In the experimental dog OA model induced by the sectioning of the ACL, treatment with ASU can reduce the development of early OA cartilage and subchondral bone lesions. The histological findings were informative with respect to the effects of ASU on the OA cartilage structural changes. In fact, dogs treated with ASU exhibited a significant decrease in indicators of cartilage matrix damage, such as the structural changes indicative of collagen network breakdown and Safranin-O staining, which indicates aggrecan degradation. In addition, treatment with ASU was found to reduce chondrocyte hyperplasia and cloning. These findings are in accordance with those of Cake and coworkers [9
] in an ovine meniscectomy model of OA.
Many proteases have been shown to play major roles in the catabolism of OA cartilage. For instance, MMP-13 has been demonstrated to play a predominant role in the degradation of collagen type II in OA cartilage [23
], whereas ADAMTS4 and ADAMTS5 are believed to be key proteases in the degradation of aggrecans [24
]. In the ACL dog model, inhibition of the synthesis of these enzymes by treatment with different drugs, such as pioglitazone (a peroxisome proliferator-activated receptor-γ agonist [26
]) and licofelone (a dual inhibitor of 5-lipoxygenase and cyclo-oxygenase [27
]), has been found to be associated with a reduction in the development of cartilage lesions. These enzymes, by cleaving the triple helix of collagen type II and core protein of the aggrecan respectively, induce major irreversible damage to the cartilage matrix structure. In so doing, they can modify the biophysical properties of cartilage and reduce its resilience to the abnormal biomechanical forces present in OA.
In the present study, treatment with ASU reduced the level of MMP-13 synthesis in the deep zone of cartilage. These findings are in accordance with a previous study in which ASU were demonstrated to reduce MMP-13 mRNA in murine chondrocytes in monolayer culture under stimulation with IL-1β [28
]. MMP-13 expression is increased in tissue that is in need of repair or remodelling, as in OA. MMP-13 was previously shown to be preferentially increased in the deep zone of cartilage [22
]. and was described as a major catabolic factor in that zone as well as in OA lesional areas [30
]. Therefore, our finding of the effect of ASU treatment on reduction in MMP-13 synthesis could explain the prevention of OA cartilage lesion development as well as the protective effect of ASU on the erosion of the calcified cartilage [19
]. On the other hand, ASU were found to have no effect on the level of synthesis of other proteases involved in cartilage matrix degradation, such as MMP-1, ADAMTS4 and ADAMTS5; these enzymes are believed to be predominantly involved in matrix degradation in the more superficial zones of cartilage. These results support the hypothesis that the reduction in collagen degradation in the deep zone of cartilage could reduce the development of OA lesions. Alternatively, the absence of effect on the above proteases may explain the mild to moderate effect that ASU had on the reduction in OA cartilage lesions.
MMP-13 is known to be involved in subchondral bone remodelling and resorption of calcified cartilage in OA [18
]. As previously demonstrated by Cake and coworkers [9
], ASU treatment was able to protect the remodelling of subchondral bone in an ovine OA model. Moreover, studies suggest that drugs that can reduce MMP-13 synthesis in cartilage and bone and prevent subchondral bone resorption in the dog ACL model could exert a disease-modifying effect in knee OA patients [18
]. Our findings demonstrate that ASU could reduce OA subchondral bone remodelling and resorption, which leads to osteopenia, a phenomenon well documented in the ACL dog OA model that occurs during the first few months after surgery [32
]. The bone volume and calcified cartilage thickness in the ASU-treated group were greater than those found in the placebo group and close to the morphometric values found in normal dogs [18
]. These findings indicate that the ASU treatment reduced the loss of subchondral bone. Moreover, ASU treatment was able to maintain subchondral bone and calcified cartilage structure close to normal values.
ASU treatment prevented the loss, but it did not increase bone surface or calcified cartilage thickness over the values found in normal dogs [18
]. These data also support the concept that the subchondral bone is the site of morphological changes that are part of the OA disease process [5
], and provide further evidence in favour of the concept that therapeutic intervention to reduce these changes may also prevent the development of cartilage lesions. This latter hypothesis is further supported by a number of published studies showing that, in ACL OA models, treatment with calcitonin [33
] and alendronate [35
] reduced bone resorption as well as cartilage degeneration. In this context, the study conducted by Henrotin and coworkers [36
], in which ASU reversed the inhibition of aggrecan and collagen synthesis in OA chondrocyte/osteoblast co-culture, is supportive of the existence and key role played by the crosstalk between cartilage and subchondral bone in OA pathophysiology.
In contrast to normal cartilage, OA cartilage produces an excess amount of nitric oxide (NO) upon iNOS (the enzyme responsible for NO production) stimulation by cytokines [37
]. High levels of nitrite/nitrate have also been found in the synovial fluid and serum of arthritis patients [39
] as well as in synovial tissue from OA patients [40
]. It has been hypothesized that NO contributes to the development of arthritic lesions [42
] by inhibiting the synthesis of cartilage matrix macromolecules [45
] and by inducing chondrocyte death [49
], which could further contribute to the reduction in extracellular matrix in OA. NO was also shown to reduce the synthesis of the IL-1 receptor antagonist in chondrocytes [38
], a process possibly responsible for the enhanced IL-1β effect on these cells. Moreover, the diffusion of NO from the superficial layer of cartilage to the deeper zone may also have contributed to increasing the level of MMP-13 synthesis at that level [51
]. An in vivo
study with N
-lysine, a potent and selective iNOS inhibitor [52
], demonstrated its therapeutic effectiveness in reducing the progression of experimental OA in the ACL dog model [53
]. The same study also demonstrated that iNOS inhibition reduced the synovial inflammation, a finding that is well in agreement with those of the present study. Moreover, ASU exhibited an inhibitory effect on iNOS, and therefore on NO production, which may provide an explanation for the protective effect of ASU. These results are in accordance with those of a study in human OA chondrocytes previously published by Henrotin and coworkers [54
The present study has limitations largely imposed by the study design. One such limitation is the duration of the study (8 weeks). A longer study would provide more information on the potential effects of ASU against the long-term development of OA. Moreover, the study design involved prophylactic use of the drug and the conclusions drawn might have been influenced by this treatment schedule. A further study in which therapeutic administration is employed would be informative and complementary to the present one. The mechanisms of action of ASU, especially their global effect on catabolic/anabolic factors, needs further investigation in order to reach a better understanding of the disease pathways that are modified by this treatment.