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RANKL Increase in Compressed Periodontal Ligament Cells from Root Resorption

Department of Orthodontics, Nihon University School of Dentistry at Matsudo, 2-870-1 Sakaecho-Nishi, Matsudo City, Chiba 271-8587, Japan

^* corresponding author, yamaguchi.masaru{at}nihon-u.ac.jp

	ABSTRACT

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The ligand receptor activator of NFB (RANKL) plays an important role in osteoclast formation. However, very little is known about the relationship between external apical root resorption during orthodontic treatment and RANKL. We hypothesized that compressive force is responsible for RANKL formation and up-regulation of osteoclastogenesis in periodontal ligament (PDL) cells from patients with severe orthodontically induced external apical root resorption. RANKL and osteoprotegerin (OPG) production, TRAP-positive cells, and resorptive pits were determined. The increase of RANKL and the decrease of OPG were greater in the severe root resorption group than in the non-resorption group. The numbers of TRAP-positive cells and resorptive pits were also increased in the severe root resorption group than in the non-resorption group. These results support the hypothesis that the compressed PDL cells obtained from tissues with severe external apical root resorption may produce a large amount of RANKL and up-regulate osteoclastogenesis.

KEY WORDS: PDL cells • compressive force • RANKL • OPG • root resorption

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
External apical root resorption is an unavoidable pathologic consequence of orthodontic tooth movement. It can be defined as an iatrogenic disorder that occurs, unpredictably, after orthodontic treatment, whereby the resorbed apical root portion is replaced with normal bone. External apical root resorption is a sterile inflammatory process that is extremely complex and involves various disparate components, including mechanical forces, tooth roots, bone, cells, surrounding matrix, and certain known biologic messengers (Brezniak and Wasserstein, 2002). Killiany (1999) reported external apical root resorption of > 3 mm to occur at a frequency of 30%, with only 5% of treated individuals found to have > 5 mm of root resorption. The etiology of external apical root resorption following orthodontic treatment is not fully understood. In the last 10 years, interestingly, it was suggested that individual susceptibility (Owman-Moll et al., 1995; Kurol et al., 1996), genetics (Harris et al. 1997, 2001; Al-Qawasmi et al., 2003; Ngan et al., 2004), and systemic factors (McNab et al., 1999) are risk factors for external apical root resorption.

The ligand receptor activator of NFB (RANKL) has been identified as a member of the membrane-associated tumor necrosis factor ligand family, and is an important regulatory molecule of osteoclastogenesis (Suda et al., 1999). Ogasawara et al.(2004) reported that RANKL was detected in osteoblasts and periodontal ligament (PDL) cells during experimental tooth movement. Osteoprotegerin (OPG) is a secreted tumor necrosis factor (TNF) receptor member that functions as a decoy receptor for RANKL, thereby inhibiting these processes and accelerating osteoclast apoptosis (Burgess et al., 1999; Lacey et al., 2000). Kanzaki et al.(2004) reported that OPG gene transfer to periodontal tissue inhibited RANKL-mediated osteoclastogenesis and inhibited experimental tooth movement. Thus, the signaling and regulation of the expression of RANKL and OPG in PDL may play critical roles in bone remodeling during orthodontic tooth movement. However, very little is known about the relationship between external apical root resorption and the production of these modulators during orthodontic tooth movement. We hypothesized that compressive force is responsible for RANKL formation and up-regulation of osteoclastogenesis in PDL cells from patients with severe orthodontically induced external apical root resorption. Thus, this study examined the effect of compressive force on the production of these cytokines and on osteoclast formation.

	MATERIALS & METHODS

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Volunteers
The teeth used for the classification of dental root resorption were the central incisors of the maxilla, and when the amount of the root resorbed was measured radiologically before and after the period of orthodontic treatment, a severe root resorption group was designated by the resorption of one-third of the tooth root, and a normal group by resorption of no more than 2 mm (Malmgren et al., 1982). The cells provided for the study were derived from the PDLs of the upper first premolars extracted from these individuals. Thus, we collected the PDL from the first premolars extracted from a large number of individuals at the start of orthodontic treatment, subcultured them, and preserved them by freezing. After the treatment was completed, the PDL cells from participants in whom severe root resorption had occurred were thawed and made available for study.

