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Icariin enhances the healing of rapid palatal expansion induced root resorption in rats
Phytomedicine 19 (2012) 1035–1041
Contents lists available at SciVerse ScienceDirect
Phytomedicine journal homepage: www.elsevier.de/phymed
Icariin enhances the healing of rapid palatal expansion induced root resorption in rats Feng Wang a,∗,1 , Zhifeng Liu a,1 , Songshan Lin a , Huaixiu Lu a , Juan Xu b a b
Department of Stomatology, Navy General Hospital, Beijing, China Department of Stomatology, General Hospital of PLA, Beijing, China
a r t i c l e
i n f o
Keywords: Rapid palatal expansion Root resorption Herba Epimedii Icariin Osteoclast RANKL OPG
a b s t r a c t Icariin has been reported to enhance bone healing and treat osteoporosis. In this study, we examined the effect of Icariin on rapid palatal expansion induced root resorption in rats. Our hypothesis is that Icariin can enhance the healing of rapid palatal expansion induced root resorption. Forty-eight male Wistar rats were divided randomly and equally into three groups (n = 16 rats each). The rats were untreated (negative control) or treated with rapid palatal expansion without (positive control) or with Icariin at 2.5 mg/kg day (Icariin-treated groups). An initial force of 50 × g was applied to the areas between the right and left upper first molars of the rats for 21 days. Eight rats were randomly chosen from each group, and the root resorption index (RRI) was determined with scanning electron microscopy (SEM). Upper first molar-centered buccal- lingual tissue slices were generated from the upper first molars and peridentium of the remaining eight rats from each group. Specimen slices were analyzed with HE and tararate-resistant acid phosphatase staining, osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) immunohistochemistry, and optical microscopy. Analyses of cell number, densitometry, and one-way analysis of variance were performed. The Icariin-treated groups displayed decreased RRI values, decreased osteoclast numbers and activity levels, and increased OPG/RANKL expression ratios. High-power SEM revealed reparative cementum in the Icariin-treated samples. Icariin regulates osteoclast differentiation via the OPG/RANKL ratio, evoking a reparative effect on rapid palatal expansion induced root resorption in rats. © 2012 Elsevier GmbH. All rights reserved.
Introduction The malocclusion caused by a narrow maxillary dental arch width, such as Class I with crowding, Class II with a V-shaped arch, and Class III with small maxilla, in the maturing patient, is commonly treated with rapid palatal expansion (RPE). This increases the posterior dentition width rapidly. However, the major complication of rapid palatal expansion is root resorption that is confined to the buccal root surface of the anchored teeth (maxillary first premolars and first molars) (Erverdi et al. 1994). Second premolars and molars were also affected when connected to the RPE appliance (Erverdi et al. 1994). Rapid maxillary expansion induced root resorption has all of the features of an inflammatory reaction. The inflammatory extent of
∗ Corresponding author at: Department of Stomatology, Navy General Hospital, 6 FuCheng Road, Beijing 100049 China. Tel.: +86 010669580; fax: +86 010669580. E-mail addresses: [email protected] (F. Wang), [email protected] (Z. Liu). 1 These authors should be considered co-first authors. 0944-7113/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2012.06.001
root resorption is determined by many factors, such as the invasiveness of the various absorptive cells, tissue sensitivity, and individual variation (Brezniak and Wasserxtein 2002). Accordingly, it is difficult to predict the development of rapid maxillary expansion induced root resorption. The mechanisms of root resorption are thought to be similar to those of bone resorption. In bone resorption processes, mechanical forces induce the activity of the primitive osteoclasts, which differentiate and enter osteoclastogenesis (Blair and Athanasou 2004). Cementoclasts, which mediate cement resorption through an acidization/degradation-associated pathway, are similar to osteoclasts in morphology, activity, functions, and features (Matsumoto 1994). Tartrate-resistant acid phosphatase (TRAP) staining is used to mark osteoclasts and cementoclasts, and can be used to identify bone and root resorption. Receptor activator of nuclear factor kappa-B ligand (RANKL) is a type-II transmembrane protein that is indispensible for osteoclast development and differentiation. Interactions between RANKL and its transmembrane receptor RANK on the osteoclast precursor surface induce osteoclast differentiation. Osteoprotegerin (OPG, also known as osteoclast inhibitor) is a receptor of tumor necrosis
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factor (TNF) and a natural inhibitor of RANKL. Osteoprotegerin inhibits alveolar bone resorption and increases osteoclast apoptosis by inhibiting the ruffling of mature osteoclasts. The level of RANKL expressed by osteoblasts (Ishii et al. 2006) and OPG are critical in the regulation of the formation of osteoclast-like cells. When the balance is inclined toward OPG, there are fewer active osteoclasts; when inclined toward RANKL, there are more active osteoclasts. According to in vivo and in vitro studies (Sasaki 2003), OPG and RANKL not only adjust terminal osteoclast differentiation, but also influence resorption. Moreover, cementoblasts can also express OPG and RANKL and can modulate osteoclast cytogenesis (Boabaid et al. 2004). No effective clinical methods are currently available to treat rapid maxillary expansion induced root resorption. Topical bisphosphonate administration can dose-dependently inhibit root resorption in orthodontically treated rats (Igarashi et al. 1996). However, the clinical utility of bisphosphonate in orthodontics is limited, since bisphosphonate is a bone-resorption inhibitor that can affect orthodontic tooth movement (Fujimura et al. 2009). Epimedii herba is one of the most frequently used herbs in formulas prescribed for the treatment of osteoporosis in China. The main active flavonoid glucoside extracted from Epimedium pubescens is