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Cyclical Tensile Force on Periodontal Ligament Cells Inhibits Osteoclastogenesis through OPG Induction

¹ Division of Orthodontics and Dentofacial Orthopedics, and
² Division of Oral Dysfunction Science, Department of Oral Health and Development Sciences, Graduate School of Dentistry, Tohoku University, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan

View larger version (47K): [in a new window]   Figure 1. Osteoclastogenesis from PBMCs. (a–f) TRAP-staining. Bar = 100 µm. (a) PBMCs cultured in 10% FBS -MEM supplemented with 1 x 10–8 M of 1,25-(OH)2D3. (b) PBMCs cultured with 50% conditioned medium of control PDL cells. (c) PBMCs cultured with 50% conditioned medium of control PDL cells in the presence of anti-TGF-beta antibodies (10.4 µg/mL). (d) PBMCs cultured with 50% conditioned medium of PDL cells under tensile force. (e) PBMCs cultured with 50% conditioned medium of PDL cells under tensile force in the presence of anti-TGF-beta antibodies (10.4 µg/mL). (f) The number of TRAP-positive multinucleated cells was counted. The data are expressed as the mean ± SD of 12 measurements of 3 wells. **p < 0.01, *p < 0.05. (g–i) Immunohistochemical staining of PBMCs for cathepsin K. Bar = 100 µm. (g) Photograph after TRAP-staining. (h) Immunohistochemical staining of PBMCs for cathepsin K. (i) Merged image of (g) and (h). (j–L) Immunohistochemical staining of PBMCs for. vß3 integrin. Bar = 100 µm (j) Photograph after TRAP-staining. (k) Immunohistochemical staining of PBMCs for vß3 integrin. (L) Merged image of (j) and (k). (m–o) Pit assay. Bar = 100 µm. (m) Negative control. (n) PBMCs cultured with 1,25(OH)2D3. (o) PBMCs cultured with the conditioned medium of tensile-stimulated PDL cells and 1,25(OH)2D3. White arrow indicates resorption pit.

View larger version (32K): [in a new window]   Figure 2. Results of semi-quantitative RT-PCR. (a) RT-PCR analysis for TGF-beta1, OPG, and RANKL in PDL cells. PDL cells were subjected to a cyclical tensile force for 48 hrs in the presence or absence of anti-TGF-beta antibodies (10 µg/mL). The results of one of three representative independent experiments are shown. (b,c) Densitometric analysis (b, RANKL; c, OPG; and TGF-beta1). The results are expressed as the ratio to the level of ß-actin expression in three independent experiments. The results are expressed as the mean ± SD. *p < 0.05.

View larger version (13K): [in a new window]   Figure 3. OPG production from stimulated PDL cells. Cyclical tensile force increased OPG production in PDL cells. The conditioned medium of PDL cells that had been stimulated by tensile force in the presence or absence of anti-TGF-beta antibodies (Hi, 40 µg/mL; Mi, 10 µg/mL; Lo, 2.5 µg/mL) was collected, and the level of OPG in the conditioned medium was measured with the use of an ELISA kit. The data are expressed as the mean ± SD of 3 independent experiments. Statistically significant between groups (p < 0.05, p < 0.01) or vs. control (*p < 0.05, **p < 0.01).

View larger version (11K): [in a new window]   Figure 4. TGF-beta production from stimulated PDL cells. Application of cyclical tensile force increased TGF-beta production in PDL cells. The conditioned medium of PDL cells that had been stimulated by tensile force (Experiment 1 - 0, 6, 24, or 48 hrs; Experiment 2 - 0, 24, 48, or 72 hrs) was collected, and the level of TGF-beta in the conditioned medium was measured with the use of an ELISA kit. The data are expressed as the mean ± SD of 3 measurements. *p < 0.05: Statistically significant vs. 0 hr.