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Effect of Alveolar Bone Mass on Mechanical Stress in Calcium-sufficient and -deficient Rats
J. Oral Biosci. 51 （1）：11−22, 2009
ORIGINAL
Effect of Alveolar Bone Mass on Mechanical Stress in Calcium−sufficient and −deficient Rats Yuko Uozumi1）§and Shinji Shimoda2） Departments of 1）Orthodontics and 2）Anatomy−1, School of Dental Medicine, Tsurumi University, 2−1−3 Tsurumi, Yokohama, Kanagawa 230−8501, Japan 〔Received on August 29, 2008；Accepted on December 14, 2008〕 Key words：bone mass／mechanical stress／alveolar bone／femur／calcium−deficient Abstract：It is clinically important to check whether sufficient supporting bone mass is maintained in subjects with a low calcium diet. In this study, alveolar and femoral bone mass were measured after tooth movement in calcium−sufficient and −deficient rats, and the correlation between mechanical stress and bone mass was examined using the serum level and histological observation. Seventy rats were divided into 2 groups：normal（NDG）and low calcium diet（LCG）groups. After feeding for 28 days, the distance of tooth movement and the alveolar and femoral bone mass were observed on days 1, 3, 5 and 7 after fitting the appliance. Tooth movement distance was large in LCG. Alveolar bone mass in NDG and LCG showed a different tendency than tooth movement；the former was increased and the latter was decreased. Body weight decreased from the start of the experiment until day 3 in both groups, and increased gradually thereafter. Femoral bone mass in NDG slightly decreased and then recovered at day 7, but such a result was not observed in LCG. These results suggest that the tooth was able to move when an orthodontic force was applied in rats, even in cases of low alveolar bone mass, although various physical influences were present.
Introduction During the last few decades, numerous studies have been conducted on bone and its calcium metabolism. These studies revealed the significance of strict regulation of the total serum calcium level and related hormones in the function and cell differentiation of osteoblasts during osteogenesis. The findings have significantly improved our understanding of many clinical bone diseases, such as osteoporosis. On the other hand, since the 1990s, much work has been done with regard to bone mass, bone mineral §
Corresponding author E−mail：uozumi−yuko@tsurumi−u.ac.jp
density and／or biochemical markers of circulating body fluids1―8）to diagnose osteoporosis and predict its resultant effects. These studies suggested that this phenomenon was the result of the physical system’s ability to adapt to mechanical stress. Investigations involving subjects with osteoporosis have been conducted in the dental field, particularly in orthodontics, because the remodeling of cranio− facial bones and calcium homeostasis are essential for tooth movement. Many orthodontics−related experimental studies therefore have been performed to clarify the relationship between orthodontic tooth movement and a low calcium diet9―13）. These experimental studies also elucidated the mechanism of bone remodeling resulting from orthodontic tooth movement in cases of low alveolar bone mass；however, it is clini-
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Y. Uozumi and S. Shimoda：Bone Mass and Mechanical Stress
cally important to check whether sufficient supporting bone mass is maintained in subjects with a low calcium diet. In fact, quantitative analysis of the bone mass in cases of osteoporosis might be useful for clinical diagnosis before orthodontic tooth movement. Although many experimental studies have been performed to elucidate the mechanism of bone remodeling, the changes in bone volume before and after tooth movement have not been clarified. The porous structure and complicated morphology of the alveolar bone, which make it difficult to analyze, may explain the small number of studies in the orthodontic field. Recently, three−dimensional and quantitative analysis with the aid of computed tomography has been employed for bone and／or muscle volume measurements14―18）. This is one of the suitable methods to analyze the variance of alveolar bone volume. The accuracy of the volume measuring method using CT has been reported previously19―21）. Basic experimental studies which evaluate alveolar bone mass before and after orthodontic tooth movement in rats fed a low calcium diet are required and it remains to be determined whether tooth movement can affect bone mass in other areas by way of calcium metabolism. Since orthodontic tooth movement is considered form of mechanical stress, it is reported that the decrease in bone mass correlates with lower mechanical stress22―24）. Against this background, we quantitatively investigated bone mass using micro−CT before and after orthodontic tooth movement in the alveolar bone and femur in both normal and low calcium diet−fed rats while monitoring serum Ca, P and ALPase levels, and discussed the correlation between mechanical stress and bone mass in both calcium−sufficient and −deficient rats.
mental period（CLEA Japan, Inc., Tokyo, Japan）. After feeding for 28 days, the appliance was fixed and tooth movement and observations were made on days 1, 3, 5 and 7. Lee et al.24）reported that remarkable bone resorption from the marrow side of the alveolar bone and a decrease in the thickness of the cortical bone was seen at day 28 with the low calcium diet. In this study, therefore, the rats were fed a low calcium diet for 28 days and tooth movement was then initiated. Okumura’s method25） was employed for tooth movement. An orthodontic metal mesh band was made for the maxillary incisor of the rats. The main wire used was 0.7 mm in diameter（Dentsply−Sankin, Tokyo）and a wire spring was bent into a helical loop （diameter 0.35 mm, Dentaurum）for tooth movement and soldered as shown in Fig. 1A. The wire spring was fixed by carving a guiding groove to prevent it from detaching（Fig. 1B）. A force of about 15 g was applied to the buccal side of the right first molar, and it was moved to the palatal side. This force is thought to be too heavy for the rat. The molar was moved for 1, 3, 5 and 7 days. Acrylic resin cement（Super−Bond, Sun Medical, Co., Ltd.）was used to install the orthodontic mesh band. Inflammation was often seen in the region due to irritation from the wire when the wire was adjacent to the cervical area. Animals that exhibited inflammation were excluded from the experiment. Tooth movement was measured from the palatal cusps of the right and left first molars with micrometer calipers, and the mean value was calculated in each experiment period.
Materials and Methods
3 ．Measurement of serum elements Seven animals were used for each experimental period for the measurement of serum Ca, P, and ALPase levels. The rats were anesthetized with ether and 4−mL blood samples were obtained by direct heart puncture. The blood serum was separated by centrifuging at 2,500 rpm for 12 min. Serum Ca, P and
1 ．Materials Seventy adult male Wistar strain rats（8 weeks old） with an average weight of 245.82±13.3 g were used in this study. They were divided into 2 groups and fed either a normal diet（NDG；Ca：1.05％）or a low calcium diet（LCG；Ca：0.005％）throughout the experi-
2 ．Measurement of body weight Body weights were measured once a week for 28 days before the appliance was fixed, and monitored every day after the appliance had been fixed.