Icariin, which has been reported to enhance bone healing and reduce osteoporosis occurrence. Icariin produces an obvious biological activity in living tissue, eliciting antiinflammatory effects (Wu et al. 2011), stimulating growth factors (Hsieh et al. 2010), and enhancing bone protein expression. Icariin can promote osteoblast proliferation, differentiation and mineralization in vitro (Ma et al. 2011). Cementoblast and osteoblast are similar in biological effects. Based on these study, we speculated that Icariin may be used to treat rapid maxillary expansion induced root resorption. This hypothesis has not been tested. Therefore, the purpose of the present study was to explore the effect of Icariin on rapid maxillary expansion induced root resorption in rats. Materials and methods Experimental animals and groups A total of 48 healthy, 8-week-old male Wistar rats (Experimental Animal Center, Academy of Military Medical Sciences), each weighing about 230 g, were divided randomly and equally into three groups (n = 16 rats each). Group I rats were untreated (negative control group). Group II rats were subjected to RME treatment for 21 days only (positive control group). Group III rats were subjected to RME treatment combined with Icariin orally administered at 2.5 mg/kg day for 21 days (Icariin-treated experimental groups). All animals were housed individually in plastic cages in a colony room. Animals were fed a standard pellet diet, and water was provided ad libitum. Animal model establishment The rats were anesthetized with an intraperitoneal injection of 70 mg/kg dormicum during the setting and adjustment of the orthodontic appliance. A uniform standardized expansion spring, made of 0.012 in. nickel–titanium (NiTi) wire, was set in each animal’s mouth between the right and left upper first molars. The spring was retained in the mouth by its own expansive force (Adachi et al. 1994). According to the manufacturer’s database, the force level of the expansion spring after activation is approximately 50 × g. The force magnitude was measured with a tension gauge (Changsha Tiantian Dental Co., Ltd.) when the appliance was set. From the first day of the expansion, Icariin (2.5 mg/kg day) was orally administered to the experimental group and a placebo to the
control group for 21 consecutive days. Icariin (purity > 99%) were purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The spring was checked every day to prevent it from dropping off. All procedures were in accordance with the National Institute of Health and Nutrition guidelines for the Care and Use of Laboratory Animals and were approved by the ethics committee of our institution (No. H201101082).
Preparation of specimens for SEM or histological analyses After 21 days of force application, all 48 rats were killed by neck break. The rats were allocated randomly and equally into two groups for further analysis by scanning electron microscopy (SEM) or histology. Specimens intended for SEM (n = 8 rats from each group) or histological analysis (n = 8 rats from each group) were fixed in 10% formaldehyde solution for 48 h. For SEM analysis, the right upper first molar, including its surrounding bone, was cut as a block. Next, the alveolar bone was removed delicately, so as to avoid any root surface damage. The molars were submerged in 1% sodium hypochlorite to eliminate any remaining periodontal ligament remnants. The molars were dried for 1 week in a 40 ◦ C incubator, placed on a retainer, plated with gold, and observed with SEM. Adobe Photoshop ME Version 7 was used to analyze the root resorption index (RRI), defined as the resorbed percentage of the buccal area of the middle palatal root of the first molar (Fig. 1). For histological analysis, fixed samples were decalcified with 15% EDTA solution for 45 days, dehydrated with alcohol, rendered transparent by xylene, and embedded in paraffin wax. Upper first molar-centered buccal-lingual tissue slices were created with a thickness of 3–5 m.
Observation of staining and root resorption TRAP staining was performed with a reagent kit (Sigma–Aldrich, US). Areas (×400) around the buccal apex of the middle palatal root were used. The number of osteoclasts that stained positive for TRAP was determined as the mean value of 3 observations by optical microscopy.
Immunohistochemical staining Immunological staining was performed with OPG and RANKL polyclonal antibodies (Santa Cruz, USA), the streptavidin–peroxidase (S–P) immunohistochemical reagent kit, and the diaminobenzidine (DAB) developing box (Beijing Zhongshan Biochemicals Co., Ltd.) according to the manufacturers’ instructions. In the negative control, phosphate-buffered saline (PBS) was substituted for the primary antibodies. Areas around the buccal apex of the middle palatal root were selected. ImageproPlus 6.0 was employed to measure the mean optical density (OD) value of the positive products from a single optical microscopy observation (×400).
Statistical analysis One-way analysis of variance (ANOVA) was performed with SPSS 13.0. Multiple intergroup comparisons were performed with the q test. The statistical significance was defined as p < 0.05.
F. Wang et al. / Phytomedicine 19 (2012) 1035–1041
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Fig. 1. Root resorption index (RRI). Black and gray refer to areas of resorption and no resorption, respectively. RRI represents black area/(black area + gray area) × 100%.
Results Electron microscopy The SEM results revealed smooth root surfaces and few root resorption pits in the negative control group (Fig. 2A). The positive control group displayed widespread root resorption lacunae. These resorption lacunae had clear-cut yet irregular rims (Fig. 2B). Root surfaces in the Icariin-treated groups were coarser than those in the negative control group, and small isolated resorption pits were
Fig. 2. SEM images of control and Icariin-treated rats (magnification 1000×). (A) Negative control group showing smooth root surfaces and few root resorption pits. (B) Positive control group showing widespread resorption lacunae with clear-cut yet irregular rims ( ). (C) Icariin-treated groups showing small isolated resorption pits and new cementum on the root surfaces.
