Immunohistochemical (IHC) Staining IHC staining was performed with a two-step detection kit (Zhongshan-Golden-Bridge-Biotechnology) as described previous- ly [27]. The primary antibodies were MMP3 (Abcam, 1:100 dilution), caspase3 (Cell Signaling Technology, 1:1000 dilution), and a-SMA (Abcam 1:100 dilution). Statistical Analysis Statistical analysis was performed using SPSS version 11.0 for Windows. All data were presented as mean 6 SEM. Following confirmation of normal data distribution, all data between the experimental and control groups were analyzed using Student’s t tests with P values ,0.05 considered to be statistically significant. Results Confirmation of Injection into Upper Compartment To confirm the injection site in the upper compartment of the TMJ, one rat was preliminarily dissected after injection of fast green solution into the upper compartment. The needle was inserted right under the root of the zygoma, beneath the temporal fossa into the upper compartment. The green stained region was mainly limited to the upper compartment (Fig. 1B). Ultrastructural Changes in Disc To determine whether MIA injected into the upper compart- ment of the TMJ could diffuse into the lower compartment, the ultrastructure of the TMJ disc was evaluated by SEM and TEM 1 day after injection of MIA or saline (Fig. 2). SEM showed that the surface of the disc in the control group was furrowed and covered by an evenly distributed gelatinous layer, whereas the intermediate zone of the disc in the MIA group lost these features and presented a limited region with a thinner and smooth surface surrounded by areas with a rough and uneven surface. From the section view of the intermediate zone, disc cells in the control group inserted into the collagen fibrils, whereas the disc cells in the MIA group were crimpled and rounded, detached from the surrounding collagen fibrils (Fig. 2A). TEM showed that the disc cells of the intermediate zone in the control group were surrounded by a dense, collagenous extracel- lular matrix (ECM) and the cell junction was tight, with ovoid- shaped mitochondria around the nucleus. However, the disc cells in the MIA group underwent morphological changes, including cell shrinkage, condensation of the cytoplasm and nucleus, cell membrane detachment from the surrounding collagen fibrils, and loosened cell junctions accompanied by the disrupted ECM. Some cells even presented features of apoptosis, such as chromatin compaction, swelling mitochondria, and vacuolar degeneration (Fig. 2B). Dose-dependent Histopathologic Changes in TMJ To understand the effects of MIA on the TMJ, the morphology of the TMJ was examined for 4 weeks after injection with saline or increasing doses of MIA (Fig. 3A). In the control TMJ, HE staining showed that the condylar cartilage was a regular alignment of multilayer chondrocytes. S.O and TB staining showed that the hypertrophic layer was stained red and metachromatically purple, respectively, indicating abundant pro- teoglycans in the condylar cartilage. In the 0.05 mg MIA group, HE staining showed that the condylar cartilage was slightly decreased in cell number and thickness as compared with the control. S.O and TB staining showed slight decreases in cartilage proteoglycans. In the 0.1 mg MIA group, discontinuousness of the hypertrophic layer with peripheral cartilage thickening was MIA Induced TMJOA in Rat observed. However, in the 0.5 mg group, HE staining showed severe discontinuity of the four-layer cartilage, regional loss of chondrocytes, peripheral proliferation and clustering of chondro- cytes, a disorganized matrix network, horizontal clefts, and subchondral bone resorption with adjacent bone marrow filled with fibroblast-like cells. TB and S.O staining showed severe loss of staining in irregularly arranged chondrocytes and enhanced staining at the periphery. In the 1 mg and 2 mg MIA groups, complete loss of chondrocytes, severe thinning of cartilage, and subchondral bone erosion were evident in the lesion, but without peripheral clustering of chondrocytes and thickening of the cartilage. Typical OA-like destruction of the cartilage and erosion of the subchondral bone were observed in the 0.5 mg MIA group [39]. Therefore, 0.5 mg was defined as the minimum effective dose of MIA for induction of typical OA-like lesions in the rat TMJ. Time-dependent Histopathologic Changes in TMJ To further characterize the development of OA, the major structures of the TMJ were evaluated after injection of 0.5 mg MIA at different time points for up to 12 weeks (Fig. 3B, C). With regard to the condyle, chondrocytes disappeared following MIA induction and the matrix was less stained within the proliferative zone in the anterior and central areas of the condyle corresponding to the load-bearing region. Additionally, scattered cells with nuclear condensation were evident after 3 days. Loss of chondrocytes in all of the cartilage layers with no matrix staining was observed by 1 week. In addition to the above features, regional osteolysis and peripheral chondrocyte proliferation with deep matrix staining were observed by 2 weeks. By 4 weeks, typical OA- like lesions were observed, as described for the 0.5 mg MIA group in the dose course. By 8 weeks, fibrosis in the lesions was evident and the subchondral bone was developing sclerosis. By 12 weeks, the condylar lesions were fully repaired by sclerotic subchondral bone and thin cartilage with disorganized chondrocytes. These changes over the 12-week period were not due to aging effects when compared with the control group. (Fig. 3B). Time-dependent changes, including synovitis, disc thinning, and the destruction of temporal fossa cartilage following MIA induction, are shown in Fig. 3C. Massive fibrin-like exudates were observed in the upper compartments of TMJs in the experimental group by 3 days to 1 week after MIA injection, but not in the control group. Abundant proliferative villi consisting of multi-layer synovial lining cells and apparent infiltrated mononu- cleated cells were present in the upper compartment by 2 weeks. The synovial villi decreased and became smaller by 4 weeks and nearly disappeared by 12 weeks. Chondrocytes were almost lost in the cartilage of the temporal fossa and intermediate zone of the disc by 3 days after MIA injection. Until 2 weeks,there were almost no further changes in the disc and temporal fossa. From 4 weeks to 12 weeks, the disc and the cartilage of the temporal fossa became thinner, but the subchondral bone of the temporal fossa remained intact and no disc perforation was observed. Radiographic Changes in Subchondral Bone To fully understand the changes in the subchondral bone after MIA-injection (0.5 mg/joint), radiographic changes in the condyle were evaluated by MicroCT scanning (Fig. 4A). On sagittal images, the bone surface of the control condyle was smooth and continuous, whereas the bone surface of the anterior and central areas of the condyle was discontinuous by 1 week after MIA injection. Multi-erosions, characterized by translucency disrupting the bone surface of the load bearing areas, grew deeper and more extensive with obvious defects from 2 to 4 weeks. By 8 weeks, the PLOS ONE | www.plosone.org 4 September 2012 | Volume 7 | Issue 9 | e45036