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Effects of Pulpectomy on the Amount of Root Resorption during Orthodontic Tooth Movement Masato Kaku, PhD, DDS, Hiromi Sumi, DDS, Hanaka Shikata, DDS, Shunichi Kojima, PhD, DDS, Masahide Motokawa, PhD, DDS, Tadashi Fujita, PhD, DDS, Kotaro Tanimoto, PhD, DDS, and Kazuo Tanne, PhD, DDS Abstract Introduction: Previous studies have revealed that orthodontic force affects dental pulp via the rupture of blood vessels and vacuolization of pulp tissues. We hypothesized that pulp tissues express inflammatory cytokines and regulators of odontoclast differentiation after excess orthodontic force. The purpose of this study was to investigate the effects of tensile force in human pulp cells and to measure inflammatory root resorption during tooth movement in pulpless rat teeth. Methods: After cyclic tensile force application in human pulp cells, gene expression and protein concentration of macrophage colony-stimulating factor, receptor activator of nuclear factor kappa-B ligand, interleukin-1 beta, and tumor necrosis factor alpha were determined by realtime polymerase chain reaction and enzyme-linked immunoassay. Moreover, the role of the stretchactivated channel was evaluated by gadolinium (Gd3+) treatment. The upper right first molars of 7-week Wistar rats were subjected to pulpectomy and root canal filling followed by mesial movement for 6 months. Results: The expression of cytokine messenger RNAs and proteins in the experimental group peaked with loading at 10-kPa tensile force after 48 hours (P < .01). Gd3+ reduced the expression of these cytokine messenger RNAs and protein concentrations (P < .01). The amount of inflammatory root resorption was significantly larger in the control teeth than the pulpectomized teeth (P < .05). Conclusions: This study shows that tensile forces in the pulp cells enhance the expression of various cytokines via the S-A channel, which may lead to inflammatory root resorption during tooth movement. It also suggests that root canal treatment is effective for progressive severe inflammatory root resorption during tooth movement. (J Endod 2014;40:372–378)
Key Words Odontclast, orthodontic tooth movement, pulp cells, pulpectomy, root resorption, tensile forces
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uring orthodontic tooth movement, apical root resorption is an undesirable side effect that is difficult to predict and repair. Massler and Malone (1) found that root resorption occurred in 86.4% of orthodontic patients, and root resorption associated with orthodontics is reported to be related to factors such as patient age, sex, and systemic conditions (2). Root resorption is also associated with inflammatory reactions by odontoclasts, which are similar to osteoclasts in morphology, activity, functions, and features (3). Previous studies have shown that macrophage colony-stimulating factor (M-CSF) plays an essential role in osteoclastogenesis (4). In addition, it was clarified that M-CSF supports osteoclast differentiation in cooperation with receptor activator of nuclear factor kappa-B ligand (RANKL) (5). Moreover, cementoblasts are able to express RANKL and can modulate osteoclast cytogenesis (6). Thus, M-CSF and RANKL are indispensable to odontoclast differentiation for root resorption, and it is important to clarify how and from where M-CSF and RANKL are released when root resorption occurs during orthodontic tooth movement. Because orthodontic force is recognized as a type of trauma, pulp tissue can be injured during orthodontic tooth movement. McDonald and Pitt Ford (7) found that human pulpal blood decreased when continuous tipping forces were applied. Miura (8) suggested that continuous heavy orthodontic forces predispose pulp tissue to pulp necrosis via rupture of blood vessels in the apical root. Furthermore, some researchers have investigated the relationship between pulp tissue and root resorption during orthodontic tooth movement. Remington et al (9) and Spurrier et al (10) showed that root resorption is more often observed in intact teeth when compared with pulpless teeth. Indeed, we have noted severe apical root resorption in intact teeth and mild root resorption in pulpectomized teeth. Based on these findings, we hypothesized that stretched and injured pulp cells express M-CSF and RANKL as well as inflammatory cytokines; as a result, inflammatory apical root resorption occurs because of the derived odontoclasts. A stretch-activated channel (S-A channel), which is an ionic channel activated by stretching, was detected in tissue-cultured embryonic chick skeletal muscle (11). Vascular endothelial cells have been reported to contain a cation-selective S-A channel permeable to calcium ions (12). It was also shown that intracellular Ca2+ increased in response to mechanical stretching via the S-A channel in human umbilical endothelial cells, and this response was blocked by gadolinium (Gd3+), an S-A channel blocker (13). Based on these findings, it was assumed that mechanical stimuli such as tensile forces in pulp cells are controlled through the S-A channel, and, consequently, osteoclast-inducing factors and inflammatory cytokines can be produced. Therefore, in this study, we examined the effects of cyclic tensile forces on the expression of M-CSF, RANKL, interleukin-1 beta (IL-1b), and tumor necrosis factor alpha (TNF-a) in human pulp cells as well as the inhibition of these actions by blockade
From the Department of Orthodontics and Craniofacial Developmental Biology, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima, Japan. Address requests for reprints to Dr Masato Kaku, Department of Orthodontics and Craniofacial Developmental Biology, Hiroshima University Graduate School of Biomedical Sciences, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8553, Japan. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2014 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2013.11.027
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Basic Research—Biology of mechanosensory S-A channels. Furthermore, we investigated differences in root resorption during orthodontic tooth movement between pulpless and intact teeth.
