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Local administration of calcitonin inhibits alveolar bone loss in an experimental periodontitis in rats
Biomedicine & Pharmacotherapy 97 (2018) 765–770
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Original article
Local administration of calcitonin inhibits alveolar bone loss in an experimental periodontitis in rats
MARK
Chie Wada-Mihara, Hiroyuki Seto, Hirofumi Ohba, Kaku Tokunaga, Jun-ichi Kido, ⁎ Toshihiko Nagata, Koji Naruishi Department of Periodontology and Endodontology, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15 Kuramoto, Tokushima 770-8504, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords: Calcitonin Experimental periodontitis Bone loss Osteoclasts
Calcitonin (CTN), a calcium regulatory hormone, promotes calcium diuresis from the kidney and suppresses bone resorption. The objective of this study was to evaluate whether the topical and intermittent application of CTN inhibits alveolar bone resorption using ligature-induced experimental periodontitis in rats. Experimental periodontitis was induced by placing a nylon ligature around maxillary molars of 8-week-old male Wistar rats for 20 days. Thirty-two rats were divided into four groups: basal sham control group, periodontitis group, periodontitis plus 0.2 U CTN (low dose), and periodontitis plus 1.0 U CTN (high dose) group. To investigate the effects of CTN on alveolar bone resorption, CTN was topically injected into the palatal gingivae every 2 days after ligature removal (day 0). Micro-computed tomography (CT) analysis was performed for linear parameter assessment of alveolar bone on day 5 and day 14. Periodontal tissues were examined histo-pathologically to assess the differences among the study groups. Micro-CT images showed that alveolar bone resorption was induced statistically around the molar of ligatured rats on day 5 and day 14. The amount of bone resorption was more severe on day 14 than that on day 5. On day 5, only high-dose CTN treatment significantly suppressed bone resorption. In addition, both doses of CTN significantly suppressed bone resorption on day 14. Histological examination clarified that there were fewer TRAP-positive cells in the CTN treatment groups than in the periodontitis group on day 5. Local administration of CTN decreased alveolar bone resorption by regulating osteoclast activation in rats with periodontitis.
1. Introduction Periodontitis is a polymicrobial infectious disease and may result in loss of teeth by inflammation-mediated bone resorption [1]. It is wellknown that periodontal inflammation arising from bacterial infection exacerbates bone destruction. Bone homeostasis is controlled by a balance between osteoblastic bone formation and osteoclastic bone resorption [2]. An imbalance between the interaction of osteoblasts and osteoclasts leads to the progression of periodontitis. Osteoclast activation and maturation is regulated by the receptor activator of nuclear factor-κB (RANK), RANK ligand (RANKL), and osteoprotegerin (OPG) [3]. Excessive activation of osteoclasts in periodontitis lesions might lead to severe alveolar bone resorption. Recently, Kim et al. reported that curcumin, a potent antioxidant drug, suppressed ovariectomy-induced bone loss, at least in part, by reducing RANKL signaling [4]. Furthermore, Nakamura et al. suggested that green tea catechin suppressed lipopolysaccharide-induced bone
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resorption by inhibiting IL-1beta production or by directly inhibiting osteoclastogenesis [5]. Drugs with a capacity to modulate inflammatory responses including bone resorption might be useful clinically because these drugs may have new therapeutic effects. Calcitonin (CTN), which is released from parafollicular cells (C cells) in the thyroid gland, is a hormone involved in bone homeostasis and the regulation of calcium metabolism [6]. In bone tissues, CTN binds to osteoclasts exclusively, exhibiting the highest expression of calcitonin receptor (CTR), and causes cessation of osteoclast activity [6,7]. Granholm et al. reported that CTN inhibits osteoclast formation in mouse hematopoietic cells by regulating RANK signaling [8]. In addition, CTN has been used for Paget’s disease, hypercalcemia of malignancy, osteogenesis imperfecta, and post-menopausal osteoporosis [9]. Although these findings suggest that bone loss can be attenuated by CTN administration, few studies have investigated the efficacy of CTN in the treatment of periodontitis. Parathyroid hormone (PTH), an anabolic agent targeting bone, is
Corresponding author. E-mail address: [email protected] (K. Naruishi).
http://dx.doi.org/10.1016/j.biopha.2017.10.165 Received 14 September 2017; Received in revised form 30 October 2017; Accepted 31 October 2017 0753-3322/ © 2017 Elsevier Masson SAS. All rights reserved.
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alveolar bone crest (ABC) of the second molar was measured using computer software.
known as an endocrine hormone secreted by parathyroid glands [10], and many studies have shown that PTH is effective in reversing bone loss due to its effect in increasing osteoblast number and activity [11,12]. Furthermore, CTN and PTH are complementary hormones involved in bone metabolism [6]. Previously, we reported that topical and intermittent administration of PTH recovered alveolar bone loss in experimental rat periodontitis [13]. In vivo experimental periodontitis is useful to clarify the pathogenesis and treatment strategies for periodontal disease. In the present study using micro-computed tomography (micro-CT) and histology, we explored the inhibitory effects of CTN on the alveolar bone resorption of rats with experimental periodontitis.
2.4. Histological analysis On day 5 and day 14, six rats from each group were sacrificed, and the maxillae were collected, dissected, and immediately fixed in 4% paraformaldehyde (Wako Pure Chemical Industries Ltd., Osaka, Japan). After fixation, samples were decalcified with 10% EDTA for 28 days and embedded in paraffin (Paraplust Plus, Sigma, St Louis, MO). The frontal sections, parallel with the mesial root of the second molar, were cut into 4 μm-thick sections. All sections were routinely stained with hematoxylin and eosin. Vertical alveolar bone loss of the second molar of the maxilla was assessed for inflammation and alveolar bone destruction was assessed by measuring the length (mm) of the CEJ to ABC between the first and second molars of the maxilla as described previously [13]. In brief, five sections were randomly obtained from each rat to observe histological changes and the measured area was determined at the square (200 × 200 μm at a magnification of 400×) of the buccal site around the alveolar bone crest. The length was measured at five sites in the square and the mean value was determined. Next, infiltrating cells were designated as inflammatory cells, including macrophages, lymphocytes, and neutrophils, based on shape and size. The total number of inflammatory cells represented the inflammatory status of the connective tissues, and the mean of the histological data was calculated as described previously [17]. For osteoclast analysis, sections were stained with a tartrate-resistant acid phosphatase (TRAP) kit (Sigma-Aldrich, St Louis, MO, USA) according to the manufacturer’s instructions. The stained sections were observed under an optical microscope (Microphoto V series, Nikon, Tokyo, Japan), and the TRAP-positive multinucleated cells were defined by cells with greater than three nuclei under a light microscope and counted according to a previous report [18]. Five sections of each group were analyzed and each measurement was conducted by an independent examiner in a blind manner. The results from each group were expressed as mean ± SD.
