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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Objective

To study the influence of tissue maturation and antioxidants on apoptosis in bovine articular cartilage induced by injurious compression.

Methods

Bovine articular cartilage disks were obtained from the femoropatellar groove of animals ages 0.5–23 months and placed in culture. Cartilage disks were preincubated overnight with the cell-permeable superoxide dismutase (SOD) mimetic Mn(III) porphyrin (0–12.5 μM) or α-tocopherol (0–50 μM) and then injured by a single unconfined compression to a final strain of 50% at a velocity of 1 mm/second. After 4 days of additional incubation, the disks were fixed and embedded for light and electron microscopy. Apoptotic cells were quantified morphologically by the appearance of nuclear blebbing on light microscopy. Biosynthetic activity was demonstrated by incorporation of radiolabeled proline. The antioxidative action of the SOD mimetic was confirmed by histologic examination of cartilage after incubation with nitroblue tetrazolium.

Results

Injurious compression induced significantly more apoptosis in cartilage disks from newborn calves (22% of cells) than in cartilage from more mature cows (2–6%). In cartilage from 22-month-old animals, the SOD mimetic reduced the percentage of apoptotic cells induced by injury in a dose-dependent manner (complete inhibition with 2.5 μM), while α-tocopherol had no effect. Neither antioxidant altered protein biosynthesis or cellular ultrastructure.

Conclusion

Our data suggest that the apoptotic response of articular cartilage to mechanical injury is affected by maturation and is mediated in part by reactive oxygen species. The antioxidative status of the tissue might be important for the prevention of mechanically induced cell death in articular cartilage.

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.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Reagents

The cell permeable SOD mimetic MnTMPyP was obtained from Alexis Biochemicals (Grünberg, Germany). Its chemical structure is shown in Figure 1. All other chemicals were obtained from Sigma (Munich, Germany) unless indicated otherwise.

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Figure 1. Chemical structure of the superoxide dismutase mimetic Manganese(III)tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride.

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Isolation and culturing of articular cartilage explants

Articular cartilage disks were obtained from the bovine femoropatellar groove, as previously described (33), from animals ranging in age from 2–3 weeks (0.5 months) to 23 months. Briefly, cartilage–bone cylinders (9 mm in diameter) were obtained by drilling perpendicular to the cartilage surface. Samples were removed and placed in a microtome holder. After removing enough cartilage to create a level surface, the top 1 mm of cartilage was sliced at a thickness of 1 mm with a microtome. Explant disks (n = 5 or 6) measuring 3 mm in diameter × 1 mm in thickness were punched out of each slice and equilibrated for 24 hours at 37°C in an atmosphere of 5% CO2 in culture medium consisting of low-glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 10 mM HEPES buffer, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 0.4 mM proline, 20 μg/ml of ascorbic acid, 100 units/ml of penicillin G, 100 μg/ml of streptomycin, and 0.25 μg/ml of amphotericin B. The explants were distributed among the different experimental groups matched by their original anatomic location in order to avoid tissue-dependent false-positive or false-negative effects.

Injurious compression

Before injurious compression, some of the explants were preincubated overnight with antioxidative substances. Injury was then applied to groups of 3 or 4 cartilage explants at a time. The explants were placed in chambers, and radially unconfined compression was applied by an incubator-housed loading device as described previously (16, 34). Controlled displacement ramps to 50% final strain were applied at a ramp velocity of 1 mm/second (corresponding to a strain rate of 100%/second) and were held at the final strain for 5 minutes. The force produced during compression was also recorded, which allowed calculation of the peak stress applied to the cartilage during the injury. After compression, the explants were cultured for another 4 days, and biochemical and microscopic analyses were performed as described below.

Histologic detection of apoptosis

After mechanical compression and subsequent culture, the cartilage explants were fixed overnight using 4% paraformaldehyde and then embedded in Paraplast. Serial sections (7 μm) were obtained sagittally through the entire thickness of the explant disks, immobilized on glass slides, and stained with Mayer's hematoxylin to quantify the percentage of cells showing nuclear blebbing. In some cases, sections were stained for the presence of TUNEL-positive cells according to the manufacturer's protocol (ApopTag peroxidase in situ apoptosis detection kit; Oncor, Gaithersburg, MD).