Materials and Methods Cell Culture Freshly extracted and intact teeth were used. These teeth were indicated for extraction for orthodontic treatment in patients aged 18–30 years. The protocol was reviewed and approved by the Ethics Committee of Hiroshima University, and informed consent was obtained from each tooth donor. Teeth were cut vertically, and pulp tissues were removed aseptically and rinsed with phosphate buffered saline (PBS). The outgrowth method was performed for culture of human dental pulp cells in accordance with a previous study (14). Explants were cut into small fragments with a sharp blade and transferred to 10-cm Petri dishes and cultured in a-Modified Eagle’s Medium (MEM) (Sigma-Aldrich, St Louis, MO) containing 10% fetal bovine serum (FBS) (Biological Industries, Kibbutz Beit-Haemek, Israel), 32 U/mL penicillin G (Meiji Seika, Tokyo, Japan), 250 mg/mL amphotericin B (Nacalai Tesque, Kyoto, Japan), and 60 mg/mL kanamycin (Meiji Seika, Tokyo, Japan) at 37 C in a humidified atmosphere of 5% CO2. The medium was changed twice a week. Cells were subcultured by treatment with 0.25% trypsin/EDTA and plated at 3 105 cells per 100-mm culture dish. For all experiments, cells between the 4th and 6th passages were used. Application of Cyclic Tensile Forces The Flexcell strain unit FX-2000 (Flexcell International Co, Hillsborough, NC) consists of a vacuum unit and a valve controlled by a computer program. Pulp cells (1 105) cultured on a flexible membrane base were subjected to cyclic tensile forces produced by computercontrolled application of sinusoidal negative pressure. Flexible membranes supporting the cultured cells were deformed by negative pressure. Application of a vacuum results in a maximum cell elongation of 20% at the periphery of wells, with the strain declining toward the center. Cells were placed in a humidified incubator under an atmosphere of 5% CO2 at 37 C. To examine the expression of M-CSF, RANKL, IL-1b, and TNF-a messenger RNAs (mRNAs) and their concentrations, a tensile force of 10 kPa was loaded at a frequency of 30 cycles per minute for 12, 24, and 48 hours. Next, we investigated the influence of various tensile forces of 1, 3, 5, 10, and 15 kPa on the amounts of M-CSF, RANKL, IL-1b, and TNF-a for 48 hours. Total RNA Extraction and Complementary DNA Synthesis Total RNA was isolated from the cell cultures using a Quickprep Total RNA extraction kit (Amersham Biosciences, Tokyo, Japan). Single-stranded complementary DNA (cDNA) was synthesized from 1 mg total RNA using Oligo(dT)20 primer (Toyobo, Osaka, Japan) and a Rever Tra Ace-a first-strand cDNA synthesis kit (Toyobo). Primers The following primers were used: M-CSF (15) 50 -GGCCATGAGAGGCAGTCCGAGGG-30 (forward), 50 -CACTGGCAGTTCCACCTGTCT GTC-30 (reverse); RANKL (16): 50 -TCAGAAGATGGCACTCACTG-30 (forward), 50 -AACATCTCCCACTGGCTGTA-30 (reverse); IL-1b (17): 50 CTCAGGTGTCCTCGAAGAAATCAA-30 (forward), 50 -GCTTTTTTGCTGT GAGTCCCG-30 (reverse); and TNF-a (18): 50 -CCCCAGGGCTCCAGGCG GTGCTTGT-30 (forward), 50 -GGAGACGGCGATGCGGCTGATGGTG-30 (reverse). G3PDH primer (Rever Tra Ace-a first-strand cDNA synthesis JOE — Volume 40, Number 3, March 2014
kit, Toyobo) was used as a control primer; 50 -ACCACAGTCCATGCCATCAC-30 (sense), 50 -TCCACCACCCTGTTGCTGTA-30 (antisense).
Real-time Quantitative Polymerase Chain Reaction Real-time polymerase chain reaction (PCR) was performed using SYBR Green I assay and an ABI Prism 7700 sequence detection system (Biosystems, Foster City, CA) from a 1-mL sample of cDNA under the following conditions: denaturation at 94 C for 15 seconds, annealing at 60 C for 30 seconds, and primer extension at 72 C for 22 seconds for 45 cycles. PCR for each sample was repeated 3 times for both the target gene and control (without tensile force). Quantitative results of real-time fluorescence PCR were assessed based on the cycle threshold value, which identifies a cycle when the fluorescence of a given sample becomes significantly different from the baseline signal. Relative quantification of the M-CSF, RANKL, IL-1b, and TNF-a signals was normalized and expressed relative to G3PDH signals. Measurement of M-CSF, RANKL, IL-1b, and TNF-a Concentrations Conditioned medium from cultured pulp cells and controls with and without the application of cyclic tensile were collected and cleared at 2000 rpm for 5 minutes. The amounts of M-CSF (Quantikine Human M-CSF Immunoassay Kit; R&D Systems, Inc, Minneapolis, MN), RANKL (BI-20452 Ampli sRANKL Human Immunoassay Kit; BiomedicaGruppe, Inc), IL-1b, and TNF-a (Quantikine Human IL-1b Immunoassay Kit, Quantikine Human TNF-a Immunoassay Kit; R&D Systems, Inc, Wien, Austria) were measured using the quantitative sandwich enzyme immunoassay technique according to the manufacturer’s instructions. Standard curves were obtained as usual, and the experiment was repeated 5 times. Blockade of the S-A Channel The role of the S-A channel in the effects of cyclic tensile forces was examined by gadolinium (Gd3+) treatment. Cells were incubated with 10 or 100 mmol/L Gd3+ chloride hexahydrate (Wako, Osaka, Japan) for 30 minutes and were prepared to assess the effects of cyclic tensile forces of 10 kPa on the amounts of M-CSF, RANKL, IL-1b, and TNF-a for 48 hours. To examine the cytotoxicity of Gd3+, cells were stained with 0.5% Trypan Blue (Sigma-Aldrich) after Gd3+ treatment. The numbers of surviving and dead cells were then counted. Experimental Animals and Treatment Seven 7-week-old Wistar rats were used in this experiment. All animals were handled in accordance with the ethical regulations for animal experiments defined by the Ethics Committee of the Hiroshima University Faculty of Dentistry. Dental pulp from the upper right first molars was exposed by drilling cavities on the central portion of the occlusal surface with a round bur (1-mm diameter). A #25 endodontic file was then used to remove pulp tissue. Canals were irrigated with 2.5% sodium hypochlorite followed by 0.9% sterile saline solution using long needles. Canals were then dried and filled with a #25 gutta-percha point and sealer. The condition of root canal filling was evaluated by dental radiographic imaging. The left first molars were used as controls. An experimental appliance with a closed-coil spring was bonded onto the upper right and left first molars and incisors. Both molars were subjected to mesial movement for 6 months. Rats were killed under general anesthesia with sodium pentobarbital. Specimens were fixed in 4% paraformaldehyde, decalcified in 14% EDTA (pH = 7.4) for 28 days, and embedded in paraffin. Premaxillary bones, including the upper molars, were cut into 7-mm thick frontal sections. Effects of Pulpectomy on Root Resorption
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Figure 1. Loading time changes in mRNAs and protein expression of (A) M-CSF, (B) RANKL, (C) IL-1b, and (D) TNF-a in pulp cells stimulated by 10-kPa tensile force. Dotted line indicates the expression of M-CSF, RANKL, IL-1b, and TNF-a mRNAs in the control group. mRNAs of M-CSF and TNF-a in the experimental groups were significantly higher after 24 and 48 hours when compared with the control group. There were significant differences between the experimental and control groups in the expression of RANKL and IL-1b mRNA after 48 hours (*P < .05 vs the control group, **P < .01 vs the control group). Protein concentrations of MCSF, RANKL, IL-1b, and TNF-a increased in a time-dependent manner. Protein expression of all factors at 48 hours showed significant differences when compared with 12 hours (*P < .05, **P < .01).
