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Effects of risedronate on cortical and trabecular bone of the mandible in glucocorticoid-treated growing rats
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Effects of risedronate on cortical and trabecular bone of the mandible in glucocorticoid-treated growing rats Yuko Fujita,a Koji Watanabe,b Shinsaku Uchikanbori,c and Kenshi Makid Kitakyushu, Japan
Introduction: This study was performed to estimate the effects of risedronate on mandibular bone density, bone structure, and bone metabolism in established glucocorticoid-induced osteoporosis in growing rats. Methods: The rats were given oral risedronate at 0, 0.5, or 1.0 mg per kilogram per day for 4 weeks afterthe administration of oral prednisolone at 30 mg per kilogram per 2 days for 6 weeks. Trabecular and cortical bone masses were analyzed by using peripheral quantitative computed tomography, and bone structure and bone formation were measured by using static and dynamic histomorphometry. Results: In trabecular bone, risedronate improved the prednisolone-induced decreases in bone cross-sectional area and bone mineral content. Risedronate increased bone density and also formed dense bone microarchitecture by reducing the bone turnover rate. In cortical bone, risedronate improved the prednisolone-induced decreases in bone cross-sectional area and bone mineral content without affecting bone density by increasing the mineralizing surface. Conclusions: Risedronate improved prednisolone-induced retardation of trabecular and cortical bone growth, but the bone turnover in these 2 sites was regulated differently in the growing rat mandibles. (Am J Orthod Dentofacial Orthop 2011;139:e267-e277)
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lucocorticoids are used to treat various diseases, such as severe asthma, juvenile rheumatoid arthritis, and chronic renal diseases,1,2 but the incidence rate of glucocorticoid-induced osteoporosis is approximately 50% in patients treated for 6 months or longer.3,4 In children, not only osteoporosis but also growth retardation occurred with chronic glucocorticoid therapy.1,5 Bone mass increases dramatically during childhood and adolescence, peaking in young
a Assistant professor, Division of Developmental Stomatognathic Function Science, Department of Growth and Development of Functions, Kyushu Dental College, Kitakyushu, Japan. b Assistant professor, Division of pediatric dentistry, Department of Human Development and Fostering, Meikai University, Saitama, Japan. c Assistant professor, Division of Developmental Stomatognathic Function Science, Department of Growth and Development of Functions, Kyushu Dental College, Kitakyushu, Japan. d Professor and chair, Division of Developmental Stomatognathic Function Science, Department of Growth and Development of Functions, Kyushu Dental College, Kitakyushu, Japan. The authors report no commercial, proprietary, or financial interest in the products or companies described in this article. Reprint requests to: Kenshi Maki, Division of Developmental Stomatognathic Function Science, Department of Growth and Development of Functions, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Japan 803-8580; e-mail, [email protected]. Submitted, January 2009; revised and accepted, May 2009. 0889-5406/$36.00 Copyright Ó 2011 by the American Association of Orthodontists. doi:10.1016/j.ajodo.2009.05.028
adulthood (the peak bone mass6) plateaus, and finally declines. The bone that must last a lifetime is made between the ages of 10 and 18 years. Patients whose bone mass does not reach the peak during childhood because of glucocorticoid-induced osteoporosis are at permanent risk of suffering fractures.7 In oral bone tissues, glucocorticoid affects mandibular bone growth and bone strength in humans and animals.8-11 However, little is known about bone metabolism and bone microarchitecture of the mandible with glucocorticoidinduced osteoporosis in children and adolescents. Furthermore, no guidelines exist for treating glucocorticoid-induced osteoporosis in children. Bisphosphonates, which are widely used to manage adults with osteoporosis as first-line therapeutic agents, are divided into 2 classes according to their chemical structure and mechanism of action.12,13 The nitrogencontaining bisphosphonates, such as alendronate and risedronate, are markedly more potent inhibitors of osteoclastic bone resorption than nonnitrogen-containing bisphosphonates, such as etidronate and clodronate.14 Several recent reports described cases of osteonecrosis of the mandible associated with nitrogen-containing bisphosphonate therapy, and controversy is ongoing regarding this issue.15,16 A few clinical studies have indicated that bisphosphonate therapy inhibited loss of long bones in children with osteogenesis imperfecta e267
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and glucocorticoid-induced osteoporosis.17,18 However, the effects of nitrogen-containing bisphosphonates on bone structure and bone formation of the mandible in cases of established glucocorticoid-induced osteoporosis during the growth phase are unknown, and the cause of bisphosphonate-induced osteonecrosis of the jaw has not been clarified. Peripheral quantitative computed tomography (pQCT) can be used to measure cortical and trabecular parts separately; this is useful for analyzing bone masses and cross-sectional areas, and also provides volumetric data.19 Recently, microcomputed tomography has been applied to quantify trabecular bone structure, such as trabecular bone volume and trabecular thickness.20 However, in the analyses withy pQCT and microcomputed tomography, we can evaluate static phenomena of bone metabolism but not those associated with bone formation because the data have a temporal component. Both static and dynamic histomorphometry can be performed with 2-dimensional evaluation by using prepared specimens. In this study, we evaluated the effects of risedronate, used frequently for treating osteoporosis, on trabecular and cortical bone density with pQCT, and bone structure and bone metabolism with static and dynamic histomorphometry in the mandibles of growing glucocorticoid-treated rats. MATERIAL AND METHODS
In total, 48 male Wistar rats, aged 5 weeks, were purchased from Charles River Japan (Kanagawa, Japan). The animals were housed individually and maintained under a 12-hour light and 12-hour dark cycle at a constant temperature of 22 C 6 1 C with humidity of 50% 6 5%. The animals were given a standard pelleted chow containing 1.14% calcium and 1.06% phosphorus (CE-2, CLEA Japan, Tokyo, Japan). The animal procedures were all approved by the Committee for the Care and Use of Laboratory Animals of Kyushu Dental College. The animals were divided randomly into 6 groups of 8 each as follows: (1) control 6 weeks; (2) prednisolone (pred) 6 weeks; (3) control 10 weeks; (4) pred 1 saline solution; (5) pred 1 low risedronate (ris); and (6) pred 1 high ris (Table I). In the pred 1 saline and pred 1 ris groups, prednisolone was administered for 6 weeks followed by vehicle or ris for 4 weeks. Prednisolone sodium succinate (Prednine, Shionogi & Co, Osaka, Japan) was administered orally at 30 mg per kilogram per day on alternate days. Risedronate sodium (Ajinomoto, Tokyo, Japan) was administered orally at 0.5 mg per kilogram per day or 1.0 mg per kilogram per starting immediately after the 6-week prednisolone regimen. We compared the control 6 weeks group with the pred
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Table I. Experimental protocol Experimental period Treatment group Control 6 weeks Pred 6 weeks Control 10 weeks Pred 1 saline solution Pred 1 low ris Pred 1 high ris
6 weeks Prednisolone vehicle (saline) Prednisolone 30 mg/kg/2 days Prednisolone vehicle (saline) Prednisolone 30 mg/kg/2 days Prednisolone 30 mg/kg/2 days Prednisolone 30 mg/kg/2 days
4 weeks — — Risedronate vehicle (saline) Risedronate vehicle (saline) Risedronate 0.5 mg/kg/day Risedronate 1.0 mg/kg/day
6 weeks group to confirm the establishment of osteoporosis induced by prednisolone administration. The effects of risedronate treatment were evaluated in the remaining 4 groups. The doses and durations of prednisolone treatment were determined from the results of a previous study that confirmed mandibular bone deterioration.11 The doses and durations of risedronate were determined from a previous study that demonstrated significant inhibition of bone loss in immobilized rats.21 Both compounds were dissolved in sterile saline solution before delivery so that the required dose was in a volume of 10 mL per kilogram of body weight. Prednisolone and vehicle (saline solution) were administered in the morning. Risedronate and vehicle (saline solution) were administered in the afternoon in animals that had fasted for 4 hours previously and 2 hours after risedronate administration. All rats were given free access to water throughout the experimental period.21 Body weight was measured weekly. At the end of the experimental period, all rats were deeply anesthetized with diethyl ether and given a lethal injection of thiamylal natrium (Isozole; Mitsubishi Pharma, Osaka, Japan) intraperitoneally at a dose of 90 mg per kilogram of body weight. For dynamic histomorphometry, all rats were given a subcutaneous injection of calcein (8 mg/kg of body weight; Kanto Chemical, Tokyo, Japan) on days 10 and 3 before they were killed. Bilateral hemi-mandibles were removed from each animal, trimmed of soft tissues, and stored in 70% ethanol. The left hemi-mandible was used for measurement of mandibular bone weight as an index of bone growth and for pQCT analysis. The right hemi-mandible was used for histopathologic evaluation and bone histomorphometry. Bone density (BD; mg/cm3), cross-sectional area (CSA; mm2), and mineral content (MC; mg/mm) of trabecular (Tr) and cortical (Ct) bone from the left
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hemi-mandible were measured by using pQCT (XCT Research SA1 series; Stratec, Medizintechnik, Pforzheim, Germany). For measurement of TrBD, TrCSA, TrBMC, CtBD, CtCSA, and CtBMC, we scanned between the center of the mesial root of the mandibular first molar that contained rich trabecular and cortical bone at 3 positions with an interval of 0.1 mm and measured the cortical component using a voxel size of 0.08 3 0.26 mm. The trabecular region was defined manually, and the cortical region was defined by setting a threshold value of 690 mg per cubic centimeter.11,22 The measurement errors for TrBD, CtBD, TrCSA, and CtCSA were calculated accordingP to the formula of DahlP berg23: Se 5 O d2/2n, where d2 is the sum of the squared differences between pairs of recordings and n is the number of duplicate measurements. The errors were 2.1 mg per cubic centimeter, 1.3 mg per cubic centimeter, 0.17 mm2, and 0.05 mm2, respectively. The trimmed right hemi-mandibles were fixed in 70% ethanol and stained with Villanueva bone stain.24 The bone tissues were then dehydrated in ethanol and embedded in methylmethacrylate. Frontal cross-sections of the mesial root of the mandibular first molar region were cut (300 mm) and polished (20 mm) with a precision bone saw (cutting grinding system, EXAKT Apparatebau, Norderstedt, Germany). The Villanueva-stained specimens were used to assess dynamic histomorphometry. After static and dynamic histomorphometric measurements, each specimen was also stained by using the Goldner method for histopathologic evaluation.25 Images of the mandibular bone were scanned under a light epifluorescence microscope (Carl Zeiss, Thornwood, NY). Histomorphometric parameters were measured at 200-times magnification by using a semiautomatic method (Osteoplan II, Kontron, Munich, Germany).26 Measurement areas are shown in Figure 1. The measured parameters for trabecular bone included total bone tissue (TV), bone volume (BV), bone surface (BS), eroded surface (ES), single-labeled surface (sLS), double-labeled surface (dLS), and the width between double fluorochrome labels (Ir.L.Th). The static histomorphometric parameters were as follows: percentage of trabecular bone volume (BV/ TV; %), trabecular number (Tb.N, 1/mm), trabecular thickness (Tb.Th, mm), trabecular separation (Tb.Sp, mm), and eroded surface (ES/BS, %). The dynamic histomorphometric parameters were as follows: mineralizing surface (MS/BS 5 [dLS1sLS/2]/BS, %), mineral apposition rate (MAR 5 Ir.L.Th/7 days; mm/day), and bone formation rate (BFR/BS 5 MAR3MS/BS30.365; mm3/cm2 per year). The measured parameters for cortical bone were induced periosteal bone surface (perimeter) and the lengths of the sLS and dLS. These data were used to calculate MS/BS, MAR, and BFR/BS. The
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Fig 1. Frontal cross-section in the mandibular first molar region with Villanueva bone staining. Original magnification: 2.5 times. The red line indicates the periosteal surface of cortical bone. The green area indicates the trabecular bone tissue area. Measurements were made of the interior of the trabecular bone tissue area (trabecular bone) and along the periosteal surface (cortical bone).