We evaluated 3–5 sections from each explant disk. Since cutting of the explants induces apoptosis at the edges of the tissue, the margins of the sections (∼150 μm thickness) were excluded. Using a Zeiss Axiophot microscope (Zeiss, Wetzlar, Germany) with a 40× objective, positive and negative cells were counted in 3 optical fields in each section (60–100 cells/field). One optical field was located in the center of the explant sections and 2 were located more peripherally, near the corners of the sections (but not including the margins). The mean value from each field was recorded. In a secondary analysis, cell apoptosis rates in the central and peripheral fields were compared. Encoded labels were used on all samples to ensure blind scoring.

Ultrastructural detection of apoptosis

To further validate the determination of apoptosis by nuclear blebbing on light microscopy, ultrastructural changes were evaluated by electron microscopy. Explants were fixed overnight at 4°C with 2.5% glutaraldehyde in phosphate buffered saline (PBS), washed, and incubated for 1 hour at room temperature with 1% OsO4. Samples were embedded in Araldite, and 50-nm–thick sections were prepared, incubated for 15 minutes with saturated uranyl acetate in 70% methanol, hydrated in descending concentrations of methanol, and incubated in a lead citrate solution in a CO2-poor atmosphere for 5 minutes. The sections were visualized using a Zeiss EM900 electron microscope.

Biosynthetic activity

The wet weight of each cartilage explant was measured before injury. After the experimental treatment, the explants were placed in fresh culture medium containing 10 μCi/ml of 3H-proline for 18 hours under free-swelling conditions. Unincorporated radiolabel was removed by washing in PBS containing 0.5 mM proline (solution replaced 3 times after 20 minutes each) and digested overnight at 65°C in 1 ml of papain solution per explant (2.125 units/ml of papain in 0.1M Na2HPO4, 0.01M sodium EDTA, 0.01ML-cysteine, pH 6.5). One hundred microliters of each sample was added to 2 ml of scintillation fluid, and radiolabel incorporation was measured in a Beckmann scintillation counter. Results were expressed as counts per minute per milligram wet weight and normalized to the radiolabel incorporation by uninjured control tissue, which was set at 100%.

Statistical analysis

To test the effect of maturation on the rate of cell apoptosis after injury, apoptosis was measured in paired samples of unloaded control and injured cartilage explants from animals of different ages. Because the resulting data included both animal-to-animal variation as well as age-related effects, a mixed-effects model (35) was required to analyze the data. Age was modeled as a fixed effect, each animal was modeled as a random effect, and the paired difference in apoptosis rate (apoptosis after injury minus apoptosis in control for each pair of matched explants) was used as the outcome measure. The model was fit by restricted maximum likelihood estimation of parameters using S-Plus software (Insightful [formerly, MathSoft], Seattle, WA).

Dose-response experiments with 4 doses of SOD mimetic and α-tocopherol were designed to test the effect of ROS scavengers on apoptosis produced by injury. The effect of dose on the paired difference in apoptosis rate between injured and control explants was tested with a linear regression model. A nonlinear response was assumed, and doses were applied over an exponential range. The data were linearized by rank transformation of both dose and outcome (i.e., dose was converted from 0, 0.0025, 0.025, and 2.5 μM SOD mimetic to 0, 1, 2, and 3) (36). Since 2 animals were used to generate the cartilage explants, and the anatomic site of each cartilage explant was not completely matched among all 8 experimental groups, we included animal and site as covariates in the model.

An experiment to test the effect of preincubation in SOD mimetic also included data from 2 animals and was analyzed by linear regression, including animal in the model as a covariate. Control and experimental groups were otherwise compared by Student's 2-tailed t-test, and differences were considered significant at α = 0.05.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Detection of apoptosis

Apoptosis was demonstrated morphologically by counting cells that exhibited nuclear blebbing, a specific morphologic indicator of apoptosis (11, 37, 38), in serial histologic sections of articular cartilage explants (Figures 2A and 2B). In some experiments, apoptotic cells were counted in histologic sections after labeling with TUNEL (Figures 2C and 2D). Nuclear blebbing was also demonstrated by electron microscopy (Figures 3A and 3B), confirming the presence of apoptosis in these samples.

