The inhibition of subchondral bone resorption in the early phase of experimental dog osteoarthritis by licofelone is associated with a reduction in the synthesis of MMP-13 and cathepsin K

Abstract

Objective: To evaluate the morphological changes that take place in the subchondral bone and calcified cartilage zone in the experimental anterior cruciate ligament (ACL) dog model of osteoarthritis (OA) and analyze concomitant changes in the level of MMP-13 and cathepsin K, as well as examine the therapeutic effects of licofelone, a lipoxygenase (LO)/cyclooxygenase (COX) inhibitor, on these morphological and biochemical changes. Methods: Experimental group 1 underwent sectioning of the ACL of the right knee with no active treatment (placebo group). Experimental groups 2 and 3 underwent sectioning of the ACL of the right knee and were administered therapeutic concentrations of licofelone (2.5 or 5.0 mg/kg/day po, respectively) for 8 weeks, beginning the day following surgery. Group 4 consisted of untreated dogs used as normal control. Specimens of subchondral bone including the calcified cartilage were selected from lesional and non-lesional areas of OA tibial plateaus. Specimens were processed for static morphometric analysis and immunohistochemical analysis for MMP-13 and cathepsin K. Results: As indicated by a reduction in bone surface and trabecular thickness, a significant loss of subchondral bone occurred in OA dogs. These changes were associated with an increased level of MMP-13 synthesis by bone cells and an increase in the osteoclast population that stained strongly positive for cathepsin K and MMP-13. Changes were much more pronounced in the specimens taken from the lesional areas. Treatment with licofelone decreased, in a dose-dependent manner, the OA bone morphological changes at the same time it reduced the level of MMP-13 in bone cells and the number of cathepsin K and MMP-13 positive osteoclasts. Conclusions: Increased bone loss and bone resorption is associated with the development of OA cartilage lesions. Licofelone treatment was found to prevent the morphological and biochemical changes seen in early experimental OA effectively. These findings may help explain the mechanisms by which this drug could exert its possible effect on the development of OA.

Introduction

The structural changes observed in osteoarthritis (OA) involve all the major tissues of the joint, including the cartilage, synovium, subchondral bone, and periarticular soft tissues, such as the muscles and ligaments [1]. Changes at the cartilage and synovial levels have been extensively described and well characterized [1], [2], [3]. The role, for instance, of the synovial membrane in cartilage degradation and other structural changes is now well established [1], [4]. There is a growing body of evidence that points to the importance of cross-talk between these tissues as being an integral part of the disease process [1], [4].

Radin and Rose [5] were among the first to point to a possible role for subchondral bone in the initiation and progression of cartilage degeneration. It was speculated that the increase in bone mass and thickness could have modified the biomechanical properties of the tissue and could have favored the appearance/progression of articular cartilage structural changes. Many studies have demonstrated that the subchondral bone is the site of several dynamic morphological changes that vary over time during the evolution of the disease and seem to be part of the disease process [3], [6], [7], [8], [9]. These changes are allied with many local abnormal biochemical pathways that are likely involved not only in the bone changes but may also contribute, in association with the anatomical changes, to cartilage degradation [10], [11], [12], [13], [14], [15], [16], [17].

Several reports have indicated that the subchondral bone remodeling that occurs in OA involves both bone resorption and bone formation. Several studies have demonstrated that, in the more advanced stage of the disease, bone formation was predominant in the different layers of the tissues [2], [6], [18]. In contrast, studies which looked at the morphology and metabolism of subchondral bone in the early phase of the disease have, in general, identified a remodeling process that primarily favors bone resorption [6], [9], [11], [19], [20], [21], [22]. Most of these findings came from experimental models allowing for a chronological evaluation of events. A recent report by Bettica et al. [9] clearly demonstrated that bone resorption is also increased in patients with progressive knee OA. Reports have also mentioned that, in the early phase of experimental OA, the subchondral plate and the underlying trabecular bone become thinner, indicating excess bone resorption [6], [9], [20], [21], [22]. These changes are associated with an increase in the number and size of the remodeling units [20]. Moreover, in experimental dog OA, osteoblasts isolated from subchondral bone were shown to produce in excess many biochemical factors which have been previously demonstrated to favor either the maturation and activation of osteoclast and/or the resorption of bone matrix [23], [24]. Such factors included urokinase plasminogen activator (uPA), insulin growth factor-1 (IGF-1), and prostaglandin E₂ (PGE₂) [10], [11], [25]. Recent reports have indicated that the subchondral bone remodeling and resorption in experimental dog OA can therefore be reduced by an NSAID inhibiting the cyclooxygenase activity [25]. This effect was associated with a reduction in the level of synthesis of several proteolytic enzymes, namely, uPA and growth factors known to be involved in bone remodeling. These findings are most interesting and very much in line with the results of a recent study that demonstrated the key role played by eicosanoids, PGE₂ and LTB₄, in mediating the abnormal synthetic activity of human OA subchondral bone osteoblasts [17].

There is still speculation as to whether the abnormal subchondral bone morphology and biochemistry are linked to the development/progression of cartilage damage. Some studies have reported that the subchondral bone changes preceded the cartilage lesions and could be responsible for the evolution of cartilage lesions [5], [7], [8]. Other studies have reported that subchondral bone changes happen simultaneously or even follow cartilage changes and, therefore, could only be a secondary phenomenon to cartilage degradation [3], [20], [22]. Obviously, the question about a possible relationship between subchondral bone remodeling and cartilage lesion changes remains of great relevance and is the subject of intense research. The possible pathways involved in such a cross-talk between these two tissues remain largely unknown at this time. However, there are several factors synthesized by subchondral bone cells known to be capable of inducing cartilage catabolic changes. For instance, TGF-β, which is synthesized in increased amounts by human OA subchondral bone cells [16], has the capacity to induce MMP-13 synthesis by chondrocytes and therefore could be responsible for the increased level of this enzyme found in the deep layers of OA cartilage [26]. This enzyme, as well as cathepsin K, can be synthesized by bone cells and has been demonstrated to be involved both in endochondral ossification and in OA cartilage degradation [27], [28].

The aims of the present study were to further characterize the morphological changes that take place in the calcified cartilage and subchondral bone in the early phase of experimental dog OA and to explore some of the mechanisms that could be responsible for those changes, more specifically, two major proteolytic enzymes involved in bone remodeling/resorption, namely, MMP-13 and cathepsin K. Moreover, the effect of in vivo treatment with therapeutic concentrations of licofelone, a lipoxygenase (LO)/cyclooxygenase (COX) inhibitor, on this process was studied; the interest being that this drug was previously demonstrated to reduce the development of experimental OA cartilage lesions.