2. Materials and methods 2.1. Animals Thirty-six male Wistar rats (200–250 g, 8 weeks-old) were purchased from CLEA Japan (Tokyo, Japan). Rats were fed with standard rodent chow and water ad libitum, and housed in a temperature-controlled room (23 ± 1 °C, 60 ± 5% humidity) under a 12-h light-dark cycle. Prior to the surgical procedures, all animals were allowed to acclimatize to the laboratory environment for 1 week. The experimental protocol was approved by the Animal Research Control Committee of the Tokushima University Graduate School. The study complied with the Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines developed by the National Centre for the Replacement, Refinement & Reduction of Animals in Research (NC3Rs) [14]. 2.2. Experimental periodontitis For experimental periodontitis induction, the cervical area of the right second molar of the rat maxilla was ligatured with sterilized nylon thread (5–0: Natsume Corporation, Tokyo, Japan) under anesthesia with sodium pentobarbital for 20 days according to a previous report [13]. The ligature was knotted on the mesial in order to confirm the ligature remained during the experimental period, and the ligature was checked every 2 days to ensure subgingival placement, and was replaced when necessary. CTN used as Elcitonin was a gift from Asahi Kasei Pharma (Tokyo, Japan). Elcitonin is a calcitonin derivative that is transformed from eel calcitonin by modifying the SeS bond into a stable CeN bond. Thirtysix rats were randomly divided into three groups as follows: ligature placement and administration of saline (N = 12), ligature placement and administration of low-dose of CTN (N = 12, 1.0 U/kg), and ligature placement and administration of high-dose of CTN (N = 12, 5.0 U/kg). The left maxillae without ligature was used as a non-ligatured control (basal sham control) and selected randomly (N = 12). CTN was administered into the subperiosteum at the right buccal and palatal gingivae of the maxillary second molar every 2 days in a volume of 50 μL for 5 or 14 days, and the injection level of CTN was used according to a previous report [15]. Solutions were prepared just before administration. The number of animals was justified using a sample size calculation based on the primary outcome variable [16].
2.5. Statistical analysis Statistical analyses were performed with SPSS Statistics version 20 (Armonk, NY). Significant differences among each group were analyzed using one-way ANOVA with Scheffe post hoc test or Tukey-Kramer HSD test, because the data were not normally distributed. In all statistical analyses, P values of less than 0.05 were considered significant. 3. Results 3.1. A volumetric quantitative analysis of alveolar bone loss No clinical signs of systemic infection or mortality were noted in any of the rats at any time. The micro-CT images show decreased alveolar bone height of the molars in ligatured rats compared to that of non-ligatured rats on both day 5 and day 14 (Fig. 1A). We confirmed that significant alveolar bone loss was found in the group of experimental periodontitis. The alveolar bone loss was inhibited by application of high-dose CTN on both day 5 and day 14. In case of low-dose CTN application, no significant difference was observed in bone loss compared to that of the periodontitis group on day 5, but significant inhibition of bone loss was observed by low-dose CTN application on day 14. The distance between CEJ and ABC represents the degree of bone resorption, and the lengths were measured in each group and quantitatively analyzed. As shown in Fig. 1B, the CEJ-ABC length of the ligatured rat group increased significantly compared to that of the nonligatured group (day 5: ligatured, 442.6 ± 67.3 μm, non-ligatured, 280.8 ± 52.4 μm, P = 0.0017; day 14: ligatured, 585.6 ± 59.4 μm, non-ligatured, 258.3 ± 21.3 μm, P < 0.0001, one-way ANOVA-
2.3. Micro-CT examination The maxillary specimens were evaluated by micro-CT imaging using a micro-CT scanner (Hitachi Medico, Tokyo, Japan). To maintain the longitudinal position of the samples, the maxillae were oriented vertically in a sample holder. Scans were performed by determining the angle based on the position of buccal and palatal cusps in the second molar. The micro-CT setting was as follows: 1024 × 1024 pixel size, 14 μm slice thickness, 8× magnification, 50 kV voltage, and 0.1 mA electrical current. Three dimensional images were produced using computer software TRI/3D-BON (Ratoc Systems Inc., Tokyo, Japan). The distance from the palatal cementum-enamel junction (CEJ) to the 766
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Fig. 1. (A) Images of micro-Computed tomography (micro-CT). Effects of calcitonin on ligation-induced alveolar bone loss examined by micro-CT in maxilla secondary molars of rats. The images have shown on day 5 and day 14, as reconstructed by the micro-CT. Experimental groups were classified as follows: Nonligatured control (basal sham control), ligatured, low-dose CTN (1.0 U/kg) and high-dose CTN (5.0 U/ kg). Arrow, the distance from the cemento-enamel junction (CEJ) to the alveolar bone crest (ABC). (B) Differences of alveolar bone levels. The statistical differences were evaluated by comparing the distance from the CEJ to the ABC in each group. Data represent mean ± SD (N = 4/group). CTN, calcitonin. Bu, buccal, Pa, palatal. *, P < 0.05; **, P < 0.01, one-way ANOVA and Scheffe test.
addition, we found that the CEJ-ABC length of the high-dose CTN group decreased significantly compared to that of the ligatured group on day 5 (day 5: high-CTN, 339.1 ± 92.2 μm, P = 0.042, one-way ANOVAScheffe test), but there was no significant difference between the lowdose CTN group and the ligatured group (day 5: low-CTN, 466.3 ± 18.3 μm, P = 0.80, one-way ANOVA-Scheffe test). On day 14, the CEJ-ABC length decreased significantly by application of both lowdose and high-dose CTN compared to that of the ligatured group (day 14: low-CTN: 316.3 ± 80.6 μm, P = 0.007; high-CTN: 359.8 ± 44.8 μm, P = 0.032, one-way ANOVA-Scheffe test). There was no significant difference between the low-dose and high-dose CTN groups on both day 5 and day 14 (day 5: P = 0.15; day 14: P = 0.83, one-way ANOVA-Scheffe test). As shown in Fig. 2D, significant infiltration of inflammatory cells was observed in the ligatured group. CTN-treated rats had a significantly lower number of inflammatory cells compared to that of ligatured rats on day 5 (low-CTN: P = 0.0002; high-CTN: P < 0.0001, Tukey-Kramer HSD test). There was a decreasing trend in the number of inflammatory cells in the group of high-CTN rats compared to that of low-CT rats on day 5 (P = 0.13, Tukey-Kramer HSD test). No inflammatory cells were observed on day 14 (data not shown). Next, the number of osteoclasts in tissue sections was determined by TRAP staining. CTN-treated rats had a significantly lower number of TRAPpositive osteoclasts per square millimeter of alveolar bone compared to that of ligatured rats on day 5 (low-CTN: P = 0.0067; high-CTN: P < 0.0001, one-way ANOVA-Scheffe test) (Fig. 2E). There was a decreasing trend in the number of TRAP-positive osteoclasts in the group of high-CTN rats compared to that of low-CT rats on day 5 (P = 0.15, one-way ANOVA-Scheffe test). No TRAP-positive cells were observed on day 14 (data not shown).