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F. Wang et al. / Phytomedicine 19 (2012) 1035–1041
Table 1 Percentage of root resorption for all groups (mean ± SD, n = 8). Root resorption, % 0.50 ± 0.06 25.12 ± 2.54a 3.40 ± 0.48a,b
Negative control Positive control Icariin-treated
a,b p < 0.05 by ANOVA compared with negative control (a) or force only positive control (b).
Table 2 Osteoclast numbers in all groups (mean ± SD, n = 8). Number of osteoclasts 0.7 ± 0.5 8.5 ± 1.4a 5.1 ± 0.8a,b
Negative control Positive control Icariin-treated
a,b p < 0.05 by ANOVA compared with negative control (a) or force only positive control (b).
seen on the root surfaces (Fig. 2C). At the same time, a large amount of new cementum was evidently found in Icariin-treated groups (Fig. 2C and Table 1). The total number and area of the resorption lacunae in the Icariin-treated groups were smaller than those in the positive control group. Histological observations Staining with HE indicated that root resorption mainly occurred at the root furcation and in the vicinity of the stress-side apex. The Icariin-treated groups showed less resorption than the positive control group (Fig. 3). TRAP staining Except for the negative control group, all groups displayed osteoclasts or osteoclastic cytoplasm at the teeth roots. The alveolar bone area in the two groups stained positive. The multinuclear cells of this area were distributed in the resorption pits of the cementum and the alveolar bone, forming a line along the pit rims. The positive chroma values of TRAP staining in the Icariin-treated groups were weaker than that in the positive control group (Fig. 4 and Table 2). These differences between the Icariin-treated groups and the positive control group were statistically significant. Fewer cells stained positive for TRAP in the Icariin-treated groups than in the positive control group. Immunohistochemical results The OPG/RANKL expression appeared as a yellow–brown staining in the cytoplasm. The major locations of positive OPG/RANKL expression were the periodontal membrane, cementum, and osteoclasts in the resorption bone pits (Fig. 5). Fig. 5 displays the optical microscopy images for OPG and RANKL staining. Table 3 summarizes the densitometric analysis of these findings. The mean OD value for OPG of the Icariin-treated groups was significantly higher Table 3 Mean OPG and RANKL OD values for all groups (mean ± SD, n = 8).
Negative control Positive control Icariin-treated
OPG OD values
RANKL OD values
0.0302 ± 0.0019 0.0410 ± 0.0031a 0.0986 ± 0.0028a,b
0.0310 ± 0.0020 0.0894 ± 0.0024a 0.0387 ± 0.0029a,b
OPG, osteoprotegerin; OD, optical density; RANKL, receptor activator of nuclear factor kappa-B ligand. a,b p < 0.05 by ANOVA compared with negative control (a), force-only positive control (b).
Fig. 3. Histological examination of compressed surfaces. (A) Negative control group showing smooth root surfaces. (B) Positive control group showing widespread resorption lacunae. (C) Icariin-treated groups showing small isolated resorption pits (HE staining stain, magnification 200×).
than that of the positive control group. The mean RANKL OD value of the Icariin-treated groups was significantly lower than that of the positive control group. Discussion In this study, we explored the effect of Icariin on rapid maxillary expansion induced root resorption in rats. The RRI values were reduced in the Icariin-treated groups. High-power SEM revealed newly developed cementum in the Icariin-treated groups. Fewer osteoclasts were seen in the groups treated with Icariin than in the positive control group. These results indicate that root resorption was less active in the Icariin-treated groups.
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Fig. 4. TRAP staining of the rat molar tooth root. (A) Negative control group showing few osteoclasts in the apex. (B) Positive control group showing substantial numbers of osteoclasts in the apex. (C) Icariin-treated groups showing fewer osteoclasts in the apex than in the positive control group (left: magnification 200×; right: magnification 400×).