terminology used is in accordance with the American Society for Bone and Mineral Research Committee on Histomorphometric Nomenclature.27 Statistical analysis
Data are presented as means and standard deviations, and statistical comparisons between the control 6 weeks and the pred 6 weeks groups were performed by using 2-tailed t tests for unpaired samples. Multiple comparisons among the 4 10-week treatment groups were performed by 1-way analysis of variance (ANOVA) with the Tukey method. The level of significance was set at 5%. RESULTS
The body weight and mandibular bone weight in the pred 6 weeks group were significantly lower than those in the control 6 weeks group, and these differences persisted after 4 weeks of treatment with saline solution. However, treatment with risedronate reversed the prednisolone-induced decrease in mandibular bone weight (Table II). The values of TrCSA, TrBMC, CtCSA, and CtBMC in the pred 6 weeks group were significantly lower than those in the control 6 weeks group, and these decreases did not improve after 4 weeks of saline-solution treatment. Although the mean value of CtBD in the pred 6 weeks group was lower than that in the control 6 weeks group, the difference was not significant. TrBD in the pred 6 weeks group was significantly higher than that in the control 6 weeks group. TrBD increased with risedronate in a dose-dependent manner, but lower doses of risedronate showed a tendency to improve TrCSA and TrBMC, and higher doses of risedronate significantly
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Table II. Body weight and mandibular bone weight Pred 1 ris Pred 1 Ris Control 6 weeks Pred 6 weeks Control 10 weeks Pred 1 vehicle (0.5 mg/kg) (1.0 mg/kg) 135.88 6 1.31 136.38 6 2.55 135.83 6 1.92 134.75 6 1.76 Initial body weight (g) 135.18 6 2.89 133.48 6 2.44 Final body weight (g) 407.65 6 11.41 368.63 6 12.09* 422.80 6 11.20 378.63 6 12.46y 384.7 6 17.35y 386.43 6 20.57y Mandibular bone weight (g) 0.43 6 0.02 0.40 6 0.02* 0.52 6 0.02 0.49 6 0.03y 0.51 6 0.02 0.50 6 0.03 Data are expressed as means 6 standard deviations. *P \0.05 vs control 6 weeks group (t test); yP \0.05 vs control 10 weeks group (ANOVA and Tukey test).
Table III. pQCT measurements of trabecular and cortical bone density, cross-sectional area, and mineral content Control 6 weeks Pred 6 weeks Control 10 weeks Pred 1 saline Pred 1 low ris Pred 1 high ris 608.51 6 38.91 646.38 6 35.28* 707.60 6 16.47 685.13 6 28.79 684.73 6 29.32 723.97 6 26.00z,§ Trabecular bone density (mg/cm3) 1.56 6 0.42 1.15 6 0.24* 1.70 6 0.38 1.22 6 0.25y Trabecular bone 1.35 6 0.47 1.19 6 0.23y cross-sectional area (mm2) Trabecular bone mineral 1.01 6 0.27 0.78 6 0.20* 1.20 6 0.26 0.83 6 0.16y 0.92 6 0.30 0.86 6 0.17y content (mg/mm) Cortical bone density (mg/cm3) 1268.86 6 9.48 1261.75 6 11.21 1301.63 6 14.65 1296.55 6 9.15 1301.45 6 8.32 1298.41 6 9.22 6.73 6 0.67 6.08 6 0.63* 7.40 6 0.52 6.74 6 0.32y 7.33 6 0.33z 7.51 6 0.57z Cortical bone cross-sectional 2 area (mm ) Cortical bone mineral 8.54 6 0.87 7.67 6 0.80* 9.62 6 0.64 8.74 6 0.36y 9.55 6 0.41z 9.75 6 0.72z content (mg/mm) Data are expressed as means 6 standard deviations. *P \0.05 vs control 6 weeks group (t test); yP \0.05 vs control 10 weeks group (ANOVA and Tukey test); zP \0.05 vs pred 1 saline group (ANOVA and Tukey test); §P \0.05 vs pred 1 high ris group (ANOVA and Tukey test).
reduced these parameters compared with the control 10 weeks group (Table III). In cortical bone, both doses of risedronate improved the prednisolone-induced decreases in CtCSA and CtBMC (Table IV). Another representative pQCT mandibular bone scan indicated that the trabecular region in the pred 6 weeks group clearly decreased compared with the control 6 weeks group, and risedronate treatment dosedependently reduced the trabecular bone area and increased the mineralization of trabecular bone (Fig 2). The results of trabecular microstructural parameters indicated a significantly higher Tb.Sp in the pred 6 weeks group relative to the control 6 weeks group, and risedronate treatment dose-dependently reduced Tb.Sp and increased Tb.N (Table V). The results of static histomorphometry of trabecular bone indicated that Ob.S/BS, Oc.S/BS, and ES/BS in the pred 6 weeks group significantly decreased compared with the control 6 weeks group (Table V), and osteoblasts with cuboidal morphology were observed in trabecular bone of the pred 6 weeks group (Fig 4). Risedronate decreased Oc.S/BS and ES/BS in a dosedependent manner. Furthermore, ES/BS, Ob.S/BS, MS/ BS, and BFR/BS were significantly lower with risedronate treatment in the control 10 weeks group (Table V). In the double-labeling study, the distance between labels
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narrowed in the risedronate-treated rats (Fig 3), and risedronate treatment altered the morphology of osteoblasts from cuboidal to a more flattened appearance compared with those in the untreated controls (Fig 4). The results of dynamic histomorphometry in cortical bone indicated that MAR and BFR/BS in the pred 6 weeks group significantly decreased compared with the control 6 weeks group, and the mean values for MS/BS and BFR/BS in the pred 1 saline group were lower than those in the control 10 weeks group; these reductions, however, were not statistically significant (Table VI). The distance between double labels was narrowed by prednisolone administration (Fig 5). Risedronate dose-dependently increased MS/BS and BFR/BS, and these values in the pred 1 high ris group were significantly elevated compared with the control 10 weeks level (Table VI). DISCUSSION
Prednisolone clearly reduced body weight and mandibular bone weight; these findings were consistent with those of other studies indicating that glucocorticoid treatment in growing rats reduced body weight and bone growth.11,28 In addition, prednisolone decreased TrCSA and TrBMC and caused deterioration of the connectivity of trabeculae (Tb.Sp). In contrast, TrBD in
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Table IV. Bone histomorphometric analysis of trabecular bone of the mandible: microarchitectural parameters BV/TV (%) Tb.Th (mm) Tb.N (1/mm) Tb.Sp (mm)
Control 6 weeks 73.03 6 7.48 367.32 6 25.22 1.90 6 0.18 149.66 6 11.90
Pred 6 weeks 68.90 6 3.25 398.75 6 18.84 1.64 6 0.17 190.89 6 19.47*
Control 10 weeks 67.37 6 0.33 350.68 6 48.98 1.99 6 0.23 177.87 6 25.72
Pred 1 saline 68.09 6 1.92 347.53 6 46.89 1.80 6 0.22 184.03 6 20.00
Pred 1 low ris 69.89 6 1.27 364.51 6 19.26 1.92 6 0.07 156.80 6 4.47
Pred 1 high ris 70.93 6 2.09 345.54 6 30.62 2.36 6 0.32z 123.33 6 15.82y,z
Data are expressed as means 6 standard deviations. BV/TV, Bone volume fraction (TV, total tissue volume); Tb.Th, trabecular thickness; Tb.N, trabecular number; Tb.Sp, trabecular separation. *P \0.05 vs control 6 weeks group (t test); yP \0.05 vs control 10 weeks group (ANOVA and Tukey test); zP \0.05 vs pred 1 saline group (ANOVA and Tukey test).