Scheffe test). There was a significant difference between day 5 and day 14 in the CEJ-ABC length of the ligatured group (P = 0.0077). In addition, we found that the CEJ-ABC length of the high-dose CTN group decreased significantly compared to that of the ligatured group on day 5 (322.0 ± 23.1 μm, P = 0.041, one-way ANOVA-Scheffe test), but there was no significant difference between the low-dose CTN and ligatured rat groups (day 5: low-dose CTN, 385.6 ± 51.1 μm, P = 0.80, one-way ANOVA-Scheffe test). On day 14, the CEJ-ABC length decreased significantly by application of both low-dose and high-dose CTN compared to that of the ligatured group (low-CTN: 412.2 ± 32.7 μm, P = 0.0006; high-CTN: 368.1 ± 40.4 μm, P < 0.0001, one-way ANOVA-Scheffe test). There was no statistical difference between the low-dose and high-dose CTN groups in CEJ-ABC length on both day 5 and day 14.
3.2. Histological changes of periodontal tissues by application of CTN As shown in Fig. 2A, prominent loss of epithelial attachment, bone resorption, and infiltration of inflammatory cells were observed in periodontal tissues of ligatured rats compared to that of non-ligatured rats on day 5. A number of TRAP positive osteoclasts lining the alveolar bone surface were visually enumerated in periodontal tissues of ligatured rats (arrows in Fig. 2A–D), and osteoclasts eroded the bone surface roughly. On the other hand, loss of epithelial attachment and bone resorption in ligatured rats was impaired slightly by application of lowdose CTN (Fig. 2A–E). Although the boundary of the connective tissue and gingival sulcus epithelium were flat in non-ligatured rats on day 5, typical features of gingival inflammation, such as elongated rete pegs into connective tissues, were observed in both ligatured and CTNtreated rats (Fig. 2A–C, E, G). On day 14, significant histological changes were not observed compared to that on day 5 (Fig. 2B). No TRAP-positive osteoclasts were observed even in the ligatured group. As shown in Fig. 2C, the CEJ-ABC length of the ligatured group increased significantly compared to that of the non-ligatured group (day 5: ligatured, 520.3 ± 65.9 μm, non-ligatured, 293.5 ± 78.8 μm, P = 0.011; day 14: ligatured, 521.8 ± 54.0 μm, non-ligatured, 285.9 ± 74.3 μm, P = 0.002, one-way ANOVA-Scheffe test). In
4. Discussion Increased alveolar bone loss, considered a hallmark of periodontitis, may cause tooth loss [1]. Based on our findings in animal models, CTN may be a potential drug with anti-bone resorption effects. Many investigators have proposed that treatment of bone destruction by a bone 767
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Fig. 2. TRAP staining of rat palatal periodontal tissues on day 5 (A) and day 14 (B). Low (left column: a, c, e, g) and high (right column: b, d, f, h) magnification of decalcified sections have shown. Each panel of right column shows magnified view of the square in left column, respectively. Experimental groups were classified as follows: Non-ligatured control (basal sham control), ligatured, low-dose CTN (1.0 U/kg) and high-dose CTN (5.0 U/kg). Scale bars, 100 μm. The arrow heads shown in low magnification section (left column) indicate cemento-enamel junction (CEJ). The arrow heads in high magnification section (right column) indicate TRAP-positive osteoclasts. Ab, alveolar bone. De, dentin. (C) Comparison of histological bone level: Length from the CEJ to the alveolar bone crest (ABC). The statistical differences were evaluated by comparing the distance from the CEJ to the ABC in each group. Data represent mean ± SD. CTN, calcitonin. *, P < 0.05; **, P < 0.01, one-way ANOVA and Scheffe test. (D) Statistical analysis for periodontal tissue inflammation. Number of inflammatory cells on day5. Inflammatory cells per visual field, counted 5 visual fields. Data represent mean ± SD. **P < 0.01, ANOVA Tukey-Kramer HSD test. (E) Number of TRAPpositive osteoclasts on day 5. TRAP-positive cells per visual field, counted 5 visual fields. Data represent mean ± SD. CTN, calcitonin. **P < 0.01, ANOVA Tukey-Kramer HSD test.
periodontitis model [20]. On the other hand, Chen et al. suggested that systemic sclerostin antibody treatment can create greater alveolar crest height in rats exposed to ligature-induced periodontitis [21] because sclerostin secreted primarily by osteocytes has been known to negatively regulate osteoblast-mediated bone formation. Further investigation is needed to determine the efficacy of systemic versus local
anabolic agent could be a potential adjuvant therapy to suppress alveolar bone loss in periodontitis. Yoshinaga et al. reported that locally administered green tea extract suppresses the onset of loss of attachment and alveolar bone resorption in a rat experimental periodontitis model [19]. Furthermore, it has been reported that locally administered tiludronic acid reduced alveolar bone loss in a rat experimental 768
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approaches might be proposed to enhance CTN effects on osteoclasts in combination with PTH, vit D, or bisphosphonates for periodontal treatment.
