| Temporomandibular disorders (TMD) are frequently seen in dental practice. One of the severe pathological changes of such disorders is the destruction of joint cartilage, known as osteoarthritis (OA), which is characterized by degradation and loss of articular cartilage, hypertrophic bone changes with osteophyte formation and subchondral bone remodeling, and, at the clinical stage, chronic inflammation of the synovial membrane. Changes of condylar cartilage have been reported in different animal models with specially created occlusal disharmony. However, to the best of our knowledge no pathological evidences that well-resembled changes in OA have been reported.Normal cartilage metabolism is characterized by a highly regulated balance between the synthesis and degradation of the various components of the extracellular matrix (ECM). Any changes in this homeostatic steady state immediately affect the healthy functioning of the articular cartilage. Several lines of evidence speak for an important role of matrix metalloproteinases (MMPs) and its inhibitors (TIMPs) in the development of progressive joint destruction. Members of the family of matrix metalloproteinases are thought to be key enzymes in the degradation of the extracellular matrix. MMP-3 (stromelysin) is secreted by fibroblasts, synovial cells and chondrocytes and is considered to be the most important protease responsible for cartilage matrix degradation because of its ability to degrade most components of the extracellular matrix such as aggrecan, basement membrane, elastin, laminin, fibronection etc. It also activates other MMPs in cartilage such as MMP-9 (gelatinase B), which takes type IV and V collagens as substrates and plays an important role in the development of OA. Tissue inhibitors of matrix metalloproteinases (TIMPs), the natural endogenous inhibitors of MMPs, can bind MMPs with a 1:1 stoichiometry. Among them, TIMP-1 that is produced by most connective tissue cells and macrophages inhibits collagenase, stromelysin and gelatinase. The imbalance between MMPs and TIMPs has been suggested as a major factor in the development of progressive joint destruction. Aggrecan, a large aggregating proteoglycan (PG) that binds to hyaluronan leading to a super molecular complex, is one of the major structural components of cartilage.In summary, MMP-3, -9, TIMP-1 and aggrecan are expressed in joint cartilage and their expressions are sensitive to degenerative changes in joint, such as in osteoarthritis. Recently, we created a disordered occlusion in rats and observed in the temporomandibular joint (TMJ) of rats a significant degradation lesion, increased chondrocyte death and thickened disc thickness in the intermediate zone. Further evaluation on the cartilage degeneration in this rat model is expected. The purpose of present study was to examine the effect of this specially created disordered occlusion on the expression of MMP-3, MMP-9, TIMP-1 and aggrecan in the induced degenerative condylar cartilage. Thirty-two 8-week-old Sprague-Dawley rats, 16 males, weighing 200±5g, and 16 females, weighing 190±5g, were provided by the animal center of the Fourth Military Medical University. The 32 rats were divided randomly into 2 experimental groups and 2 sham-operated control groups, each group containing 4 males and 4 females. In the experimental group, the first and third molar of left maxilla and right mandible were moved medially and distally respectively. All sham-operated control rats were subjected to all of the above procedures except for those keeping the elastic rubber and self-curing resin between molars.Experimental animals, together with their age-matched controls, were sacrificed at the end of 8th or 12th week after the start of experiment. The results were shown as following:(1) The surface of the condyle cartilage in control group was smooth and intact. Four layers were recognized, that were fibrous, proliferative, hypertrophic and endochondral ossification layer. Proteoglycan distribute evenly in the extracellular matrix in proliferative and hypertrophic layers.PG loss and disarrangement of cellular disposition characterized by pyknotic nucleus, and condensed cytoplasm, which failed to fill the lacuna, were observed in 4 and 6 out of 8 joints in 8- and 12-wk-female Exp_Subgroups, and in 2 out of 8 joints in 12-wk-male Exp_Subgroups. The degraded areas varied greatly in size and were often larger in 12-wk than in 8-wk subgroups, and in female than in male subgroups. Nevertheless, repairing proliferation characterized by conjunctive invagination of chondrocytes in the hypertrophic layer that penetrated into the subchondral bone in