Treatment with Icariin increased OPG and decreased RANKL expression in the experimental groups, thereby reducing the number and activity of osteoclasts and naturally reducing root resorption. There are at least three potential reasons for this results. First, Icariin can up-regulate OPG expression and down-regulate RANKL expression by culturing with osteoblasts (Hsieh et al. 2010). Second, Icariin can influence stem cells in the periodontal membrane. For example, Icariin can promote bone marrow stromal cells to differentiate into osteoblast (Chen et al. 2007). Third, the mechanical stimulus elicits the strong release of inflammatory factors, such as tumor necrosis factor-␣ (TNF-␣) and prostaglandin E2 (PGE2 ), from the weakened periodontal ligament. These inflammatory factors can stimulate RANKL expression. Icariin can reduce the levels of these inflammatory factors, thereby reducing RANKL expression and osteoclast differentiation (Hsieh et al. 2011). Prolonged treatment with Icariin previously have demonstrated that Icariin improves maturation and mineralization of osteoblasts in vitro, and suppresses osteoclastogenesis and inhibits bone
resorption activity in vivo and in vitro (Zhao et al. 2008; Qin et al. 2008; Hsieh et al. 2011). Cultured with osteoblasts, Icariin increases alkaline phosphatase activity and affects the mRNA expression of type-I collagen COL1␣2 gene (Ma et al. 2011), which regulate the mineralization process. In vitro Icariin is a bone anabolic agent that may exert its osteogenic effects through the induction of BMP-2 and NO synthesis, subsequently regulating Cbfa1/Runx2, OPG, and RANKL gene expressions (Hsieh et al. 2010). This effect may contribute to its action on the induction of osteoblasts proliferation and differentiation, resulting in bone formation. Recently Icariin was shown to stimulate bone marrow stromal cells differentiation into osteoblast (Chen et al. 2007). These findings suggest that Icariin may be advantageous for stem cell and tissue engineering applications, and has great potential in hard tissue repair and regeneration. The different phylogenic status between humans and lower animals such as mice, rats, and rabbits makes it difficult to extrapolate results from animal models to the clinic. The growth pattern of cementum in lower animals involves continuous eruption, with cementum being formed throughout their lifetime. However, the
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Fig. 5. OPG/RANKL immunohistochemical staining of the rat molar tooth root. (A) Apical region of the middle palatal root of orthodontic tooth movement. (E) Negative control experiment using nonimmune IgG instead of the primary antibody. (B–D) Immunoreactivity for OPG on day 21. Untreated specimens (B) show weak immunoreactivity for OPG compared to the other groups. Specimens are shown after treatment with RPE force (50 × g) without (C) or with Icariin (D). Part D shows strong staining for OPG compared to part C on the compressed side. (F–H) Immunoreactivity for RANKL at day 21. Untreated specimens (F) show weak immunoreactivity for RANKL compared to the other groups. Specimens are shown after treatment with RPE force (50 × g) without (G) or with Icariin (H). Part G shows strong staining for RANKL compared to part H on the compressed side. PDL, periodontal ligament; C, cementum. Magnification is ×40 (A) or ×400 (B–H).
study of root resorption in an adequate sample size of higher animals (e.g., monkeys) is prohibitively expensive. As a result, the rat model for RME induced root resorption remains widely used.
Many plant-derived natural products have been used in traditional medicine for the treatment of various diseases. Herba Epimedii is a traditional Chinese herbal medicine, which has been commonly used as tonic, aphrodisiac and anti-rheumatic in China
F. Wang et al. / Phytomedicine 19 (2012) 1035–1041
Fig. 6. Chemical structure of Icariin.
for thousands of years. Its physical and functional characteristics have been thoroughly documented in the Chinese pharmacopoeia 2005. Icariin [molecular formula: C33 H40 O15 ; molecular weight: 676.67; chemical structure (Fig. 6)] is the main active flavonoid glucoside isolated from Epimedium pubescens. Our dosage is lower than the document .So, our dosage is much safer. So far, Icariin is considered to be noncarcinogenic and has no known deleterious effects. These findings may help to promote the development of convenient, abundant and low cost herbs to prevent tooth resorption and improve human health and quality of life. Additional studies on this process are recommended. Conclusion Icariin has a reparative effect on RME induced root resorption in rats by modulating the OPG/RANKL ratio and osteoclast differentiation. Therefore, the extremely low cost of Icariin and its high abundance make it appealing for RME induced root resorption. Acknowledgement This work was supported by National Natural Science Funds of China (grant no. 81070837, China). References Adachi, H., Igarashi, K., Mitani, H., Shinoda, H., 1994. Effects of topical administration of a bisphosphonate (risedronate) on orthodontic tooth movements in rats. Journal of Dental Research 73 (8), 1478–1484.