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Figure 2. Light microscopy of cartilage tissue explants from 23-month-old cows. A and B, Mayer's hematoxylin–stained sections. In contrast to normal cells in unloaded explants (arrow in A), paraffin sections of explants 4 days after injurious compression showed cells with prominent nuclear blebbing (arrow in B). Inset, Higher magnification view. C and D, TUNEL-stained sections. Unloaded tissue showed no positive nuclei (arrow in C), whereas TUNEL-positive nuclei (brown) were present after injurious compression of tissue (D), and nuclear blebbing was present as well (arrow in D). Inset, Higher magnification view. E and F, Cryosections after incubation for 1 hour with nitroblue tetrazolium (NBT). Cartilage incubated with NBT only (E) showed strong transformation of NBT into the blue dye formazan in the cytoplasm of the cells (arrowheads in E). Inset, Higher magnification view. This was reversed by the addition of 2.5 μM superoxide dismutase mimetic (arrowheads in F). Bars = 25 μm.

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Figure 3. Ultrastructure of articular chondrocytes from 23-month-old cows 4 days after injurious compression. A, Nucleus of a chondrocyte from uninjured control tissue. B, Apoptotic nucleus of an injured chondrocyte, showing nuclear blebbing (arrow). C, Incubation with the superoxide dismutase mimetic (2.5 μM) inhibits nuclear blebbing of the cells and has no toxic influence on the ultrastructure of articular chondrocytes as compared with control tissue. Arrows in A and C show cell organelles, such as the Golgi apparatus and the rough endoplasmic reticulum. Bars = 1.2 μm.

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There was no significant difference between the percentage of cells positive for apoptosis by TUNEL staining versus nuclear blebbing in injured cartilage explants from 6-month-old calves (mean ± SD nuclear blebbing 7.3 ± 2.0% versus TUNEL staining 8.0 ± 1.9%; n = 3 injured cartilage explants from 2 animals). However, consistent with previous reports, we observed in preliminary experiments that the results of the TUNEL assay were highly sensitive to small changes in the protocol, so we primarily quantified apoptosis by histomorphometric examination of chondrocytes on light microscopy in all the results reported below.

Influence of tissue maturation on apoptosis produced by injurious compression

A single axial compression to a final strain of 50% at a velocity of 1 mm/second induced mean peak stresses of 17 MPa in tissue from newborns and 25–29 MPa in tissue from more mature animals (6–23 months old).

Cell apoptosis induced by injurious compression was measured by the presence of nuclear blebbing in the central microscopic fields of cartilage from 5 animals of various ages (Figure 4). Nuclear blebbing was present in 22% of the cells in injured cartilage from newborn (0.5-month-old) calves, compared with 5.9% (6 months), 5.4% (16 months), 2.3% (20 months) and 5.7% (23 months) of tissue from more mature animals. Due to the nonlinear response of apoptosis with age, age was dichotomized as newborn versus more mature (6–23 months) for the statistical analysis. The change in apoptosis after injury was significantly decreased in the more mature cartilage compared with the newborn cartilage (mean ± SEM –18 ± 3%; P = 0.01 in a mixed-effects model).

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Figure 4. Percentage of cells showing nuclear blebbing 4 days after injurious compression. The increase in apoptosis after injury was higher in cartilage from newborn cows (age 0.5 months) compared with cartilage from more mature cows (ages 6–23 months) (P = 0.01 by mixed-effects analysis). Values are the mean and SEM of 18 unloaded and injured cartilage disks from 5 animals.