administration, but clinical use of novel agents, such as CTN, may prevent inflammatory periodontal bone loss. Osteoporosis is a disease characterized by reduction of bone mass and poor bone strength in post-menopausal women [22]. CTN has long been used as a therapeutic agent for osteoporosis because of the antiresorptive properties via directly reducing osteoclastic resorption, and thus leads to an increase in bone mineral density and bone strength [23]. Based on clinical use, we speculated that CTN may inhibit alveolar bone loss induced by periodontal infection, even though bone resorption induced by osteoporosis depends on hormonal impairment such as estrogen deficiency. There are many methods to induce in vivo periodontitis experimentally [24,25], and we selected an experimental model using simple ligation by nylon thread without periodontal pathogens because inflammatory cells, such as neutrophils and macrophages, arise sufficiently in periodontal tissues [13,26]. We considered that this model may simulate mild inflammation, but our histological findings show significant infiltration of leukocytes in periodontal tissues of ligatured rats. In addition, micro-CT has been used as a fast, precise nondestructive, analytical procedure to measure bone volume within defects. Our micro-CT images showed marked resorption of alveolar bone of ligatured rats. Because no clinical signs of systemic infection or mortality were noted in any of the rats at any time, these findings indicate that our experimental periodontitis model is suitable to evaluate novel treatment strategies of periodontitis using CTN. Systemic injections of eel CTN, a synthetic eel CTN analogue, inhibit formalin-induced nociceptive hypersensitivity (hyperalgesia and allodynia) in rats [15]. Therefore, in our in vivo experimental periodontitis model, we used synthetic eel CTN. Importantly, compared to the ligatured group, rats given CTN had less bone loss, and rats given a high dose of CTN had significantly decreased bone loss even on day 5. Histological findings proved that CTN inhibited bone resorption induced by ligation. In addition, the number of TRAP-positive osteoclasts decreased significantly in rats given a high dose of CTN on day 5, and positive cells disappeared on day 14 even from groups given a low dose of CTN. Because TRAP-positive osteoclasts resorb bone, these results suggest that inhibition of bone loss by CTN depends on direct impairment of osteoclast activation. Osteoclasts typically are multinucleated and express TRAP and the CTN receptor (CTR). Previously, Kim et al. reported that monocyte chemoattractant protein-1 (MCP-1) is induced by RANKL and promotes osteoclast fusion in multinuclear cells [27]. In addition, CTN inhibited MCP-1-mediated osteoclast fusion through the inhibition of its own receptor CTR. Quinn et al. suggested that CTN strongly inhibits CTR expression in osteoclasts, which results in inhibition of osteoclast differentiation [28]. In our experimental periodontitis model, MCP-1 may appear in periodontal tissues by reflecting inflammation cascades, which results in osteoclast inactivation. Furthermore, Spolidorio et al. recently reported that CTN inhibited cyclosporine A-induced alveolar bone loss via a decrease of circulating inflammatory cytokines, such as IL-1 and IL-6, in a rat model [29]. Inhibition of several inflammatory cytokines by CTN might induce indirect effects on the inhibition of alveolar bone loss. To clarify the chemokine/cytokine-mediated responses of osteoclasts regulated by CTN, further experiments will be needed in the future. Topical and intermittent administration of PTH recovered alveolar bone loss in rat experimental periodontitis [13]. Because PTH increased alkaline phosphatase activity and bone nodule formation in fetal rat osteoblastic cells, the target cells may be osteoblasts, but not osteoclasts [11,12]. In addition, vitamin D (vit D) increased calcium and phosphate uptake to mask the effects of PTH [30]. It is well-known that a combination of CTN and vit D is a standard regimen for treating post-menopausal osteoporosis clinically. Tanaka et al. reported that combination therapy with CTN and bisphosphonates appears to be an effective treatment for osteoporosis patients [31]. Thus, there is a lot of research focused on understanding how agents are useful and how they cause beneficial or harmful effects in novel treatment strategies. New
5. Conclusion We demonstrated that ligature procedures induced marked alveolar bone loss around the molars, and greater bone recovery was observed in the local CTN-treated rats. In the short term, local administration of CTN may have a highly anti-resorptive effect in experimental periodontitis. However, CTN may have limited usefulness in situations where sustained anti-resorptive action is needed because osteoclasts tend to become resistant to the action of CTN due to receptor downregulation and reactivate bone resorption [27,32]. More research is needed before CTN can be used in the preclinical practice and for clinical therapy of periodontitis. Further studies are needed to clarify its anti-inflammatory roles, effective dose, and side effects. We hope that dentists may have medication to control the activities of bone-related cells to allow novel treatment strategies for unwanted bone loss by periodontal inflammation in the future. Conflict of interest The authors declare no conflicts of interest related to this study. Acknowledgements The authors are grateful to the staff of Tokushima University Graduate School. This study was supported by a Grant-in-Aid for Scientific Research (B) (No. 15H05054) from the Japan Society for the Promotion of Science. References [1] R.C. Page, S. Offenbacher, H.E. Schroeder, G.J. Seymour, K.S. Kornman, Advances in the pathogenesis of periodontitis: summary of developments, clinical implications and future directions, Periodontol. 2000 14 (1997) 216–248. [2] Z. Schwartz, J. Goultschin, D.D. Dean, B.D. Boyan, Mechanisms of alveolar bone destruction in periodontitis, Periodontol. 2000 14 (1997) 158–172. [3] B.F. Boyce, Advances in the regulation of osteoclasts and osteoclast functions, J. Dent. Res. 92 (2013) 860–867. [4] W.K. Kim, K. Ke, O.J. Sul, H.J. Kim, S.H. Kim, M.H. Lee, et al., Curcumin protects against ovariectomy-induced bone loss and decreases osteoclastogenesis, J. Cell. Biochem. 112 (2011) 3159–3166. [5] H. Nakamura, T. Ukai, A. Yoshimura, Y. Kozuka, H. Yoshioka, Y. Yoshinaga, et al., Green tea catechin inhibits lipopolysaccharide-induced bone resorption in vivo, J. Periodontal Res. 45 (2010) 23–30. [6] F.J. de Paula, C.J. Rosen, Back to the future: revisiting parathyroid hormone and calcitonin control of bone remodeling, Horm. Metab. Res. 42 (2010) 299–306. [7] S. Wallach, J.B. Carstens Jr, L.V. Avioli, Calcitonin, osteoclasts, and bone turnover, Calcif. Tissue Res. 47 (1990) 388–391. [8] S. Granholm, P. Lundberg, U.H. Lerner, Calcitonin inhibits osteoclast formation in mouse haematopoetic cells independently of transcriptional regulation by receptor activator of NF-κB and c-Fos, J. Endocrinol. 195 (2007) 415–427. [9] M. Zaidi, B.S. Moonga, E. Abe, Calcitonin and bone formation: a knockout full of surprises, J. Clin. Invest. 110 (2002) 1769–1771. [10] Y. Fei, M.M. Hurley, Role of fibroblast growth factor 2 and Wnt signaling in anabolic effects of parathyroid hormone on bone formation, J. Cell. Physiol. 227 (2012) 3539–3545. [11] E.M. Greenfield, Anabolic effects of intermittent PTH on osteoblasts, Curr. Mol. Pharmacol. 5 (2012) 127–134. [12] N.S. Datta, A.B. Abou-Samra, PTH and PTHrP signaling in osteoblasts, Cell Signal. 21 (2009) 1245–1254. [13] K. Tokunaga, H. Seto, H. Ohba, C. Mihara, H. Hama, M. Horibe, et al., Topical and intermittent application of parathyroid hormone recovers alveolar bone loss in rat experimental periodontitis, J. Periodontal Res. 46 (2011) 655–662. [14] C. Kilkenny, W.J. Browne, I.C. Cuthill, M. Emerson, D.G. Altman, Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research, PLoS Biol. 8 (2010) e1000412. [15] H. Umeno, T. Nagasawa, N. Yamazaki, Y. Kuraishi, Antinociceptive effects of repeated systemic injections of calcitonin in formalin-induced hyperalgesic rats, Pharmacol. Biochem. Behav. 55 (1996) 151–156. [16] J. Kim, B.S. Seo, How to calculate sample size and why, Clin. Orthopedic Surg. 5 (2013) 235–242. [17] K. Irie, T. Tomofuji, N. Tamaki, T. Sanbe, D. Ekuni, T. Azuma, et al., Effects of
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Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Original article
Local administration of calcitonin inhibits alveolar bone loss in an experimental periodontitis in rats
MARK
Chie Wada-Mihara, Hiroyuki Seto, Hirofumi Ohba, Kaku Tokunaga, Jun-ichi Kido, ⁎ Toshihiko Nagata, Koji Naruishi Department of Periodontology and Endodontology, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15 Kuramoto, Tokushima 770-8504, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords: Calcitonin Experimental periodontitis Bone loss Osteoclasts
Calcitonin (CTN), a calcium regulatory hormone, promotes calcium diuresis from the kidney and suppresses bone resorption. The objective of this study was to evaluate whether the topical and intermittent application of CTN inhibits alveolar bone resorption using ligature-induced experimental periodontitis in rats. Experimental periodontitis was induced by placing a nylon ligature around maxillary molars of 8-week-old male Wistar rats for 20 days. Thirty-two rats were divided into four groups: basal sham control group, periodontitis group, periodontitis plus 0.2 U CTN (low dose), and periodontitis plus 1.0 U CTN (high dose) group. To investigate the effects of CTN on alveolar bone resorption, CTN was topically injected into the palatal gingivae every 2 days after ligature removal (day 0). Micro-computed tomography (CT) analysis was performed for linear parameter assessment of alveolar bone on day 5 and day 14. Periodontal tissues were examined histo-pathologically to assess the differences among the study groups. Micro-CT images showed that alveolar bone resorption was induced statistically around the molar of ligatured rats on day 5 and day 14. The amount of bone resorption was more severe on day 14 than that on day 5. On day 5, only high-dose CTN treatment significantly suppressed bone resorption. In addition, both doses of CTN significantly suppressed bone resorption on day 14. Histological examination clarified that there were fewer TRAP-positive cells in the CTN treatment groups than in the periodontitis group on day 5. Local administration of CTN decreased alveolar bone resorption by regulating osteoclast activation in rats with periodontitis.
