimulates proteoglycan synthesis. These differences may be attributable to differences in the loading regimens between the two studies. Specifically, Imler and coworkers (30) applied 1 Hz compression of 3% oscillatory strain on top of a 10% static offset, whereas in the present study, we applied intermittent compression of 0.1 MPa at 0.5 Hz. In other studies, Upton and coworkers (53) showed that sinusoidal compression of the meniscus for 24 h at 0.1 MPa, 0.5 Hz decreased mRNA levels for decorin (∼2.1-fold difference) and type II collagen (∼4-fold difference) but had no effect on gene expression for type I collagen or aggrecan. Taken together with the results of the present study, these findings suggest that the effects of mechanical compression on matrix biosynthesis occur, at least in part, at the posttranscriptional level.
The magnitude of mechanical stress used in our model system was selected to simulate physiological levels of tissue deformation (strain), believed to be on the order of 10–15% (48). The duration of these mechanical loading regimens (24 h) likely exceeds that encountered during normal activities. It is important to note that these experiments were primarily designed to measure matrix metabolism and the production of inflammatory mediators in response to a steady-state mechanical stimulus. Our previous studies have shown that this mechanical regimen significantly increases NO and PGE2 production by porcine meniscus or articular cartilage without loss of cell viability (17, 19).
In other studies, cyclic tensile stretch of fibrochondrocytes in monolayer was noted to suppress the catabolic action of IL-1 on fibrochondrocytes and chondrocytes by inhibiting the expression of NOS2 and NO production (1, 20, 60). These studies demonstrated that cyclic tension is a potent antagonist of IL-1 actions and exerts its effect via transcriptional regulation of the IL-1 response element. These results are consistent with our findings that dynamic compression decreased the stimulation of NO production by IL-1. It is important to note that compression of cells in an explant, as we have used, will likely expose the cells to a significantly different mechanical environment at the cellular level (24, 26) compared with tensile stretch in monolayer.
In the absence of mechanical loading, we observed that IL-1 increased the rate of protein synthesis. The meniscus is composed primarily of type I collagen; therefore incorporation of [3H]proline is likely to reflect predominantly collagen I synthesis. This finding is in general agreement with previous reports that IL-1 can increase collagen I and III gene expression and decrease collagen II expression (22). Cao and associates (5) also showed that IL-1 reduced collagen synthesis, whereas total protein synthesis was unaffected as measured by [3H]proline incorporation in meniscal culture. They also noted that inhibition of NO production suppressed the synthesis of collagen and proteoglycan by menisci but, similar to our findings, protected proteoglycan from the catabolic effects of interleukin-1. Similar to these findings, a previous study of chondrocytes compressed in agarose showed that dynamic strain counteracted the effects of IL-1 on articular chondrocytes by suppressing NO synthesis (8).
Our data confirmed that meniscal cells produce NO spontaneously and that NO production is increased by dynamic compression (19). However, protein synthesis was not affected by inhibiting NOS. These findings are in general agreement with previous reports showing that the endogenous synthesis of NO does not affect the synthesis of noncollagenous protein in articular cartilage (6). This study also reported that NO fails to reduce the abundance of Col2lA mRNA and, furthermore, that blocking the induction of NO by chondrocytes cultured with IL-1 restores translational and posttranslational processes but fails to increase mRNA abundance (6). These findings imply that the alterations in collagen synthesis rates may occur without significantly affecting levels of mRNAs encoding the various collagen α chains (5). This finding is consistent with a recent report showing that 24 h of dynamic compression does not alter collagen I gene mRNA levels (53), although protein synthesis rates may be altered.
In the presence of IL-1, dynamic compression did not increase protein or proteoglycan synthesis. This finding suggests that an inflammatory environment may alter the physiological response of menisci to mechanical stress. Of particular interest was the finding that the inhibition of NO synthesis by the NOS2 inhibitor 1400W restored the stimulatory biosynthetic response to dynamic compression. In the absence of IL-1, however, NO did not appear to play a role in the mechanical stimulation of biosynthetic activity. Of interest was the finding that 1400W significantly abrogated the stimulation of proteoglycan release in response to IL-1, dynamic compression, or both stimuli together. These findings suggest that actions of IL-1 on mechanically stimulated biosynthesis may involve a separate mechanism compared with mechanically induced proteoglycan breakdown.
Our data thus provide further evidence of the interaction between mechanical stress and the actions of the catabolic cytokine IL-1 and the role of NO in this process (1, 8). Although the mechanisms of these interactions are not fully understood, it appears that the antagonistic influences of IL-1 on mechanically stimulated biosynthesis are mediated via the NOS2 pathway. These findings are of particular relevance in light of recent studies showing that selective inhibition of NOS2 reduces the progression of osteoarthritis in an in vivo model of altered joint loading (44). In conclusion, both inflammation and mechanical stress are important factors in matrix turnover in the meniscus. Alterations in the distribution and magnitude of stress in the menisci may have important metabolic and biomechanical consequences on joint physiology and function.
We thank Maureen Upton and Dr. Lori Setton for valuable insights and discussion on this project, and Robert Nielsen and Stephen Johnson for excellent technical assistance.
This research was supported by Veterans Affairs Rehabilitation Research and Development Service; National Institutes of Health Grants AR-48182, AR-47920, AR-43876, and AG-15768; the North Carolina Biotechnology Center; and Flexcell International.
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