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solution（pH 7.4, 4℃）for about 4 weeks, dehydrated in a graded alcohol series, and embedded in paraffin. Bucco−lingual serial sections of thickness 5 μm were prepared for hematoxylin and eosin （HE） staining. 2 ）Micro−CT scan Following dissection and fixation with 10％ neutral formalin of the samples, the maxilla, including the right first molar, and femur were scanned using micro−CT （MCT−100 MFZ, Hitachi Medical Co., Tokyo, Japan）with operating exposure parameters of 50 kV and 0.1 mA and slice thickness 26 μm and 13 μm in order to obtain appropriate sections.
Fig. 1 Schema of tooth movement appliance A ：Appliance. The right first molar was moved towards the palatal side. M1, first molar, M2, second molar, M3；third molar. B ：Observation area in histological sections. The wire spring was fixed by carving a guiding groove to prevent it from detaching. P, Palatal；B, Buccal；Arrow, Direction of force
ALPase levels were measured by the OCPC method, by the molybdic acid direct technique, and by the method of JSCC standardization, respectively. 4 ．Sample analysis 1 ）Histological and histo−chemical observations The rats were anesthetized with ether and then perfused through the left ventricle with 0.1 M phosphate buffer solution for about 2 min, followed by the fixative for 20 min. The fixative solution consisted of 4％ paraformaldehyde in 0.1 M phosphate buffer solution, pH 7.4. After perfusion, the maxilla and femur were removed. The maxilla was divided along the median palate. The specimens were then immersed in fresh fixative solution overnight. After fixation, the specimens were decalcified with 10％ EDTA−Na2
5 ．Measurement of bone mass In the actual experiment, we employed micro−CT to measure bone mass, because it was considered an ；however, there are very few accurate method19,20） micro−CT geometrical techniques for volume measurement21）. The geometrical measurement of both alveolar and femoral bone mass was as follows. 1 ）Alveolar bone The corrected reconstructed micro−CT data were stored in DICOM （digital imaging and communications in medicine）files and 3D images were then constructed from the original reconstructed （i. e., DICOM）data with the aid of Zed View（LEXI, Tokyo, Japan） and ExaVision Lite （ZIOSOFT, Inc., Tokyo, Japan） software. For bone volume measurements, common binary values were used in reconstructed original slice DICOM data sets to extract the alveolar bone area with aid of software Zed View. Following the 3D construction of the alveolar bone, including the tooth, the geometrical measurement was developed to divide the maxillary alveolar bone from the upper right first molar with RapidformTM2004 software（The Standard Software for 3D Scanners, INUS Technology, Inc., Korea）. The details of geometrical points26）and planes are described in Fig. 2. The reconstructed image was cut out according to the plane of（Ⅰ）−（Ⅴ）, and tooth regions were then removed. After this procedure, the bone mass and
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Y. Uozumi and S. Shimoda：Bone Mass and Mechanical Stress
Fig. 2 Measurement method of alveolar bone mass The measured alveolar bone on 3D images was processed and cut according to［a］ through［g］ . Standardized points and planes are as follows： Point A, paracone；Point B, disto−palatal root apex；Point C, disto−buccal root apex；Point D, mesio−palatal root apex；Point E, anterocone；Point F, metacone；Point G, mesial root of mesial side uppermost alveolar crest；Point H, bucco−distal root of distal side uppermost alveolar crest. 1 , plane passing through point A, B and C；Plane 2 , plane passing through Plane 3 , plane passing through point A, E and F. point A, B and D；Plane 1 on the mesial side and intersecting point Mesial plane（Ⅰ） , translation of plane 1 on the distal side and intersecting G；Distal plane（Ⅱ） , translation of plane 2； point H；Palatal plane（Ⅲ） , translation on the palatal side 1.4 mm from plane 2 ；Lower Buccal plane（Ⅳ） , translation on the buccal side 2.2 mm from plane 3 and intersected point G. base plane（Ⅴ）, translation from plane Mesiodistal distance, minimum distance between mesial plane（Ⅰ）and distal plane （Ⅱ） .
mesiodistal distance of alveolar bone were measured with software measuring tools. Since the longitudinal size of each sample might be different, the alveolar bone mass was represented as the ratio of the volume of alveolar bone to mesiodistal distance （Vol／Ave. Dis）；defined in standardized points and planes in Fig. 2. 2 ）Femur The reconstructed micro−CT data of the middle region of the femur were stored in DICOM format,
and then the volume and distance were measured by constructing 3D images. Average diameters of horizontal femur sections were measured in eight directions for each image. Femoral bone mass was represented as the ratio of femur volume to average diameter（Vol／Ave. Dia）. 6 ．Statistical analysis The chronological changes in tooth movement, bone mass, and body weight were assessed by two− way ANOVA. Serum level values were analyzed by
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Fig. 4 Quantity of tooth movement A significant difference, ＊p＜0.05 was observed between NDG and LCG on day 7.
ence of feeding and time by two−way ANOVA（p＜ 0.05） .
Fig. 3 Body weight change A ：Body weight change before starting tooth movement for 28 days. B ：Body weight change until day 7 of tooth movement. No significant difference between groups（p ＜0.05）.
2 ．Amount of tooth movement Considerable tooth movement was initially observed in LCG. A significant difference in the amount of tooth movement was observed（p＜0.05）in LCG and NDG 7 days after tooth movement（Fig. 4）. Statistically significant differences were observed in the amount of tooth movement with time by two−way ANOVA. Furthermore, alternate interactions were observed in the difference of feeding and time.
Results
3 ．Histological observation 1 ）Before tooth movement （after 28 days of feeding） In LCG, differences in the size of medullary cavities were observed in the alveolar bone and resorption from the marrow side was marked. In comparison with NDG, the periodontal ligament fibers were scattered and irregular. The osteoblasts and fibroblasts exhibited atrophy and shrinkage, and bone cavities were also enlarged. In addition, the bundle bone was thin and more irregular than in NDG（Fig. 5）.