All cases were considered to be Angle class I crowding extraction cases. Ten patients were assigned to groups as follows: the severe root resorption group (two males, three females; 18–25 yrs old, 22.5 ± 2.8 yrs); and the non-resorption group (two males, three females; 17–26 yrs old, 23.2 ± 3.3 yrs). Treatment periods were, for the severe root resorption group, 22.8 ± 2.8 mos, and for the non-resorption group, 22.4 ± 3.3 mos. Moving distance of the central incisor root was, for the severe root resorption group, 1.61 ± 1.28 mm, and for the non-resorption group, 1.59 ± 1.33 mm. The root apex shapes in both groups were normal, not blunt, eroded, pointed, bent, or bottle-shaped (Mirabella and Artun, 1995). Class II elastic bands were not used in either group.

Cell Culture
Human PDL cells were prepared according to a modification of the method of Somerman et al.(1988), as described previously (Yamaguchi et al., 1996). Briefly, PDL tissue samples from the roots of premolars extracted from 10 patients during the course of orthodontic treatment were obtained, after informed consent was received from the donors. This study was conducted according to a protocol reviewed by the ethics committee of Nihon University School of Dentistry at Matsudo (#04-021).

Application of Compressive Forces
To reproduce the conditions of pressure during orthodontic tooth movement, we performed the following in vitro experiment, in accordance with the method developed by Kanzaki et al.(2002). PDL cells were continuously compressed by a uniform compression method as a model of pressure at the site of orthodontic movement. PDL cells were subjected to 0.5, 1.0, 2.0, or 3.0 g/cm² of compression force for 48 hrs (Yamaguchi et al., 2004).

RANKL and OPG Determination
The amounts of soluble RANKL (sRANKL) and OPG released from PDL cells into the culture medium were determined in duplicate with the use of commercially available enzyme-linked immunosorbent assay kits (Quantikine, R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Tartrate-resistant Acid Phosphatase (TRAP) Staining and Pit Formation on Dentin Slices after the Addition of Conditioned Medium
Human osteoclast precursor cells (Hokudo, Sapporo, Japan) were cultured in commercial medium (-MEM supplemented with 10 unit/mL of penicillin, 10 µg/mL of gentamicin, and 10% fetal calf serum) with both macrophage colony-stimulating factor (M-CSF) and RANKL (both at 10 ng/mL) (Hokudo) at 37°C for 8 days. After the osteoclasts matured, the medium was changed to that obtained from PDL cells subjected to compressive-force-conditioned medium (2.0 g/cm²) for 12 hrs. After incubation for 48 hrs, cells were stained positive with tartrate-resistant acid phosphatase (TRAP), with the use of a commercial staining kit (Sigma Co., St. Louis, MO, USA). A pit formation assay, as well as TRAP staining, was also carried out under the same experimental conditions. The dentin slices were sonicated in 1 M of ammonia water for cell removal, then washed and dried. After being dried, the slices were mounted onto stubs and sputter-coated with platinum for scanning electron microscopic examination (HCP-2 Hitachi, Tokyo, Japan). We measured the resorption area on dentin slices.

Co-culture of Compressed PDL Cells and Human Osteoclast Precursor Cells
After compressive force (2.0 g/cm²) was applied to the PDL cells for 12 hrs, the glass cylinder was removed carefully. Human osteoclast precursor cells were subsequently co-cultured with the PDL cells in commercial medium (-MEM supplemented with 10 unit/mL of penicillin, 10 µg/mL of gentamicin, and 10% fetal calf serum) with both macrophage colony-stimulating factor (M-CSF) and RANKL (both at 10 ng/mL) (Hokudo) at 37°C for 8 days. Then, TRAP staining was carried out.

Western-blot Analysis of RANKL
PDL cells cultured under compression (2.0 g/cm², 12 hrs) were solubilized in a lysis buffer and Western-blotted. The primary antibody used in this experiment was goat polyclonal anti-RANKL antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The second antibody was anti-goat IgG-horseradish peroxidase (Santa Cruz Biotechnology, Inc.). After the membrane was washed thoroughly, the reactive bands were visualized with H₂O₂-chloronaphthol reagents.

Statistical Methods
Values were calculated as the mean ± standard deviation (SD). Statistical significance was determined by Student’s t test (Figs. 1–3). Data were subjected to two-way analysis of variance (ANOVA) and were then presented as indicated in Figs. 1 and 2.

