Induction of osteoclast-like cells derived from the synovial lavage fluids of patients with temporomandibular joint disorders H. Takano, D.D.S., W. Ariyoshi,

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Induction of osteoclast-like cells derived from the synovial lavage fluids of patients with temporomandibular joint disorders H. Takano, D.D.S., W. Ariyoshi, D.D.S., Ph.D., T. Kanno, D.D.S., Ph.D., E. Fukuhara, D.D.S., Ph.D., H. Ichimiya, D.D.S., Ph.D., T. Matayoshi, D.D.S., Ph.D., T. Goto, D.D.S., Ph.D., T. Takahashi, D.D.S., Ph.D. Osteoarthritis and Cartilage Volume 15, Issue 3, Pages 291-299 (March 2007) DOI: 10.1016/j.joca.2006.08.001 Copyright © 2006 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Phase-contrast photomicrograph of TMJ SF cells cultured for 2 weeks (A) and 4 weeks (B) in the presence of 20ng/ml rh M-CSF and 10−7M 1,25(OH)2D3. After 4 weeks, the culture showed a predominance of fibroblastic cells (B). Original magnification, ×100. Osteoarthritis and Cartilage 2007 15, 291-299DOI: (10.1016/j.joca.2006.08.001) Copyright © 2006 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 May-Grunwald–Giemsa staining (A) and TRAP (B) of multinucleated giant cells after removal of the fibroblastic cells. The cells induced in this culture system were multinucleated and TRAP-positive. Original magnification, ×200. Osteoarthritis and Cartilage 2007 15, 291-299DOI: (10.1016/j.joca.2006.08.001) Copyright © 2006 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Examination of resorption areas formed by multinucleated bone-resorbing giant cells on Osteologic discs, as seen by phase-contrast microscopy (A, B, black arrows). Giant cells were present near the pits (A, white arrows). Original magnification, ×200. Osteoarthritis and Cartilage 2007 15, 291-299DOI: (10.1016/j.joca.2006.08.001) Copyright © 2006 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Examination of the resorption pits formed by multinucleated bone-resorbing giant cells on dentin slices. Lectin staining of lacunar resorption of dentin slices on which multinucleated bone-resorbing giant cells were incubated (A). SEM (B). Bar=50μm in (A); 20μm in (B). Osteoarthritis and Cartilage 2007 15, 291-299DOI: (10.1016/j.joca.2006.08.001) Copyright © 2006 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 The stimulation of fibroblastic cells from the SF with 10−7M 1,25(OH)2D3 or 10−7M PGE2 increased the expression of RANKL mRNA (341bp PCR product), as shown by RT-PCR. Lane 1, control (no stimulation); Lane 2, 10−7M 1,25(OH)2D3; Lane 3, 10−8M 1,25(OH)2D3; Lane 4, 10−7M PGE2; Lane 5, 10−8M PGE2. Osteoarthritis and Cartilage 2007 15, 291-299DOI: (10.1016/j.joca.2006.08.001) Copyright © 2006 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Immunocytochemical examination of fibroblast-like cells from the SF of patients with TMD. All of the fibroblast-like cells were positive for prolyl 4-hydroxylase (A) and vimentin (B). Macrophage marker (C) and dendritic cell marker (D) showed negative staining. Original magnification, ×200. Osteoarthritis and Cartilage 2007 15, 291-299DOI: (10.1016/j.joca.2006.08.001) Copyright © 2006 Osteoarthritis Research Society International Terms and Conditions

Fig. 7 Examination of capability of supporting osteoclast formation on fibroblast-like cells from the SF of patients with TMD in co-cultured with peripheral blood monocyte. Numerous TRAP-positive multinucleated cells were appeared in co-culture of fibroblast-like cells and peripheral blood monocyte (A). Culture of only peripheral blood monocytes under the same condition, there were only a few two nuclei-cells (B). Osteoarthritis and Cartilage 2007 15, 291-299DOI: (10.1016/j.joca.2006.08.001) Copyright © 2006 Osteoarthritis Research Society International Terms and Conditions

Fig. 8 Effect of OPG on osteoclastogenesis in co-culture of fibroblast-like cells and peripheral blood monocytes. Inhibitory effects of TRAP-positive multinucleated giant cells formation in response to 10ng/ml and 100ng/ml of OPG in this culture system. Each value represents mean±SD. *P<0.05 vs absence of OPG. Osteoarthritis and Cartilage 2007 15, 291-299DOI: (10.1016/j.joca.2006.08.001) Copyright © 2006 Osteoarthritis Research Society International Terms and Conditions

Fig. 9 ELISA for sRANKL in SF. Each detectable sRANKL concentration (pg/ml) shown was calculated per 1mg of SF total protein. Each value represents mean±SD. n=5; *P<0.05 vs control. Osteoarthritis and Cartilage 2007 15, 291-299DOI: (10.1016/j.joca.2006.08.001) Copyright © 2006 Osteoarthritis Research Society International Terms and Conditions