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Clodronate Inhibits PGE2 Production in Compressed Periodontal Ligament Cells
1 Divisions of Orthodontics and Dentofacial Orthopedics, Correspondence: * corresponding author, igarashi{at}mail.tains.tohoku.ac.jp
Periodontal ligament (PDL) cells play an essential role in orthodontic tooth movement. We recently reported that clodronate, a non-N-containing bisphosphonate, strongly inhibited tooth movement in rats, and thus could be a useful adjunct for orthodontic treatment. However, it is not clear how clodronate affects the responses of PDL cells to orthodontic force. In this study, we hypothesized that clodronate prevents the mechanical stress-induced production of prostaglandin E2 (PGE2), interleukin-1β (IL-1β), and nitric oxide (NO) in human PDL cells. A compressive stimulus caused a striking increase in PGE2 production, while the responses of IL-1β and NO were less marked. Clodronate concentration-dependently inhibited the stress-induced production of PGE2. Clodronate also strongly inhibited stress-induced gene expression for COX-2 and RANKL. These results suggest that the inhibitory effects of clodronate on tooth movement and osteoclasts may be due, at least in part, to the inhibition of COX-2-dependent PGE2 production and RANKL expression in PDL cells.
Key Words: clodronate • periodontal ligament cell • mechanical stress • prostaglandin E2
Periodontal ligament cells play an essential role in orthodontic tooth movement, and mechanically induced bone resorption is known to be a rate-limiting step (Rygh, 1987; Igarashi et al., 1994). Recently, we demonstrated that clodronate, a non-N-containing bisphosphonate that has been used to treat various metabolic bone diseases associated with excessive bone resorption (Plosker and Goa, 1994; Fleisch, 2000), strongly inhibited bone resorption induced by orthodontic mechanical stress and tooth movement in rats, suggesting that it could be a useful adjunct for orthodontic treatment (Liu et al., 2004). A previous study has demonstrated the direct inhibitory action of clodronate on osteoclastic bone resorption, i.e., clodronate induces apoptosis of osteoclasts through incorporation into the cells (Frith et al., 2001). Although the number of osteoclasts on the pressure side of the periodontal ligament decreased in clodronate-treated animals, the mechanism of action in this process has not yet been determined. The purpose of the present study was to clarify how clodronate affects the responses of periodontal ligament cells to orthodontic force, especially those leading to bone resorption. In this study, we hypothesized that clodronate prevents the mechanical stress-induced production of prostaglandin E2 (PGE2), interleukin 1β (IL-1β), and nitric oxide (NO) in cultured human periodontal ligament cells, which are known to play important roles in the bone-resorptive responses to orthodontic mechanical stimulation (Yamasaki et al., 1980; Chumbley and Tuncay, 1986; Saito et al., 1991; Zhou et al., 1997; Alhashimi et al., 2001; Iwasaki et al., 2001; Hayashi et al., 2002; Shirazi et al., 2002).
The protocol for the experiment was approved by the Research Ethics Committee of Tohoku University Graduate School of Dentistry, and informed consent was obtained from all patients.
Drug
Compression of Primary Human Periodontal Ligament Cells For the experiment, periodontal ligament cells were seeded on 35-mm wells in a six-well plate at a density of 3 x 105 cells/dish and cultured until they were confluent. They were then transferred to 2 mL of fresh medium that contained a specific concentration of clodronate and cultured for an additional 24 hrs. After the pre-culture, the cells were continuously compressed according to the method described previously (Kanzaki et al., 2002). Briefly, compressive force was applied directly to periodontal ligament cells by the placement of a custom-made glass cylinder (diameter, 30.3 mm; height, 14.8 mm; thickness, 2.0 mm) that contained lead granules over a confluent cell layer in the well. We adjusted the force magnitude by adding or reducing the granules. In the present study, the cells were subjected to 2.0 g/cm2 of compressive force for 48 hrs. After the experiment, total RNA was extracted from each culture with the use of the QuickPrep Total RNA Extraction Kit (Pharmacia Biotech, Uppsala, Sweden). The culture medium was also withdrawn and stored at –20°C for determination of PGE2, IL-1β , and NO. The concentrations of PGE2 and IL-1β were measured with respective specific enzyme immunoassay kits (for PGE2, RPN222, Amersham Pharmacia Biotech, Piscataway, NJ, USA; for IL-1β, QLB00, R&D Systems, Inc., Minneapolis, MN, USA). We evaluated NO production by measuring nitrite and nitrate concentrations in the medium using the HPLC-Griess method (Ohta et al., 1994). Since responsiveness of cultured human periodontal ligament cells varies depending on their sources, the experiment was repeated, and each single experiment was performed with cells from a different subject.
Semi-quantitative Reverse-transcription Polymerase Chain-reaction (RT-PCR) Assays for Cyclo-oxygenase-2 (COX-2) and Receptor Activator Nuclear Factor
Statistical Analysis
Clodronate showed different effects on PGE2, IL-1β , and NO production in periodontal ligament cells induced by compressive mechanical stress (Fig. 1  ). The compression of cells at 2.0 g/cm2 for 48 hrs caused nearly a 30-fold increase in PGE2 release (Fig. 1A), while the increase was not significant for IL-1β (Fig. 1B) and only minimal for NO (Fig. 1C). Clodronate (5, 25, 125 µM) concentration-dependently inhibited the mechanical stress-induced increase in PGE2 production in periodontal ligament cells (Fig. 1A). The inhibitory effect of clodronate on NO production was significant only at the highest concentration (125 µM) (Fig. 1C).