Fig 2. Representative pQCT cross-sectional transverse scans of the mandibular first molar region.
the pred 6 weeks rats increased significantly compared with the untreated controls. The major action of glucocorticoids on bone is to reduce osteoblast numbers and function, leading to suppression of bone formation.29 Furthermore, glucocorticoids might function secondarily to increase bone resorption. Glucocorticoids increase the expression of receptor activator of nuclear factor LB ligand (RANK-L) and decrease the expression of its soluble decoy receptor osteoprotegerin (OPG) in stromal and osteoblastic cells.30 Glucocorticoids might also have direct effects on osteoclasts by suppressing the expression of autocrine cytokines, such as interferon b, which normally exerts inhibitory effects on osteoclastogenesis.31
In this study, prednisolone significantly increased bone resorption (Oc.S/BS and ES/BS) but also showed a tendency to increase bone formation (Ob.S/BS and MS/BS). Although this trend was not significant, the differences were inconsistent with those of previous studies. One possible explanation for those observations is that we simply altered a delicate balance between the activities of osteoblasts and osteoclasts, which generally act in tandem to establish an equilibrium between bone buildup and resorption, respectively. Nakamura et al32 reported that activated osteoblasts with a cuboidal shape were often observed near sites of osteoclast resorption in the trabecular bone of OPG–/–mice,
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Table V. Bone histomorphometry of trabecular bone of the mandible: formative and resorptive variables Ob.S/BS (%) MAR (mm/day) MS/BS (%) BFR/BS (mm3/cm2/y) Oc.S/BS (%) ES/BS (%)
Control 6 weeks 6.00 6 3.62 1.69 6 0.14 19.25 6 8.86 14.78 6 4.73 0.21 6 0.18 1.56 6 0.32
Pred 6 weeks 12.66 6 3.62* 1.33 6 0.20 25.67 6 4.07 12.31 6 0.82 1.49 6 0.51* 5.12 6 1.39*
Control 10 weeks 3.19 6 1.47 1.23 6 0.07 5.72 6 0.32 2.56 6 0.23 0.72 6 0.09 2.46 6 0.50
Pred 1 saline 1.20 6 0.30 1.17 6 0.17 3.97 6 2.00 1.63 6 0.63 0.65 6 0.29 3.21 6 0.32
Pred 1 low ris 0.71 6 0.65y 1.30 6 0.58 1.92 6 0.68y 0.94 6 0.51y 0.56 6 0.09 2.01 6 0.46z
Pred 1 high ris 1.06 6 0.38y 0.94 6 0.03 3.94 6 0.20 1.36 6 0.11y 0.35 6 0.10 1.09 6 0.17y,z
Data are expressed as means 6 standard deviations. *P \0.05 vs control 6 weeks group (t test); yP \0.05 vs control 10 weeks group (ANOVA and Tukey test); zP \0.05 vs pred 1 saline group (ANOVA and Tukey test).
Fig 3. Fluorescent micrographs showing double-labeled mineralization in the trabecular bone of the mandible. Arrows indicate calcein that was taken into the bone formation area. Original magnification: 200 times.
indicating that bone formation is tightly coupled with bone resorption. In this study, the presence of similar formative osteoblasts in the trabecular bone from the pred 6 weeks rats suggested that prednisolone-treated growing rats tend to exhibit higher turnover rates in trabecular bone. Furthermore, we used pQCT, which
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measures the degree of bone mineralization.33 We considered that the increase in TrBD by prednisolone could be explained in terms of increased mineralization by prednisolone at sites where osteoblastic bone formation was most active in trabecular regions. The decreases in TrCSA and TrBMC by prednisolone, however, could
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Fig 4. Histologic evaluations of trabecular bone of the mandible. Original magnification: 400 times. Arrows indicate osteoblasts along the bone surface.
have been the result of increased bone resorption by prednisolone, which in turn accelerated compensatory increases in bone formation. In a previous study, chronic glucocorticoid treatment increased the rate of orthodontic tooth movement in rats and suggested that the orthodontic force level should be reduced in patients receiving chronic glucocorticoid treatment.34 This might be due to the increase in bone resorption and the deterioration of the trabecular architecture by glucocorticoid treatment. In cortical bone regions, prednisolone decreased CtCSA and CtBMC by suppressing the BFR/BS, whereas the bone turnover rate had little effect on CtBD. This suppression caused a decrease in the MAR, generally considered to be an indicator of cellular activity. Thus, a unique suppressive effect of prednisolone on bone formation was found for the periosteal surface in the mandible. These results were consistent with those of recent clinical studies indicating that glucocorticoid treatment has little or no effect on bone mineral density despite an increased fracture risk, and that the cause of glucocorticoid-induced bone deterioration is the decrease in bone quality, such as bone geometry and bone metabolism.35,36
In this study, risedronate treatment was started after 6 weeks of prednisolone administration. In many studies, however, both glucocorticoid and bisphosphonate had been given for the whole experimental period to evaluate the preventive effects of bisphosphonate on glucocorticoid-induced deterioration of bone structure in animals.28,37,38 However, little is actually known regarding whether risedronate improves the bone mass of the mandible or whether sufficient peak bone mass can be accumulated with glucocorticoid administration. In addition, few studies have examined the effects of risedronate on mandibular bone formation in young animals. Therefore, prednisolone was not given for the whole 10-week experimental period to investigate the effects of risedronate treatment alone on prednisoloneinduced osteoporosis of the mandible in young rats. Risedronate treatment improved the deterioration of mandibular bone growth induced by prednisolone. However, differential effects of risedronate on bone formation were seen between trabecular and cortical bone. Risedronate dose-dependently reduced bone resorption (Oc.S/BS and ES/BS). With regard to bone formation, the mean values of Ob.S/BS, BFR/BS, and MS/BS in the risedronate treatment groups were lower than those
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Table VI. Bone histomorphometry of cortical bone of the mandible: formative variables MAR (mm/day) MS/BS (%) BFR/BS (mm3/cm2/y)
Control 6 weeks 3.20 6 0.22 65.53 6 0.87 76.66 6 4.86
Pred 6 weeks 2.68 6 0.24* 60.74 6 7.58 59.32 6 3.63*
Control 10 weeks 2.25 6 0.02 46.13 6 4.38 37.86 6 3.98
Pred 1 saline 2.34 6 0.22 37.55 6 2.31 32.17 6 5.02
Pred 1 low ris 2.17 6 0.08 54.43 6 7.63 42.96 6 4.59
Pred 1 high ris 2.36 6 0.43 67.78 6 3.64y,z 58.22 6 7.61y,z
Data are expressed as means 6 standard deviations. *P \0.05 vs control 6 weeks group (t test); yP \0.05 vs control 10 weeks group (ANOVA and Tukey test); zP \0.05 vs pred 1 saline group (ANOVA and Tukey test).
Fig 5. Fluorescent micrographs showing double-labeled mineralization in the periosteal surface of cortical bone. Arrows indicate calcein that was taken into the bone formation area. Original magnification: 200 times.
in the saline-treatment group, suggesting that risedronate tended to decrease bone formation. Furthermore, risedronate treatment altered the morphology of osteoblasts from cuboidal to flat as in bone-lining cells. These results were similar to those of 2 previous studies indicating that risedronate reduced bone formation in ovariectomized rats39 and caused dedifferentiation of active osteoblasts to flatter cells, more typical of bone lining cells in OPG–/–mice.32 Furthermore, risedronate
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dose-dependently increased TrBD and formed dense trabecular bone micro-architecture by decreasing bone turnover, but the higher dose of risedronate did not improve TrCSA or TrBMC. Therefore, we postulated that bisphosphonate-induced osteonecrosis of the jaw might have been due to excessive doses of bisphosphonates that suppressed trabecular bone turnover, which reduced new trabecular bone tissue formation and caused the accumulation of old trabeculae, although no gross
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histopathologic findings of osteonecrosis were found in the rat mandibles. Two experimental studies with small animals also showed the positive effects of bisphosphonates on bone healing after tooth movement or distraction osteogenesis. Adachi et al40 reported that risedronate inhibited the relapse of moved teeth by inhibiting alveolar bone resorption in rats, and Tekin et al41 reported that alendronate increased bone mineral density and accelerated bone healing on distraction osteogenesis of rabbit mandibles. In a study of long-term bisphosphonate therapy, Odvina et al42 discussed the possible adverse effects of long-term alendronate therapy for patients with postmenopausal osteoporosis who sustained spontaneous nonvertebral fractures; some showed delayed healing and histologic evidence of a marked reduction in bone turnover. These findings suggest that short-term bisphosphonate treatment did not prevent healing of the mandible after orthodontic and surgical treatment, although longterm bisphosphonate treatment might cause detrimental changes in bone. Because the prevalence of osteonecrosis is more frequent in the jaws, we believe that the osteonecrosis is caused by other factors that are specific for jaw bones, in addition to the marked suppression of bone turnover. Interestingly, Rizzoli et al43 reported that previous histologic studies on bisphosphonate-induced osteonecrosis of the jaw all showed pronounced inflammatory changes. In addition, patients administered glucocorticoids show increased susceptibility to infectious diseases because glucocorticoids have a powerful immunosuppressive effect.5 Therefore, osteonecrosis of the jaw can occur more frequently when bisphosphonates are given with glucocorticoids because bacterial infection leads to exacerbation of inflammation in the mandible. However, few studies have been conducted regarding the relationship between the analysis of bacteria, such as those involved in periodontal disease and osteonecrosis of the jaw. Further studies are needed to determine whether anaerobic bacteria are involved in these effects. In cortical bone, risedronate inhibited prednisoloneinduced decreases in CtCSA and CtBMC. Furthermore, risedronate treatment increased MS/BS and BFR/BS. Consistent with our findings, bisphosphonate counteracted glucocorticoid-induced bone loss of longitudinal bones in rats.28 In contrast, risedronate suppressed periosteal osteoblast activity independent of resorption in rats’ longitudinal bones.44 Several studies have suggested that bisphosphonates have a stimulatory effect on osteoblast proliferation and differentiation in vitro.45-47 However, there is little evidence to indicate that risedronate increases bone formation in vivo.