1. Introduction Periodontitis is a polymicrobial infectious disease and may result in loss of teeth by inflammation-mediated bone resorption [1]. It is wellknown that periodontal inflammation arising from bacterial infection exacerbates bone destruction. Bone homeostasis is controlled by a balance between osteoblastic bone formation and osteoclastic bone resorption [2]. An imbalance between the interaction of osteoblasts and osteoclasts leads to the progression of periodontitis. Osteoclast activation and maturation is regulated by the receptor activator of nuclear factor-κB (RANK), RANK ligand (RANKL), and osteoprotegerin (OPG) [3]. Excessive activation of osteoclasts in periodontitis lesions might lead to severe alveolar bone resorption. Recently, Kim et al. reported that curcumin, a potent antioxidant drug, suppressed ovariectomy-induced bone loss, at least in part, by reducing RANKL signaling [4]. Furthermore, Nakamura et al. suggested that green tea catechin suppressed lipopolysaccharide-induced bone
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resorption by inhibiting IL-1beta production or by directly inhibiting osteoclastogenesis [5]. Drugs with a capacity to modulate inflammatory responses including bone resorption might be useful clinically because these drugs may have new therapeutic effects. Calcitonin (CTN), which is released from parafollicular cells (C cells) in the thyroid gland, is a hormone involved in bone homeostasis and the regulation of calcium metabolism [6]. In bone tissues, CTN binds to osteoclasts exclusively, exhibiting the highest expression of calcitonin receptor (CTR), and causes cessation of osteoclast activity [6,7]. Granholm et al. reported that CTN inhibits osteoclast formation in mouse hematopoietic cells by regulating RANK signaling [8]. In addition, CTN has been used for Paget’s disease, hypercalcemia of malignancy, osteogenesis imperfecta, and post-menopausal osteoporosis [9]. Although these findings suggest that bone loss can be attenuated by CTN administration, few studies have investigated the efficacy of CTN in the treatment of periodontitis. Parathyroid hormone (PTH), an anabolic agent targeting bone, is
Corresponding author. E-mail address: [email protected] (K. Naruishi).
http://dx.doi.org/10.1016/j.biopha.2017.10.165 Received 14 September 2017; Received in revised form 30 October 2017; Accepted 31 October 2017 0753-3322/ © 2017 Elsevier Masson SAS. All rights reserved.
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alveolar bone crest (ABC) of the second molar was measured using computer software.
known as an endocrine hormone secreted by parathyroid glands [10], and many studies have shown that PTH is effective in reversing bone loss due to its effect in increasing osteoblast number and activity [11,12]. Furthermore, CTN and PTH are complementary hormones involved in bone metabolism [6]. Previously, we reported that topical and intermittent administration of PTH recovered alveolar bone loss in experimental rat periodontitis [13]. In vivo experimental periodontitis is useful to clarify the pathogenesis and treatment strategies for periodontal disease. In the present study using micro-computed tomography (micro-CT) and histology, we explored the inhibitory effects of CTN on the alveolar bone resorption of rats with experimental periodontitis.
2.4. Histological analysis On day 5 and day 14, six rats from each group were sacrificed, and the maxillae were collected, dissected, and immediately fixed in 4% paraformaldehyde (Wako Pure Chemical Industries Ltd., Osaka, Japan). After fixation, samples were decalcified with 10% EDTA for 28 days and embedded in paraffin (Paraplust Plus, Sigma, St Louis, MO). The frontal sections, parallel with the mesial root of the second molar, were cut into 4 μm-thick sections. All sections were routinely stained with hematoxylin and eosin. Vertical alveolar bone loss of the second molar of the maxilla was assessed for inflammation and alveolar bone destruction was assessed by measuring the length (mm) of the CEJ to ABC between the first and second molars of the maxilla as described previously [13]. In brief, five sections were randomly obtained from each rat to observe histological changes and the measured area was determined at the square (200 × 200 μm at a magnification of 400×) of the buccal site around the alveolar bone crest. The length was measured at five sites in the square and the mean value was determined. Next, infiltrating cells were designated as inflammatory cells, including macrophages, lymphocytes, and neutrophils, based on shape and size. The total number of inflammatory cells represented the inflammatory status of the connective tissues, and the mean of the histological data was calculated as described previously [17]. For osteoclast analysis, sections were stained with a tartrate-resistant acid phosphatase (TRAP) kit (Sigma-Aldrich, St Louis, MO, USA) according to the manufacturer’s instructions. The stained sections were observed under an optical microscope (Microphoto V series, Nikon, Tokyo, Japan), and the TRAP-positive multinucleated cells were defined by cells with greater than three nuclei under a light microscope and counted according to a previous report [18]. Five sections of each group were analyzed and each measurement was conducted by an independent examiner in a blind manner. The results from each group were expressed as mean ± SD.