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Brezniak, N., Wasserxtein, A., 2002. Orthodontically induced inflammatory root resorption. Part I: the basic science aspects [J]. Angle Orthodontist 72 (2), 175–179. Blair, H.C., Athanasou, N.A., 2004. Recent advances in osteoclast biology and pathological bone resorption [J]. Histology and Histopathology 19 (1), 189–199. Boabaid, F., Berry, J.E., Koh, A.J., Somerman, M.J., McCauley, L.K., 2004. The role of parathyroid hormone-related protein in the regulation of osteoclastogenesis by cementoblasts. Journal of Periodontology 75, 1247–1254. Chen, K.M., Ma, H.P., Ge, B.F., Liu, L.Y., Ma, L.P., Bai, M.H., Wang, Y., 2007. Icariin enhances the osteogenic differentiation of bone marrow stromal cells but has no effects on the differentiation of newborn calvarial osteoblasts of rats. Pharmazie 62, 785–789. Erverdi, N., Okar, I., Kücükkeles, N., Arbak, S., 1994. A comparison of two rapid palatal expansion techniques from the point of root resorption. American Journal of Orthodontics and Dentofacial Orthopedics 106, 47–51. Fujimura, Y., Kitaura, H., Yoshimatsu, M., Eguchi, T., Kohara, H., Morita, Y., Yoshida, N., 2009. Influence of bisphosphonates on orthodontic tooth movement in mice. European Journal of Orthodontics 31, 572–577. Hsieh, T.P., Sheu, S.Y., Sun, J.S., Chen, M.H., Liu, M.H., 2010. Icariin isolated from Epimedium pubescens regulates osteoblasts anabolism through BMP-2, SMAD4, and Cbfa1expression. Phytomedicine 17, 414–423. Hsieh, T.P., Sheu, S.Y., Sun, J.S., Chen, M.H., 2011. Icariin inhibits osteoclast differentiation and bone resorption by suppression of MAPKs/NF-B regulated HIF-1␣ and PGE2 synthesis. Phytomedicine 18, 176–185. Ishii, M., Iwai, K., Koike, M., Ohshima, S., Kudo-Tanaka, E., Ishii, T., Mima, T., Katada, Y., Miyatake, K., Uchiyama, Y., Saeki, Y., 2006. RANKL-induced expression of tetraspanin CD9 in lipid raft membrane microdomain is essential for cell fusion during osteoclastogenesis. Journal of Bone and Mineral Research 21 (6), 965–976. Igarashi, K., Adachi, H., Mitani, H., Shinoda, H., 1996. Inhibitory effect of topical administration of a bisphosphonate (risedronate) on root resorption incident to orthodontic tooth movement in rats. Journal of Dental Research 75 (9), 1644–1649. Matsumoto, Y., 1994. Morphological and functional properties of odontoclasts on dentine resorption. Kokubyo Gakkai Zasshi (The Journal of the Stomatological Society), Japan 61 (1), 123–143. Ma, H.P., Ming, L.G., Ge, B.F., Zhai, Y.K., Song, P., Xian, C.J., Chen, K.M., 2011. Icariin is more potent than genistein in promoting osteoblast differentiation and mineralization in vitro. Journal of Cellular Biochemistry 112, 916–923. Qin, L.P., Han, T., Zhang, Q.Y., Cao, D.P., Nian, H., Rahman, K., Zheng, H.C., 2008. Antiosteoporotic chemical constituents from Er-Xian decoction, a traditional Chinese herbal formula. Journal of Ethnopharmacology 118, 271–279. Sasaki, T., 2003. Differentiation and functions of osteoclasts and odontoclasts in mineralized tissue resorption. Microscopy Research and Technique 61 (6), 483–495. Wu, J.F., Du, J., Xu, C.Q., Le, J.J., Liu, B.J., Xu, Y.Z., Dong, J.C., 2011. In vivo and in vitro anti-inflammatory effects of a novel derivative of icariin. Immunopharmacology and Immunotoxicology 33 (1), 49–54. Zhao, J.Y., Ohba, S., Shinkai, M., Chung, U., Nagamune, T., 2008. Icariin induces osteogenic differentiation in vitro in a BMP- and Runx2-dependent manner. Biochemical and Biophysical Research Communications 369, 444–448.
Contents lists available at SciVerse ScienceDirect
Phytomedicine journal homepage: www.elsevier.de/phymed
Icariin enhances the healing of rapid palatal expansion induced root resorption in rats Feng Wang a,∗,1 , Zhifeng Liu a,1 , Songshan Lin a , Huaixiu Lu a , Juan Xu b a b
Department of Stomatology, Navy General Hospital, Beijing, China Department of Stomatology, General Hospital of PLA, Beijing, China
a r t i c l e
i n f o
Keywords: Rapid palatal expansion Root resorption Herba Epimedii Icariin Osteoclast RANKL OPG
a b s t r a c t Icariin has been reported to enhance bone healing and treat osteoporosis. In this study, we examined the effect of Icariin on rapid palatal expansion induced root resorption in rats. Our hypothesis is that Icariin can enhance the healing of rapid palatal expansion induced root resorption. Forty-eight male Wistar rats were divided randomly and equally into three groups (n = 16 rats each). The rats were untreated (negative control) or treated with rapid palatal expansion without (positive control) or with Icariin at 2.5 mg/kg day (Icariin-treated groups). An initial force of 50 × g was applied to the areas between the right and left upper first molars of the rats for 21 days. Eight rats were randomly chosen from each group, and the root resorption index (RRI) was determined with scanning electron microscopy (SEM). Upper first molar-centered buccal- lingual tissue slices were generated from the upper first molars and peridentium of the remaining eight rats from each group. Specimen slices were analyzed with HE and tararate-resistant acid phosphatase staining, osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) immunohistochemistry, and optical microscopy. Analyses of cell number, densitometry, and one-way analysis of variance were performed. The Icariin-treated groups displayed decreased RRI values, decreased osteoclast numbers and activity levels, and increased OPG/RANKL expression ratios. High-power SEM revealed reparative cementum in the Icariin-treated samples. Icariin regulates osteoclast differentiation via the OPG/RANKL ratio, evoking a reparative effect on rapid palatal expansion induced root resorption in rats. © 2012 Elsevier GmbH. All rights reserved.