Sections were stained with hematoxylin-eosin, and the mesial roots of the first molars were observed under a light microscope. Five sections at 35-mm intervals within the most central section for each specimen were subjected to histologic analysis. At the apex area (measuring range = 700 1200 mm2), the amount of root resorption for whole dentin was measured using digitized photomicrographs captured on a computer (NIH Image, Bethesda, MD).
48 hours, and the differences were significant (P < .05 and P < .01 respectively, Fig. 1A–D). The expression of M-CSF, RANKL, IL-1b, and TNF-a mRNAs in pulp cells was assessed when various tensile forces of 1, 3, 5, 10, and 15 kPa had been applied for 48 hours. M-CSF, RANKL, IL-1b, and TNF-a mRNAs in the experimental groups reached maximum levels of 3.3-, 2.0-, 5.1-, and 3.1-fold at 10 kPa tensile force (Fig. 2A–D).
Statistical Treatment Statistical significance of mRNA expression and protein concentration was evaluated using analysis of variance followed by the Fisher procedure. We used the Student t test to evaluate statistical differences in the area of root resorption. A confidence level of P < .05 was defined as being statistically significant.
Protein Expression The protein concentrations of M-CSF, RANKL, IL-1b, and TNF-a after 10 kPa tensile force application increased time dependently and reached a maximum at 48 hours (75.3, 19.0, 2.5, and 3.0 pg/mL, respectively). The protein concentration at 48 hours showed a significant difference when compared with 12 hours (M-CSF, RANKL, and IL-1b: P < .01; TNF-a: P < .05; Fig. 1). Protein expression of all factors reached a maximum with loading at 10 kPa tensile force at 48 hours. The protein concentration with 10 kPa tensile force showed significant differences when compared with 1, 3, 5, and 15 kPa (P < .01, Fig. 2).
Results Effects of Various Duration and Tensile Forces on the Expression of M-CSF, RANKL, IL-1b, and TNF-a mRNAs mRNAs of all factors showed time-dependent increases and reached maximum levels after 48 hours. The expression of M-CSF mRNA was 2.7- and 3.3-fold higher than control levels after 24 and 48 hours. There were significant differences in the amounts of M-CSF mRNA when compared with controls and after 48 hours (P < .01). The expression of RANKL and IL-1b mRNAs peaked at 2.0- and 5.0-fold, respectively, after 48 hours. There were significant differences in the amount of RANKL and IL-1b mRNAs when compared with controls and after 48 hours (P < .01). The expression of TNF-a mRNA was 2.3- and 3.1-fold higher than control levels after 24 and 374
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Gadolinium (Gd3+) Treatment Gd3+ at 10 and 100 mmol/L reduced the expression of all factor mRNAs under 10 kPa cyclic tensile force, showing significant differences when compared with non-Gd3+ treatment (P < .05 or .01, Fig. 3A–D). A quantitative sandwich enzyme immunoassay technique showed that 10 and 100 mmol/L Gd3+ treatment significantly reduced the expression of all factor proteins (P < .01, Fig. 3E–H). No significant decreases in the number of surviving cells were observed in the Gd3+ treatment group when compared with the nontreatment group. JOE — Volume 40, Number 3, March 2014
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Figure 2. Changes in mRNAs and protein expression of (A) M-CSF, (B) RANKL, (C) IL-1b, and (D) TNF-a in pulp cells stimulated by various tensile forces for 48 hours. The expression of M-CSF, RANKL, IL-1b, and TNF-a mRNAs in pulp cells was assessed after various tensile forces (1, 3, 5, 10, and 15 kPa) were applied for 48 hours. The dotted line indicates the expression of M-CSF, RANKL, IL-1b, and TNF-a mRNAs in the control group. mRNAs of all factors in the experimental groups increased gradually and peaked with loading at a 10-kPa tensile force, showing significant differences from controls (*P < .05 vs the control group, **P < .01 vs the control group). Protein concentrations of M-CSF, RANKL, IL-1b, and TNF-a peaked at a 10-kPa tensile force (*P < .05, **P < .01).
Histologic Examination and Amount of Root Resorption After experimental tooth movement for 6 months, apical root resorption was observed both in control and pulpless teeth. Both cementum and dentin were more markedly resorbed in the control group than in the pulpless group (Fig. 4A and B). The root resorption area in the control group was around 10%, but it was around 6% in the pulpless group; this difference was significant (P < .05, Fig. 4C).
Discussion Orthodontic tooth movement often leads to external root resorption (eg, cervical invasive root resorption), which is an aggressively destructive form of external root resorption characterized by the invasion of root dentin by fibrovascular tissue and clastic resorbing cells (19). Previous studies have suggested numerous risk factors for root resorption, including systemic factors, treatment mechanics, treatment period, age (20), root shape, and density of alveolar bone (21). In these articles, the authors concluded that the direct cause of the root resorption is outside resorption by contact between the root and alveolar bone. On the other hand, some authors have investigated the relation between pulp and root resorption. After orthodontic treatment, it was reported that root resorption in pulpectomized tooth was less marked than in vital teeth (9, 10) although the mechanism was unclear. Dental pulp is surrounded by hard dental tissue. The vitality of pulp tissue is influenced by blood vessels from the alveolar bone through the apical foramen. Graber (22) found that strong forces are able to traumatize apical vessels, and Salzmann (23) also reported that pulpal disturbance during orthodontic treatment may be the result of tipping of the teeth, during which the apical blood vessels are stretched. JOE — Volume 40, Number 3, March 2014
McDonald and Pitt Ford (7) found that human pulpal blood decreased when continuous tipping forces were applied. Miura (8) suggested that continuous heavy orthodontic forces can predispose pulp tissue to pulp necrosis through the rupture of bleed vessels in the apical root. Thus, it is possible that iatrogenic reactions occur in the apical pulp if blood vessels and pulp tissue are stretched and ruptured. In this study, we aimed to investigate the mechanisms of inflammatory root resorption regarding pulpal reactions. The mechanisms of root resorption are thought to be similar to those of bone resorption, and odontoclasts act on the resorption of the cementum and dentin through an acidification/degradationassociated pathway (3). A previous study showed that severe deficiency of osteoclasts in osteopetrotic mice can be cured by injections of recombinant human M-CSF (4). The direct action of M-CSF on osteoclast lineage cells was exhibited by the expression of the receptor for M-CSF (ie, c-fms) in osteoclasts both in vitro (24) and in vivo (25). Based on these findings, it appears that M-CSF plays an essential role in the differentiation of osteoclasts. In addition, it became clear that M-CSF supports osteoclast differentiation in cooperation with RANKL (5). Cytokines such as IL-1b and TNF-a are synthesized at the site of inflammation. In vitro studies have shown that IL-1b stimulates the differentiation and bone-resorbing activity of osteoclasts (26). TNF-a promotes the fusion of monocytes into osteoclasts while inhibiting the differentiation of osteoblasts from progenitor cells (27). In this study, both the expression of mRNAs and the protein concentration of M-CSF, RANKL, IL-1b, and TNF-a in the pulp cells increased gradually with tensile force and reached a maximum on the loading at 10 kPa after 48 hours. Conventional orthodontic force on the maxillary canines is around 60 g. Iwasaki et al (28) converted this force into compressive stress on the distal
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Figure 3. The effects of Gd3+ on the (A–D) expression of M-CSF, RANKL, IL-1b, and TNF-a mRNA and (E–H) protein expression in pulp cells stimulated by a 10-kPa tensile force for 48 hours. The dotted line indicates the expression of M-CSF, RANKL, IL-1b, and TNF-a mRNAs in the control group. M-CSF, RANKL, IL1b, and TNF-a mRNA levels were significantly reduced in the 10- and 100-mmol/L Gd3+ treatment groups (*P < .05, **P < .01). Significant decreases in M-CSF, RANKL, IL-1b, and TNF-a concentrations were observed in the 10- and 100-mmol/L Gd3+ treatment groups when compared with the untreated groups (**P < .01).