2. Materials and methods 2.1. Animals Thirty-six male Wistar rats (200–250 g, 8 weeks-old) were purchased from CLEA Japan (Tokyo, Japan). Rats were fed with standard rodent chow and water ad libitum, and housed in a temperature-controlled room (23 ± 1 °C, 60 ± 5% humidity) under a 12-h light-dark cycle. Prior to the surgical procedures, all animals were allowed to acclimatize to the laboratory environment for 1 week. The experimental protocol was approved by the Animal Research Control Committee of the Tokushima University Graduate School. The study complied with the Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines developed by the National Centre for the Replacement, Refinement & Reduction of Animals in Research (NC3Rs) [14]. 2.2. Experimental periodontitis For experimental periodontitis induction, the cervical area of the right second molar of the rat maxilla was ligatured with sterilized nylon thread (5–0: Natsume Corporation, Tokyo, Japan) under anesthesia with sodium pentobarbital for 20 days according to a previous report [13]. The ligature was knotted on the mesial in order to confirm the ligature remained during the experimental period, and the ligature was checked every 2 days to ensure subgingival placement, and was replaced when necessary. CTN used as Elcitonin was a gift from Asahi Kasei Pharma (Tokyo, Japan). Elcitonin is a calcitonin derivative that is transformed from eel calcitonin by modifying the SeS bond into a stable CeN bond. Thirtysix rats were randomly divided into three groups as follows: ligature placement and administration of saline (N = 12), ligature placement and administration of low-dose of CTN (N = 12, 1.0 U/kg), and ligature placement and administration of high-dose of CTN (N = 12, 5.0 U/kg). The left maxillae without ligature was used as a non-ligatured control (basal sham control) and selected randomly (N = 12). CTN was administered into the subperiosteum at the right buccal and palatal gingivae of the maxillary second molar every 2 days in a volume of 50 μL for 5 or 14 days, and the injection level of CTN was used according to a previous report [15]. Solutions were prepared just before administration. The number of animals was justified using a sample size calculation based on the primary outcome variable [16].
2.5. Statistical analysis Statistical analyses were performed with SPSS Statistics version 20 (Armonk, NY). Significant differences among each group were analyzed using one-way ANOVA with Scheffe post hoc test or Tukey-Kramer HSD test, because the data were not normally distributed. In all statistical analyses, P values of less than 0.05 were considered significant. 3. Results 3.1. A volumetric quantitative analysis of alveolar bone loss No clinical signs of systemic infection or mortality were noted in any of the rats at any time. The micro-CT images show decreased alveolar bone height of the molars in ligatured rats compared to that of non-ligatured rats on both day 5 and day 14 (Fig. 1A). We confirmed that significant alveolar bone loss was found in the group of experimental periodontitis. The alveolar bone loss was inhibited by application of high-dose CTN on both day 5 and day 14. In case of low-dose CTN application, no significant difference was observed in bone loss compared to that of the periodontitis group on day 5, but significant inhibition of bone loss was observed by low-dose CTN application on day 14. The distance between CEJ and ABC represents the degree of bone resorption, and the lengths were measured in each group and quantitatively analyzed. As shown in Fig. 1B, the CEJ-ABC length of the ligatured rat group increased significantly compared to that of the nonligatured group (day 5: ligatured, 442.6 ± 67.3 μm, non-ligatured, 280.8 ± 52.4 μm, P = 0.0017; day 14: ligatured, 585.6 ± 59.4 μm, non-ligatured, 258.3 ± 21.3 μm, P < 0.0001, one-way ANOVA-
2.3. Micro-CT examination The maxillary specimens were evaluated by micro-CT imaging using a micro-CT scanner (Hitachi Medico, Tokyo, Japan). To maintain the longitudinal position of the samples, the maxillae were oriented vertically in a sample holder. Scans were performed by determining the angle based on the position of buccal and palatal cusps in the second molar. The micro-CT setting was as follows: 1024 × 1024 pixel size, 14 μm slice thickness, 8× magnification, 50 kV voltage, and 0.1 mA electrical current. Three dimensional images were produced using computer software TRI/3D-BON (Ratoc Systems Inc., Tokyo, Japan). The distance from the palatal cementum-enamel junction (CEJ) to the 766
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Fig. 1. (A) Images of micro-Computed tomography (micro-CT). Effects of calcitonin on ligation-induced alveolar bone loss examined by micro-CT in maxilla secondary molars of rats. The images have shown on day 5 and day 14, as reconstructed by the micro-CT. Experimental groups were classified as follows: Nonligatured control (basal sham control), ligatured, low-dose CTN (1.0 U/kg) and high-dose CTN (5.0 U/ kg). Arrow, the distance from the cemento-enamel junction (CEJ) to the alveolar bone crest (ABC). (B) Differences of alveolar bone levels. The statistical differences were evaluated by comparing the distance from the CEJ to the ABC in each group. Data represent mean ± SD (N = 4/group). CTN, calcitonin. Bu, buccal, Pa, palatal. *, P < 0.05; **, P < 0.01, one-way ANOVA and Scheffe test.
addition, we found that the CEJ-ABC length of the high-dose CTN group decreased significantly compared to that of the ligatured group on day 5 (day 5: high-CTN, 339.1 ± 92.2 μm, P = 0.042, one-way ANOVAScheffe test), but there was no significant difference between the lowdose CTN group and the ligatured group (day 5: low-CTN, 466.3 ± 18.3 μm, P = 0.80, one-way ANOVA-Scheffe test). On day 14, the CEJ-ABC length decreased significantly by application of both lowdose and high-dose CTN compared to that of the ligatured group (day 14: low-CTN: 316.3 ± 80.6 μm, P = 0.007; high-CTN: 359.8 ± 44.8 μm, P = 0.032, one-way ANOVA-Scheffe test). There was no significant difference between the low-dose and high-dose CTN groups on both day 5 and day 14 (day 5: P = 0.15; day 14: P = 0.83, one-way ANOVA-Scheffe test). As shown in Fig. 2D, significant infiltration of inflammatory cells was observed in the ligatured group. CTN-treated rats had a significantly lower number of inflammatory cells compared to that of ligatured rats on day 5 (low-CTN: P = 0.0002; high-CTN: P < 0.0001, Tukey-Kramer HSD test). There was a decreasing trend in the number of inflammatory cells in the group of high-CTN rats compared to that of low-CT rats on day 5 (P = 0.13, Tukey-Kramer HSD test). No inflammatory cells were observed on day 14 (data not shown). Next, the number of osteoclasts in tissue sections was determined by TRAP staining. CTN-treated rats had a significantly lower number of TRAPpositive osteoclasts per square millimeter of alveolar bone compared to that of ligatured rats on day 5 (low-CTN: P = 0.0067; high-CTN: P < 0.0001, one-way ANOVA-Scheffe test) (Fig. 2E). There was a decreasing trend in the number of TRAP-positive osteoclasts in the group of high-CTN rats compared to that of low-CT rats on day 5 (P = 0.15, one-way ANOVA-Scheffe test). No TRAP-positive cells were observed on day 14 (data not shown).