1 ．Body weight The body weights of both NDG and LCG increased gradually until the start of tooth movement；however, the decrease started and stopped 3 days after placing the appliance, and then started to rebound or was maintained at the same level until the end of the experiment（Fig. 3）. Alternate interactions were observed in the differ-
2 ）After tooth movement i ）NDG In NDG, the extension of some periodontal ligament fibers was observed, the surface of the alveolar bone which faced the periodontal ligament was smooth and the thickness of the bundle bone remained constant on day 1 of tooth movement. On days 3 and 5, significant extension of the periodontal
one−way ANOVA followed by Tukey’s test. Two−way ANOVA was used to investigate the co−effect of each LCG and NDG with variance. Differences were considered statistically significant at p＜0.05. These analyses were carried out using SPSS（SPSS, Tokyo, Japan）.
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Y. Uozumi and S. Shimoda：Bone Mass and Mechanical Stress
ligament was observed, which was maintained until day 7. Alveolar bone, facing the periodontal ligament, exhibited a rough surface （Fig. 6, NDG）；thus, changes in the periodontal ligament space by tooth movement were observed. Although the three−dimensional bone volume changes were not difficult to estimate, there was almost no significant difference in H. E.-stained sections.
according to tooth movement are shown in Fig. 8B. Femoral bone mass of LCG was approximately 80％ that of NDG throughout most of the experiment. It did not change significantly, although a small increase was observed；it was 77％ that of NDG at the beginning of tooth movement, increased to 82％ by day 3, and then decreased to 79％ toward the end of the experiment.
ii ）LCG In LCG, extension of the periodontal ligament was observed, as in NDG；however, further bone resorption from the marrow side was observed on day 3 and 5. Slightly thickened cortical bone was observed on 6, LCG）. day 7（Fig.
6 ．Serum Ca, P and ALPase levels Serum Ca and P levels did not significantly differ between the two groups throughout the experimental period. LCG showed a high value for serum ALPase with a significant difference after placing the appliance compared with NDG. The serum Ca level did not change significantly in either group；however, a slight decrease was observed on day 5 and 7 of tooth movement in LCG, and a significant difference was observed between day 3 and 7 （p＜0.05）. Furthermore, the difference between serum Ca levels before tooth movement and （Fig. 9−Ca）. on day 5 in NDG was significant（p＜0.05） The serum P level of NDG decreased significantly compared to the values before and after placing the appliance and the serum P level of LCG had also slightly decreased by day 3 of tooth movement（p＜ 0.05）（Fig. 9−P）. Regarding the serum ALPase level, although LCG showed quite high activity in comparison with NDG, both groups showed a decrease in activity at the beginning of tooth movement；the lowest value was seen on days 3 or 5, and the value had risen by day 7 . （Fig. 9−ALP）
4 ．Alveolar bone mass The daily change in alveolar bone mass tended to increase slightly at the beginning of the experiment （days 1 to 3 after the start of tooth movement）and reached baseline levels toward day 7 in NDG；however, LCG showed the opposite pattern：a decreasing tendency until day 5 after placing the appliance fol7A）. lowed by an increase toward day 7（Fig. The difference between two groups was statistically significant at every time point；however, the daily change in alveolar bone mass was not significantly different（p＜0.05）. Bone mass ratios of NDG to LCG in alveolar bone according to tooth movement are shown in Fig. 7B. Alveolar bone mass of LCG was 50 to 70％ that of NDG：at the beginning of tooth movement, alveolar bone mass of LCG was 67％ that of NDG；it then decreased to 53％ by day 3 and increased to 75％ toward the end of the experiment（Fig. 7B） . 5 ．Femoral bone mass In NDG, femoral bone mass slightly decreased on day 1 of tooth movement, but recovery was seen by day 7. In LCG, on the other hand, a significant change in femoral bone mass was not seen （p＜0.05）；it maintained an almost constant value or was slightly increased, though a slight decrease was seen on day 5 （Fig. 8A）. Bone mass ratios of NDG to LCG in the femur
Discussion We regarded orthodontic tooth movement as a form of mechanical stress in the present study. Changes in alveolar and femoral bone mass were observed over time. Additionally, the relationship between the influence of mechanical stress and the time elapsed for each set of analyzed data was examined. The results clearly show that even with low bone mass, such as 70％ normal bone mass, teeth were moved by orthodontic force, and the supporting alveolar bone recov-
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Fig. 5 Histological sections before tooth movement at 28 days. Bone resorption from the marrow side is observed （arrow）. 1a：NDG, 2a：LCG, H. E. staining, Bar represents 0.5 mm 1b and 2b：Higher magnification of periodontal tissue in 1a and 2a. The periodontal ligament fibers are scattered and irregular, and the osteoblasts and fibroblasts exhibit atrophy and shrinkage in LCG（2b） . The bone lacunae are also enlarged in LCG（2b, arrowhead）. B, alveolar bone；PDL, periodontal ligament；D, dentin；b, Bar represents 50 μm
Fig. 6 Histological sections after tooth movement Histological changes in periodontal tissue and its adaptation to tooth movement are clearly observed with time；however, it is difficult to judge the total alveolar bone mass from the histological or two− dimensional views. A thin alveolar bone wall facing the tooth root remains on day 7 in LCG（2d） . 1：NDG, 2：LCG, 1a and 2a：day 1, 1b and 2b：day 3, 1c and 2c：day 5, 1d and 2d：day 7, HE staining, Bar represents 0.5 mm
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Y. Uozumi and S. Shimoda：Bone Mass and Mechanical Stress
Fig. 7 Change in alveolar bone mass After tooth movement, a slight increase in NDG and a decrease in LCG of alveolar bone mass are observed. A ：Daily change in alveolar bone mass. B ：Bone mass ratios of NDG to LCG in alveolar bone. Data were assessed by two−way ANOVA, ＊ p＜0.05.