Introduction The malocclusion caused by a narrow maxillary dental arch width, such as Class I with crowding, Class II with a V-shaped arch, and Class III with small maxilla, in the maturing patient, is commonly treated with rapid palatal expansion (RPE). This increases the posterior dentition width rapidly. However, the major complication of rapid palatal expansion is root resorption that is confined to the buccal root surface of the anchored teeth (maxillary first premolars and first molars) (Erverdi et al. 1994). Second premolars and molars were also affected when connected to the RPE appliance (Erverdi et al. 1994). Rapid maxillary expansion induced root resorption has all of the features of an inflammatory reaction. The inflammatory extent of
∗ Corresponding author at: Department of Stomatology, Navy General Hospital, 6 FuCheng Road, Beijing 100049 China. Tel.: +86 010669580; fax: +86 010669580. E-mail addresses: [email protected] (F. Wang), [email protected] (Z. Liu). 1 These authors should be considered co-first authors. 0944-7113/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2012.06.001
root resorption is determined by many factors, such as the invasiveness of the various absorptive cells, tissue sensitivity, and individual variation (Brezniak and Wasserxtein 2002). Accordingly, it is difficult to predict the development of rapid maxillary expansion induced root resorption. The mechanisms of root resorption are thought to be similar to those of bone resorption. In bone resorption processes, mechanical forces induce the activity of the primitive osteoclasts, which differentiate and enter osteoclastogenesis (Blair and Athanasou 2004). Cementoclasts, which mediate cement resorption through an acidization/degradation-associated pathway, are similar to osteoclasts in morphology, activity, functions, and features (Matsumoto 1994). Tartrate-resistant acid phosphatase (TRAP) staining is used to mark osteoclasts and cementoclasts, and can be used to identify bone and root resorption. Receptor activator of nuclear factor kappa-B ligand (RANKL) is a type-II transmembrane protein that is indispensible for osteoclast development and differentiation. Interactions between RANKL and its transmembrane receptor RANK on the osteoclast precursor surface induce osteoclast differentiation. Osteoprotegerin (OPG, also known as osteoclast inhibitor) is a receptor of tumor necrosis
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factor (TNF) and a natural inhibitor of RANKL. Osteoprotegerin inhibits alveolar bone resorption and increases osteoclast apoptosis by inhibiting the ruffling of mature osteoclasts. The level of RANKL expressed by osteoblasts (Ishii et al. 2006) and OPG are critical in the regulation of the formation of osteoclast-like cells. When the balance is inclined toward OPG, there are fewer active osteoclasts; when inclined toward RANKL, there are more active osteoclasts. According to in vivo and in vitro studies (Sasaki 2003), OPG and RANKL not only adjust terminal osteoclast differentiation, but also influence resorption. Moreover, cementoblasts can also express OPG and RANKL and can modulate osteoclast cytogenesis (Boabaid et al. 2004). No effective clinical methods are currently available to treat rapid maxillary expansion induced root resorption. Topical bisphosphonate administration can dose-dependently inhibit root resorption in orthodontically treated rats (Igarashi et al. 1996). However, the clinical utility of bisphosphonate in orthodontics is limited, since bisphosphonate is a bone-resorption inhibitor that can affect orthodontic tooth movement (Fujimura et al. 2009). Epimedii herba is one of the most frequently used herbs in formulas prescribed for the treatment of osteoporosis in China. The main active flavonoid glucoside extracted from Epimedium pubescens is Icariin, which has been reported to enhance bone healing and reduce osteoporosis occurrence. Icariin produces an obvious biological activity in living tissue, eliciting antiinflammatory effects (Wu et al. 2011), stimulating growth factors (Hsieh et al. 2010), and enhancing bone protein expression. Icariin can promote osteoblast proliferation, differentiation and mineralization in vitro (Ma et al. 2011). Cementoblast and osteoblast are similar in biological effects. Based on these study, we speculated that Icariin may be used to treat rapid maxillary expansion induced root resorption. This hypothesis has not been tested. Therefore, the purpose of the present study was to explore the effect of Icariin on rapid maxillary expansion induced root resorption in rats. Materials and methods Experimental animals and groups A total of 48 healthy, 8-week-old male Wistar rats (Experimental Animal Center, Academy of Military Medical Sciences), each weighing about 230 g, were divided randomly and equally into three groups (n = 16 rats each). Group I rats were untreated (negative control group). Group II rats were subjected to RME treatment for 21 days only (positive control group). Group III rats were subjected to RME treatment combined with Icariin orally administered at 2.5 mg/kg day for 21 days (Icariin-treated experimental groups). All animals were housed individually in plastic cages in a colony room. Animals were fed a standard pellet diet, and water was provided ad libitum. Animal model establishment The rats were anesthetized with an intraperitoneal injection of 70 mg/kg dormicum during the setting and adjustment of the orthodontic appliance. A uniform standardized expansion spring, made of 0.012 in. nickel–titanium (NiTi) wire, was set in each animal’s mouth between the right and left upper first molars. The spring was retained in the mouth by its own expansive force (Adachi et al. 1994). According to the manufacturer’s database, the force level of the expansion spring after activation is approximately 50 × g. The force magnitude was measured with a tension gauge (Changsha Tiantian Dental Co., Ltd.) when the appliance was set. From the first day of the expansion, Icariin (2.5 mg/kg day) was orally administered to the experimental group and a placebo to the
control group for 21 consecutive days. Icariin (purity > 99%) were purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The spring was checked every day to prevent it from dropping off. All procedures were in accordance with the National Institute of Health and Nutrition guidelines for the Care and Use of Laboratory Animals and were approved by the ethics committee of our institution (No. H201101082).
Preparation of specimens for SEM or histological analyses After 21 days of force application, all 48 rats were killed by neck break. The rats were allocated randomly and equally into two groups for further analysis by scanning electron microscopy (SEM) or histology. Specimens intended for SEM (n = 8 rats from each group) or histological analysis (n = 8 rats from each group) were fixed in 10% formaldehyde solution for 48 h. For SEM analysis, the right upper first molar, including its surrounding bone, was cut as a block. Next, the alveolar bone was removed delicately, so as to avoid any root surface damage. The molars were submerged in 1% sodium hypochlorite to eliminate any remaining periodontal ligament remnants. The molars were dried for 1 week in a 40 ◦ C incubator, placed on a retainer, plated with gold, and observed with SEM. Adobe Photoshop ME Version 7 was used to analyze the root resorption index (RRI), defined as the resorbed percentage of the buccal area of the middle palatal root of the first molar (Fig. 1). For histological analysis, fixed samples were decalcified with 15% EDTA solution for 45 days, dehydrated with alcohol, rendered transparent by xylene, and embedded in paraffin wax. Upper first molar-centered buccal-lingual tissue slices were created with a thickness of 3–5 m.