The application of compressive force to periodontal ligament cells also caused a more than two-fold increase in mRNA expression for both COX-2 and RANKL (Figs. 2A, 2B ). Clodronate (5, 25, 125 µM) significantly inhibited these responses (Fig. 2B).
Although the responsiveness of periodontal ligament cells to compression varied between and among experiments (individuals), the inhibitory effects of clodronate on stress-induced PGE2, COX-2, and RANKL were reproducible (Table ).
Clodronate is a non-N-containing bisphosphonate that possesses potential anti-inflammatory activity as well as anti-bone-resorptive activity (Österman et al., 1995; Richards et al., 2001). It has been shown that clodronate inhibits the production of pro-inflammatory molecules, including IL-1β (Pennanen et al., 1995; Makkönen et al., 1999), NO (Makkönen et al., 1996; 1999), and PGE2 (Felix et al., 1981; Igarashi et al., 1997) in macrophages and/or osteoblastic cells. The present results clearly demonstrated that clodronate could also prevent the mechanical stress-induced production of PGE2 by periodontal ligament cells, which is one of the most important signaling molecules in the responses of periodontal ligament to orthodontic force (Yamasaki et al., 1980; Saito et al., 1991; Kanzaki et al., 2002). The compressive stimulus caused a striking increase in PGE2 production, while responses were less marked for IL-1β and NO. Clodronate significantly inhibited the mechanical stress-induced production of PGE2 in a concentration-dependent manner. Furthermore, clodronate strongly inhibited stress-induced gene expression for COX-2 and RANKL. Prostaglandins have been shown to play a crucial role in osteoclast formation induced by orthodontic mechanical stress (Yamasaki et al., 1980; Sandy and Harris, 1984; Zhou et al., 1997). Recently, Kanzaki et al.(2002) demonstrated that compressive force stimulates osteoclastogenesis in the co-culture of peripheral blood mononuclear cells with periodontal ligament cells, by increasing the expression of RANKL in periodontal ligament cells. RANKL is known to be an essential factor in the differentiation and activation of osteoclasts (Suda et al., 1999). It has also been demonstrated that this increase in RANKL expression paralleled that in COX-2 expression and was dependent on PGE2 production (Kanzaki et al., 2002). Clodronate inhibited all of these responses in compressed periodontal ligament cells, suggesting that it may have decreased RANKL expression in these cells by inhibiting the COX-2-dependent production of PGE2. At present, the mechanism by which clodronate inhibits COX-2 expression in periodontal ligament cells is not known. Although NO and IL-1 have been shown to induce COX-2 in osteoblastic cells (Buttery et al., 2002; Pilbeam et al., 2002), their involvement is not likely, since the effects of mechanical stress with or without clodronate on the production of these molecules were only minimal or insignificant.
In our previous in vivo study, the number of osteoclasts on the pressure side of the periodontal ligament decreased in clodronate-injected animals (Liu et al., 2004), indicating that clodronate may have either inhibited the recruitment of osteoclasts, promoted osteoclast apoptosis, or both (Rogers et al., 2000). The present in vitro results suggest that clodronate may have impaired the ability of periodontal ligament cells to support osteoclast formation by decreasing RANKL expression. It is also possible that the decreased expression of RANKL promoted osteoclast apoptosis, and hence decreased the number of osteoclasts, since RANKL has been shown to act as a survival factor and to prevent apoptosis of osteoclasts (Lacey et al., 2000). Osteoclast apoptosis has been considered to be a major mechanism of action for the inhibition of bone resorption by this bisphosphonate (Halasy-Nagy et al., 2001). Frith et al.(2001) demonstrated that clodronate is incorporated into osteoclasts and metabolized to adenosine 5'-(β, In conclusion, the present results suggest that the inhibitory effects of clodronate on orthodontic tooth movement and osteoclasts may be due in part to the inhibition of COX-2-dependent PGE2 production, which leads to decreased RANKL expression in periodontal ligament cells subjected to orthodontic mechanical stress.
This research was supported by a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Science, Sports, and Culture of Japan (No. 13672138).
4 present address, Department of Orthodontics, Dalian University Stomatology Hospital, Dalian, China  Received for publication August 8, 2005. Revision received April 23, 2006. Accepted for publication May 3, 2006.
Journal of Dental Research, Vol. 85, No. 8,
757-760 (2006)
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ABSTRACT
INTRODUCTION
-MEM supplemented with 10% FBS, antibiotics, and 1 x 10–8 M 1
B Ligand (RANKL) Gene Expression
P < 0.05 compared with load. 
-dichloromethylene) triphosphate, which may induce apoptosis in these cells. In addition to the formation of this ATP analogue, the inhibition of RANKL expression in supporting cells like periodontal ligament cells might also be involved in the induction of apoptosis in osteoclasts.