Fig. 8 Change in femur bone mass After tooth movement, slight decreases are observed in NDG, but not in LCG. A ：Daily change in femur bone mass. B ：Bone mass ratios of NDG to LCG in femur. Data were assessed by two−way ANOVA, ＊ p＜0.05.
ered up to 75％ normal bone mass （i. e., the same mass as before placing the appliance） . Additionally, NDG and LCG showed completely different bone mass tendencies concerning tooth movement. The experimental design of this study was intended to simulate and reproduce a previous osteoporosis experiment9,10,13）. This was considered mostly appropriate to observe the influence of mechanical stress on bone mass because i ）the quantity of tooth movement of NDG as a reaction in an animal experiment was in good agreement with conventional ii ）the large tooth movement initially reports22）, and observed was caused by low bone mass in LCG. Furthermore, the detailed histological observations of hematoxylin−eosin staining were consistent with previous histological descriptions23）related to orthodon-
tic tooth movement. Alveolar bone mass in the NDG showed a slight increase after mechanical stress, while femoral bone mass showed a slight decrease. Regarding the relation between mechanical stress and bone mass, it is generally reported that a decrease in bone mass correlates with lower mechanical stress27―29）. This phenomenon―that is, an increase in bone mass resulting from severe mechanical stress―has also been reported in fracture−related studies1―8）. The observed result in NDG was in good agreement with this phenomenon, as alveolar bone mass showed a slight tendency to increase with added mechanical stress （described in Results）. Furthermore, previous fracture−related studies reported that bone mass decreased in various skeletal
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Fig. 9 Comparison of serum level values Serum Ca and P levels did not significantly differ between groups throughout the experimental period. LCG showed a significantly higher value of serum ALPase after placing the appliance. Data were analyzed by one−way ANOVA followed by Tukey’s test, ＊p＜ 0.05. Ca：Serum Ca, P：Serum P, ALP：Serum ALPase
sites that were far from the applied mechanical stress1,7）. In this study, a similar phenomenon was also observed with a decrease of bone mass in the femur. From the many orthopedic studies related to fracture, it can be assumed that this phenomenon is the result of the physical system’s ability to adapt to mechanical stress. For this reason, studies on fracture prediction in cases of osteoporosis have been actively conducted to assess the biochemical markers of bone metabolism3,4,7）. As the background of these fracture researches, recent fundamental knowledge must be gathered in
relation to the intra− and inter−cell signal system that governs the system controlling physical activity, such as the induction of stress proteins（HSP；heat shock protein）, which has been reported32）as a heat shock response, the hypothalamic−pituitary−adrenal （HPA） system in neuroscience33,34）, and reports on 35） of RANKL（receptor activation of NF−κB ligand） osteoblasts as the bone metabolic control factor. However, LCG rats with low bone volume showed a completely different tendency―that is, a plateau or slight decrease in alveolar bone mass after mechanical stress was applied―in contrast to the slight
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Y. Uozumi and S. Shimoda：Bone Mass and Mechanical Stress
increase observed in the femur. This result was considerably different from NDG. In addition, in the present study, serum calcium, phosphorus ALPase levels and body weight showed quite different results in LCG, as follows： i ）Serum calcium level in LCG was almost the same as that in NDG. ii ）Serum phosphate level in LCG was slightly lower than that in NDG. iii ）Serum ALPase levels were maintained at a higher level in LCG. iv ）Body weight in both LCG and NDG decreased after placing the appliance（from days 1 to 4）；however, it recovered thereafter. The constant serum calcium level, high serum ALPase levels, and the recovery of body weight may indicate that high level bone turnover occurred with the calcium ion supply via feeding, and that the serum calcium level must be maintained prior to adaptation against mechanical stress, even in the case of low bone mass, since one of the most important aspects of bone is the storage and supply of calcium ions for all cell activities. Additionally, previous studies have considered that the reason for weight loss when using the appliance22,36） was eating disorders and mastication dysfunction；however, these results are contradicted by the increase of body weight during days 5―7 of tooth movement, as shown in this study. The increase in body weight should be explained by another physical reaction. The results of the present study suggest that i） for low alveolar bone mass, the bone mass required to support tooth movement may not be available once the mechanical stress is applied；however, ii ）bone mass recovers due to the influence of constant serum calcium levels on mechanical stress, even with low bone mass；and iii ） even bone mass far from the applied mechanical stress may be affected by circulating body fluids or other factors. We conclude, therefore, that tooth movement by orthodontic force was possible even with low alveolar bone mass in rats, although various physical influences were involved. However, for better understanding of the assess-
ment of physical activity in relation to mechanical stress, more basic research is needed not only on calcium metabolism and related factors but also regarding the possible roles played by stress factors in the physical system. Acknowledgements We would like to thank Professors A. Hirashita and K. Kawasaki for their support and encouragement throughout the present study. Part of the study was supported by a grant from the Japanese Ministry of Education, Culture, Sports, Science and Technology （Academic Frontier Project, 2004―2008,“High−Tech Research Center”Project, 2005―2009）. References 1）Cummings, S. R., Black, D. M. and Nevitt, M. C.： Bone density at various sites for prediction of hip fractures. Lancet 341：72―75, 1993. 2）Melton, L. J. Ⅲ., Actinson, E. J., O’Fallon, W. M., Wahner, H. W. and Riggs, B. L.：Long−term fracture prediction by bone mineral density assessed at different skeletal sites. J. Bone Miner. Res. 8：1227― 1233, 1993. 3）Akesson, K., Liunghall, S., Jonsson, B., Sernbo, I., Johnell, O., Gardsell, P. and Obrant, K. J.：Assessment of biochemical markers of bone metabolism in relation to the occurrence of fracture：A retrospective and prospective population−based study of women. J. Bone Miner. Res. 10：1823―1829, 1995. 4）Garnero, P., Hausherr, E., M.−C, Chapuy., Marcelli, C., Grandjean, H., Muller, C., Cormier, C., Breart, G., Meunier, P. J. and Delmas, P. D.：Markers of bone resorption predict hip fracture in elderly women： The EPIDOS prospective study. J. Bone Miner. Res. 11：1531―1538, 1996. 5）Specker, B. L.：Evidence for an interation between calcium intake and physical activity on changes in bone mineral density. J. Bone Miner. Res. 11：1539― 1544, 1996. 6）Ingle, B. M., Hay, S. M., Bottjer, H. M. and Eastell, R.：Changes in bone mass and bone turnover following distal forearm fracture. Osteoporos. Int. 10：399 ―407, 1999. 7）Margareta, H., Jan, S. and Nils, D.：Biochemical bone markers and bone density in hip fracture