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Therefore, whether bisphosphonates stimulate or suppress osteoblast proliferation and differentiation, or both, is not yet clear, or whether the balance between proliferation and differentiation is just sensitive, similar to that between formation and resorption. A possible explanation for our findings is that the increase in sLS was greater than the decrease in dLS with risedronate in the bones of growing rats. Risedronate, however, had no significant effect on CtBD. Consistent with our findings, risedronate prevented bone density loss of the femoral distal metaphysis without affecting bone density of the femoral diaphysis in sciatic neurectomized rats.48 Our findings suggest that risedronate had a positive effect on bone geometry but not on cortical bone density of growing rats. CONCLUSIONS
Risedronate improved prednisolone-induced retardation of trabecular and cortical bone growth, but the bone turnover in these 2 sites is regulated differently in the growing rat’s mandible. In trabecular bone, risedronate improved the prednisolone-induced decrease in bone cross-sectional area and bone mineral content, and increased bone density by suppressing bone turnover. However, in cortical bone, risedronate increased the mineralizing surface and improved prednisoloneinduced decreases in bone cross-sectional area and bone mineral content with no effect on bone density. We thank Kiichi Nonaka (Elk Corporation Research Laboratory, Tokyo, Japan) and Hisashi Murayama (Kureha Special Laboratory, Tokyo, Japan) for technical assistance in the bone histomorphometry and helpful discussions. REFERENCES 1. Burnham JM, Shults J, Petit MA, Semeao E, Beck TJ, Zemel BS, et al. Alterations in proximal femur geometry in children treated with glucocorticoids for Crohn disease or nephrotic syndrome: impact of the underlying disease. J Bone Miner Res 2007;22: 551-9. 2. Falcini F, Trapani S, Civinini R, Capone A, Ermini M, Bartolozzi G. The primary role of steroids on the osteoporosis in juvenile rheumatoid patients evaluated by dual energy x-ray absorptiometry. J Endocrinol Invest 1996;19:165-9. 3. Reid IR. Glucocorticoid-induced osteoporosis: assessment and treatment. J Clin Densitom 1998;1:65-73. 4. van Staa TP, Leufkens HG, Abenhaim L, Zhang B, Cooper C. Oral corticosteroids and fracture risk: relationship to daily and cumulative doses. Rheumatology (Oxford) 2000;39: 1383-9. 5. Mushtaq T, Ahmed SF. The impact of corticosteroids on growth and bone health. Arch Dis Child 2002;87:93-6. 6. Osteoporosis prevention, diagnosis, and therapy. NIH Consens Statement 2000;17:1-45.
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7. Rabinovich CE. Osteoporosis: a pediatric perspective. Arthritis Rheum 2004;50:1023-5. 8. Komerik N, Akkaya A, Yildiz M, Buyukkaplan US, Kuru L. Oral health in patients on inhaled corticosteroid treatment. Oral Dis 2005;11:303-8. 9. von Wowern N, Klausen B, Olgaard K. Steroid-induced mandibular bone loss in relation to marginal periodontal changes. J Clin Periodontol 1992;19:182-6. 10. Eratalay YK, Simmons DJ, El-Mofty SK, Rosenberg GD, Nelson W, Haus E, et al. Bone growth in the rat mandible following every-day or alternate-day methylprednisolone treatment schedules. Arch Oral Biol 1981;26:769-77. 11. Kimura E, Nishioka T, Hasegawa K, Maki K. Effects of bisphosphonate on the mandible of rats in the growing phase with steroidinduced osteoporosis. Oral Dis 2007;13:544-9. 12. Boivin GY, Chavassieux PM, Santora AC, Yates J, Meunier PJ. Alendronate increases bone strength by increasing the mean degree of mineralization of bone tissue in osteoporotic women. Bone 2000;27:687-94. 13. Sorensen OH, Crawford GM, Mulder H, Hosking DJ, Gennari C, Mellstrom D, et al. Long-term efficacy of risedronate: a 5-year placebo-controlled clinical experience. Bone 2003;32:120-6. 14. van Beek ER, Cohen LH, Leroy IM, Ebetino FH, L€owik CW, Papapoulos SE. Differentiating the mechanisms of antiresorptive action of nitrogen containing bisphosphonates. Bone 2003;33: 805-11. 15. Marx RE, Cillo JE Jr, Ulloa JJ. Oral bisphosphonate-induced osteonecrosis: risk factors, prediction of risk using serum CTX testing, prevention, and treatment. J Oral Maxillofac Surg 2007;65: 2397-410. 16. Pazianas M, Miller P, Blumentals WA, Bernal M, Kothawala P. A review of the literature on osteonecrosis of the jaw in patients with osteoporosis treated with oral bisphosphonates: prevalence, risk factors, and clinical characteristics. Clin Ther 2007; 29:1548-58. 17. Poyrazoglu S, Gunoz H, Darendeliler F, Bas F, Tutunculer F, Eryilmaz SK, et al. Successful results of pamidronate treatment in children with osteogenesis imperfecta with emphasis on the interpretation of bone mineral density for local standards. J Pediatr Orthop 2008;28:483-7. 18. Inoue Y, Shimojo N, Suzuki S, Arima T, Tomiita M, Minagawa M, et al. Efficacy of intravenous alendronate for the treatment of glucocorticoid-induced osteoporosis in children with autoimmune diseases. Clin Rheumatol 2008;27:909-12. 19. Ferretti JL, Capozza RF, Zanchetta JR. Mechanical validation of a tomographic (pQCT) index for noninvasive estimation of rat femur bending strength. Bone 1996;18:97-102. 20. R€ uegsegger P, Koller B, M€ uller R. A microtomographic system for the nondestructive evaluation of bone architecture. Calcif Tissue Int 1996;58:24-9. 21. Mosekilde L, Thomsen JS, Mackey MS, Phipps RJ. Treatment with risedronate or alendronate prevents hind-limb immobilizationinduced loss of bone density and strength in adult female rats. Bone 2000;27:639-45. 22. Maki K, Nishioka T, Nishida I, Ushijima S, Kimura M. Effect of zinc on rat mandibles during growth. Am J Orthod Dentofacial Orthop 2002;122:410-3. 23. Dahlberg G. Statistical methods for medical and biological students. London, United Kingdom: Allen & Unwin; 1940. p. 122-32. 24. Villanueva AR. A bone stain for osteoid seams in fresh, unembedded, mineralized bone. Stain Technol 1974;49:1-8. 25. Goldner J. A modification of the Masson trichrome technique for routine laboratory purposes. Am J Pathol 1938;14:237-43.
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26. Manaka RC, Malluche HH. A program package for quantitative analysis of histologic structure and remodeling dynamics of bone. Comput Programs Biomed 1981;13:191-201. 27. Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, et al. Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee. J Bone Miner Res 1987; 2:595-610. 28. Iwamoto J, Seki A, Takeda T, Sato Y, Yamada H, Shen CL, et al. Preventive effects of risedronate and calcitriol on cancellous osteopenia in rats treated with high-dose glucocorticoid. Exp Anim 2006;55:349-55. 29. O’Brien CA, Jia D, Plotkin LI, Bellido T, Powers CC, Stewart SA, et al. Glucocorticoids act directly on osteoblasts and osteocytes to induce their apoptosis and reduce bone formation and strength. Endocrinology 2004;145:1835-41. 30. Canalis E, Bilezikian JP, Angeli A, Giustina A. Perspectives on glucocorticoid-induced osteoporosis. Bone 2004;34:593-8. 31. Takuma A, Kaneda T, Sato T, Ninomiya S, Kumegawa M, Hakeda Y. Dexamethasone enhances osteoclast formation synergistically with transforming growth factor-beta by stimulating the priming of osteoclast progenitors for differentiation into osteoclasts. J Biol Chem 2003;278:4467-74. 32. Nakamura M, Udagawa N, Matsuura S, Mogi M, Nakamura H, Horiuchi H, et al. Osteoprotegerin regulates bone formation through a coupling mechanism with bone resorption. Endocrinology 2003;144:5441-9. 33. Rosen HN, Chen V, Cittadini A, Greenspan SL, Douglas PS, Moses AC, et al. Treatment with growth hormone and IGF-I in growing rats increases bone mineral content but not bone mineral density. J Bone Miner Res 1995;10:1352-8. 34. Kalia S, Melsen B, Verna C. Tissue reaction to orthodontic tooth movement in acute and chronic corticosteroid treatment. Orthod Craniofac Res 2004;7:26-34. 35. Kumagai S, Kawano S, Atsumi T, Inokuma S, Okada Y, Kanai Y, et al. Vertebral fracture and bone mineral density in women receiving high dose glucocorticoids for treatment of autoimmune diseases. J Rheumatol 2005;32:863-9. 36. Van Staa TP, Laan RF, Barton IP, Cohen S, Reid DM, Cooper C. Bone density threshold and other predictors of vertebral fracture in patients receiving oral glucocorticoid therapy. Arthritis Rheum 2003;48:3224-9. 37. Dalle Carbonare L, Bertoldo F, Valenti MT, Zordan S, Sella S, Fassina A, et al. Risedronate prevents the loss of microarchitecture in glucocorticoid-induced osteoporosis in rats. J Endocrinol Invest 2007;30:739-46. 38. Wimalawansa SJ, Simmons DJ. Prevention of corticosteroidinduced bone loss with alendronate. Proc Soc Exp Biol Med 1998;217:162-7. 39. Hunziker J, Wronski TJ, Miller SC. Mandibular bone formation rates in aged ovariectomized rats treated with anti-resorptive agents alone and in combination with intermittent parathyroid hormone. J Dent Res 2000;79:1431-8. 40. Adachi H, Igarashi K, Mitani H, Shinoda H. Effects of topical administration of a bisphosphonate (risedronate) on orthodontic tooth movements in rats. J Dent Res 1994;73:1478-86. 41. Tekin U, T€ uz HH, Onder E, Ozkaynak O, Korkusuz P. Effects of alendronate on rate of distraction in rabbit mandibles. J Oral Maxillofac Surg 2008;66:2042-9. 42. Odvina CV, Zerwekh JE, Rao DS, Maalouf N, Gottschalk FA, Pak CY. Severely suppressed bone turnover: a potential complication of alendronate therapy. J Clin Endocrinol Metab 2005; 90:1294-301.