Scheffe test). There was a significant difference between day 5 and day 14 in the CEJ-ABC length of the ligatured group (P = 0.0077). In addition, we found that the CEJ-ABC length of the high-dose CTN group decreased significantly compared to that of the ligatured group on day 5 (322.0 ± 23.1 μm, P = 0.041, one-way ANOVA-Scheffe test), but there was no significant difference between the low-dose CTN and ligatured rat groups (day 5: low-dose CTN, 385.6 ± 51.1 μm, P = 0.80, one-way ANOVA-Scheffe test). On day 14, the CEJ-ABC length decreased significantly by application of both low-dose and high-dose CTN compared to that of the ligatured group (low-CTN: 412.2 ± 32.7 μm, P = 0.0006; high-CTN: 368.1 ± 40.4 μm, P < 0.0001, one-way ANOVA-Scheffe test). There was no statistical difference between the low-dose and high-dose CTN groups in CEJ-ABC length on both day 5 and day 14.
3.2. Histological changes of periodontal tissues by application of CTN As shown in Fig. 2A, prominent loss of epithelial attachment, bone resorption, and infiltration of inflammatory cells were observed in periodontal tissues of ligatured rats compared to that of non-ligatured rats on day 5. A number of TRAP positive osteoclasts lining the alveolar bone surface were visually enumerated in periodontal tissues of ligatured rats (arrows in Fig. 2A–D), and osteoclasts eroded the bone surface roughly. On the other hand, loss of epithelial attachment and bone resorption in ligatured rats was impaired slightly by application of lowdose CTN (Fig. 2A–E). Although the boundary of the connective tissue and gingival sulcus epithelium were flat in non-ligatured rats on day 5, typical features of gingival inflammation, such as elongated rete pegs into connective tissues, were observed in both ligatured and CTNtreated rats (Fig. 2A–C, E, G). On day 14, significant histological changes were not observed compared to that on day 5 (Fig. 2B). No TRAP-positive osteoclasts were observed even in the ligatured group. As shown in Fig. 2C, the CEJ-ABC length of the ligatured group increased significantly compared to that of the non-ligatured group (day 5: ligatured, 520.3 ± 65.9 μm, non-ligatured, 293.5 ± 78.8 μm, P = 0.011; day 14: ligatured, 521.8 ± 54.0 μm, non-ligatured, 285.9 ± 74.3 μm, P = 0.002, one-way ANOVA-Scheffe test). In
4. Discussion Increased alveolar bone loss, considered a hallmark of periodontitis, may cause tooth loss [1]. Based on our findings in animal models, CTN may be a potential drug with anti-bone resorption effects. Many investigators have proposed that treatment of bone destruction by a bone 767
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Fig. 2. TRAP staining of rat palatal periodontal tissues on day 5 (A) and day 14 (B). Low (left column: a, c, e, g) and high (right column: b, d, f, h) magnification of decalcified sections have shown. Each panel of right column shows magnified view of the square in left column, respectively. Experimental groups were classified as follows: Non-ligatured control (basal sham control), ligatured, low-dose CTN (1.0 U/kg) and high-dose CTN (5.0 U/kg). Scale bars, 100 μm. The arrow heads shown in low magnification section (left column) indicate cemento-enamel junction (CEJ). The arrow heads in high magnification section (right column) indicate TRAP-positive osteoclasts. Ab, alveolar bone. De, dentin. (C) Comparison of histological bone level: Length from the CEJ to the alveolar bone crest (ABC). The statistical differences were evaluated by comparing the distance from the CEJ to the ABC in each group. Data represent mean ± SD. CTN, calcitonin. *, P < 0.05; **, P < 0.01, one-way ANOVA and Scheffe test. (D) Statistical analysis for periodontal tissue inflammation. Number of inflammatory cells on day5. Inflammatory cells per visual field, counted 5 visual fields. Data represent mean ± SD. **P < 0.01, ANOVA Tukey-Kramer HSD test. (E) Number of TRAPpositive osteoclasts on day 5. TRAP-positive cells per visual field, counted 5 visual fields. Data represent mean ± SD. CTN, calcitonin. **P < 0.01, ANOVA Tukey-Kramer HSD test.
periodontitis model [20]. On the other hand, Chen et al. suggested that systemic sclerostin antibody treatment can create greater alveolar crest height in rats exposed to ligature-induced periodontitis [21] because sclerostin secreted primarily by osteocytes has been known to negatively regulate osteoblast-mediated bone formation. Further investigation is needed to determine the efficacy of systemic versus local
anabolic agent could be a potential adjuvant therapy to suppress alveolar bone loss in periodontitis. Yoshinaga et al. reported that locally administered green tea extract suppresses the onset of loss of attachment and alveolar bone resorption in a rat experimental periodontitis model [19]. Furthermore, it has been reported that locally administered tiludronic acid reduced alveolar bone loss in a rat experimental 768
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approaches might be proposed to enhance CTN effects on osteoclasts in combination with PTH, vit D, or bisphosphonates for periodontal treatment.
