The etiology of osteoarthritis (OA), a degenerative joint disease, is still not fully understood. Along with several other factors, mechanical overload and cell death may be important contributors to the degeneration of articular cartilage (1–3). Chondrocyte death must have important consequences in cartilage, since these cells represent 1–10% of the tissue volume and have a very low regenerative capacity (4). Since each cell is responsible for the maintenance of its surrounding extracellular matrix, cell death could play a significant role in the degradative activity that leads to OA.
In addition, it has been proposed that apoptotic cell death occurs in OA cartilage and may play a causative role in the pathogenesis of OA (5–8). In particular, Hashimoto et al (7) reported that destabilization of rabbit knee joints induces apoptosis in articular cartilage and that the prevalence of apoptotic cells is significantly correlated with the grade of OA. This theory remains a subject of controversy, since other investigators were unable to confirm this finding (9), and the finding may have been attributable to false-positive results from the TUNEL assay (10–13).
However, several studies have shown that in vitro mechanical injury by various compressive loading protocols can cause significant apoptotic cell death (11, 14, 15). Investigators from our group have shown that injurious compression of newborn bovine cartilage can induce apoptosis, as assessed by both TUNEL and nuclear morphology (that is, the presence of nuclear disintegration or blebbing), accompanied by cartilage swelling, release of matrix proteoglycan, and loss of the anabolic response to low-amplitude dynamic compression in the remaining cells (14, 16). We were therefore interested in using this model to investigate additional aspects of mechanically induced apoptosis. Since degenerative diseases are correlated with age, and it is known that biomechanical and biochemical properties of articular cartilage vary at different stages of age and maturity (17, 18), we hypothesized that the apoptotic response of the tissue to mechanical injury might also be affected by the maturation of the tissue.
In addition to characterizing this apoptotic response in order to develop insights into the mechanotransduction of mechanical injury, the induction of programmed cell death is clinically interesting, since it may be possible to prevent cell death after traumatic joint injury (19). We were therefore interested in testing whether apoptosis could be inhibited in our model. Several investigators have shown that reactive oxygen species (ROS) and especially superoxides are involved in some of the pathways leading to programmed cell death (20–23). As a result, antioxidative substances have been shown to inhibit apoptosis in a number of cell types (22, 24–26), including chondrocytes (27).
We therefore hypothesized that antioxidative scavenger mechanisms would influence the induction of apoptosis by mechanical injury. This hypothesis is supported by a recent report by investigators from our group that a diet enriched in vitamins and selenium increased the expression of antioxidative enzymes in articular cartilage and significantly reduced the incidence of mechanically induced OA in the STR/1N mouse (28).
To test this hypothesis in our bovine model, we used 2 molecules that are considered to have different antioxidative functions. Manganese(III)tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride (MnTMPyP) is a molecule with superoxide dismutase (SOD) mimetic properties (29–31), and α-tocopherol (vitamin E) inhibits peroxidation of membrane molecules by hydroxyl radicals (32). The objectives of this study were therefore to test the influence of tissue maturation and the antioxidative scavengers MnTMPyP and α-tocopherol on apoptosis induced by injurious compression of bovine articular cartilage.
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It has now been demonstrated by several groups of investigators that mechanical compressive injury can induce apoptotic death in chondrocytes under certain circumstances in vitro (11, 14, 15). We have shown here that there is a significant effect of tissue maturation on the apoptotic cell death produced by injurious compression, with much higher cell death in newborn bovine cartilage. We found that the apoptotic cell death can be inhibited by incubation with the superoxide dismutase mimetic MnTMPyP, demonstrating that this response to mechanical injury is mediated at least partly by the generation of reactive oxygen species.
The reason for the increased apoptotic cell death after mechanical injury in the most immature tissue is not known, but this observation is consistent with that of Tew et al (10), who reported that cell death after cutting bovine cartilage explants was much higher in newborn tissue than in mature tissue. It seems very likely that the increased cell death we observed in newborn cartilage is related to the increased metabolic activity and mitotic rate of this tissue (40–42), as hypothesized by Tew et al.