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43. Rizzoli R, Burlet N, Cahall D, Delmas PD, Eriksen EF, Felsenberg D, et al. Osteonecrosis of the jaw and bisphosphonate treatment for osteoporosis. Bone 2008;42:841-7. 44. Iwata K, Li J, Follet H, Phipps RJ, Burr DB. Bisphosphonates suppress periosteal osteoblast activity independently of resorption in rat femur and tibia. Bone 2006;39:1053-8. 45. von Knoch F, Jaquiery C, Kowalsky M, Schaeren S, Alabre C, Martin I, et al. Effects of bisphosphonates on proliferation and
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osteoblast differentiation of human bone marrow stromal cells. Biomaterials 2005;26:6941-9. 46. Frediani B, Spreafico A, Capperucci C, Chellini F, Gambera D, Ferrata P, et al. Long-term effects of neridronate on human osteoblastic cell cultures. Bone 2004;35:859-69. 47. Plotkin LI, Weinstein RS, Parfitt AM, Roberson PK, Manolagas SC, Bellido T. Prevention of osteocyte and osteoblast apoptosis by bisphosphonates and calcitonin. J Clin Invest 1999;104:1363-74.
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Effects of risedronate on cortical and trabecular bone of the mandible in glucocorticoid-treated growing rats Yuko Fujita,a Koji Watanabe,b Shinsaku Uchikanbori,c and Kenshi Makid Kitakyushu, Japan
Introduction: This study was performed to estimate the effects of risedronate on mandibular bone density, bone structure, and bone metabolism in established glucocorticoid-induced osteoporosis in growing rats. Methods: The rats were given oral risedronate at 0, 0.5, or 1.0 mg per kilogram per day for 4 weeks afterthe administration of oral prednisolone at 30 mg per kilogram per 2 days for 6 weeks. Trabecular and cortical bone masses were analyzed by using peripheral quantitative computed tomography, and bone structure and bone formation were measured by using static and dynamic histomorphometry. Results: In trabecular bone, risedronate improved the prednisolone-induced decreases in bone cross-sectional area and bone mineral content. Risedronate increased bone density and also formed dense bone microarchitecture by reducing the bone turnover rate. In cortical bone, risedronate improved the prednisolone-induced decreases in bone cross-sectional area and bone mineral content without affecting bone density by increasing the mineralizing surface. Conclusions: Risedronate improved prednisolone-induced retardation of trabecular and cortical bone growth, but the bone turnover in these 2 sites was regulated differently in the growing rat mandibles. (Am J Orthod Dentofacial Orthop 2011;139:e267-e277)
G
lucocorticoids are used to treat various diseases, such as severe asthma, juvenile rheumatoid arthritis, and chronic renal diseases,1,2 but the incidence rate of glucocorticoid-induced osteoporosis is approximately 50% in patients treated for 6 months or longer.3,4 In children, not only osteoporosis but also growth retardation occurred with chronic glucocorticoid therapy.1,5 Bone mass increases dramatically during childhood and adolescence, peaking in young
a Assistant professor, Division of Developmental Stomatognathic Function Science, Department of Growth and Development of Functions, Kyushu Dental College, Kitakyushu, Japan. b Assistant professor, Division of pediatric dentistry, Department of Human Development and Fostering, Meikai University, Saitama, Japan. c Assistant professor, Division of Developmental Stomatognathic Function Science, Department of Growth and Development of Functions, Kyushu Dental College, Kitakyushu, Japan. d Professor and chair, Division of Developmental Stomatognathic Function Science, Department of Growth and Development of Functions, Kyushu Dental College, Kitakyushu, Japan. The authors report no commercial, proprietary, or financial interest in the products or companies described in this article. Reprint requests to: Kenshi Maki, Division of Developmental Stomatognathic Function Science, Department of Growth and Development of Functions, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Japan 803-8580; e-mail, [email protected]. Submitted, January 2009; revised and accepted, May 2009. 0889-5406/$36.00 Copyright Ó 2011 by the American Association of Orthodontists. doi:10.1016/j.ajodo.2009.05.028
adulthood (the peak bone mass6) plateaus, and finally declines. The bone that must last a lifetime is made between the ages of 10 and 18 years. Patients whose bone mass does not reach the peak during childhood because of glucocorticoid-induced osteoporosis are at permanent risk of suffering fractures.7 In oral bone tissues, glucocorticoid affects mandibular bone growth and bone strength in humans and animals.8-11 However, little is known about bone metabolism and bone microarchitecture of the mandible with glucocorticoidinduced osteoporosis in children and adolescents. Furthermore, no guidelines exist for treating glucocorticoid-induced osteoporosis in children. Bisphosphonates, which are widely used to manage adults with osteoporosis as first-line therapeutic agents, are divided into 2 classes according to their chemical structure and mechanism of action.12,13 The nitrogencontaining bisphosphonates, such as alendronate and risedronate, are markedly more potent inhibitors of osteoclastic bone resorption than nonnitrogen-containing bisphosphonates, such as etidronate and clodronate.14 Several recent reports described cases of osteonecrosis of the mandible associated with nitrogen-containing bisphosphonate therapy, and controversy is ongoing regarding this issue.15,16 A few clinical studies have indicated that bisphosphonate therapy inhibited loss of long bones in children with osteogenesis imperfecta e267
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and glucocorticoid-induced osteoporosis.17,18 However, the effects of nitrogen-containing bisphosphonates on bone structure and bone formation of the mandible in cases of established glucocorticoid-induced osteoporosis during the growth phase are unknown, and the cause of bisphosphonate-induced osteonecrosis of the jaw has not been clarified. Peripheral quantitative computed tomography (pQCT) can be used to measure cortical and trabecular parts separately; this is useful for analyzing bone masses and cross-sectional areas, and also provides volumetric data.19 Recently, microcomputed tomography has been applied to quantify trabecular bone structure, such as trabecular bone volume and trabecular thickness.20 However, in the analyses withy pQCT and microcomputed tomography, we can evaluate static phenomena of bone metabolism but not those associated with bone formation because the data have a temporal component. Both static and dynamic histomorphometry can be performed with 2-dimensional evaluation by using prepared specimens. In this study, we evaluated the effects of risedronate, used frequently for treating osteoporosis, on trabecular and cortical bone density with pQCT, and bone structure and bone metabolism with static and dynamic histomorphometry in the mandibles of growing glucocorticoid-treated rats. MATERIAL AND METHODS
In total, 48 male Wistar rats, aged 5 weeks, were purchased from Charles River Japan (Kanagawa, Japan). The animals were housed individually and maintained under a 12-hour light and 12-hour dark cycle at a constant temperature of 22 C 6 1 C with humidity of 50% 6 5%. The animals were given a standard pelleted chow containing 1.14% calcium and 1.06% phosphorus (CE-2, CLEA Japan, Tokyo, Japan). The animal procedures were all approved by the Committee for the Care and Use of Laboratory Animals of Kyushu Dental College. The animals were divided randomly into 6 groups of 8 each as follows: (1) control 6 weeks; (2) prednisolone (pred) 6 weeks; (3) control 10 weeks; (4) pred 1 saline solution; (5) pred 1 low risedronate (ris); and (6) pred 1 high ris (Table I). In the pred 1 saline and pred 1 ris groups, prednisolone was administered for 6 weeks followed by vehicle or ris for 4 weeks. Prednisolone sodium succinate (Prednine, Shionogi & Co, Osaka, Japan) was administered orally at 30 mg per kilogram per day on alternate days. Risedronate sodium (Ajinomoto, Tokyo, Japan) was administered orally at 0.5 mg per kilogram per day or 1.0 mg per kilogram per starting immediately after the 6-week prednisolone regimen. We compared the control 6 weeks group with the pred
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Table I. Experimental protocol Experimental period Treatment group Control 6 weeks Pred 6 weeks Control 10 weeks Pred 1 saline solution Pred 1 low ris Pred 1 high ris
6 weeks Prednisolone vehicle (saline) Prednisolone 30 mg/kg/2 days Prednisolone vehicle (saline) Prednisolone 30 mg/kg/2 days Prednisolone 30 mg/kg/2 days Prednisolone 30 mg/kg/2 days