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In contrast, when nuclear blebbing of cells in the more peripheral corner fields was measured, the change in apoptosis after injury was not significantly affected by age (1 ± 4%; P = 0.87 in a mixed-effects model). This was attributable to differences in apoptosis rates between cells in the central field and cells in the peripheral field, which varied with maturity. In newborn tissue, the percentage of apoptotic cells in the central fields of the explants was higher than that in the peripheral fields (mean ± SEM rate in central minus peripheral fields 7.1% ± 3.2%, n = 3 animals). In contrast, in the more mature tissue (6–23 months), apoptosis was more common in the peripheral fields (rate in central minus peripheral fields –7.5% ± 1.4%, n = 4 animals).

MnTMPyP inhibition of apoptosis produced by injurious compression

A control experiment was performed to confirm the antioxidative action of the SOD mimetic on cartilage. Explants were incubated for 90 minutes with 1.25 mg/ml of nitroblue tetrazolium (NBT) in Hanks' buffer with or without 2.5 μM SOD mimetic. NBT is transformed predominantly by superoxide anions produced by living cells into the blue dye formazan (39). Cryosections of cartilage explants incubated with NBT alone were strongly stained (Figure 2E), while addition of the SOD mimetic inhibited the formation of formazan (Figure 2F).

Over the range of doses used in these studies, neither SOD mimetic or α-tocopherol had any significant effect on proline incorporation by explants of unloaded cartilage from 23-month-old cows after 3 days of incubation (data not shown). Furthermore, incubation of injured cartilage in SOD mimetic did not alter proline incorporation (mean ± SEM control 100 ± 9%, loaded tissue 95 ± 8%, loaded tissue plus SOD mimetic 98 ± 9%, n = 4 explants per condition, from 2 animals; data normalized to control). Finally, electron microscopy revealed no obvious change in ultrastructural cell morphology of unloaded cartilage (Figure 3A), unloaded cartilage incubated with α-tocopherol (results not shown), or injured cartilage incubated with 2.5 μM SOD mimetic (Figure 3C).

In cartilage from 22-month-old cows, the SOD mimetic reduced the percentage of mechanically induced apoptotic cells significantly in a dose-dependent manner (P < 0.001 for linear effect of rank-transformed dose on rank-transformed change in apoptosis after injury, by linear regression, adjusted for animal and site) (Figure 5), while α-tocopherol (concentrations up to 50 μM) had no effect on the apoptotic response (data not shown). A 2.5 μM concentration of SOD mimetic was needed in order to decrease the apoptotic response to the level in uninjured controls.

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Figure 5. Effect of a superoxide dismutase mimetic on apoptosis 4 days after injurious compression of cartilage explants from 22-month-old cows. The superoxide dismutase mimetic had a significant and dose-dependent effect on the increase in apoptosis produced by injury (P < 0.001 for linear effect of rank-transformed dose on rank-transformed change in apoptosis after injury, adjusted for animal and cartilage site). Values are the mean and SEM of 10 unloaded and injured cartilage disks from 2 animals.

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To determine whether the presence of the SOD mimetic was required during injurious compression in order to inhibit apoptosis, cartilage from 22-month-old cows was incubated in 2.5 μM SOD mimetic starting either before injury or immediately after injury. Control tissues were not loaded but were incubated in SOD mimetic in parallel with the experimental tissues. Nuclear blebbing was measured after 4 days of culture following injury. The following mean (±SEM) increases in the apoptosis rate (calculated as the apoptosis rate after injury minus the apoptosis rate in uninjured cartilage with the same SOD mimetic treatment) were seen: 11.0 ± 1.8% without SOD mimetic, 1.4 ± 0.1% with SOD mimetic after injury, and 0.5 ± 0.1% with SOD mimetic before and after injury (n = 7 explants per condition, from 2 animals).

Since the equal-variance assumption of linear regression did not appear to be valid, the outcome measure (change in apoptosis after injury) was rank-transformed. Treatment with SOD mimetic before and after injury significantly reduced apoptosis after injury. In addition, there was a small, but statistically significant, reduction in apoptosis for treatment before and after injury compared with treatment only after injury (P < 0.001 for all 3 comparisons, by linear regression, adjusted for animal).

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

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.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

The authors would like to thank Rita Kirsch, Claudia Kremling, and Frank Lichte for excellent technical support.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES