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Iowa Orthop J. 2007; 27: 1–8. | PMCID: PMC2150661 |
Copyright © 2007, The Iowa Orthopaedic Journal Antioxidants Block Cyclic Loading Induced Chondrocyte Death BR Beecher, JA Martin, DR Pedersen, AD Heiner, and JA Buckwalter Department of Orthopaedics and Rehabilitation, University of Iowa, Iowa City, IA 52242 Articular cartilage in congruous joints benefits from the moderate stresses and strains associated with normal cyclic loading. However, loading of joints with surface incongruities can lead to local stress and strain elevation at “step-off” sites where cartilage is not fully buttressed by surrounding matrix. Excessive stresses and strains predicted to occur at such sites may induce apoptosis, a process thought to promote cartilage degeneration and osteoarthritis (OA) through chondrocyte attrition. We hypothesized that the induction of apoptosis is mediated by oxidants, and that antioxidants can reduce elevated stress-induced chondrocyte attrition. To test this we exposed cylindrical cartilage explants from human articular cartilage to radially unconfined cyclic axial compression (3600 cycles, 1 Hz, 50% duty cycle) using two different physiologic loads (2MPa and 5 MPa). We found that 30% of chondrocytes in the superficial zone died within 24 hours of exposure to loading with 5 MPa axial compression, whereas mortality was limited to less than 15% with 2 MPa axial compression. Similarly, lactate accumulation in the medium was suppressed by compression with 5 MPa, but not 2 MPa. Approximately 80% of cell death induced by 5 MPa compression was blocked by pre-incubation of the explants in a variety of anti-oxidants including vitamin E, n-acetyl cysteine (NAC), and a superoxide dismutase mimetic (SOD). SOD and NAC also prevented the suppression of lactate secretion after 5 MPa compression. These observations support the hypothesis that the harmful effects of abnormal cyclic loading are mediated by oxidants and suggest that treatments to prevent OA may include methods of minimizing oxidative damage to chondrocytes. High-energy joint injuries that disrupt articular surfaces often lead within a few years to post-traumatic OA. 13,15,32 Although the overall risk for post-traumatic OA varies among different joints and with patient age at the time of injury, mechanical factors play a significant role in determining outcome. 4,5,32 These factors include the energy delivered to the articular surface at the time of injury and, in joints with residual surface incongruity or instability, the potential for chronically abnormal mechanical stress. The development of treatments to forestall post-traumatic OA may be aided by a better understanding of the biologic responses to mechanical conditions in injured joints. 20,38,40The effects of mechanical injury on cartilage stability have been investigated in a number of in vitro studies using cartilage from various animals and from humans. Physical effects of impact loading were demonstrated by Jeffrey et al. 22 This study of bovine cartilage showed a linear increase in chondrocyte death three days after impact, indicating a partial loss of ECM integrity. A follow-up study showed that biosynthetic activity, as assessed by metabolic radiolabeling, was significantly impaired in the days post-impact. 23 Similar results were found in canine cartilage after impact: Hexosamine content in impacted cartilage declined after impact loading, coincident with structural disruption and death of chondrocytes. 9 Proteoglycan synthesis was also impaired and water content declined in bovine cartilage exposed to severe impacts of 25-75 MPa. 2 These authors also reported the detachment of cartilage surrounding an impact site as a significant source of destabilizing collateral damage. Other authors studying the metabolic and physical effects of impact loading reported that proteoglycan synthesis declined at 24 hours post-injury in concert with loss of chondrocyte viability, collagen network disruption, and increased tissue hydration. 39 They suggested that the threshold for these effects was near 20 MPa and that loads equal to or greater than that caused irrevocable structural damage. Though not as severe as the effects of impact loading, cyclic loading may also contribute to matrix damage and instability. 11 Lin et al., reported that cyclic loading with 5 MPa (0.5 Hz) for 1 or more hours caused increased stromelysin-1 (MMP-3), proteoglycan degradation and collagen damage in cyclically load-injured articular cartilage. 30 These studies demonstrate unequivocally that mechanical insult is associated with chondrocyte death, either by apoptosis or necrosis, loss of biosynthetic activity, and with structural disruption of the cartilage ECM. Several studies show an association between mechanical stress and increased production of reactive oxygen species (ROS), as well as decreased antioxidant capacity. 