4 weeks — — Risedronate vehicle (saline) Risedronate vehicle (saline) Risedronate 0.5 mg/kg/day Risedronate 1.0 mg/kg/day
6 weeks group to confirm the establishment of osteoporosis induced by prednisolone administration. The effects of risedronate treatment were evaluated in the remaining 4 groups. The doses and durations of prednisolone treatment were determined from the results of a previous study that confirmed mandibular bone deterioration.11 The doses and durations of risedronate were determined from a previous study that demonstrated significant inhibition of bone loss in immobilized rats.21 Both compounds were dissolved in sterile saline solution before delivery so that the required dose was in a volume of 10 mL per kilogram of body weight. Prednisolone and vehicle (saline solution) were administered in the morning. Risedronate and vehicle (saline solution) were administered in the afternoon in animals that had fasted for 4 hours previously and 2 hours after risedronate administration. All rats were given free access to water throughout the experimental period.21 Body weight was measured weekly. At the end of the experimental period, all rats were deeply anesthetized with diethyl ether and given a lethal injection of thiamylal natrium (Isozole; Mitsubishi Pharma, Osaka, Japan) intraperitoneally at a dose of 90 mg per kilogram of body weight. For dynamic histomorphometry, all rats were given a subcutaneous injection of calcein (8 mg/kg of body weight; Kanto Chemical, Tokyo, Japan) on days 10 and 3 before they were killed. Bilateral hemi-mandibles were removed from each animal, trimmed of soft tissues, and stored in 70% ethanol. The left hemi-mandible was used for measurement of mandibular bone weight as an index of bone growth and for pQCT analysis. The right hemi-mandible was used for histopathologic evaluation and bone histomorphometry. Bone density (BD; mg/cm3), cross-sectional area (CSA; mm2), and mineral content (MC; mg/mm) of trabecular (Tr) and cortical (Ct) bone from the left
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hemi-mandible were measured by using pQCT (XCT Research SA1 series; Stratec, Medizintechnik, Pforzheim, Germany). For measurement of TrBD, TrCSA, TrBMC, CtBD, CtCSA, and CtBMC, we scanned between the center of the mesial root of the mandibular first molar that contained rich trabecular and cortical bone at 3 positions with an interval of 0.1 mm and measured the cortical component using a voxel size of 0.08 3 0.26 mm. The trabecular region was defined manually, and the cortical region was defined by setting a threshold value of 690 mg per cubic centimeter.11,22 The measurement errors for TrBD, CtBD, TrCSA, and CtCSA were calculated accordingP to the formula of DahlP berg23: Se 5 O d2/2n, where d2 is the sum of the squared differences between pairs of recordings and n is the number of duplicate measurements. The errors were 2.1 mg per cubic centimeter, 1.3 mg per cubic centimeter, 0.17 mm2, and 0.05 mm2, respectively. The trimmed right hemi-mandibles were fixed in 70% ethanol and stained with Villanueva bone stain.24 The bone tissues were then dehydrated in ethanol and embedded in methylmethacrylate. Frontal cross-sections of the mesial root of the mandibular first molar region were cut (300 mm) and polished (20 mm) with a precision bone saw (cutting grinding system, EXAKT Apparatebau, Norderstedt, Germany). The Villanueva-stained specimens were used to assess dynamic histomorphometry. After static and dynamic histomorphometric measurements, each specimen was also stained by using the Goldner method for histopathologic evaluation.25 Images of the mandibular bone were scanned under a light epifluorescence microscope (Carl Zeiss, Thornwood, NY). Histomorphometric parameters were measured at 200-times magnification by using a semiautomatic method (Osteoplan II, Kontron, Munich, Germany).26 Measurement areas are shown in Figure 1. The measured parameters for trabecular bone included total bone tissue (TV), bone volume (BV), bone surface (BS), eroded surface (ES), single-labeled surface (sLS), double-labeled surface (dLS), and the width between double fluorochrome labels (Ir.L.Th). The static histomorphometric parameters were as follows: percentage of trabecular bone volume (BV/ TV; %), trabecular number (Tb.N, 1/mm), trabecular thickness (Tb.Th, mm), trabecular separation (Tb.Sp, mm), and eroded surface (ES/BS, %). The dynamic histomorphometric parameters were as follows: mineralizing surface (MS/BS 5 [dLS1sLS/2]/BS, %), mineral apposition rate (MAR 5 Ir.L.Th/7 days; mm/day), and bone formation rate (BFR/BS 5 MAR3MS/BS30.365; mm3/cm2 per year). The measured parameters for cortical bone were induced periosteal bone surface (perimeter) and the lengths of the sLS and dLS. These data were used to calculate MS/BS, MAR, and BFR/BS. The
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Fig 1. Frontal cross-section in the mandibular first molar region with Villanueva bone staining. Original magnification: 2.5 times. The red line indicates the periosteal surface of cortical bone. The green area indicates the trabecular bone tissue area. Measurements were made of the interior of the trabecular bone tissue area (trabecular bone) and along the periosteal surface (cortical bone).
terminology used is in accordance with the American Society for Bone and Mineral Research Committee on Histomorphometric Nomenclature.27 Statistical analysis
Data are presented as means and standard deviations, and statistical comparisons between the control 6 weeks and the pred 6 weeks groups were performed by using 2-tailed t tests for unpaired samples. Multiple comparisons among the 4 10-week treatment groups were performed by 1-way analysis of variance (ANOVA) with the Tukey method. The level of significance was set at 5%. RESULTS
The body weight and mandibular bone weight in the pred 6 weeks group were significantly lower than those in the control 6 weeks group, and these differences persisted after 4 weeks of treatment with saline solution. However, treatment with risedronate reversed the prednisolone-induced decrease in mandibular bone weight (Table II). The values of TrCSA, TrBMC, CtCSA, and CtBMC in the pred 6 weeks group were significantly lower than those in the control 6 weeks group, and these decreases did not improve after 4 weeks of saline-solution treatment. Although the mean value of CtBD in the pred 6 weeks group was lower than that in the control 6 weeks group, the difference was not significant. TrBD in the pred 6 weeks group was significantly higher than that in the control 6 weeks group. TrBD increased with risedronate in a dose-dependent manner, but lower doses of risedronate showed a tendency to improve TrCSA and TrBMC, and higher doses of risedronate significantly
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Table II. Body weight and mandibular bone weight Pred 1 ris Pred 1 Ris Control 6 weeks Pred 6 weeks Control 10 weeks Pred 1 vehicle (0.5 mg/kg) (1.0 mg/kg) 135.88 6 1.31 136.38 6 2.55 135.83 6 1.92 134.75 6 1.76 Initial body weight (g) 135.18 6 2.89 133.48 6 2.44 Final body weight (g) 407.65 6 11.41 368.63 6 12.09* 422.80 6 11.20 378.63 6 12.46y 384.7 6 17.35y 386.43 6 20.57y Mandibular bone weight (g) 0.43 6 0.02 0.40 6 0.02* 0.52 6 0.02 0.49 6 0.03y 0.51 6 0.02 0.50 6 0.03 Data are expressed as means 6 standard deviations. *P \0.05 vs control 6 weeks group (t test); yP \0.05 vs control 10 weeks group (ANOVA and Tukey test).
Table III. pQCT measurements of trabecular and cortical bone density, cross-sectional area, and mineral content Control 6 weeks Pred 6 weeks Control 10 weeks Pred 1 saline Pred 1 low ris Pred 1 high ris 608.51 6 38.91 646.38 6 35.28* 707.60 6 16.47 685.13 6 28.79 684.73 6 29.32 723.97 6 26.00z,§ Trabecular bone density (mg/cm3) 1.56 6 0.42 1.15 6 0.24* 1.70 6 0.38 1.22 6 0.25y Trabecular bone 1.35 6 0.47 1.19 6 0.23y cross-sectional area (mm2) Trabecular bone mineral 1.01 6 0.27 0.78 6 0.20* 1.20 6 0.26 0.83 6 0.16y 0.92 6 0.30 0.86 6 0.17y content (mg/mm) Cortical bone density (mg/cm3) 1268.86 6 9.48 1261.75 6 11.21 1301.63 6 14.65 1296.55 6 9.15 1301.45 6 8.32 1298.41 6 9.22 6.73 6 0.67 6.08 6 0.63* 7.40 6 0.52 6.74 6 0.32y 7.33 6 0.33z 7.51 6 0.57z Cortical bone cross-sectional 2 area (mm ) Cortical bone mineral 8.54 6 0.87 7.67 6 0.80* 9.62 6 0.64 8.74 6 0.36y 9.55 6 0.41z 9.75 6 0.72z content (mg/mm) Data are expressed as means 6 standard deviations. *P \0.05 vs control 6 weeks group (t test); yP \0.05 vs control 10 weeks group (ANOVA and Tukey test); zP \0.05 vs pred 1 saline group (ANOVA and Tukey test); §P \0.05 vs pred 1 high ris group (ANOVA and Tukey test).