View larger version (20K): [in this window] [in a new window]   Figure 1. Effects of incubation time with compressive force on sRANKL (A) and OPG (B) production in conditioned media of human PDL cells. Production of these cytokines was assayed as described in MATERIALS & METHODS. Data are expressed as the mean ± SD of 5 independent cultures. Cytokine production was changed by application of compressive force (2.0 g/cm2), and the pattern was time-dependent (p < 0.001, by two-way ANOVA). Significantly different from the corresponding control at each incubation time: *p < 0.001. Significantly different from non-resorption (with compressive force) at each incubation time: #p < 0.001.

View larger version (24K): [in this window] [in a new window]   Figure 2. Effects of different magnitudes of compressive force on sRANKL (A) and OPG (B) production by human PDL cells. Human PDL cells were cultured with or without the indicated magnitudes of compressive force for 12 hrs. Data are expressed as the mean ± SD of 5 independent cultures. The cytokines were changed by application of compressive force in a force-magnitude-dependent manner (p < 0.001, by two-way ANOVA). Significantly different from the corresponding control: *p < 0.001. Significantly different from non-resorption (with compressive force) at each magnitude: #p < 0.001.

View larger version (12K): [in this window] [in a new window]   Figure 3. The number of TRAP-positive cells and the pit formation on dentin slices on the addition of conditioned medium or by co-culture of osteoclast precursor cells and compressed PDL cells. (A,B) Human osteoclast precursor cells cultured in commercial medium (-MEM supplemented with 10 unit/mL of penicillin, 10 µg/mL of gentamicin, and 10% fetal calf serum) with both macrophage colony-stimulating factor (M-CSF) and RANKL (both at 10 ng/mL) for 8 days. After the osteoclasts matured, the medium was changed to that obtained from human PDL cells after incubation with or without compressive-force-conditioned medium (2.0 g/cm2) for 12 hrs. The numbers of TRAP-positive cells (A) and resorption pits (B) were significantly increased by the conditioned medium (with compressive force) as compared with those in the respective controls (without compressive force). Further, the numbers of TRAP-positive cells and resorption pits were more abundant in the severe root resorption group (with compressive force) than in the non-resorption group (with compressive force). (C) Co-culture with compressed PDL cells (2.0 g/cm2, 12 hrs) activated the differentiation of osteoclast precursor cells into mature osteoclasts for 8 days. The number of TRAP-positive cells in the compressed PDL cells was significantly differenr from that in the non-compressed PDL cells in both the severe root resorption and non-resorption groups. Furthermore, the increase was greater in the severe root resorption group than in the non-resorption group. We measured the numbers of TRAP-positive cells and resorption pits, and subjected them to statistical analysis. Data are expressed as the mean ± SD of 5 independent cultures. Significantly different from the corresponding control (*p < 0.001). Significantly different from non-resorption (with compressive force) (#p < 0.001).

	RESULTS

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Effects of Different Compressive Forces on Cytokine Production from PDL Cells
When compressive force ranging from 0.5 to 2.0 g/cm² was applied to PDL cells for 12 hrs, the secretions of sRANKL in both the severe root resorption and non-resorption groups showed significant increases at all compressive forces, as compared with the corresponding control cells (p < 0.001), in a force-dependent manner, up to 2.0 g/cm² (p < 0.001, two-way ANOVA), while the secretions of OPG showed significant decreases at all compressive forces as compared with the control (p < 0.001), in a force-dependent manner, up to 2.0 g/cm² (p < 0.001, two-way ANOVA). A comparison between datasets revealed that the secretions in both the severe root resorption and non-resorption groups were significantly changed in PDL cells subjected to compressive force at all magnitudes, as compared with those in the corresponding control cells (p < 0.001). Furthermore, the increase of sRANKL and the decrease of OPG were greater in the severe root resorption group than in the non-resorption group (Fig. 2).

The Number of TRAP-positive Cells (A,C) and the Pit Formation on Dentin Slices (B) on the Addition of Conditioned Medium or by Co-culture of Osteoclast Precursor Cells and Compressed PDL Cells
The number of TRAP-positive cells and the resorption pit area were significantly stimulated by the conditioned medium from cultures with compressive force in both the severe root resorption and non-resorption groups, but the stimulation rate was much higher in the severe root resorption group (TRAP-positive cells, 3.8 times; resorption pit area, 6.0 times) than that in the non-resorption group (TRAP-positive cells, 2.3 times; resorption pit area, 3.2 times). Furthermore, the increase was greater in the severe root resorption group than in the non-resorption group (TRAP-positive cells, 1.8 times; resorption pit area, 1.9 times; p < 0.001), whereas there was no significant difference between the controls (without compressive force) of the severe root resorption and non-resorption groups (Figs. 3A, 3B).