administration, but clinical use of novel agents, such as CTN, may prevent inflammatory periodontal bone loss. Osteoporosis is a disease characterized by reduction of bone mass and poor bone strength in post-menopausal women [22]. CTN has long been used as a therapeutic agent for osteoporosis because of the antiresorptive properties via directly reducing osteoclastic resorption, and thus leads to an increase in bone mineral density and bone strength [23]. Based on clinical use, we speculated that CTN may inhibit alveolar bone loss induced by periodontal infection, even though bone resorption induced by osteoporosis depends on hormonal impairment such as estrogen deficiency. There are many methods to induce in vivo periodontitis experimentally [24,25], and we selected an experimental model using simple ligation by nylon thread without periodontal pathogens because inflammatory cells, such as neutrophils and macrophages, arise sufficiently in periodontal tissues [13,26]. We considered that this model may simulate mild inflammation, but our histological findings show significant infiltration of leukocytes in periodontal tissues of ligatured rats. In addition, micro-CT has been used as a fast, precise nondestructive, analytical procedure to measure bone volume within defects. Our micro-CT images showed marked resorption of alveolar bone of ligatured rats. Because no clinical signs of systemic infection or mortality were noted in any of the rats at any time, these findings indicate that our experimental periodontitis model is suitable to evaluate novel treatment strategies of periodontitis using CTN. Systemic injections of eel CTN, a synthetic eel CTN analogue, inhibit formalin-induced nociceptive hypersensitivity (hyperalgesia and allodynia) in rats [15]. Therefore, in our in vivo experimental periodontitis model, we used synthetic eel CTN. Importantly, compared to the ligatured group, rats given CTN had less bone loss, and rats given a high dose of CTN had significantly decreased bone loss even on day 5. Histological findings proved that CTN inhibited bone resorption induced by ligation. In addition, the number of TRAP-positive osteoclasts decreased significantly in rats given a high dose of CTN on day 5, and positive cells disappeared on day 14 even from groups given a low dose of CTN. Because TRAP-positive osteoclasts resorb bone, these results suggest that inhibition of bone loss by CTN depends on direct impairment of osteoclast activation. Osteoclasts typically are multinucleated and express TRAP and the CTN receptor (CTR). Previously, Kim et al. reported that monocyte chemoattractant protein-1 (MCP-1) is induced by RANKL and promotes osteoclast fusion in multinuclear cells [27]. In addition, CTN inhibited MCP-1-mediated osteoclast fusion through the inhibition of its own receptor CTR. Quinn et al. suggested that CTN strongly inhibits CTR expression in osteoclasts, which results in inhibition of osteoclast differentiation [28]. In our experimental periodontitis model, MCP-1 may appear in periodontal tissues by reflecting inflammation cascades, which results in osteoclast inactivation. Furthermore, Spolidorio et al. recently reported that CTN inhibited cyclosporine A-induced alveolar bone loss via a decrease of circulating inflammatory cytokines, such as IL-1 and IL-6, in a rat model [29]. Inhibition of several inflammatory cytokines by CTN might induce indirect effects on the inhibition of alveolar bone loss. To clarify the chemokine/cytokine-mediated responses of osteoclasts regulated by CTN, further experiments will be needed in the future. Topical and intermittent administration of PTH recovered alveolar bone loss in rat experimental periodontitis [13]. Because PTH increased alkaline phosphatase activity and bone nodule formation in fetal rat osteoblastic cells, the target cells may be osteoblasts, but not osteoclasts [11,12]. In addition, vitamin D (vit D) increased calcium and phosphate uptake to mask the effects of PTH [30]. It is well-known that a combination of CTN and vit D is a standard regimen for treating post-menopausal osteoporosis clinically. Tanaka et al. reported that combination therapy with CTN and bisphosphonates appears to be an effective treatment for osteoporosis patients [31]. Thus, there is a lot of research focused on understanding how agents are useful and how they cause beneficial or harmful effects in novel treatment strategies. New
5. Conclusion We demonstrated that ligature procedures induced marked alveolar bone loss around the molars, and greater bone recovery was observed in the local CTN-treated rats. In the short term, local administration of CTN may have a highly anti-resorptive effect in experimental periodontitis. However, CTN may have limited usefulness in situations where sustained anti-resorptive action is needed because osteoclasts tend to become resistant to the action of CTN due to receptor downregulation and reactivate bone resorption [27,32]. More research is needed before CTN can be used in the preclinical practice and for clinical therapy of periodontitis. Further studies are needed to clarify its anti-inflammatory roles, effective dose, and side effects. We hope that dentists may have medication to control the activities of bone-related cells to allow novel treatment strategies for unwanted bone loss by periodontal inflammation in the future. Conflict of interest The authors declare no conflicts of interest related to this study. Acknowledgements The authors are grateful to the staff of Tokushima University Graduate School. This study was supported by a Grant-in-Aid for Scientific Research (B) (No. 15H05054) from the Japan Society for the Promotion of Science. References [1] R.C. Page, S. Offenbacher, H.E. Schroeder, G.J. Seymour, K.S. Kornman, Advances in the pathogenesis of periodontitis: summary of developments, clinical implications and future directions, Periodontol. 2000 14 (1997) 216–248. [2] Z. Schwartz, J. Goultschin, D.D. Dean, B.D. Boyan, Mechanisms of alveolar bone destruction in periodontitis, Periodontol. 2000 14 (1997) 158–172. [3] B.F. Boyce, Advances in the regulation of osteoclasts and osteoclast functions, J. Dent. Res. 92 (2013) 860–867. [4] W.K. Kim, K. Ke, O.J. Sul, H.J. Kim, S.H. Kim, M.H. Lee, et al., Curcumin protects against ovariectomy-induced bone loss and decreases osteoclastogenesis, J. Cell. Biochem. 112 (2011) 3159–3166. [5] H. Nakamura, T. Ukai, A. Yoshimura, Y. Kozuka, H. Yoshioka, Y. Yoshinaga, et al., Green tea catechin inhibits lipopolysaccharide-induced bone resorption in vivo, J. Periodontal Res. 45 (2010) 23–30. [6] F.J. de Paula, C.J. Rosen, Back to the future: revisiting parathyroid hormone and calcitonin control of bone remodeling, Horm. Metab. Res. 42 (2010) 299–306. [7] S. Wallach, J.B. Carstens Jr, L.V. Avioli, Calcitonin, osteoclasts, and bone turnover, Calcif. Tissue Res. 47 (1990) 388–391. [8] S. Granholm, P. Lundberg, U.H. Lerner, Calcitonin inhibits osteoclast formation in mouse haematopoetic cells independently of transcriptional regulation by receptor activator of NF-κB and c-Fos, J. Endocrinol. 195 (2007) 415–427. [9] M. Zaidi, B.S. Moonga, E. Abe, Calcitonin and bone formation: a knockout full of surprises, J. Clin. Invest. 110 (2002) 1769–1771. [10] Y. Fei, M.M. Hurley, Role of fibroblast growth factor 2 and Wnt signaling in anabolic effects of parathyroid hormone on bone formation, J. Cell. Physiol. 227 (2012) 3539–3545. [11] E.M. Greenfield, Anabolic effects of intermittent PTH on osteoblasts, Curr. Mol. Pharmacol. 5 (2012) 127–134. [12] N.S. Datta, A.B. Abou-Samra, PTH and PTHrP signaling in osteoblasts, Cell Signal. 21 (2009) 1245–1254. [13] K. Tokunaga, H. Seto, H. Ohba, C. Mihara, H. Hama, M. Horibe, et al., Topical and intermittent application of parathyroid hormone recovers alveolar bone loss in rat experimental periodontitis, J. Periodontal Res. 46 (2011) 655–662. [14] C. Kilkenny, W.J. Browne, I.C. Cuthill, M. Emerson, D.G. Altman, Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research, PLoS Biol. 8 (2010) e1000412. [15] H. Umeno, T. Nagasawa, N. Yamazaki, Y. Kuraishi, Antinociceptive effects of repeated systemic injections of calcitonin in formalin-induced hyperalgesic rats, Pharmacol. Biochem. Behav. 55 (1996) 151–156. [16] J. Kim, B.S. Seo, How to calculate sample size and why, Clin. Orthopedic Surg. 5 (2013) 235–242. [17] K. Irie, T. Tomofuji, N. Tamaki, T. Sanbe, D. Ekuni, T. Azuma, et al., Effects of
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