reduced these parameters compared with the control 10 weeks group (Table III). In cortical bone, both doses of risedronate improved the prednisolone-induced decreases in CtCSA and CtBMC (Table IV). Another representative pQCT mandibular bone scan indicated that the trabecular region in the pred 6 weeks group clearly decreased compared with the control 6 weeks group, and risedronate treatment dosedependently reduced the trabecular bone area and increased the mineralization of trabecular bone (Fig 2). The results of trabecular microstructural parameters indicated a significantly higher Tb.Sp in the pred 6 weeks group relative to the control 6 weeks group, and risedronate treatment dose-dependently reduced Tb.Sp and increased Tb.N (Table V). The results of static histomorphometry of trabecular bone indicated that Ob.S/BS, Oc.S/BS, and ES/BS in the pred 6 weeks group significantly decreased compared with the control 6 weeks group (Table V), and osteoblasts with cuboidal morphology were observed in trabecular bone of the pred 6 weeks group (Fig 4). Risedronate decreased Oc.S/BS and ES/BS in a dosedependent manner. Furthermore, ES/BS, Ob.S/BS, MS/ BS, and BFR/BS were significantly lower with risedronate treatment in the control 10 weeks group (Table V). In the double-labeling study, the distance between labels
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narrowed in the risedronate-treated rats (Fig 3), and risedronate treatment altered the morphology of osteoblasts from cuboidal to a more flattened appearance compared with those in the untreated controls (Fig 4). The results of dynamic histomorphometry in cortical bone indicated that MAR and BFR/BS in the pred 6 weeks group significantly decreased compared with the control 6 weeks group, and the mean values for MS/BS and BFR/BS in the pred 1 saline group were lower than those in the control 10 weeks group; these reductions, however, were not statistically significant (Table VI). The distance between double labels was narrowed by prednisolone administration (Fig 5). Risedronate dose-dependently increased MS/BS and BFR/BS, and these values in the pred 1 high ris group were significantly elevated compared with the control 10 weeks level (Table VI). DISCUSSION
Prednisolone clearly reduced body weight and mandibular bone weight; these findings were consistent with those of other studies indicating that glucocorticoid treatment in growing rats reduced body weight and bone growth.11,28 In addition, prednisolone decreased TrCSA and TrBMC and caused deterioration of the connectivity of trabeculae (Tb.Sp). In contrast, TrBD in
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Table IV. Bone histomorphometric analysis of trabecular bone of the mandible: microarchitectural parameters BV/TV (%) Tb.Th (mm) Tb.N (1/mm) Tb.Sp (mm)
Control 6 weeks 73.03 6 7.48 367.32 6 25.22 1.90 6 0.18 149.66 6 11.90
Pred 6 weeks 68.90 6 3.25 398.75 6 18.84 1.64 6 0.17 190.89 6 19.47*
Control 10 weeks 67.37 6 0.33 350.68 6 48.98 1.99 6 0.23 177.87 6 25.72
Pred 1 saline 68.09 6 1.92 347.53 6 46.89 1.80 6 0.22 184.03 6 20.00
Pred 1 low ris 69.89 6 1.27 364.51 6 19.26 1.92 6 0.07 156.80 6 4.47
Pred 1 high ris 70.93 6 2.09 345.54 6 30.62 2.36 6 0.32z 123.33 6 15.82y,z
Data are expressed as means 6 standard deviations. BV/TV, Bone volume fraction (TV, total tissue volume); Tb.Th, trabecular thickness; Tb.N, trabecular number; Tb.Sp, trabecular separation. *P \0.05 vs control 6 weeks group (t test); yP \0.05 vs control 10 weeks group (ANOVA and Tukey test); zP \0.05 vs pred 1 saline group (ANOVA and Tukey test).
Fig 2. Representative pQCT cross-sectional transverse scans of the mandibular first molar region.
the pred 6 weeks rats increased significantly compared with the untreated controls. The major action of glucocorticoids on bone is to reduce osteoblast numbers and function, leading to suppression of bone formation.29 Furthermore, glucocorticoids might function secondarily to increase bone resorption. Glucocorticoids increase the expression of receptor activator of nuclear factor LB ligand (RANK-L) and decrease the expression of its soluble decoy receptor osteoprotegerin (OPG) in stromal and osteoblastic cells.30 Glucocorticoids might also have direct effects on osteoclasts by suppressing the expression of autocrine cytokines, such as interferon b, which normally exerts inhibitory effects on osteoclastogenesis.31
In this study, prednisolone significantly increased bone resorption (Oc.S/BS and ES/BS) but also showed a tendency to increase bone formation (Ob.S/BS and MS/BS). Although this trend was not significant, the differences were inconsistent with those of previous studies. One possible explanation for those observations is that we simply altered a delicate balance between the activities of osteoblasts and osteoclasts, which generally act in tandem to establish an equilibrium between bone buildup and resorption, respectively. Nakamura et al32 reported that activated osteoblasts with a cuboidal shape were often observed near sites of osteoclast resorption in the trabecular bone of OPG–/–mice,
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Table V. Bone histomorphometry of trabecular bone of the mandible: formative and resorptive variables Ob.S/BS (%) MAR (mm/day) MS/BS (%) BFR/BS (mm3/cm2/y) Oc.S/BS (%) ES/BS (%)
Control 6 weeks 6.00 6 3.62 1.69 6 0.14 19.25 6 8.86 14.78 6 4.73 0.21 6 0.18 1.56 6 0.32
Pred 6 weeks 12.66 6 3.62* 1.33 6 0.20 25.67 6 4.07 12.31 6 0.82 1.49 6 0.51* 5.12 6 1.39*
Control 10 weeks 3.19 6 1.47 1.23 6 0.07 5.72 6 0.32 2.56 6 0.23 0.72 6 0.09 2.46 6 0.50
Pred 1 saline 1.20 6 0.30 1.17 6 0.17 3.97 6 2.00 1.63 6 0.63 0.65 6 0.29 3.21 6 0.32
Pred 1 low ris 0.71 6 0.65y 1.30 6 0.58 1.92 6 0.68y 0.94 6 0.51y 0.56 6 0.09 2.01 6 0.46z
Pred 1 high ris 1.06 6 0.38y 0.94 6 0.03 3.94 6 0.20 1.36 6 0.11y 0.35 6 0.10 1.09 6 0.17y,z
Data are expressed as means 6 standard deviations. *P \0.05 vs control 6 weeks group (t test); yP \0.05 vs control 10 weeks group (ANOVA and Tukey test); zP \0.05 vs pred 1 saline group (ANOVA and Tukey test).
Fig 3. Fluorescent micrographs showing double-labeled mineralization in the trabecular bone of the mandible. Arrows indicate calcein that was taken into the bone formation area. Original magnification: 200 times.
indicating that bone formation is tightly coupled with bone resorption. In this study, the presence of similar formative osteoblasts in the trabecular bone from the pred 6 weeks rats suggested that prednisolone-treated growing rats tend to exhibit higher turnover rates in trabecular bone. Furthermore, we used pQCT, which
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measures the degree of bone mineralization.33 We considered that the increase in TrBD by prednisolone could be explained in terms of increased mineralization by prednisolone at sites where osteoblastic bone formation was most active in trabecular regions. The decreases in TrCSA and TrBMC by prednisolone, however, could
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Fig 4. Histologic evaluations of trabecular bone of the mandible. Original magnification: 400 times. Arrows indicate osteoblasts along the bone surface.
have been the result of increased bone resorption by prednisolone, which in turn accelerated compensatory increases in bone formation. In a previous study, chronic glucocorticoid treatment increased the rate of orthodontic tooth movement in rats and suggested that the orthodontic force level should be reduced in patients receiving chronic glucocorticoid treatment.34 This might be due to the increase in bone resorption and the deterioration of the trabecular architecture by glucocorticoid treatment. In cortical bone regions, prednisolone decreased CtCSA and CtBMC by suppressing the BFR/BS, whereas the bone turnover rate had little effect on CtBD. This suppression caused a decrease in the MAR, generally considered to be an indicator of cellular activity. Thus, a unique suppressive effect of prednisolone on bone formation was found for the periosteal surface in the mandible. These results were consistent with those of recent clinical studies indicating that glucocorticoid treatment has little or no effect on bone mineral density despite an increased fracture risk, and that the cause of glucocorticoid-induced bone deterioration is the decrease in bone quality, such as bone geometry and bone metabolism.35,36
In this study, risedronate treatment was started after 6 weeks of prednisolone administration. In many studies, however, both glucocorticoid and bisphosphonate had been given for the whole experimental period to evaluate the preventive effects of bisphosphonate on glucocorticoid-induced deterioration of bone structure in animals.28,37,38 However, little is actually known regarding whether risedronate improves the bone mass of the mandible or whether sufficient peak bone mass can be accumulated with glucocorticoid administration. In addition, few studies have examined the effects of risedronate on mandibular bone formation in young animals. Therefore, prednisolone was not given for the whole 10-week experimental period to investigate the effects of risedronate treatment alone on prednisoloneinduced osteoporosis of the mandible in young rats. Risedronate treatment improved the deterioration of mandibular bone growth induced by prednisolone. However, differential effects of risedronate on bone formation were seen between trabecular and cortical bone. Risedronate dose-dependently reduced bone resorption (Oc.S/BS and ES/BS). With regard to bone formation, the mean values of Ob.S/BS, BFR/BS, and MS/BS in the risedronate treatment groups were lower than those
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Table VI. Bone histomorphometry of cortical bone of the mandible: formative variables MAR (mm/day) MS/BS (%) BFR/BS (mm3/cm2/y)
Control 6 weeks 3.20 6 0.22 65.53 6 0.87 76.66 6 4.86
Pred 6 weeks 2.68 6 0.24* 60.74 6 7.58 59.32 6 3.63*
Control 10 weeks 2.25 6 0.02 46.13 6 4.38 37.86 6 3.98
Pred 1 saline 2.34 6 0.22 37.55 6 2.31 32.17 6 5.02
Pred 1 low ris 2.17 6 0.08 54.43 6 7.63 42.96 6 4.59
Pred 1 high ris 2.36 6 0.43 67.78 6 3.64y,z 58.22 6 7.61y,z
Data are expressed as means 6 standard deviations. *P \0.05 vs control 6 weeks group (t test); yP \0.05 vs control 10 weeks group (ANOVA and Tukey test); zP \0.05 vs pred 1 saline group (ANOVA and Tukey test).