16,18,19,27,33,34 Although low levels of oxidants are required for normal cartilage metabolism, exposure to elevated ROS levels is associated with chondrocyte death and matrix degeneration. 6,10,21,37,41 There is evidence to suggest that apoptosis associated with intra-articular fractures is due, at least in part, to elevated levels of ROS induced by impact loading. 25,26 Apoptosis and the resulting reduction in cell density is thought to play a significant role in the development of post-traumatic OA. 24Exogenous antioxidants provide some protection to chondrocytes from the harmful effects of elevated oxi-dants. 8,14 There is evidence that a superoxide dismutase (SOD) mimetic combined with methotrexate protects rats with collagen-induced arthritis from the development of OA. 12 Previous studies in our laboratory established that mechanical compression in an unconfined configuration leads to extensive chondrocyte death and apoptosis in cartilage explants. Cell death was blocked by the free radical scavenger n-acetyl cysteine (NAC), suggesting that oxidative damage mediated the harmful effects of compression. 34 Another published study indicated that apoptotic chondrocyte death induced by a single high impact load could be inhibited by treatment with a SOD mimetic that detoxifies oxygen free radicals, but not by the anti-lipid peroxidation activity of a-tocopherol (vitamin E). 26 These findings suggested that lipid oxidation plays a minor role in impact-induced death. Nitric oxide (NO) also appears to play a role in oxidative apoptosis: The nitric oxide synthase inhibitor N-Nitro-L-arginine methyl ester (L-NAME) has been shown to inhibit chondrocyte apoptosis when oxygen free radicals are present. 28,31 One study suggests that a peroxnitrite scavenger decreases GAG loss in articular cartilage implicating an oxidative mechanism for GAG breakdown. 1Estimates of contact stresses at cartilage surfaces in ankle and knee joints during routine physical activity range from a few to several MPa. 3,36 Within this range of contact stresses it is thought that articular cartilage strain in normal, congruent joints rarely exceeds 15%. 7 However, in incongruent joints, the same moderate contact stresses can lead to local strains of 30% or greater at “step-off” sites due to the partial loss of transverse support normally provided by surrounding matrix. 35 Repetitive exposure to strains near this magnitude has been shown to induce chondrocyte apoptosis in cyclically-compressed cartilage explants. 11 We hypothesized that apoptosis induced by cyclic axial compression is mediated by oxidants, and is therefore subject to inhibition by antioxidants. To test this we determined the effects of antioxidants on chondrocyte viability in human cartilage explants exposed to unconfined cyclic axial compression (1 Hz, 3600 cycles) in a mechanically-active bioreactor. 17,34 Compression amplitude was varied (2 MPa or 5 MPa) to produce near physiologic strains (10-15%), and super-physiologic strains (20-30%), representing conditions that might occur in and around step-off sites in vivo. Human cartilage explants (4 mm diameter) were harvested from non-osteoarthritic ankle joints from 10 donors. The explants were incubated in culture medium (40% Dulbecco's modified Eagle medium, 40% Ham's F12, 10% alpha-MEM, 10% fetal bovine serum) in a 5% O 2, 5% CO 2, 90% N 2 atmosphere at 37°C. Some explants were incubated in antioxidants for 24 hours prior to mechanical stress exposure. These antioxidants included n-acetyl-cysteine (NAC) at a concentration of 2.5 mM, superoxide dismutase (SOD) (MnTBAP) at a concentration of 50 μM, catalase ( Aspergillus niger)at a concentration of 15 mg/mL, and vitamin E at a concentration of 100 μM. Additional explants were incubated for 24 hours with the nitric oxide (NO) synthase inhibitor N-Nitro-L-Arginine Methyl Ester (L-NAME), which blocks NO-induced apop-tosis. L-NAME was used at a concentration of 1.0 mM. Untreated controls were also included. All explants were placed in the bioreactor for mechanical stress treatment. The bioreactor is a mechanically active culture device capable of imposing variable shear stress states at quasi-physiologic levels. 17,34 Here, the bioreactor was used to apply unconfined cyclic axial compression to cartilage explants (30-40 MPa/sec loading, 3600 cycles at 1 Hz, 50% duty cycle) to simulate the lack of support at joint reduction incongruities. The amplitude of compression was either 2 MPa or 5 MPa. Non-compressed controls were included in each experiment. Data obtained from DVRTs and caliper measurements of initial and final explant height were used to calculate Green-Lagrange strains over 3,600 cycles of loading. Strain versus cycle number plots revealed that the rate of change in overall strain, and the greatest peak-to-peak strains were greatest over the first few hundred cycles of loading. The rate of change declined significantly after 1000 cycles, when near maximum steady