the posterior part of the condyle cartilage was observed in 1 and 2 joints, respectively, from the 8-wk and 12-wk male ECDO subgroups, but not in the female Exp_Subgroups and the sham-operated Con_Groups. Almost all the changes located exclusively in the hypertrophic layer beneath the posterior disc attachment to condyle. (2) In Con_Group, mild MMP-3 immunoreactivity located mainly in the chondrocytes of inferior hypertrophic layer and less pronounced in the articular surface and proliferative layer.In Exp_Group, MMP-3 immunopositive cells were observed in the superior hypertrophic layer in girdle shape. There were no differences in expression intensity between 8- and 12-wk subgroups (P > 0.05). The MMP-3 immunopositive cells were identified within the seriously degradation and proliferation areas beneath the posterior disc attachment. Take treatment as the main effect, we found that the ratio of the number of MMP-3 immunopositive cells in Exp_Group was higher than that in Con_Group (P = 0.000). The interaction of sex and treatment outcome (P = 0.007) was significant. According to the SNK-q post-test, remarkably statistically significant differences were observed between female Exp_Subgroups and their age-matched Con_Groups in both 8- and 12-wk subgroups, and between 12-wk-male Exp_Subgroup and its age-matched Con_Group (P < 0.05), but not between the 8-wk-male Exp_Subgroup and its age-matched controls (P > 0.05).(3)In Con_Group, the MMP-9 immunopositive cells were mainly located in the proliferative and the superior hypertrophic layers but scarcely in the inferior hepertrophic layers. Compared to MMP-3, the immunohistochemistry reactivity of MMP-9 was weaker and the number of positive cells was fewer.In the Exp_Group, weaker immunoreactivity was found within the area of coagulation necrosis, empty cartilage lacuna, and other cell-deranged parts of cartilage, but stronger immunoreactivity was observed adjacent to these degraded areas. Within the proliferative area, the immunoreactivity was obvious. Generally the expression of MMP-9 in Exp_Group was stronger and more extensive than in Con_Group (P = 0.000). The interaction of sex and treatment (P = 0.024) was significant. In female rats, the percentage of MMP-9 immunopositive cells in 12-wk (P < 0.05), but not in 8-wk (P > 0.05) Exp_Subgroup was higher than its age-matched Con_Group. No significant difference was found between the male Exp_Groups and their age-matched controls (P > 0.05).(4) In the Con_Group, the TIMP-1 immunopositive cells were observed in almost all the layers of the joint, obviously in the proliferative and the hypertrophic layer, the same position where MMPs expressed. The expression was similar between sexes and time points.In Exp_Group, the TIMP-1 immunoreactivity was much stronger and the percentage of the number of positive cells was higher than in Con_Group (P = 0.000). Strong immunoreactivity was observed within or adjacent to the degradation and proliferative lesions. The main effect of treatment (P = 0.000), time point (P = 0.000), sex (P = 0.012), and the interactions of sex and time point (P = 0.017), the time point and treatment (P = 0.001) were significant. According to the SNK-q post-test, statistically significant difference was observed between 12-wk Exp_Subgroups and their age-matched Con_Groups (P < 0.05) in both male and female rats and between 12-wk-male and 12-wk-female Exp_Subgroups whereas no such difference was found in 8-wk subgroups.(5) In the Con_Group, the immunoreactivity of aggrecan distributed evenly in the extracellular matrix of proliferative and hypertrophic layers, stronger in 8-wk than in 12-wk subgroups in both sexes (P < 0.05).In the Exp_Group, the immunoreactivity of aggrecan was observed mainly in the hypertrophic layer. Immunoreactivity in the degeneration lesions was weaker than in Con_Group. In the proliferative area, the immunostaining distributed similar to Con_Group. There was significant main effect of sex (P = 0.006) and treatment (P = 0.000). According to the SNK-q post-test, the 12-wk-female Exp_Group showed less immunoreactivity than the age-matched Con_Group (P < 0.05). No such difference was found between other sex and age-matched Exp_ and Con_Groups.Conclusion(1) The changes of occlusion may induce the remodeling activities of temporomandibular cartilage.(2) MMP-3,-9 and TIMP-1 may play important roles in this progress.(3) Increased or reduced immunoreactivity of aggrecan were found in male and female experimental groups respectively. The changes of its distribution reflect the remodeling activities of condylar cartilage. |
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