Observation of staining and root resorption TRAP staining was performed with a reagent kit (Sigma–Aldrich, US). Areas (×400) around the buccal apex of the middle palatal root were used. The number of osteoclasts that stained positive for TRAP was determined as the mean value of 3 observations by optical microscopy.
Immunohistochemical staining Immunological staining was performed with OPG and RANKL polyclonal antibodies (Santa Cruz, USA), the streptavidin–peroxidase (S–P) immunohistochemical reagent kit, and the diaminobenzidine (DAB) developing box (Beijing Zhongshan Biochemicals Co., Ltd.) according to the manufacturers’ instructions. In the negative control, phosphate-buffered saline (PBS) was substituted for the primary antibodies. Areas around the buccal apex of the middle palatal root were selected. ImageproPlus 6.0 was employed to measure the mean optical density (OD) value of the positive products from a single optical microscopy observation (×400).
Statistical analysis One-way analysis of variance (ANOVA) was performed with SPSS 13.0. Multiple intergroup comparisons were performed with the q test. The statistical significance was defined as p < 0.05.
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Fig. 1. Root resorption index (RRI). Black and gray refer to areas of resorption and no resorption, respectively. RRI represents black area/(black area + gray area) × 100%.
Results Electron microscopy The SEM results revealed smooth root surfaces and few root resorption pits in the negative control group (Fig. 2A). The positive control group displayed widespread root resorption lacunae. These resorption lacunae had clear-cut yet irregular rims (Fig. 2B). Root surfaces in the Icariin-treated groups were coarser than those in the negative control group, and small isolated resorption pits were
Fig. 2. SEM images of control and Icariin-treated rats (magnification 1000×). (A) Negative control group showing smooth root surfaces and few root resorption pits. (B) Positive control group showing widespread resorption lacunae with clear-cut yet irregular rims ( ). (C) Icariin-treated groups showing small isolated resorption pits and new cementum on the root surfaces.
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Table 1 Percentage of root resorption for all groups (mean ± SD, n = 8). Root resorption, % 0.50 ± 0.06 25.12 ± 2.54a 3.40 ± 0.48a,b
Negative control Positive control Icariin-treated
a,b p < 0.05 by ANOVA compared with negative control (a) or force only positive control (b).
Table 2 Osteoclast numbers in all groups (mean ± SD, n = 8). Number of osteoclasts 0.7 ± 0.5 8.5 ± 1.4a 5.1 ± 0.8a,b
Negative control Positive control Icariin-treated
a,b p < 0.05 by ANOVA compared with negative control (a) or force only positive control (b).
seen on the root surfaces (Fig. 2C). At the same time, a large amount of new cementum was evidently found in Icariin-treated groups (Fig. 2C and Table 1). The total number and area of the resorption lacunae in the Icariin-treated groups were smaller than those in the positive control group. Histological observations Staining with HE indicated that root resorption mainly occurred at the root furcation and in the vicinity of the stress-side apex. The Icariin-treated groups showed less resorption than the positive control group (Fig. 3). TRAP staining Except for the negative control group, all groups displayed osteoclasts or osteoclastic cytoplasm at the teeth roots. The alveolar bone area in the two groups stained positive. The multinuclear cells of this area were distributed in the resorption pits of the cementum and the alveolar bone, forming a line along the pit rims. The positive chroma values of TRAP staining in the Icariin-treated groups were weaker than that in the positive control group (Fig. 4 and Table 2). These differences between the Icariin-treated groups and the positive control group were statistically significant. Fewer cells stained positive for TRAP in the Icariin-treated groups than in the positive control group. Immunohistochemical results The OPG/RANKL expression appeared as a yellow–brown staining in the cytoplasm. The major locations of positive OPG/RANKL expression were the periodontal membrane, cementum, and osteoclasts in the resorption bone pits (Fig. 5). Fig. 5 displays the optical microscopy images for OPG and RANKL staining. Table 3 summarizes the densitometric analysis of these findings. The mean OD value for OPG of the Icariin-treated groups was significantly higher Table 3 Mean OPG and RANKL OD values for all groups (mean ± SD, n = 8).
Negative control Positive control Icariin-treated
OPG OD values
RANKL OD values
0.0302 ± 0.0019 0.0410 ± 0.0031a 0.0986 ± 0.0028a,b
0.0310 ± 0.0020 0.0894 ± 0.0024a 0.0387 ± 0.0029a,b
OPG, osteoprotegerin; OD, optical density; RANKL, receptor activator of nuclear factor kappa-B ligand. a,b p < 0.05 by ANOVA compared with negative control (a), force-only positive control (b).
