Injurious Loading of Articular Cartilage Compromises Chondrocyte Respiratory Function

Abstract

Objective: To determine whether repeatedly overloading healthy cartilage disrupts mitochondrial function in a manner similar to that associated with osteoarthritis (OA) pathogenesis.

Methods: We exposed normal articular cartilage on bovine osteochondral explants to 1 day or 7 consecutive days of cyclic axial compression (0.25 MPa or 1.0 MPa at 0.5 Hz for 3 hours) and evaluated the effects on chondrocyte viability, ATP concentration, reactive oxygen species (ROS) production, indicators of oxidative stress, respiration, and mitochondrial membrane potential.

Results: Neither 0.25 MPa nor 1.0 MPa of cyclic compression caused extensive chondrocyte death, macroscopic tissue damage, or overt changes in stress-strain behavior. After 1 day of loading, differences in respiratory activities between the 0.25 MPa and 1.0 MPa groups were minimal; however, after 7 days of loading, respiratory activity and ATP levels were suppressed in the 1.0 MPa group relative to the 0.25 MPa group, an effect prevented by pretreatment with 10 mM N-acetylcysteine. These changes were accompanied by increased proton leakage and decreased mitochondrial membrane potential, as well as by increased ROS formation, as indicated by dihydroethidium staining and glutathione oxidation.

Conclusion: Repeated overloading leads to chondrocyte oxidant-dependent mitochondrial dysfunction. This mitochondrial dysfunction may contribute to destabilization of cartilage during various stages of OA in distinct ways by disrupting chondrocyte anabolic responses to mechanical stimuli.

Figure 1. Representative strain profiles demonstrate no overt changes in stress-strain behavior over one week with healthy or injurious loads

Loading tissue at 0.25 MPa, shown in panel A, or 1.0 MPa, shown in panel B, for 5400 cycles at 0.5 Hz induces maximum tissue strains ranging from 15% to 23% and 43% to 56%, respectively, in both single day and seven day loading specimens. Strain occurring over a single cycle during 0.25 MPa loading did not exceed 7% of the original thickness while these strains in 1.0 MPa loaded tissue did not exceed 12%. This mechanical behavior of both 0.25 and 1.0 MPa loaded explants was not significantly altered by the seventh day of loading, shown in grey, relative to single day loading, shown in black. This suggests that the loading procedure utilized did not overtly alter the mechanical properties of the bovine cartilage.

Figure 2. One week of heavy loading increases endpoints indicative of oxidative stress in chondrocytes

DHE oxidation in live chondrocytes following a week of cyclic loading was significantly higher in explants loaded with 1.0 MPa than in explants loaded with 0.25 MPa, (A, red, quantified in B). Of particular interest are chondrocytes staining more lightly for Calcein with significant DHE staining (A, arrows). No observable changes in the viable chondrocyte density were detected as indicated by Calcein Green AM staining (A, green). In association with increased live cell oxidation of DHE, GSSG levels were significantly increased after a week of overloading (C normalized to matched controls in D), an effect reversed by NAC addition. Data represent the mean of at least three images per sample, standard deviation shown is for n = 5 for all groups except NAC where n = 4, p-values as indicated. These data indicate NAC-responsive cellular oxidative stress following repeated injurious loading.

Figure 3. Repeated injurious loading compromises mitochondrial function to a similar but not identical degree to changes observed in osteoarthritis

As previously shown, human osteoarthritic chondrocytes display increased basal and maximum mitochondrial oxygen consumption, relative to intact tissue, as well as a strong trend towards a decrease in spare respiratory capacity (A) and an increase in proton leakage (B), indicating mitochondrial dysfunction. After single day cyclic loading, bovine chondrocytes demonstrate no significant differences between 0.25 and 1.0 MPa loading (C). After 7 loading sessions, significant losses in basal respiration, maximum respiration, and spare respiratory capacity were observed in the overloaded explants (D). We also observed significant increases in proton leakage associated with overloading, similar to those observed previously in arthritic chondrocytes (18) though less significant (B). We expressed proton leakage as a % of basal function, a likely source of this distinction. Data represent the mean of at least 4 wells per explant, standard error of the mean of n = 5 for bovine data and n = 18 for human data, p-values as indicated. These data suggest that damage similar to that observed in human arthritic chondrocytes is occurring within the mitochondria of overloaded cartilage.

Figure 4. Mitochondrial membrane potential is depressed after one week of injurious loading

Sagittal sections of the loaded area of each explant were assayed immediately after harvest for mitochondrial membrane potential using JC-1 confocal microscopy. Similar to respiratory effects, single day overloading has no impact upon mitochondrial membrane potential compared to normally loaded controls (quantified in A, representative pictures in B). By contrast, overloading for one week appears to depress mitochondrial membrane potential to levels consistent with the losses in mitochondrial function described above (18). Data represent the mean of at least three images per sample, standard error of the mean of n = 4 is shown, p-values as indicated. These data suggest that the losses in respiration observed in extracellular flux measurements are present immediately at harvest and also indicate a disruption of overall mitochondrial function consistent with osteoarthritis.

Figure 5. N-acetylcysteine protects against metabolic dysfunction associated with overloading

Pretreatment with 10 mM NAC 2 hours prior to loading with NAC maintained during loading was able to protect against loss of basal mitochondrial respiration (A). This was associated with prevention of increases in proton leakage caused by overloading (B). ECAR data obtained from this study suggest a trend towards decreased in glycolytic function (C); however, ATP content, a stronger indicator of anabolic activity, was decreased with overloading (D). In both ECAR and ATP measurements, NAC prevented deficits incurred by overloading. Seahorse XF96 data represent the mean of at least 4 wells per sample, standard error of the mean of n = 4 is shown, p-values as indicated. ATP measurement shown with standard deviations of n = 4, p-value as indicated. These data suggest that the losses in respiration observed are occurring as a result of oxidative damage to the chondrocytes.