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Intermittent Force Induces High RANKL Expression in Human Periodontal Ligament Cells

¹ Division of Orofacial Functions and Orthodontics, and
² Division of Anatomy, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, 803-8580, Japan

View larger version (52K): [in this window] [in a new window]   Figure 1. Compressive force and cell damage. (A) Isolated cells were seeded in culture dishes and incubated at 37°C in an atmosphere of 5% CO2. We applied compression by changing the amount of medium. The gravity of medium was 1.01 g/mL and the cross-section area of the cylindrical tube was 5.7 cm2. The forces produced by the medium were 0.17 g/cm2 (0.96 mL; control), 2.0 g/m2 (11.3 mL), and 5.0 g/cm2 (28.3 mL). Control force was the same as the force by 1.5 mL medium in a 35-mm dish. (B) Phase-contrast images of PDL cells under compressive force for 4 days. The cells were cultured (a) without additional medium (control) or with (b) 2.0 g/cm2 intermittent force (2.0 g i-force), (c) 2.0 g/cm2 continuous force (2.0 g c-force), (d) 5.0 g/cm2 i-force, or (e) 5.0 g/cm2 c-force. Atrophic cells appear as bright cells (arrows). Bars = 20 µm. (C) Cell damage assessed based on the activity of LDH released from the cells. N = 15 samples each. Data are presented as means ± SD. *Significant difference (p < 0.01).

View larger version (42K): [in this window] [in a new window]   Figure 2. PCR analysis of OPG, RANKL, and IL-1β mRNA expression with compressive forces. Specificities of each primer were confirmed by a BLAST-assisted Internet search. (A) PCR products of OPG mRNA in unstimulated PDL cells by amplification for 20, 25, 30, 35, or 40 cycles. Since the amount of OPG mRNA expression was dependent on the cycle number between 25 and 35, 30-cycle amplification was used at an optimal cycle for semi-quantitative analysis of OPG mRNA expression. (B) OPG, RANKL, and IL-1β mRNA expressions in PDL cells under intermittent force (i-force) or continuous force (c-force). Force was applied at 2.0 or 5.0 g/cm2 for 2 to 4 days. Total RNA was analyzed by RT-PCR. MW indicates molecular-weight markers. (C) OPG, RANKL, and IL-1β mRNA expression relative to GAPDH expression was analyzed with the use of NIH Image software. The band intensities of OPG expression in the two-day control and RANKL or IL-1β expression at 2 days with 2.0 g/cm2 i-force were assigned values of 1.

View larger version (46K): [in this window] [in a new window]   Figure 3. RANKL expressions through IL-1 receptor 1. (A) IL-1 receptor I mRNA expression was confirmed by RT-PCR with primers specific for IL-1 receptor I. (B) Exhibition of IL-1 receptor I protein (80 kDa) in PDL cells was confirmed by Western blot analysis. Bands were detected with the use of the chemiluminescent horseradish peroxidase substrate. (C) To examine the involvement of IL-1β and the IL-1β receptor in the expression of RANKL in cells under compression force, we added 100 ng/mL IL-1 receptor antagonist (IL-1Ra) (ProSpec-Tany, TechnoGene, Rehovot, Israel) to the culture medium when the compressive force was applied. The effects of the IL-1 receptor antagonist on RANKL expression in cells subjected to an intermittent force (i-force) (8 hrs per day for 4 days) are shown. Neither control cells nor cells subjected to an i-force in the presence of IL-1 receptor antagonist expressed RANKL. MW indicates molecular-weight markers.

View larger version (24K): [in this window] [in a new window]   Figure 4. Schematic of a proposed mechanism by which compressive force induces osteoclastogenesis in PDL cells. First, compressive force stimulates IL-1β expression. Second, IL-1β is released and binds to the IL-1β receptor on the cells. Third, IL-1β acts through its receptor to signal the induction of RANKL expression. Up-regulated RANKL expression and down-regulated OPG expression then induce osteoclastogenesis in osteoclast progenitors.

Journal of Dental Research, Vol. 86, No. 7, 623-628 (2007)
DOI: 10.1177/154405910708600708


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