Fig 5. Fluorescent micrographs showing double-labeled mineralization in the periosteal surface of cortical bone. Arrows indicate calcein that was taken into the bone formation area. Original magnification: 200 times.
in the saline-treatment group, suggesting that risedronate tended to decrease bone formation. Furthermore, risedronate treatment altered the morphology of osteoblasts from cuboidal to flat as in bone-lining cells. These results were similar to those of 2 previous studies indicating that risedronate reduced bone formation in ovariectomized rats39 and caused dedifferentiation of active osteoblasts to flatter cells, more typical of bone lining cells in OPG–/–mice.32 Furthermore, risedronate
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dose-dependently increased TrBD and formed dense trabecular bone micro-architecture by decreasing bone turnover, but the higher dose of risedronate did not improve TrCSA or TrBMC. Therefore, we postulated that bisphosphonate-induced osteonecrosis of the jaw might have been due to excessive doses of bisphosphonates that suppressed trabecular bone turnover, which reduced new trabecular bone tissue formation and caused the accumulation of old trabeculae, although no gross
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histopathologic findings of osteonecrosis were found in the rat mandibles. Two experimental studies with small animals also showed the positive effects of bisphosphonates on bone healing after tooth movement or distraction osteogenesis. Adachi et al40 reported that risedronate inhibited the relapse of moved teeth by inhibiting alveolar bone resorption in rats, and Tekin et al41 reported that alendronate increased bone mineral density and accelerated bone healing on distraction osteogenesis of rabbit mandibles. In a study of long-term bisphosphonate therapy, Odvina et al42 discussed the possible adverse effects of long-term alendronate therapy for patients with postmenopausal osteoporosis who sustained spontaneous nonvertebral fractures; some showed delayed healing and histologic evidence of a marked reduction in bone turnover. These findings suggest that short-term bisphosphonate treatment did not prevent healing of the mandible after orthodontic and surgical treatment, although longterm bisphosphonate treatment might cause detrimental changes in bone. Because the prevalence of osteonecrosis is more frequent in the jaws, we believe that the osteonecrosis is caused by other factors that are specific for jaw bones, in addition to the marked suppression of bone turnover. Interestingly, Rizzoli et al43 reported that previous histologic studies on bisphosphonate-induced osteonecrosis of the jaw all showed pronounced inflammatory changes. In addition, patients administered glucocorticoids show increased susceptibility to infectious diseases because glucocorticoids have a powerful immunosuppressive effect.5 Therefore, osteonecrosis of the jaw can occur more frequently when bisphosphonates are given with glucocorticoids because bacterial infection leads to exacerbation of inflammation in the mandible. However, few studies have been conducted regarding the relationship between the analysis of bacteria, such as those involved in periodontal disease and osteonecrosis of the jaw. Further studies are needed to determine whether anaerobic bacteria are involved in these effects. In cortical bone, risedronate inhibited prednisoloneinduced decreases in CtCSA and CtBMC. Furthermore, risedronate treatment increased MS/BS and BFR/BS. Consistent with our findings, bisphosphonate counteracted glucocorticoid-induced bone loss of longitudinal bones in rats.28 In contrast, risedronate suppressed periosteal osteoblast activity independent of resorption in rats’ longitudinal bones.44 Several studies have suggested that bisphosphonates have a stimulatory effect on osteoblast proliferation and differentiation in vitro.45-47 However, there is little evidence to indicate that risedronate increases bone formation in vivo.
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Therefore, whether bisphosphonates stimulate or suppress osteoblast proliferation and differentiation, or both, is not yet clear, or whether the balance between proliferation and differentiation is just sensitive, similar to that between formation and resorption. A possible explanation for our findings is that the increase in sLS was greater than the decrease in dLS with risedronate in the bones of growing rats. Risedronate, however, had no significant effect on CtBD. Consistent with our findings, risedronate prevented bone density loss of the femoral distal metaphysis without affecting bone density of the femoral diaphysis in sciatic neurectomized rats.48 Our findings suggest that risedronate had a positive effect on bone geometry but not on cortical bone density of growing rats. CONCLUSIONS
Risedronate improved prednisolone-induced retardation of trabecular and cortical bone growth, but the bone turnover in these 2 sites is regulated differently in the growing rat’s mandible. In trabecular bone, risedronate improved the prednisolone-induced decrease in bone cross-sectional area and bone mineral content, and increased bone density by suppressing bone turnover. However, in cortical bone, risedronate increased the mineralizing surface and improved prednisoloneinduced decreases in bone cross-sectional area and bone mineral content with no effect on bone density. We thank Kiichi Nonaka (Elk Corporation Research Laboratory, Tokyo, Japan) and Hisashi Murayama (Kureha Special Laboratory, Tokyo, Japan) for technical assistance in the bone histomorphometry and helpful discussions. REFERENCES 1. Burnham JM, Shults J, Petit MA, Semeao E, Beck TJ, Zemel BS, et al. Alterations in proximal femur geometry in children treated with glucocorticoids for Crohn disease or nephrotic syndrome: impact of the underlying disease. J Bone Miner Res 2007;22: 551-9. 2. Falcini F, Trapani S, Civinini R, Capone A, Ermini M, Bartolozzi G. The primary role of steroids on the osteoporosis in juvenile rheumatoid patients evaluated by dual energy x-ray absorptiometry. J Endocrinol Invest 1996;19:165-9. 3. Reid IR. Glucocorticoid-induced osteoporosis: assessment and treatment. J Clin Densitom 1998;1:65-73. 4. van Staa TP, Leufkens HG, Abenhaim L, Zhang B, Cooper C. Oral corticosteroids and fracture risk: relationship to daily and cumulative doses. Rheumatology (Oxford) 2000;39: 1383-9. 5. Mushtaq T, Ahmed SF. The impact of corticosteroids on growth and bone health. Arch Dis Child 2002;87:93-6. 6. Osteoporosis prevention, diagnosis, and therapy. NIH Consens Statement 2000;17:1-45.
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26. Manaka RC, Malluche HH. A program package for quantitative analysis of histologic structure and remodeling dynamics of bone. Comput Programs Biomed 1981;13:191-201. 27. Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, et al. Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee. J Bone Miner Res 1987; 2:595-610. 28. Iwamoto J, Seki A, Takeda T, Sato Y, Yamada H, Shen CL, et al. Preventive effects of risedronate and calcitriol on cancellous osteopenia in rats treated with high-dose glucocorticoid. Exp Anim 2006;55:349-55. 29. O’Brien CA, Jia D, Plotkin LI, Bellido T, Powers CC, Stewart SA, et al. Glucocorticoids act directly on osteoblasts and osteocytes to induce their apoptosis and reduce bone formation and strength. Endocrinology 2004;145:1835-41. 30. Canalis E, Bilezikian JP, Angeli A, Giustina A. Perspectives on glucocorticoid-induced osteoporosis. Bone 2004;34:593-8. 31. Takuma A, Kaneda T, Sato T, Ninomiya S, Kumegawa M, Hakeda Y. Dexamethasone enhances osteoclast formation synergistically with transforming growth factor-beta by stimulating the priming of osteoclast progenitors for differentiation into osteoclasts. J Biol Chem 2003;278:4467-74. 32. Nakamura M, Udagawa N, Matsuura S, Mogi M, Nakamura H, Horiuchi H, et al. Osteoprotegerin regulates bone formation through a coupling mechanism with bone resorption. Endocrinology 2003;144:5441-9. 33. Rosen HN, Chen V, Cittadini A, Greenspan SL, Douglas PS, Moses AC, et al. Treatment with growth hormone and IGF-I in growing rats increases bone mineral content but not bone mineral density. J Bone Miner Res 1995;10:1352-8. 34. Kalia S, Melsen B, Verna C. Tissue reaction to orthodontic tooth movement in acute and chronic corticosteroid treatment. Orthod Craniofac Res 2004;7:26-34. 35. Kumagai S, Kawano S, Atsumi T, Inokuma S, Okada Y, Kanai Y, et al. Vertebral fracture and bone mineral density in women receiving high dose glucocorticoids for treatment of autoimmune diseases. J Rheumatol 2005;32:863-9. 36. Van Staa TP, Laan RF, Barton IP, Cohen S, Reid DM, Cooper C. Bone density threshold and other predictors of vertebral fracture in patients receiving oral glucocorticoid therapy. Arthritis Rheum 2003;48:3224-9. 37. Dalle Carbonare L, Bertoldo F, Valenti MT, Zordan S, Sella S, Fassina A, et al. Risedronate prevents the loss of microarchitecture in glucocorticoid-induced osteoporosis in rats. J Endocrinol Invest 2007;30:739-46. 38. Wimalawansa SJ, Simmons DJ. Prevention of corticosteroidinduced bone loss with alendronate. Proc Soc Exp Biol Med 1998;217:162-7. 39. Hunziker J, Wronski TJ, Miller SC. Mandibular bone formation rates in aged ovariectomized rats treated with anti-resorptive agents alone and in combination with intermittent parathyroid hormone. J Dent Res 2000;79:1431-8. 40. Adachi H, Igarashi K, Mitani H, Shinoda H. Effects of topical administration of a bisphosphonate (risedronate) on orthodontic tooth movements in rats. J Dent Res 1994;73:1478-86. 41. Tekin U, T€ uz HH, Onder E, Ozkaynak O, Korkusuz P. Effects of alendronate on rate of distraction in rabbit mandibles. J Oral Maxillofac Surg 2008;66:2042-9. 42. Odvina CV, Zerwekh JE, Rao DS, Maalouf N, Gottschalk FA, Pak CY. Severely suppressed bone turnover: a potential complication of alendronate therapy. J Clin Endocrinol Metab 2005; 90:1294-301.
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43. Rizzoli R, Burlet N, Cahall D, Delmas PD, Eriksen EF, Felsenberg D, et al. Osteonecrosis of the jaw and bisphosphonate treatment for osteoporosis. Bone 2008;42:841-7. 44. Iwata K, Li J, Follet H, Phipps RJ, Burr DB. Bisphosphonates suppress periosteal osteoblast activity independently of resorption in rat femur and tibia. Bone 2006;39:1053-8. 45. von Knoch F, Jaquiery C, Kowalsky M, Schaeren S, Alabre C, Martin I, et al. Effects of bisphosphonates on proliferation and
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