21 patients. Acta. Ortho. Scand. 71：409―413, 2000. 8）Sugimoto, H., Yamada, H., Terada, N. and Yamaji, T.： Bone mineral density of the femoral condyle and lumber spine in femoral neck fracture patients. Cent. Jpn. J. Orthop. Traumat. 48：557―558, 2005. 9）Imazu, H.：Histo−biochemical Changes during tooth movement under controlled fed rat. J. Gifu Dent. Soc. 11：401―423, 1984. 10）Itooka, T.：Effect of low−calcium diet on tooth movement in the rat. Bull. Josai Dent. Univ. 16：346― 368, 1987. 11）Ohya, K., Amano, H. and Ogura, H.：Alveolar bone resorption process in molar tooth region in calcium− deficient rats. Bull. Tokyo Med. Dent. Univ. 59：585 ―593, 1992. 12）Oikawa, Y., Yoshida, A., Morioka, T., Lan, H. S., Seino, Y. and Miura, H.：Effects of tooth movement on the alveolar bone in osteoporotic rats. Dent. J. Iwate Med. Univ. 29：93―102, 2004. 13）Mitsuya, K.：The effect of high protein low calcium diet on tooth movement in the rat. J. Jpn. Orthod. Soc. 41：46―70, 1982. 14）Balto, K., Muller, R., Carrington, D. C., Dobeck, J. and Stashenko, P.：Quantification of periapical bone destruction in mice by micro−computed tomography. J. Dent. Res. 79：35―40, 2000. 15）Verna, C., Dalstra, M. and Melsen, B.：The rate and the type of orthodontic tooth movement is influenced by bone turnover in a rat model. Eur. J. Orthod. 22： 343―352, 2000. 16）Toyooka, E., Tanaka, M., Kono, S., Ozawa, H. and Ejiri, S.：Changes of rat alveolar bone following loss of antagonistic tooth and the influence of estrogen deficiency on the changes. Analysis using Micro focus X−Ray Computed Tomography. J. Jpn. Soc. Bone Morpho. 11：53―61, 2001. 17）Irie, K., Sakakura, Y., Tsuruga, E., Hosokawa, Y. and Yajima, T.：Three−dimensional Changes of the Mandible and Alveolar Bone in the Ovariectomized Rat Examined by Micro−focus Computed Tomography. J. Jpn. Assoc. Periodontol. 46：288―293, 2004. 18）Chan, H. P., Zachary, R. A., Mario, T., Qiming, J., Jia, C., Jaclynn, M. K., Steven, A. G. and William, V. G.： Three−dimensional micro−computed tomographic imaging of alveolar bone in experimental bone loss or repair. J. Periodontol. 78：273―281, 2007. 19）Kawahara, H., Shimoda, S., Kobayashi, K. and Kawasaki, K.：Validation Study of 3DI Measurements by Spiral CT. J. Jpn. Soc. Oral Implant. 13：321―327,
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Kinosaki, M., Mochizuki, S., Tomoyasu, A., Yano, K., Goto, M. and Murakami, A.：Osteoclast differentiation factor is a ligand for osteoprotegrin／osteoclastogenesis−inhibitory factor and is identical to TRANCE／RANKL. Proc. Natl. Acad. Sci. USA. 95： 3597―3602, 1998. 36）Sakamoto, R.：Calcium kinetics in the pressure and the tension sides incidental to the tooth movement. Orthod. Waves−Jpn. Ed. 65：1―10, 2006.
ORIGINAL
Effect of Alveolar Bone Mass on Mechanical Stress in Calcium−sufficient and −deficient Rats Yuko Uozumi1）§and Shinji Shimoda2） Departments of 1）Orthodontics and 2）Anatomy−1, School of Dental Medicine, Tsurumi University, 2−1−3 Tsurumi, Yokohama, Kanagawa 230−8501, Japan 〔Received on August 29, 2008；Accepted on December 14, 2008〕 Key words：bone mass／mechanical stress／alveolar bone／femur／calcium−deficient Abstract：It is clinically important to check whether sufficient supporting bone mass is maintained in subjects with a low calcium diet. In this study, alveolar and femoral bone mass were measured after tooth movement in calcium−sufficient and −deficient rats, and the correlation between mechanical stress and bone mass was examined using the serum level and histological observation. Seventy rats were divided into 2 groups：normal（NDG）and low calcium diet（LCG）groups. After feeding for 28 days, the distance of tooth movement and the alveolar and femoral bone mass were observed on days 1, 3, 5 and 7 after fitting the appliance. Tooth movement distance was large in LCG. Alveolar bone mass in NDG and LCG showed a different tendency than tooth movement；the former was increased and the latter was decreased. Body weight decreased from the start of the experiment until day 3 in both groups, and increased gradually thereafter. Femoral bone mass in NDG slightly decreased and then recovered at day 7, but such a result was not observed in LCG. These results suggest that the tooth was able to move when an orthodontic force was applied in rats, even in cases of low alveolar bone mass, although various physical influences were present.
Introduction During the last few decades, numerous studies have been conducted on bone and its calcium metabolism. These studies revealed the significance of strict regulation of the total serum calcium level and related hormones in the function and cell differentiation of osteoblasts during osteogenesis. The findings have significantly improved our understanding of many clinical bone diseases, such as osteoporosis. On the other hand, since the 1990s, much work has been done with regard to bone mass, bone mineral §
Corresponding author E−mail：uozumi−yuko@tsurumi−u.ac.jp
density and／or biochemical markers of circulating body fluids1―8）to diagnose osteoporosis and predict its resultant effects. These studies suggested that this phenomenon was the result of the physical system’s ability to adapt to mechanical stress. Investigations involving subjects with osteoporosis have been conducted in the dental field, particularly in orthodontics, because the remodeling of cranio− facial bones and calcium homeostasis are essential for tooth movement. Many orthodontics−related experimental studies therefore have been performed to clarify the relationship between orthodontic tooth movement and a low calcium diet9―13）. These experimental studies also elucidated the mechanism of bone remodeling resulting from orthodontic tooth movement in cases of low alveolar bone mass；however, it is clini-
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Y. Uozumi and S. Shimoda：Bone Mass and Mechanical Stress
cally important to check whether sufficient supporting bone mass is maintained in subjects with a low calcium diet. In fact, quantitative analysis of the bone mass in cases of osteoporosis might be useful for clinical diagnosis before orthodontic tooth movement. Although many experimental studies have been performed to elucidate the mechanism of bone remodeling, the changes in bone volume before and after tooth movement have not been clarified. The porous structure and complicated morphology of the alveolar bone, which make it difficult to analyze, may explain the small number of studies in the orthodontic field. Recently, three−dimensional and quantitative analysis with the aid of computed tomography has been employed for bone and／or muscle volume measurements14―18）. This is one of the suitable methods to analyze the variance of alveolar bone volume. The accuracy of the volume measuring method using CT has been reported previously19―21）. Basic experimental studies which evaluate alveolar bone mass before and after orthodontic tooth movement in rats fed a low calcium diet are required and it remains to be determined whether tooth movement can affect bone mass in other areas by way of calcium metabolism. Since orthodontic tooth movement is considered form of mechanical stress, it is reported that the decrease in bone mass correlates with lower mechanical stress22―24）. Against this background, we quantitatively investigated bone mass using micro−CT before and after orthodontic tooth movement in the alveolar bone and femur in both normal and low calcium diet−fed rats while monitoring serum Ca, P and ALPase levels, and discussed the correlation between mechanical stress and bone mass in both calcium−sufficient and −deficient rats.
