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Increase in RANKL: OPG Ratio in Synovia of Patients with Temporomandibular Joint Disorder

T. Wakita1,2, M. Mogi1, K. Kurita2, M. Kuzushima1, and A. Togari1,*

1 Department of Pharmacology and
2 Department of Oral and Maxillofacial Surgery-I, School of Dentistry, Aichi-Gakuin University, Nagoya 464–8650, Japan


Figure 1
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  		Figure 1. Plots depicting RANKL (A, pg/mg protein) and OPG (B, pg/mg protein) concentrations, and the RANKL:OPG ratio (C) in the synovial fluid of joints with disk displacement with reduction (DDwR), joints with disk displacement without reduction (DDw/oR), and osteoarthritis (OA) in patients and control participants. The content of RANKL in the synovial fluid was determined by an ELISA, and that of OPG, by a commercially available ELISA. Data are expressed as the mean ± SEM. Data from control (Cont; n = 13), DDwR (n = 25), DDw/oR (n = 39), and osteoarthritis (OA; n = 53) participants were analyzed. Fisher’s protected least-significant-difference post hoc test was used when multiple groups were compared with a single control group. **P < 0.01, ***P < 0.005 (compared with the control), and #P < 0.05, ##P < 0.01, and ###P < 0.005, as indicated by the brackets.

 

Figure 2
Figure 2
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  		Figure 2. RANKL+OPG induces osteoclast formation from human peripheral monocytes in the presence of M-CSF. Human peripheral blood mononuclear cells were isolated, washed, and cultured for 6 days with M-CSF (100 ng/mL) in 24-well plates. According to the estimated value of the RANKL:OPG ratio in the control (0.07), DDwR (0.30), DDw/oR (0.43), and OA (0.77) participants, we prepared the respective mixtures of recombinant human RANKL (PeproTeck, London, UK) and OPG (OPG/Fc chimera, R&D Systems, Minneapolis, MN, USA) in {alpha}-MEM. With RANKL fixed at 30 ng/mL in the sample, OPG was added to the sample at a final concentration of 400 ng/mL (Cont), 100 ng/mL (DDwR), 70 ng/mL (DDw/oR), or 40 ng/mL (OA). Then the mixture was used in the osteoclast formation assay by PBMC (TRAP staining), and to the pit-formation assay, in the presence of M-CSF (100 ng/mL). TRAP-positive cells containing 3 or more nuclei were counted as osteoclasts.

Values are expressed as the mean ± SEM (number/well) of 3 cultures. *P < 0.05, **P < 0.01, ***P < 0.005 (compared with the control), and ###P < 0.005 as indicated by the brackets. Experiments were repeated 3 times, and similar results were obtained. Bar = 200 µm.

Pit-forming activity of OCLs formed in the cultures was assayed on dentin slices (diameter, 10 mm) in 24-well plates. After having been cultured for 21 days, the adherent cells were removed from the slices, and the resorption pits on the slices were then stained with Mayer’s hematoxylin. Osteoclastic bone-resorption activity was measured in terms of the pit area formed by osteoclasts. Values are mean ± SEM of 3 cultures. **P < 0.01 compared with control groups. ##P < 0.01 as indicated by the brackets. Bar = 500 µm. Each experiment was repeated 3 times, and the data shown are representative of 3 independent experiments. Arrows point to osteoclasts. Original magnification, 40x for TRAP staining, and 100x for pit-formation assay.

 

Figure 3
Figure 3
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  		Figure 3. Synovial fluid induces osteoclast formation from human peripheral monocytes in the presence of M-CSF. The synovial fluids were de-salted by dialysis, lyophilized, and concentrated up to the original concentration (around 100-fold). Then, the reconstituted samples were applied to the osteoclast formation assay by PBMC, and to the pit-formation assay, in the presence of M-CSF (100 ng/mL). **P < 0.01, ***P < 0.005 compared with control groups. #P < 0.05, ##P < 0.01, and ###P <0.005, as indicated by the brackets. Experiments were repeated 3 times with similar results. Arrows point to osteoclasts. Original magnification, 40x for TRAP staining (bar = 200 µm), and 100x for pit-formation assay (bar = 500 µm).

 

Figure 4
Figure 4
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  		Figure 4. Anti-RANKL IgG or OPG blocks OA-synovial fluid-induced osteoclast formation. The reconstituted synovial fluid (OA) was applied to the osteoclast formation assay using PBMC, and to the pit-formation assay, in the presence of M-CSF (100 ng/mL), along with anti-human RANKL IgG (10 µg/mL, PeproTeck, London, UK) or human recombinant OPG (200 ng/mL, R&D Systems). ***P < 0.005 compared with the data obtained in the absence of either additive. Experiments were repeated 3 times with similar results. Arrows point to osteoclasts. Original magnification, 40x for TRAP staining (bar = 200 µm), and 100x for pit-formation assay (Bar = 500 µm).

 




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