Transforming growth factor beta 3 involved in the pathogenesis of synovial chondromatosis of temporomandibular joint

Abstract

Synovial chondromatosis (SC) of temporomandibular joint is rare proliferative disorder featured by the formation of cartilaginous nodules in synovium and joint space. Transforming growth factor beta 3 (TGF-β3) is closely related to chondrogenic differentiation, and might participate in pathogenesis of SC. We discovered that increased quantity of synoviocytes and blood vessels were observed in SC synovium. The vessel wall and sublining fibroblasts were stained positively by the antibodies against TGF-β3, fibroblast growth factor 2 (FGF-2), and CD34. In loose bodies (LBs), TGF-β3 was mainly expressed in chondrocytes and FGF-2 was expressed in chondrocytes, fibroblasts, and vessel walls. Expressions of TGF-β1, TGF-β3, FGF-2, Sox9, Wnt-4, Foxc2, and VEGF-A mRNA were significantly higher in SC synovium. Stimulation of TGF-β3 on synoviocytes increased alkaline phosphatase (ALP) activity and expressions of chondrogenic genes (Sox9, Col2α1, Aggrecan, Wnt-4, and Wnt-11), osteogenic genes (Runx2, Foxc2, osteocalcin, and Col1α1), and VEGF-A, but failed to influence FGF-2 expression. However, the addition of FGF-2 increased TGF-β3 expression. In conclusion, TGF-β3 existed in synovium and LBs of SC, and was responsible for the pathogenesis of SC.

Figure 1. Histological and IHC observations for TMJ synovium facing the articular cavity.

HE staining for control synovium (a) and SC synovium (b). IHC observation of TGF-β3 (c), FGF-2 (d), and CD34 (e) in SC synovium. Control synovium was featured by layers of fibroblast-like synoviocytes located in the lining layer and a few fibroblasts and blood vessels in the sublining layer. The SC synovium was characterized by increased amount of fibroblasts and blood vessels in the sublining layer. The vessels wall and sublining fibroblasts were stained positively by the antibodies against TGF-β3, FGF-2, and CD34. * represents for articular cavity. Scale bars: (a~e): 50 μm.

Figure 2. Histological and IHC observations for two types of LBs.

One type was the single cartilaginous nodule (a–d), and the other type was numerous nodules composing a large nodule (e–h). In the first type, a thick synovium containing increased quantity of synoviocytes and bloods vessels covered the LB (a and b). In the second type, a connective tissue containing blood vessels and fibroblasts separated these small nodules (e and f). TGF-β3 was expressed mainly in chondrocytes of LBs (c and g) while the FGF-2 was expressed in both chondrocytes and the wall of blood vessels (d and h). b and f are the amplification of the rectangle in a and e, respectively. Scale bars: (a and e): 400 μm; (b~d) and (f~h): 100 μm.

Figure 3. RT-PCR (a) and semi-quantitative evaluation (b) for analyzing the genes expressions in control and SC synovium.

*P < 0.05, **P < 0.01 with respect to the control. Semi-quantitative values are presented as mean ± standard deviation of target gene/β-actin. The assay was performed in triplicate under the same experiment conditions. Full-length gels are presented in Supplementary Figure 1.

Figure 4. Realtime PCR for analyzing the genes expressions of SC synoviocytes cultured with or without TGF-β3 for 6 days.

*P < 0.05, **P < 0.01 with respect to the control. The assay was performed in triplicate under the same experiment conditions.

Figure 5. Realtime PCR for determining the interaction between TGF-β3 and FGF-2, and the ALP activity assay.

The addition of FGF-2 in the culture medium for SC synoviocytes increased the TGF-β3 gene expression by 16.9-fold. However, the addition of TGF-β3 did not influence the FGF-2 expression significantly. The ALP activities in the two groups with and without the addition of TGF-β3 were 18.1 and 9.6 U/gprot, respectively. The assay was performed in triplicate under the same experiment conditions.