Fig. 3. Histological examination of compressed surfaces. (A) Negative control group showing smooth root surfaces. (B) Positive control group showing widespread resorption lacunae. (C) Icariin-treated groups showing small isolated resorption pits (HE staining stain, magnification 200×).
than that of the positive control group. The mean RANKL OD value of the Icariin-treated groups was significantly lower than that of the positive control group. Discussion In this study, we explored the effect of Icariin on rapid maxillary expansion induced root resorption in rats. The RRI values were reduced in the Icariin-treated groups. High-power SEM revealed newly developed cementum in the Icariin-treated groups. Fewer osteoclasts were seen in the groups treated with Icariin than in the positive control group. These results indicate that root resorption was less active in the Icariin-treated groups.
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Fig. 4. TRAP staining of the rat molar tooth root. (A) Negative control group showing few osteoclasts in the apex. (B) Positive control group showing substantial numbers of osteoclasts in the apex. (C) Icariin-treated groups showing fewer osteoclasts in the apex than in the positive control group (left: magnification 200×; right: magnification 400×).
Treatment with Icariin increased OPG and decreased RANKL expression in the experimental groups, thereby reducing the number and activity of osteoclasts and naturally reducing root resorption. There are at least three potential reasons for this results. First, Icariin can up-regulate OPG expression and down-regulate RANKL expression by culturing with osteoblasts (Hsieh et al. 2010). Second, Icariin can influence stem cells in the periodontal membrane. For example, Icariin can promote bone marrow stromal cells to differentiate into osteoblast (Chen et al. 2007). Third, the mechanical stimulus elicits the strong release of inflammatory factors, such as tumor necrosis factor-␣ (TNF-␣) and prostaglandin E2 (PGE2 ), from the weakened periodontal ligament. These inflammatory factors can stimulate RANKL expression. Icariin can reduce the levels of these inflammatory factors, thereby reducing RANKL expression and osteoclast differentiation (Hsieh et al. 2011). Prolonged treatment with Icariin previously have demonstrated that Icariin improves maturation and mineralization of osteoblasts in vitro, and suppresses osteoclastogenesis and inhibits bone
resorption activity in vivo and in vitro (Zhao et al. 2008; Qin et al. 2008; Hsieh et al. 2011). Cultured with osteoblasts, Icariin increases alkaline phosphatase activity and affects the mRNA expression of type-I collagen COL1␣2 gene (Ma et al. 2011), which regulate the mineralization process. In vitro Icariin is a bone anabolic agent that may exert its osteogenic effects through the induction of BMP-2 and NO synthesis, subsequently regulating Cbfa1/Runx2, OPG, and RANKL gene expressions (Hsieh et al. 2010). This effect may contribute to its action on the induction of osteoblasts proliferation and differentiation, resulting in bone formation. Recently Icariin was shown to stimulate bone marrow stromal cells differentiation into osteoblast (Chen et al. 2007). These findings suggest that Icariin may be advantageous for stem cell and tissue engineering applications, and has great potential in hard tissue repair and regeneration. The different phylogenic status between humans and lower animals such as mice, rats, and rabbits makes it difficult to extrapolate results from animal models to the clinic. The growth pattern of cementum in lower animals involves continuous eruption, with cementum being formed throughout their lifetime. However, the
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Fig. 5. OPG/RANKL immunohistochemical staining of the rat molar tooth root. (A) Apical region of the middle palatal root of orthodontic tooth movement. (E) Negative control experiment using nonimmune IgG instead of the primary antibody. (B–D) Immunoreactivity for OPG on day 21. Untreated specimens (B) show weak immunoreactivity for OPG compared to the other groups. Specimens are shown after treatment with RPE force (50 × g) without (C) or with Icariin (D). Part D shows strong staining for OPG compared to part C on the compressed side. (F–H) Immunoreactivity for RANKL at day 21. Untreated specimens (F) show weak immunoreactivity for RANKL compared to the other groups. Specimens are shown after treatment with RPE force (50 × g) without (G) or with Icariin (H). Part G shows strong staining for RANKL compared to part H on the compressed side. PDL, periodontal ligament; C, cementum. Magnification is ×40 (A) or ×400 (B–H).
study of root resorption in an adequate sample size of higher animals (e.g., monkeys) is prohibitively expensive. As a result, the rat model for RME induced root resorption remains widely used.
Many plant-derived natural products have been used in traditional medicine for the treatment of various diseases. Herba Epimedii is a traditional Chinese herbal medicine, which has been commonly used as tonic, aphrodisiac and anti-rheumatic in China
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Fig. 6. Chemical structure of Icariin.
for thousands of years. Its physical and functional characteristics have been thoroughly documented in the Chinese pharmacopoeia 2005. Icariin [molecular formula: C33 H40 O15 ; molecular weight: 676.67; chemical structure (Fig. 6)] is the main active flavonoid glucoside isolated from Epimedium pubescens. Our dosage is lower than the document .So, our dosage is much safer. So far, Icariin is considered to be noncarcinogenic and has no known deleterious effects. These findings may help to promote the development of convenient, abundant and low cost herbs to prevent tooth resorption and improve human health and quality of life. Additional studies on this process are recommended. Conclusion Icariin has a reparative effect on RME induced root resorption in rats by modulating the OPG/RANKL ratio and osteoclast differentiation. Therefore, the extremely low cost of Icariin and its high abundance make it appealing for RME induced root resorption. Acknowledgement This work was supported by National Natural Science Funds of China (grant no. 81070837, China). References Adachi, H., Igarashi, K., Mitani, H., Shinoda, H., 1994. Effects of topical administration of a bisphosphonate (risedronate) on orthodontic tooth movements in rats. Journal of Dental Research 73 (8), 1478–1484.
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