Co-culture with compressed PDL cells (2.0 g/cm², 12 hrs) activated the differentiation of osteoclast precursor cells into mature osteoclasts after 8 days. The number of TRAP-positive cells in the compressed PDL cells was significantly different from that of non-compressed PDL cells in both the severe root resorption and non-resorption groups. Furthermore, the increase was greater in the severe root resorption group than in the non-resorption group (1.8 times, p < 0.001), whereas there was no significant difference between the controls (without compressive force) of the severe root resorption and non-resorption groups (Fig. 3C).

Western-blot Analysis of RANKL
The production of RANKL (40 KDa) was significantly stimulated by compressive force in both the severe root resorption and non-resorption groups (Fig. 4). Furthermore, the increase of RANKL was greater in the severe root resorption group than in the non-resorption group (Fig. 4).

View larger version (28K): [in this window] [in a new window]   Figure 4. The expression of RANKL protein increased with compressive force. PDL cells obtained from normal resorption and severe resorption groups were exposed to compressive force (2.0 g/cm2) for 12 hrs, and then whole-cell lysate from the cells was subjected to Western blot analysis with antibody against RANKL.

	DISCUSSION

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Grimaud et al.(2003) demonstrated that the RANKL/OPG ratio was significantly increased in patients suffering from severe osteolysis, compared with the control group, and that this imbalance is involved in bone resorption mechanisms. Kanzaki et al.(2001) reported that human PDL cells may regulate osteoclastogenesis by opposing mechanisms which stimulate resorptive activity by RANKL and inhibition by OPG. Kanzaki et al.(2002) reported that RANKL mRNA expression is induced in compressed PDL cells in a force-dependent manner up to 2.0 g/cm², and in a time-dependent manner for up to 48 hrs. Our laboratory also reported that compressive force increased the production of RANKL and decreased that of OPG in human PDL cells in vitro (Nishijima et al., 2006). These results lend support to the findings of the present study. Furthermore, Lossdörfer et al.(2002) reported that RANKL immunoreactivity was detected in odontoblasts and PDL fibroblasts in human deciduous teeth undergoing root resorption. Fukushima et al.(2003) also demonstrated that PDL cells derived from roots of resorbing deciduous teeth expressed increased levels of RANKL but decreased levels of OPG, while PDL cells isolated from roots of either stable/non-resorbing deciduous or permanent teeth expressed OPG but not RANKL. These findings and our present results, taken together, suggest that compressed PDL cells obtained from the severe root resorption group may stimulate external apical root resorption by the action of large amounts of RANKL and the decrease of OPG production.

A recent study found that prostaglandin (PG) E₂ enhanced RANKL-stimulated formation of osteoclasts in spleen cells and marrow stromal cells (Wani et al.,1999; Kanematsu et al., 2000). Further, Kanzaki et al.(2002) demonstrated that RANKL up-regulation in compressed PDL cells was dependent on PGE₂. Therefore, the regulation of osteoclastogenesis-supporting activity may be due to PGE₂ stimulation in the culture media. Further, many pro-inflammatory cytokines—including interleukin (IL)-1ß, IL-6, and TNF-—activate bone resorption (Feldmann et al., 1996; Kobayashi et al., 2000; Chenoufi et al., 2001; Palmblad et al., 2001) and are able to induce osteoclastic activation via a mechanism independent of RANKL (Palmqvist et al., 2002). Further studies are necessary to confirm the relationship among RANKL, PGE₂, and pro-inflammatory cytokines.

In conclusion, compressed PDL cells obtained from patients with severe external apical root resorption produced large amounts of RANKL, and decreased the production of OPG, and also stimulated osteoclast formation. These results suggest that the PDL cells subjected to compressive force may play an important role in the occurrence of severe external apical root resorption during orthodontic treatment.

	ACKNOWLEDGMENTS

 
This research was supported by a Grant-in-Aid (No. C:14571970) for Scientific Research from the Japan Society for the Promotion of Science, and by Nihon University Individual Research Grant for 2005 (No. 05-119 to M. Yamaguchi).

Received September 8, 2005; Last revision April 24, 2006; Accepted May 3, 2006

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