aspect of the canines and showed that the stress was 13 kPa. Therefore, during clinical orthodontic tooth movement, the stretched and injured pulp cells probably express these bone-resorbing cytokines; thus, inflammatory apical root resorption may occur because of derived odontoclasts. Furthermore, our results indicated that light orthodontic forces should be applied to reduce the risk of inflammatory root resorption as advocated in previous studies (29). However, in this study, there 376
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were no data to show the relationship between odontoclast activity and up-regulation of inflammatory cytokines during orthodontic tooth movement. Therefore, further in vivo studies into these factors are necessary. An S-A channel was first reported in tissue-cultured embryonic chick skeletal muscle (11). It was shown that stretching cellular membranes increased Ca2+ concentrations in human umbilical endothelial JOE — Volume 40, Number 3, March 2014
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Figure 4. Histologic examination and amount of root resorption. Root resorption after experimental tooth movement is indicated by the black line. Measuring range at the apex area is indicated by the frame (700 1200 mm2). (A) Control group (intact tooth), (B) pulpless group, and (C) rate of root resorption in the control and pulpless groups (*P < .05).
cells cultured on silicon membranes. The Ca2+ response disappeared when extracellular Ca2+ was removed or after treatment with Gd3+, which is a potent blocker of S-A channels. It was also shown that orienting and elongating responses of cultured endothelial cells to cyclic stretching were inhibited by the removal of external Ca2+ or by the addition of Gd3+ (30). Therefore, it was concluded that cell-elongating responses are mediated by Ca2+-permeable S-A channels present on the cell membrane. In this study, Gd3+ treatment inhibited the expression of M-CSF, RANKL, IL-1b, and TNF-a genes and proteins in a dosedependent manner after tensile force application. Moreover, no significant decreases in the number of surviving cells were observed in the Gd3+ treatment group when compared with the nontreatment group, thus confirming that the effects of Gd3+ are not caused by cytotoxicity. These results strongly suggest that tensile forces through the apical foramen to the pulp cells by orthodontic treatment enhance the expresJOE — Volume 40, Number 3, March 2014
sion of M-CSF, RANKL, IL-1b, and TNF-a via S-A channels, and as a result, odontoclastic inflammatory root resorption occurs. Apical root resorption during orthodontic treatment is an unfavorable issue, and its etiology must be clarified. We herein showed that dental pulp tissue plays an important role in the processes of inflammatory apical root resorption during orthodontic tooth movement. Thus, root canal treatment may be effective for progressive severe inflammatory root resorption during orthodontic tooth movement.
Acknowledgments Supported by a Grant-in-Aid (No. 22592285) for Basic Research (C) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The authors deny any conflicts of interest related to this study. Effects of Pulpectomy on Root Resorption
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Basic Research—Biology References 1. Massler M, Malone AJ. Root resorption in human permanent teeth: a roentgenographic study. Am J Orthod Dentofacial Orthop 1954;40:619–33. 2. Brezniak N, Wasserstein A. Root resorption after orthodontic treatment: part 1. Literature review. Am J Orthod Dentofacial Orthop 1993;103:62–6. 3. Matsumoto Y. Morphological and functional properties of odontoclasts on dentine resorption. Kokubyo Gakkai Zasshi 1994;61:123–43. 4. Kodama H, Yamasaki A, Nose M, et al. Congenital osteoclast deficiency in osteopetrotic (op/op) mice is cured by injections of macrophage colony-stimulating factor. J Exp Med 1991;173:269–72. 5. Yasuda H, Shima N, Nakagawa N, et al. Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/ RANKL. Proc Natl Acad Sci U S A 1998;95:3597–602. 6. Boabaid F, Berry JE, Koh AJ, et al. The role of parathyroid hormone-related protein in the regulation of osteoclastogenesis by cementoblasts. J Periodontol 2004;75:1247–54. 7. McDonald F, Pitt Ford TR. Blood flow changes in permanent maxillary canines during retraction. Eur J Orthod 1994;16:1–9. 8. Miura F. Effect of orthodontic force on blood circulation in periodontal membrane. In: Cook JT, ed. Transactions of Third International Orthodontics Congress. London: Granada Publishing Ltd; 1973:35–41. 9. Remington DN, Joondeph DR, Artun J, et al. Long-term evaluation of root resorption occurring during orthodontic treatment. Am J Orthod Dentofacial Orthop 1989;96: 43–6. 10. Spurrier SW, Hall SH, Joondeph DR, et al. A comparison of apical root resorption during orthodontic treatment in endodontically treated and vital teeth. Am J Orthod Dentofacial Orthop 1990;97:130–4. 11. Guharay F, Sachs F. Stretch-activated single ion channel currents in tissue-cultured embryonic chick skeletal muscle. J Physiol 1984;352:685–701. 12. Lansman JB, Hallam TJ, Rink TJ. Single stretch-activated ion channels in vascular endothelial cells as mechanotransducers. Nature 1987;325:811–3. 13. Naruse K, Sokabe M. Involvement of stretch-activated ion channels in Ca2+ mobilization to mechanical stretch in endothelial cells. Am J Physiol 1993;264:C1037–44. 14. Abedini S, Kaku M, Kawata T, et al. Effects of cryopreservation with a newlydeveloped magnetic field programmed freezer on periodontal ligament cells and pulp tissues. Cryobiology 2011;62:181–7. 15. Baek KH, Oh KW, Lee WY, et al. Association of oxidative stress with postmenopausal osteoporosis and the effects of hydrogen peroxide on osteoclast formation in human bone marrow cell cultures. Calcif Tissue Int 2010;87:226–35.