mental period（CLEA Japan, Inc., Tokyo, Japan）. After feeding for 28 days, the appliance was fixed and tooth movement and observations were made on days 1, 3, 5 and 7. Lee et al.24）reported that remarkable bone resorption from the marrow side of the alveolar bone and a decrease in the thickness of the cortical bone was seen at day 28 with the low calcium diet. In this study, therefore, the rats were fed a low calcium diet for 28 days and tooth movement was then initiated. Okumura’s method25） was employed for tooth movement. An orthodontic metal mesh band was made for the maxillary incisor of the rats. The main wire used was 0.7 mm in diameter（Dentsply−Sankin, Tokyo）and a wire spring was bent into a helical loop （diameter 0.35 mm, Dentaurum）for tooth movement and soldered as shown in Fig. 1A. The wire spring was fixed by carving a guiding groove to prevent it from detaching（Fig. 1B）. A force of about 15 g was applied to the buccal side of the right first molar, and it was moved to the palatal side. This force is thought to be too heavy for the rat. The molar was moved for 1, 3, 5 and 7 days. Acrylic resin cement（Super−Bond, Sun Medical, Co., Ltd.）was used to install the orthodontic mesh band. Inflammation was often seen in the region due to irritation from the wire when the wire was adjacent to the cervical area. Animals that exhibited inflammation were excluded from the experiment. Tooth movement was measured from the palatal cusps of the right and left first molars with micrometer calipers, and the mean value was calculated in each experiment period.
Materials and Methods
3 ．Measurement of serum elements Seven animals were used for each experimental period for the measurement of serum Ca, P, and ALPase levels. The rats were anesthetized with ether and 4−mL blood samples were obtained by direct heart puncture. The blood serum was separated by centrifuging at 2,500 rpm for 12 min. Serum Ca, P and
1 ．Materials Seventy adult male Wistar strain rats（8 weeks old） with an average weight of 245.82±13.3 g were used in this study. They were divided into 2 groups and fed either a normal diet（NDG；Ca：1.05％）or a low calcium diet（LCG；Ca：0.005％）throughout the experi-
2 ．Measurement of body weight Body weights were measured once a week for 28 days before the appliance was fixed, and monitored every day after the appliance had been fixed.
13
solution（pH 7.4, 4℃）for about 4 weeks, dehydrated in a graded alcohol series, and embedded in paraffin. Bucco−lingual serial sections of thickness 5 μm were prepared for hematoxylin and eosin （HE） staining. 2 ）Micro−CT scan Following dissection and fixation with 10％ neutral formalin of the samples, the maxilla, including the right first molar, and femur were scanned using micro−CT （MCT−100 MFZ, Hitachi Medical Co., Tokyo, Japan）with operating exposure parameters of 50 kV and 0.1 mA and slice thickness 26 μm and 13 μm in order to obtain appropriate sections.
Fig. 1 Schema of tooth movement appliance A ：Appliance. The right first molar was moved towards the palatal side. M1, first molar, M2, second molar, M3；third molar. B ：Observation area in histological sections. The wire spring was fixed by carving a guiding groove to prevent it from detaching. P, Palatal；B, Buccal；Arrow, Direction of force
ALPase levels were measured by the OCPC method, by the molybdic acid direct technique, and by the method of JSCC standardization, respectively. 4 ．Sample analysis 1 ）Histological and histo−chemical observations The rats were anesthetized with ether and then perfused through the left ventricle with 0.1 M phosphate buffer solution for about 2 min, followed by the fixative for 20 min. The fixative solution consisted of 4％ paraformaldehyde in 0.1 M phosphate buffer solution, pH 7.4. After perfusion, the maxilla and femur were removed. The maxilla was divided along the median palate. The specimens were then immersed in fresh fixative solution overnight. After fixation, the specimens were decalcified with 10％ EDTA−Na2
5 ．Measurement of bone mass In the actual experiment, we employed micro−CT to measure bone mass, because it was considered an ；however, there are very few accurate method19,20） micro−CT geometrical techniques for volume measurement21）. The geometrical measurement of both alveolar and femoral bone mass was as follows. 1 ）Alveolar bone The corrected reconstructed micro−CT data were stored in DICOM （digital imaging and communications in medicine）files and 3D images were then constructed from the original reconstructed （i. e., DICOM）data with the aid of Zed View（LEXI, Tokyo, Japan） and ExaVision Lite （ZIOSOFT, Inc., Tokyo, Japan） software. For bone volume measurements, common binary values were used in reconstructed original slice DICOM data sets to extract the alveolar bone area with aid of software Zed View. Following the 3D construction of the alveolar bone, including the tooth, the geometrical measurement was developed to divide the maxillary alveolar bone from the upper right first molar with RapidformTM2004 software（The Standard Software for 3D Scanners, INUS Technology, Inc., Korea）. The details of geometrical points26）and planes are described in Fig. 2. The reconstructed image was cut out according to the plane of（Ⅰ）−（Ⅴ）, and tooth regions were then removed. After this procedure, the bone mass and
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Y. Uozumi and S. Shimoda：Bone Mass and Mechanical Stress
Fig. 2 Measurement method of alveolar bone mass The measured alveolar bone on 3D images was processed and cut according to［a］ through［g］ . Standardized points and planes are as follows： Point A, paracone；Point B, disto−palatal root apex；Point C, disto−buccal root apex；Point D, mesio−palatal root apex；Point E, anterocone；Point F, metacone；Point G, mesial root of mesial side uppermost alveolar crest；Point H, bucco−distal root of distal side uppermost alveolar crest. 1 , plane passing through point A, B and C；Plane 2 , plane passing through Plane 3 , plane passing through point A, E and F. point A, B and D；Plane 1 on the mesial side and intersecting point Mesial plane（Ⅰ） , translation of plane 1 on the distal side and intersecting G；Distal plane（Ⅱ） , translation of plane 2； point H；Palatal plane（Ⅲ） , translation on the palatal side 1.4 mm from plane 2 ；Lower Buccal plane（Ⅳ） , translation on the buccal side 2.2 mm from plane 3 and intersected point G. base plane（Ⅴ）, translation from plane Mesiodistal distance, minimum distance between mesial plane（Ⅰ）and distal plane （Ⅱ） .