Since the cartilage from more mature animals is thinner than that from newborns, the mature cartilage explants included tissue from relatively deeper zones. Zonal differences may therefore also have contributed to the results. Several investigators have reported that cells in the superficial zone are more sensitive to mechanically induced cell death than cells in deeper zones (10, 43). However, in the process of producing flat explant surfaces, we remove the superficial zone, and since differences in sensitivity to cell death seem much less pronounced in the middle and deep zones, this seems unlikely to explain the changes in cell death with maturation that we observed.
In addition, the differences in apoptosis with maturation may be partly explained by associated differences in the biomechanics and biochemical composition of the cartilage. Our observation that there was more apoptosis in the central region of newborn cartilage disks but not in more mature tissue suggests a hypothesis for how this could occur. As newborn bovine cartilage matures, the most prominent changes appear to be an increase in stiffness and a higher collagen content (17). Therefore, the radially unconfined compression applied in our studies would be expected to produce more radial bulging of the disks of cartilage from newborns. This could explain the distribution of cell death in newborn cartilage, since this radial strain would be at a minimum at the top and bottom edges of the cartilage, where the peripheral sections exhibited less cell death. In contrast, it is interesting to note that the peak stresses produced by injurious compression to 50% strain at 1 mm/second were higher in the mature cartilage (consistent with a stiffer tissue), suggesting that peak stress was not the mechanical parameter responsible for the decrease in the apoptotic response with tissue maturity.
In our in vitro experiment with more mature articular cartilage from the 2-year-old cows, we found that MnTMPyP, a Mn(III) porphyrin SOD mimetic, prevented the apoptotic cell death produced by injurious compression, with complete inhibition by a concentration of 2.5 μM. Control experiments confirmed that the observed prevention of apoptosis was not a consequence of reduced metabolic activity or cellular toxicity, as assessed by protein synthesis and cellular ultrastructure. To our knowledge, this is the first report that chondrocyte death after mechanical injury is mediated at least in part via the generation of ROS. A linkage between mechanical injury and ROS would be consistent with the report by Kaiki et al (44) that injection of hydrogen peroxide acted synergistically with the activity of running to produce OA in rat knees. Although the origin of the ROS here remains unknown, MnTMPyP inhibited a large proportion of apoptosis even when added to the explants after injurious compression. So, it is likely that the ROS are primarily generated at some point after injury in the pathway that leads to apoptosis.
In contrast to the effect of the SOD mimetic MnTMPyP, there was no influence of α-tocopherol on apoptosis induced by injurious compression. Alpha-tocopherol is not able to scavenge superoxides or hydrogen peroxide, but it inhibits the peroxidation of membrane molecules by hydroxyl radicals (32), suggesting that the role of superoxide scavenging may be particularly important in the cell death seen here. However, further studies would be needed to specifically identify any particular ROS-mediated pathway, since although scavenging of superoxides has been shown to be a major function of the Mn(III) porphyrins in living cells (29, 30), these molecules can also scavenge molecules that are descended from superoxides, such as peroxynitrite (45) or hydrogen peroxide (46).
The in vivo effect of a traumatic injury of the joint or cartilage on chondrocyte viability is not yet clear. The absolute levels of apoptosis generated by the injurious compression model used in the present study should not be interpreted as a simulation of the levels that occur clinically, since there are important differences in mechanical conditions. However, several initial clinical investigations have recently reported a substantial increase in apoptotic cell death in fragments obtained after intraarticular fracture (19, 47) and in biopsied cartilage obtained after joint injuries (48). Although those studies appear to have relied primarily on TUNEL staining (a method sensitive to false-positive staining), the induction of apoptosis after a discrete event in vivo would certainly be important to investigate as a possible target for pharmacologic intervention. It has been shown by several groups of investigators that cartilage apoptosis can be prevented by caspase inhibitors (15, 49, 50). The results of the present investigation, together with the evidence that a diet enriched in antioxidants can reduce the development of mechanically induced OA in an animal model (28), suggest that the antioxidative status of the tissue may have importance as another possible target for the prevention of chondrocyte death and OA.