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16. Heider U, Zavrski I, Jakob C, et al. Expression of receptor activator of NF-kappaB ligand (RANKL) mRNA in human multiple myeloma cells. J Cancer Res Clin Oncol 2004;130:469–74. 17. Tomoyasu Y, Yamaguchi T, Tajima A, et al. External apical root resorption and the interleukin-1b gene polymorphism in the Japanese population. Orthodontic Waves 2009;68:152–7. 18. Li X, Chen BD. Histone deacetylase inhibitor M344 inhibits cell proliferation and induces apoptosis in human THP-1 leukemia cells. Am J Biomed Sci 2009;9: 352–63. 19. Thonen A, Peltomaki T, Patcas R, et al. Occurrence of cervical invasive root resorption in first and second molar teeth of orthodontic patients eight years after bracket removal. J Endod 2013;39:27–30. 20. Linge BO, Linge L. Apical root resorption in upper anterior teeth. Eur J Orthod 1983; 5:173–83. 21. Kaley J, Phillips C. Factors related to root resorption in edgewise practice. Angle Orthod 1991;61:125–32. 22. Graber TM. Orthodontics Principles and Practice, 2nd ed. Philadelphia: WB Saunders; 1967. 23. Salzmann JA. Practice of Orthodontics, vol. 2. Philadelphia: JB Lippincott; 1966. 24. Kodama H, Nose M, Niida S, et al. Essential role of macrophage colony-stimulating factor in the osteoclast differentiation supported by stromal cells. J Exp Med 1991; 173:1291–4. 25. Hofstetter W, Wetterwald A, Cecchini MC, et al. Detection of transcripts for the receptor for macrophage colony-stimulating factor, c-fms, in murine osteoclasts. Proc Natl Acad Sci U S A 1992;89:9637–41. 26. Jimi E, Nakamura I, Duong LT, et al. Interleukin 1 induces multinucleation and bone-resorbing activity of osteoclasts in the absence of osteoblasts/stromal cells. Exp Cell Res 1999;247:84–93. 27. Nanes MS. Tumor necrosis factor-a: molecular and cellular mechanisms in skeletal pathology. Gene 2003;321:1–15. 28. Iwasaki LR, Haack JE, Nickel JC, et al. Human tooth movement in response to continuous stress of low magnitude. Am J Orthod Dentofacial Orthop 2000;117: 175–83. 29. Gonzales C, Hotokezaka H, Yoshimatsu M, et al. Force magnitude and duration effects on amount of tooth movement and root resorption in the rat molar. Angle Orthod 2008;78:502–9. 30. Naruse K, Yamada T, Sokabe M. Involvement of SA channels in orienting response of cultured endothelial cells to cyclic stretch. Am J Physiol 1998; 274:H1532–8.
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Effects of Pulpectomy on the Amount of Root Resorption during Orthodontic Tooth Movement Masato Kaku, PhD, DDS, Hiromi Sumi, DDS, Hanaka Shikata, DDS, Shunichi Kojima, PhD, DDS, Masahide Motokawa, PhD, DDS, Tadashi Fujita, PhD, DDS, Kotaro Tanimoto, PhD, DDS, and Kazuo Tanne, PhD, DDS Abstract Introduction: Previous studies have revealed that orthodontic force affects dental pulp via the rupture of blood vessels and vacuolization of pulp tissues. We hypothesized that pulp tissues express inflammatory cytokines and regulators of odontoclast differentiation after excess orthodontic force. The purpose of this study was to investigate the effects of tensile force in human pulp cells and to measure inflammatory root resorption during tooth movement in pulpless rat teeth. Methods: After cyclic tensile force application in human pulp cells, gene expression and protein concentration of macrophage colony-stimulating factor, receptor activator of nuclear factor kappa-B ligand, interleukin-1 beta, and tumor necrosis factor alpha were determined by realtime polymerase chain reaction and enzyme-linked immunoassay. Moreover, the role of the stretchactivated channel was evaluated by gadolinium (Gd3+) treatment. The upper right first molars of 7-week Wistar rats were subjected to pulpectomy and root canal filling followed by mesial movement for 6 months. Results: The expression of cytokine messenger RNAs and proteins in the experimental group peaked with loading at 10-kPa tensile force after 48 hours (P < .01). Gd3+ reduced the expression of these cytokine messenger RNAs and protein concentrations (P < .01). The amount of inflammatory root resorption was significantly larger in the control teeth than the pulpectomized teeth (P < .05). Conclusions: This study shows that tensile forces in the pulp cells enhance the expression of various cytokines via the S-A channel, which may lead to inflammatory root resorption during tooth movement. It also suggests that root canal treatment is effective for progressive severe inflammatory root resorption during tooth movement. (J Endod 2014;40:372–378)
Key Words Odontclast, orthodontic tooth movement, pulp cells, pulpectomy, root resorption, tensile forces
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uring orthodontic tooth movement, apical root resorption is an undesirable side effect that is difficult to predict and repair. Massler and Malone (1) found that root resorption occurred in 86.4% of orthodontic patients, and root resorption associated with orthodontics is reported to be related to factors such as patient age, sex, and systemic conditions (2). Root resorption is also associated with inflammatory reactions by odontoclasts, which are similar to osteoclasts in morphology, activity, functions, and features (3). Previous studies have shown that macrophage colony-stimulating factor (M-CSF) plays an essential role in osteoclastogenesis (4). In addition, it was clarified that M-CSF supports osteoclast differentiation in cooperation with receptor activator of nuclear factor kappa-B ligand (RANKL) (5). Moreover, cementoblasts are able to express RANKL and can modulate osteoclast cytogenesis (6). Thus, M-CSF and RANKL are indispensable to odontoclast differentiation for root resorption, and it is important to clarify how and from where M-CSF and RANKL are released when root resorption occurs during orthodontic tooth movement. Because orthodontic force is recognized as a type of trauma, pulp tissue can be injured during orthodontic tooth movement. McDonald and Pitt Ford (7) found that human pulpal blood decreased when continuous tipping forces were applied. Miura (8) suggested that continuous heavy orthodontic forces predispose pulp tissue to pulp necrosis via rupture of blood vessels in the apical root. Furthermore, some researchers have investigated the relationship between pulp tissue and root resorption during orthodontic tooth movement. Remington et al (9) and Spurrier et al (10) showed that root resorption is more often observed in intact teeth when compared with pulpless teeth. Indeed, we have noted severe apical root resorption in intact teeth and mild root resorption in pulpectomized teeth. Based on these findings, we hypothesized that stretched and injured pulp cells express M-CSF and RANKL as well as inflammatory cytokines; as a result, inflammatory apical root resorption occurs because of the derived odontoclasts. A stretch-activated channel (S-A channel), which is an ionic channel activated by stretching, was detected in tissue-cultured embryonic chick skeletal muscle (11). Vascular endothelial cells have been reported to contain a cation-selective S-A channel permeable to calcium ions (12). It was also shown that intracellular Ca2+ increased in response to mechanical stretching via the S-A channel in human umbilical endothelial cells, and this response was blocked by gadolinium (Gd3+), an S-A channel blocker (13). Based on these findings, it was assumed that mechanical stimuli such as tensile forces in pulp cells are controlled through the S-A channel, and, consequently, osteoclast-inducing factors and inflammatory cytokines can be produced. Therefore, in this study, we examined the effects of cyclic tensile forces on the expression of M-CSF, RANKL, interleukin-1 beta (IL-1b), and tumor necrosis factor alpha (TNF-a) in human pulp cells as well as the inhibition of these actions by blockade
From the Department of Orthodontics and Craniofacial Developmental Biology, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima, Japan. Address requests for reprints to Dr Masato Kaku, Department of Orthodontics and Craniofacial Developmental Biology, Hiroshima University Graduate School of Biomedical Sciences, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8553, Japan. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2014 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2013.11.027
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Basic Research—Biology of mechanosensory S-A channels. Furthermore, we investigated differences in root resorption during orthodontic tooth movement between pulpless and intact teeth.