mesiodistal distance of alveolar bone were measured with software measuring tools. Since the longitudinal size of each sample might be different, the alveolar bone mass was represented as the ratio of the volume of alveolar bone to mesiodistal distance （Vol／Ave. Dis）；defined in standardized points and planes in Fig. 2. 2 ）Femur The reconstructed micro−CT data of the middle region of the femur were stored in DICOM format,
and then the volume and distance were measured by constructing 3D images. Average diameters of horizontal femur sections were measured in eight directions for each image. Femoral bone mass was represented as the ratio of femur volume to average diameter（Vol／Ave. Dia）. 6 ．Statistical analysis The chronological changes in tooth movement, bone mass, and body weight were assessed by two− way ANOVA. Serum level values were analyzed by
15
Fig. 4 Quantity of tooth movement A significant difference, ＊p＜0.05 was observed between NDG and LCG on day 7.
ence of feeding and time by two−way ANOVA（p＜ 0.05） .
Fig. 3 Body weight change A ：Body weight change before starting tooth movement for 28 days. B ：Body weight change until day 7 of tooth movement. No significant difference between groups（p ＜0.05）.
2 ．Amount of tooth movement Considerable tooth movement was initially observed in LCG. A significant difference in the amount of tooth movement was observed（p＜0.05）in LCG and NDG 7 days after tooth movement（Fig. 4）. Statistically significant differences were observed in the amount of tooth movement with time by two−way ANOVA. Furthermore, alternate interactions were observed in the difference of feeding and time.
Results
3 ．Histological observation 1 ）Before tooth movement （after 28 days of feeding） In LCG, differences in the size of medullary cavities were observed in the alveolar bone and resorption from the marrow side was marked. In comparison with NDG, the periodontal ligament fibers were scattered and irregular. The osteoblasts and fibroblasts exhibited atrophy and shrinkage, and bone cavities were also enlarged. In addition, the bundle bone was thin and more irregular than in NDG（Fig. 5）.
1 ．Body weight The body weights of both NDG and LCG increased gradually until the start of tooth movement；however, the decrease started and stopped 3 days after placing the appliance, and then started to rebound or was maintained at the same level until the end of the experiment（Fig. 3）. Alternate interactions were observed in the differ-
2 ）After tooth movement i ）NDG In NDG, the extension of some periodontal ligament fibers was observed, the surface of the alveolar bone which faced the periodontal ligament was smooth and the thickness of the bundle bone remained constant on day 1 of tooth movement. On days 3 and 5, significant extension of the periodontal
one−way ANOVA followed by Tukey’s test. Two−way ANOVA was used to investigate the co−effect of each LCG and NDG with variance. Differences were considered statistically significant at p＜0.05. These analyses were carried out using SPSS（SPSS, Tokyo, Japan）.
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Y. Uozumi and S. Shimoda：Bone Mass and Mechanical Stress
ligament was observed, which was maintained until day 7. Alveolar bone, facing the periodontal ligament, exhibited a rough surface （Fig. 6, NDG）；thus, changes in the periodontal ligament space by tooth movement were observed. Although the three−dimensional bone volume changes were not difficult to estimate, there was almost no significant difference in H. E.-stained sections.
according to tooth movement are shown in Fig. 8B. Femoral bone mass of LCG was approximately 80％ that of NDG throughout most of the experiment. It did not change significantly, although a small increase was observed；it was 77％ that of NDG at the beginning of tooth movement, increased to 82％ by day 3, and then decreased to 79％ toward the end of the experiment.
ii ）LCG In LCG, extension of the periodontal ligament was observed, as in NDG；however, further bone resorption from the marrow side was observed on day 3 and 5. Slightly thickened cortical bone was observed on 6, LCG）. day 7（Fig.
6 ．Serum Ca, P and ALPase levels Serum Ca and P levels did not significantly differ between the two groups throughout the experimental period. LCG showed a high value for serum ALPase with a significant difference after placing the appliance compared with NDG. The serum Ca level did not change significantly in either group；however, a slight decrease was observed on day 5 and 7 of tooth movement in LCG, and a significant difference was observed between day 3 and 7 （p＜0.05）. Furthermore, the difference between serum Ca levels before tooth movement and （Fig. 9−Ca）. on day 5 in NDG was significant（p＜0.05） The serum P level of NDG decreased significantly compared to the values before and after placing the appliance and the serum P level of LCG had also slightly decreased by day 3 of tooth movement（p＜ 0.05）（Fig. 9−P）. Regarding the serum ALPase level, although LCG showed quite high activity in comparison with NDG, both groups showed a decrease in activity at the beginning of tooth movement；the lowest value was seen on days 3 or 5, and the value had risen by day 7 . （Fig. 9−ALP）
4 ．Alveolar bone mass The daily change in alveolar bone mass tended to increase slightly at the beginning of the experiment （days 1 to 3 after the start of tooth movement）and reached baseline levels toward day 7 in NDG；however, LCG showed the opposite pattern：a decreasing tendency until day 5 after placing the appliance fol7A）. lowed by an increase toward day 7（Fig. The difference between two groups was statistically significant at every time point；however, the daily change in alveolar bone mass was not significantly different（p＜0.05）. Bone mass ratios of NDG to LCG in alveolar bone according to tooth movement are shown in Fig. 7B. Alveolar bone mass of LCG was 50 to 70％ that of NDG：at the beginning of tooth movement, alveolar bone mass of LCG was 67％ that of NDG；it then decreased to 53％ by day 3 and increased to 75％ toward the end of the experiment（Fig. 7B） . 5 ．Femoral bone mass In NDG, femoral bone mass slightly decreased on day 1 of tooth movement, but recovery was seen by day 7. In LCG, on the other hand, a significant change in femoral bone mass was not seen （p＜0.05）；it maintained an almost constant value or was slightly increased, though a slight decrease was seen on day 5 （Fig. 8A）. Bone mass ratios of NDG to LCG in the femur
Discussion We regarded orthodontic tooth movement as a form of mechanical stress in the present study. Changes in alveolar and femoral bone mass were observed over time. Additionally, the relationship between the influence of mechanical stress and the time elapsed for each set of analyzed data was examined. The results clearly show that even with low bone mass, such as 70％ normal bone mass, teeth were moved by orthodontic force, and the supporting alveolar bone recov-
17
Fig. 5 Histological sections before tooth movement at 28 days. Bone resorption from the marrow side is observed （arrow）. 1a：NDG, 2a：LCG, H. E. staining, Bar represents 0.5 mm 1b and 2b：Higher magnification of periodontal tissue in 1a and 2a. The periodontal ligament fibers are scattered and irregular, and the osteoblasts and fibroblasts exhibit atrophy and shrinkage in LCG（2b） . The bone lacunae are also enlarged in LCG（2b, arrowhead）. B, alveolar bone；PDL, periodontal ligament；D, dentin；b, Bar represents 50 μm
Fig. 6 Histological sections after tooth movement Histological changes in periodontal tissue and its adaptation to tooth movement are clearly observed with time；however, it is difficult to judge the total alveolar bone mass from the histological or two− dimensional views. A thin alveolar bone wall facing the tooth root remains on day 7 in LCG（2d） . 1：NDG, 2：LCG, 1a and 2a：day 1, 1b and 2b：day 3, 1c and 2c：day 5, 1d and 2d：day 7, HE staining, Bar represents 0.5 mm
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Y. Uozumi and S. Shimoda：Bone Mass and Mechanical Stress
Fig. 7 Change in alveolar bone mass After tooth movement, a slight increase in NDG and a decrease in LCG of alveolar bone mass are observed. A ：Daily change in alveolar bone mass. B ：Bone mass ratios of NDG to LCG in alveolar bone. Data were assessed by two−way ANOVA, ＊ p＜0.05.