strain was reached. Linear regression analysis over early (75 to 250 cycles) and late (1,000 to 3,500 cycles) intervals was used to derive slopes and y-intercepts to characterize the rate of change in overall strain, the maximum strain within each cycle, and maximum overall strain. These parameters were calculated for individual explants and the data were pooled to find means for 5 MPa compression (n = 35) and 2 MPa compression (n = 41). Student's t-testwas used to determinethe significance of the differences between these groups. Following mechanical compression, exptants (n = 6 per group) were removed from the bioreactor and incxirjated overnight in cakem AM to stain viable chondrocytes end eihiblum nomodimee ta stain nonviable chondrocytes. Explants were cryoembedded and an aliquot of the medium was removed for lactate assay, which was performed using a colorimetric assay kit according to the manufacturer's directions (Biomedical Research Services). Explants (6 per group) were cryosectioned and imaged with 488 nm light using an Olympus BX60 epifluoresence microscope equipped with a stepper motor-driven stage. Individual frames were tiled to form high-resolution composite images. Viable cells (calcein-stained) and nonviable cells (ethidium homodi-mer stained) were counted using a custom designed automated MATLAB based image analysis program to determine percent viability. The program automatically identifies and counts fluorescent-labeled cells in highresolution composite images of full thickness cartilage sections (4 mm wide x 1-3 mm thick). The program also automatically segments images into superficial (top 15%), middle (16%-45%), and deep (46-100%) zones. Percent viability was normalized to non-stressed controls in each experimental group. Duplicate sections were analyzed from each of the 6 explants representing an experimental group (12 sections total). Replicate oyosotions from explante were stained for aeoetosis using a commercral in situ TUNEL assay kit wtth terramethyl rhodamamine-or fluorscein isothiocyanate-labeled dUTP suitable for epifluorescence imaging (Roche). The stains were imaged and analyzed as described above for viability stains. Each experiment was performed with at least 6 explants. Kruskal-Wallis One Way Analysis of Variance on Ranks was used to evaluate the statistical significance of differences between treatment groups. Representative plots illustrate strain behavior of explants over 3600 cycles of 2 MPa or 5 MPa axial compression (). Maximum overall strain averaged 19 ± 4.5% with 2 MPa compression and 27 ± 3.4% with 5 MPa, a significant difference (p < 0.001). Dynamic strain increased significantly (p <0.001) from 5.9 ± 2.7% with 2 MPa compression to 9.0 ± 2.6% with 5 MPa axial compression. Fluorescence stains for chondrocyte viability revealed that most chondrocytes (>85%) in all cartilage zones were alive in control explants and in explants after 2 MPa compression, but there was significant loss of viability in explants after 5 MPa compression (). The greatest losses occurred in the superficial zone, where viability was reduced to 40%. However, there was also significant death in the middle zone, in which viability was reduced to 55% (). Post-compression histological assays for DNA fragmentation (TUNEL reaction) revealed that much of the cell death observed in viability assays was attributable to apoptosis (). The frequency of TUNEL reaction positTe cells in uncompressed controls was less than 5% in all zones but increased to >20% in the superficial and middle zones after 5 MPa axial compression. NAC pretreatment reduced the numberofposttive cellsto lees than 10% in both zones. The primarily superficial/middle zone distribution of TUNEL reaction-positive chondro-cyte0was simitar to the pattern or deCd cell0 detected by calcein AM/ethidium homodimer staining. Chondrocyte mortality induced by high shear stress was dramatically affected by pre-incubation with antioxidants prior to stress treatment (). Post-compression viability in NAC-, vitamin E-, and SOD-treated explants increased in the superficial zone from ~40% to ~80%, a statistically significant change (p<0.005). The same reagents improved viability significantly (from 53% to >80%) in the middle zone, whereas only NAC improved viability in the deep zone (from 83% to >87%) (). L-NAME, a nitric oxide synthase inhibito r that has been shown to block chondrocyte apoptosis, had effects similar to those of antioxidants. Catalase appeared to have little if any beneficial effects in the superficial zone, as post-stress viabilities remained low (60%) in the presence of the enzyme. Lactate accumulation in the culture medium was measured to determine compression and antioxidant effects on chondrocyte metabolism (). Compression with 5 MPa caused a significant reduction in lactate (p < 0.05) relative to unloaded controls, but compression with 2 MPa had no significant effect. Lactate production in explants