Materials and Methods Cell Culture Freshly extracted and intact teeth were used. These teeth were indicated for extraction for orthodontic treatment in patients aged 18–30 years. The protocol was reviewed and approved by the Ethics Committee of Hiroshima University, and informed consent was obtained from each tooth donor. Teeth were cut vertically, and pulp tissues were removed aseptically and rinsed with phosphate buffered saline (PBS). The outgrowth method was performed for culture of human dental pulp cells in accordance with a previous study (14). Explants were cut into small fragments with a sharp blade and transferred to 10-cm Petri dishes and cultured in a-Modified Eagle’s Medium (MEM) (Sigma-Aldrich, St Louis, MO) containing 10% fetal bovine serum (FBS) (Biological Industries, Kibbutz Beit-Haemek, Israel), 32 U/mL penicillin G (Meiji Seika, Tokyo, Japan), 250 mg/mL amphotericin B (Nacalai Tesque, Kyoto, Japan), and 60 mg/mL kanamycin (Meiji Seika, Tokyo, Japan) at 37 C in a humidified atmosphere of 5% CO2. The medium was changed twice a week. Cells were subcultured by treatment with 0.25% trypsin/EDTA and plated at 3 105 cells per 100-mm culture dish. For all experiments, cells between the 4th and 6th passages were used. Application of Cyclic Tensile Forces The Flexcell strain unit FX-2000 (Flexcell International Co, Hillsborough, NC) consists of a vacuum unit and a valve controlled by a computer program. Pulp cells (1 105) cultured on a flexible membrane base were subjected to cyclic tensile forces produced by computercontrolled application of sinusoidal negative pressure. Flexible membranes supporting the cultured cells were deformed by negative pressure. Application of a vacuum results in a maximum cell elongation of 20% at the periphery of wells, with the strain declining toward the center. Cells were placed in a humidified incubator under an atmosphere of 5% CO2 at 37 C. To examine the expression of M-CSF, RANKL, IL-1b, and TNF-a messenger RNAs (mRNAs) and their concentrations, a tensile force of 10 kPa was loaded at a frequency of 30 cycles per minute for 12, 24, and 48 hours. Next, we investigated the influence of various tensile forces of 1, 3, 5, 10, and 15 kPa on the amounts of M-CSF, RANKL, IL-1b, and TNF-a for 48 hours. Total RNA Extraction and Complementary DNA Synthesis Total RNA was isolated from the cell cultures using a Quickprep Total RNA extraction kit (Amersham Biosciences, Tokyo, Japan). Single-stranded complementary DNA (cDNA) was synthesized from 1 mg total RNA using Oligo(dT)20 primer (Toyobo, Osaka, Japan) and a Rever Tra Ace-a first-strand cDNA synthesis kit (Toyobo). Primers The following primers were used: M-CSF (15) 50 -GGCCATGAGAGGCAGTCCGAGGG-30 (forward), 50 -CACTGGCAGTTCCACCTGTCT GTC-30 (reverse); RANKL (16): 50 -TCAGAAGATGGCACTCACTG-30 (forward), 50 -AACATCTCCCACTGGCTGTA-30 (reverse); IL-1b (17): 50 CTCAGGTGTCCTCGAAGAAATCAA-30 (forward), 50 -GCTTTTTTGCTGT GAGTCCCG-30 (reverse); and TNF-a (18): 50 -CCCCAGGGCTCCAGGCG GTGCTTGT-30 (forward), 50 -GGAGACGGCGATGCGGCTGATGGTG-30 (reverse). G3PDH primer (Rever Tra Ace-a first-strand cDNA synthesis JOE — Volume 40, Number 3, March 2014
kit, Toyobo) was used as a control primer; 50 -ACCACAGTCCATGCCATCAC-30 (sense), 50 -TCCACCACCCTGTTGCTGTA-30 (antisense).
Real-time Quantitative Polymerase Chain Reaction Real-time polymerase chain reaction (PCR) was performed using SYBR Green I assay and an ABI Prism 7700 sequence detection system (Biosystems, Foster City, CA) from a 1-mL sample of cDNA under the following conditions: denaturation at 94 C for 15 seconds, annealing at 60 C for 30 seconds, and primer extension at 72 C for 22 seconds for 45 cycles. PCR for each sample was repeated 3 times for both the target gene and control (without tensile force). Quantitative results of real-time fluorescence PCR were assessed based on the cycle threshold value, which identifies a cycle when the fluorescence of a given sample becomes significantly different from the baseline signal. Relative quantification of the M-CSF, RANKL, IL-1b, and TNF-a signals was normalized and expressed relative to G3PDH signals. Measurement of M-CSF, RANKL, IL-1b, and TNF-a Concentrations Conditioned medium from cultured pulp cells and controls with and without the application of cyclic tensile were collected and cleared at 2000 rpm for 5 minutes. The amounts of M-CSF (Quantikine Human M-CSF Immunoassay Kit; R&D Systems, Inc, Minneapolis, MN), RANKL (BI-20452 Ampli sRANKL Human Immunoassay Kit; BiomedicaGruppe, Inc), IL-1b, and TNF-a (Quantikine Human IL-1b Immunoassay Kit, Quantikine Human TNF-a Immunoassay Kit; R&D Systems, Inc, Wien, Austria) were measured using the quantitative sandwich enzyme immunoassay technique according to the manufacturer’s instructions. Standard curves were obtained as usual, and the experiment was repeated 5 times. Blockade of the S-A Channel The role of the S-A channel in the effects of cyclic tensile forces was examined by gadolinium (Gd3+) treatment. Cells were incubated with 10 or 100 mmol/L Gd3+ chloride hexahydrate (Wako, Osaka, Japan) for 30 minutes and were prepared to assess the effects of cyclic tensile forces of 10 kPa on the amounts of M-CSF, RANKL, IL-1b, and TNF-a for 48 hours. To examine the cytotoxicity of Gd3+, cells were stained with 0.5% Trypan Blue (Sigma-Aldrich) after Gd3+ treatment. The numbers of surviving and dead cells were then counted. Experimental Animals and Treatment Seven 7-week-old Wistar rats were used in this experiment. All animals were handled in accordance with the ethical regulations for animal experiments defined by the Ethics Committee of the Hiroshima University Faculty of Dentistry. Dental pulp from the upper right first molars was exposed by drilling cavities on the central portion of the occlusal surface with a round bur (1-mm diameter). A #25 endodontic file was then used to remove pulp tissue. Canals were irrigated with 2.5% sodium hypochlorite followed by 0.9% sterile saline solution using long needles. Canals were then dried and filled with a #25 gutta-percha point and sealer. The condition of root canal filling was evaluated by dental radiographic imaging. The left first molars were used as controls. An experimental appliance with a closed-coil spring was bonded onto the upper right and left first molars and incisors. Both molars were subjected to mesial movement for 6 months. Rats were killed under general anesthesia with sodium pentobarbital. Specimens were fixed in 4% paraformaldehyde, decalcified in 14% EDTA (pH = 7.4) for 28 days, and embedded in paraffin. Premaxillary bones, including the upper molars, were cut into 7-mm thick frontal sections. Effects of Pulpectomy on Root Resorption
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Figure 1. Loading time changes in mRNAs and protein expression of (A) M-CSF, (B) RANKL, (C) IL-1b, and (D) TNF-a in pulp cells stimulated by 10-kPa tensile force. Dotted line indicates the expression of M-CSF, RANKL, IL-1b, and TNF-a mRNAs in the control group. mRNAs of M-CSF and TNF-a in the experimental groups were significantly higher after 24 and 48 hours when compared with the control group. There were significant differences between the experimental and control groups in the expression of RANKL and IL-1b mRNA after 48 hours (*P < .05 vs the control group, **P < .01 vs the control group). Protein concentrations of MCSF, RANKL, IL-1b, and TNF-a increased in a time-dependent manner. Protein expression of all factors at 48 hours showed significant differences when compared with 12 hours (*P < .05, **P < .01).
Sections were stained with hematoxylin-eosin, and the mesial roots of the first molars were observed under a light microscope. Five sections at 35-mm intervals within the most central section for each specimen were subjected to histologic analysis. At the apex area (measuring range = 700 1200 mm2), the amount of root resorption for whole dentin was measured using digitized photomicrographs captured on a computer (NIH Image, Bethesda, MD).
48 hours, and the differences were significant (P < .05 and P < .01 respectively, Fig. 1A–D). The expression of M-CSF, RANKL, IL-1b, and TNF-a mRNAs in pulp cells was assessed when various tensile forces of 1, 3, 5, 10, and 15 kPa had been applied for 48 hours. M-CSF, RANKL, IL-1b, and TNF-a mRNAs in the experimental groups reached maximum levels of 3.3-, 2.0-, 5.1-, and 3.1-fold at 10 kPa tensile force (Fig. 2A–D).
Statistical Treatment Statistical significance of mRNA expression and protein concentration was evaluated using analysis of variance followed by the Fisher procedure. We used the Student t test to evaluate statistical differences in the area of root resorption. A confidence level of P < .05 was defined as being statistically significant.
Protein Expression The protein concentrations of M-CSF, RANKL, IL-1b, and TNF-a after 10 kPa tensile force application increased time dependently and reached a maximum at 48 hours (75.3, 19.0, 2.5, and 3.0 pg/mL, respectively). The protein concentration at 48 hours showed a significant difference when compared with 12 hours (M-CSF, RANKL, and IL-1b: P < .01; TNF-a: P < .05; Fig. 1). Protein expression of all factors reached a maximum with loading at 10 kPa tensile force at 48 hours. The protein concentration with 10 kPa tensile force showed significant differences when compared with 1, 3, 5, and 15 kPa (P < .01, Fig. 2).
Results Effects of Various Duration and Tensile Forces on the Expression of M-CSF, RANKL, IL-1b, and TNF-a mRNAs mRNAs of all factors showed time-dependent increases and reached maximum levels after 48 hours. The expression of M-CSF mRNA was 2.7- and 3.3-fold higher than control levels after 24 and 48 hours. There were significant differences in the amounts of M-CSF mRNA when compared with controls and after 48 hours (P < .01). The expression of RANKL and IL-1b mRNAs peaked at 2.0- and 5.0-fold, respectively, after 48 hours. There were significant differences in the amount of RANKL and IL-1b mRNAs when compared with controls and after 48 hours (P < .01). The expression of TNF-a mRNA was 2.3- and 3.1-fold higher than control levels after 24 and 374
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Gadolinium (Gd3+) Treatment Gd3+ at 10 and 100 mmol/L reduced the expression of all factor mRNAs under 10 kPa cyclic tensile force, showing significant differences when compared with non-Gd3+ treatment (P < .05 or .01, Fig. 3A–D). A quantitative sandwich enzyme immunoassay technique showed that 10 and 100 mmol/L Gd3+ treatment significantly reduced the expression of all factor proteins (P < .01, Fig. 3E–H). No significant decreases in the number of surviving cells were observed in the Gd3+ treatment group when compared with the nontreatment group. JOE — Volume 40, Number 3, March 2014
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Figure 2. Changes in mRNAs and protein expression of (A) M-CSF, (B) RANKL, (C) IL-1b, and (D) TNF-a in pulp cells stimulated by various tensile forces for 48 hours. The expression of M-CSF, RANKL, IL-1b, and TNF-a mRNAs in pulp cells was assessed after various tensile forces (1, 3, 5, 10, and 15 kPa) were applied for 48 hours. The dotted line indicates the expression of M-CSF, RANKL, IL-1b, and TNF-a mRNAs in the control group. mRNAs of all factors in the experimental groups increased gradually and peaked with loading at a 10-kPa tensile force, showing significant differences from controls (*P < .05 vs the control group, **P < .01 vs the control group). Protein concentrations of M-CSF, RANKL, IL-1b, and TNF-a peaked at a 10-kPa tensile force (*P < .05, **P < .01).