compressed with 5 MPa was maintained at control levels when NAC or SOD was used for pre-treatment, but not when catalase was used. Chondrocyte viability after 3600 cycles of compression with 5 MPa was significantly higher in explants treated with a variety of antioxidants than in untreated controls exposed to the same compression regime. Post-stress viability in the superficial zones of anti-oxidant-treated explants was near 80%, twice as high as in controls. These findings implicate oxidative stress in the harmful effects of cyclic mechanical stress in articular cartilage and show for the first time that antioxidants protect chondrocytes from those effects. The TUNEL reaction indicated that compression with 5 MPa induced apoptosis in the superficial and middle zones of cartilage explants. In cyclically compressed explants, nearly 20% of chondrocytes in these zones were positive for the TUNEL reaction, five times higher than in controls. Treatment with NAC before mechanical stress exposure reduced apoptosis to less than 10%. These findings indicate that apoptosis contributes significantly to cyclic compression-induced chondrocyte death and suggest that antioxidants minimize cell death by inhibiting apoptosis. The exact mechanism(s) of inhibition are still unclear: Antioxidants might act by mitigating the oxidative damage that would otherwise initiate the apoptosis cascade. Alternatively, antioxidants may block the activation of mitogen activated kinases or other signal transduction pathways involved in the expression of apoptosis-related genes. Further work is needed to determine if this is the case and to assess the effects of antieeidants on chondrocyte necrosis, which might also contribute to cell attrition induced by cyclic compression. Lactate assays revealed that 5 MPa compression suppressed overall glycolytic activity in explants. These findings paralleled the results of viability assays, suggesting that much of the reduction in lactate production was associated with chondrocyte bss. Howeveathe magnituae of redurtioniii lactate (~50% loss) was greater than might be predicted Crom cell losses alone (32% +/− 7%). This suggests that 5 MPa compression inhibited glycolysis in chondrocytes that survived compression. 29Vitamin E, NAC, and SOD all showed similar anti-apoptosis activity in explants exposed to compression with 5 MPa, strongly indicating a role for oxidative damage in this process. The effects of antioxidants were comparable to the effects of L-NAME, the nitric oxide synthase inhibitor that has been shown to inhibit IL-1-induced apoptosis in chondrocytes. 32 Although the most significant form(s) of oxidative damage induced by cyclic compression are uncertain, the anti-apoptosis activity of vitamin E suggests that, unlike blunt impact injury, cyclic stress induces lipid peroxidation. 26Compression with 5 MPa was associated with increased accumulated strain, as well as increased peak-to-peak stiffness, which correlated with increased chondrocyte death. Dead cells were found mainly in the superficial zone and the middle zones. The zone-specific distribution of these effects are likely related to zonal variations in the composition of the cartilage extracellular matrix: the relatively low concentration of proteoglycans in the superficial and middle zones leads to reduced compressive stiffness and higher local stains than in the proteoglycan-rich deep zone. 7Catalase did not protect chondrocytes in explants from mechanical stress effects. The reasons for this are unclear since catalase is expected to help detoxify hydrogen peroxide, a potentially damaging oxidant produced by chondrocytes. Although it is conceivable that H2O2 is not generatedduring cyclic compression, the failure of catalaseto improveviabik'ty could be due to more prosaic issuesrelated toits function under the culture conditions we used: the molecular weight of catalase (250 kDa) is more than 10 times as large as the other antioxidants we tested. Thus, it is possible that the inability of catalase to penetrate the cartilage matrix may have impaired any potential chondrocyte-sparing effects. The mechanisms by which excessive mechanical forces contribute to OA are unknown, stymieing development of effective methods for prevention or treatment. The findings of this study support the hypothesis that the harmful effects of excessive stress and strain are mediated du oxtdaate. These observations suggest that methods of minimizing acute oxidative damage, including local administration of antioxidants, might decrease the risk for OA, particularly under abnormal mechanical stress conditions that exist at sites of residual articular incongruities following joint trauma. Supporte d by award P50 AR48939, National Institutes off Health Specialized Center of Research for Osteoarthritis 1. Bezerra MM, Brain SD, Greenacre S, Jeronimo SM, de Melo LB, Keeble J, da Rocha FA. 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