Histologic Examination and Amount of Root Resorption After experimental tooth movement for 6 months, apical root resorption was observed both in control and pulpless teeth. Both cementum and dentin were more markedly resorbed in the control group than in the pulpless group (Fig. 4A and B). The root resorption area in the control group was around 10%, but it was around 6% in the pulpless group; this difference was significant (P < .05, Fig. 4C).
Discussion Orthodontic tooth movement often leads to external root resorption (eg, cervical invasive root resorption), which is an aggressively destructive form of external root resorption characterized by the invasion of root dentin by fibrovascular tissue and clastic resorbing cells (19). Previous studies have suggested numerous risk factors for root resorption, including systemic factors, treatment mechanics, treatment period, age (20), root shape, and density of alveolar bone (21). In these articles, the authors concluded that the direct cause of the root resorption is outside resorption by contact between the root and alveolar bone. On the other hand, some authors have investigated the relation between pulp and root resorption. After orthodontic treatment, it was reported that root resorption in pulpectomized tooth was less marked than in vital teeth (9, 10) although the mechanism was unclear. Dental pulp is surrounded by hard dental tissue. The vitality of pulp tissue is influenced by blood vessels from the alveolar bone through the apical foramen. Graber (22) found that strong forces are able to traumatize apical vessels, and Salzmann (23) also reported that pulpal disturbance during orthodontic treatment may be the result of tipping of the teeth, during which the apical blood vessels are stretched. JOE — Volume 40, Number 3, March 2014
McDonald and Pitt Ford (7) found that human pulpal blood decreased when continuous tipping forces were applied. Miura (8) suggested that continuous heavy orthodontic forces can predispose pulp tissue to pulp necrosis through the rupture of bleed vessels in the apical root. Thus, it is possible that iatrogenic reactions occur in the apical pulp if blood vessels and pulp tissue are stretched and ruptured. In this study, we aimed to investigate the mechanisms of inflammatory root resorption regarding pulpal reactions. The mechanisms of root resorption are thought to be similar to those of bone resorption, and odontoclasts act on the resorption of the cementum and dentin through an acidification/degradationassociated pathway (3). A previous study showed that severe deficiency of osteoclasts in osteopetrotic mice can be cured by injections of recombinant human M-CSF (4). The direct action of M-CSF on osteoclast lineage cells was exhibited by the expression of the receptor for M-CSF (ie, c-fms) in osteoclasts both in vitro (24) and in vivo (25). Based on these findings, it appears that M-CSF plays an essential role in the differentiation of osteoclasts. In addition, it became clear that M-CSF supports osteoclast differentiation in cooperation with RANKL (5). Cytokines such as IL-1b and TNF-a are synthesized at the site of inflammation. In vitro studies have shown that IL-1b stimulates the differentiation and bone-resorbing activity of osteoclasts (26). TNF-a promotes the fusion of monocytes into osteoclasts while inhibiting the differentiation of osteoblasts from progenitor cells (27). In this study, both the expression of mRNAs and the protein concentration of M-CSF, RANKL, IL-1b, and TNF-a in the pulp cells increased gradually with tensile force and reached a maximum on the loading at 10 kPa after 48 hours. Conventional orthodontic force on the maxillary canines is around 60 g. Iwasaki et al (28) converted this force into compressive stress on the distal
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Figure 3. The effects of Gd3+ on the (A–D) expression of M-CSF, RANKL, IL-1b, and TNF-a mRNA and (E–H) protein expression in pulp cells stimulated by a 10-kPa tensile force for 48 hours. The dotted line indicates the expression of M-CSF, RANKL, IL-1b, and TNF-a mRNAs in the control group. M-CSF, RANKL, IL1b, and TNF-a mRNA levels were significantly reduced in the 10- and 100-mmol/L Gd3+ treatment groups (*P < .05, **P < .01). Significant decreases in M-CSF, RANKL, IL-1b, and TNF-a concentrations were observed in the 10- and 100-mmol/L Gd3+ treatment groups when compared with the untreated groups (**P < .01).
aspect of the canines and showed that the stress was 13 kPa. Therefore, during clinical orthodontic tooth movement, the stretched and injured pulp cells probably express these bone-resorbing cytokines; thus, inflammatory apical root resorption may occur because of derived odontoclasts. Furthermore, our results indicated that light orthodontic forces should be applied to reduce the risk of inflammatory root resorption as advocated in previous studies (29). However, in this study, there 376
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were no data to show the relationship between odontoclast activity and up-regulation of inflammatory cytokines during orthodontic tooth movement. Therefore, further in vivo studies into these factors are necessary. An S-A channel was first reported in tissue-cultured embryonic chick skeletal muscle (11). It was shown that stretching cellular membranes increased Ca2+ concentrations in human umbilical endothelial JOE — Volume 40, Number 3, March 2014
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Figure 4. Histologic examination and amount of root resorption. Root resorption after experimental tooth movement is indicated by the black line. Measuring range at the apex area is indicated by the frame (700 1200 mm2). (A) Control group (intact tooth), (B) pulpless group, and (C) rate of root resorption in the control and pulpless groups (*P < .05).
cells cultured on silicon membranes. The Ca2+ response disappeared when extracellular Ca2+ was removed or after treatment with Gd3+, which is a potent blocker of S-A channels. It was also shown that orienting and elongating responses of cultured endothelial cells to cyclic stretching were inhibited by the removal of external Ca2+ or by the addition of Gd3+ (30). Therefore, it was concluded that cell-elongating responses are mediated by Ca2+-permeable S-A channels present on the cell membrane. In this study, Gd3+ treatment inhibited the expression of M-CSF, RANKL, IL-1b, and TNF-a genes and proteins in a dosedependent manner after tensile force application. Moreover, no significant decreases in the number of surviving cells were observed in the Gd3+ treatment group when compared with the nontreatment group, thus confirming that the effects of Gd3+ are not caused by cytotoxicity. These results strongly suggest that tensile forces through the apical foramen to the pulp cells by orthodontic treatment enhance the expresJOE — Volume 40, Number 3, March 2014
sion of M-CSF, RANKL, IL-1b, and TNF-a via S-A channels, and as a result, odontoclastic inflammatory root resorption occurs. Apical root resorption during orthodontic treatment is an unfavorable issue, and its etiology must be clarified. We herein showed that dental pulp tissue plays an important role in the processes of inflammatory apical root resorption during orthodontic tooth movement. Thus, root canal treatment may be effective for progressive severe inflammatory root resorption during orthodontic tooth movement.
Acknowledgments Supported by a Grant-in-Aid (No. 22592285) for Basic Research (C) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The authors deny any conflicts of interest related to this study. Effects of Pulpectomy on Root Resorption
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