Fig. 8 Change in femur bone mass After tooth movement, slight decreases are observed in NDG, but not in LCG. A ：Daily change in femur bone mass. B ：Bone mass ratios of NDG to LCG in femur. Data were assessed by two−way ANOVA, ＊ p＜0.05.
ered up to 75％ normal bone mass （i. e., the same mass as before placing the appliance） . Additionally, NDG and LCG showed completely different bone mass tendencies concerning tooth movement. The experimental design of this study was intended to simulate and reproduce a previous osteoporosis experiment9,10,13）. This was considered mostly appropriate to observe the influence of mechanical stress on bone mass because i ）the quantity of tooth movement of NDG as a reaction in an animal experiment was in good agreement with conventional ii ）the large tooth movement initially reports22）, and observed was caused by low bone mass in LCG. Furthermore, the detailed histological observations of hematoxylin−eosin staining were consistent with previous histological descriptions23）related to orthodon-
tic tooth movement. Alveolar bone mass in the NDG showed a slight increase after mechanical stress, while femoral bone mass showed a slight decrease. Regarding the relation between mechanical stress and bone mass, it is generally reported that a decrease in bone mass correlates with lower mechanical stress27―29）. This phenomenon―that is, an increase in bone mass resulting from severe mechanical stress―has also been reported in fracture−related studies1―8）. The observed result in NDG was in good agreement with this phenomenon, as alveolar bone mass showed a slight tendency to increase with added mechanical stress （described in Results）. Furthermore, previous fracture−related studies reported that bone mass decreased in various skeletal
19
Fig. 9 Comparison of serum level values Serum Ca and P levels did not significantly differ between groups throughout the experimental period. LCG showed a significantly higher value of serum ALPase after placing the appliance. Data were analyzed by one−way ANOVA followed by Tukey’s test, ＊p＜ 0.05. Ca：Serum Ca, P：Serum P, ALP：Serum ALPase
sites that were far from the applied mechanical stress1,7）. In this study, a similar phenomenon was also observed with a decrease of bone mass in the femur. From the many orthopedic studies related to fracture, it can be assumed that this phenomenon is the result of the physical system’s ability to adapt to mechanical stress. For this reason, studies on fracture prediction in cases of osteoporosis have been actively conducted to assess the biochemical markers of bone metabolism3,4,7）. As the background of these fracture researches, recent fundamental knowledge must be gathered in
relation to the intra− and inter−cell signal system that governs the system controlling physical activity, such as the induction of stress proteins（HSP；heat shock protein）, which has been reported32）as a heat shock response, the hypothalamic−pituitary−adrenal （HPA） system in neuroscience33,34）, and reports on 35） of RANKL（receptor activation of NF−κB ligand） osteoblasts as the bone metabolic control factor. However, LCG rats with low bone volume showed a completely different tendency―that is, a plateau or slight decrease in alveolar bone mass after mechanical stress was applied―in contrast to the slight
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Y. Uozumi and S. Shimoda：Bone Mass and Mechanical Stress
increase observed in the femur. This result was considerably different from NDG. In addition, in the present study, serum calcium, phosphorus ALPase levels and body weight showed quite different results in LCG, as follows： i ）Serum calcium level in LCG was almost the same as that in NDG. ii ）Serum phosphate level in LCG was slightly lower than that in NDG. iii ）Serum ALPase levels were maintained at a higher level in LCG. iv ）Body weight in both LCG and NDG decreased after placing the appliance（from days 1 to 4）；however, it recovered thereafter. The constant serum calcium level, high serum ALPase levels, and the recovery of body weight may indicate that high level bone turnover occurred with the calcium ion supply via feeding, and that the serum calcium level must be maintained prior to adaptation against mechanical stress, even in the case of low bone mass, since one of the most important aspects of bone is the storage and supply of calcium ions for all cell activities. Additionally, previous studies have considered that the reason for weight loss when using the appliance22,36） was eating disorders and mastication dysfunction；however, these results are contradicted by the increase of body weight during days 5―7 of tooth movement, as shown in this study. The increase in body weight should be explained by another physical reaction. The results of the present study suggest that i） for low alveolar bone mass, the bone mass required to support tooth movement may not be available once the mechanical stress is applied；however, ii ）bone mass recovers due to the influence of constant serum calcium levels on mechanical stress, even with low bone mass；and iii ） even bone mass far from the applied mechanical stress may be affected by circulating body fluids or other factors. We conclude, therefore, that tooth movement by orthodontic force was possible even with low alveolar bone mass in rats, although various physical influences were involved. However, for better understanding of the assess-
ment of physical activity in relation to mechanical stress, more basic research is needed not only on calcium metabolism and related factors but also regarding the possible roles played by stress factors in the physical system. Acknowledgements We would like to thank Professors A. Hirashita and K. Kawasaki for their support and encouragement throughout the present study. Part of the study was supported by a grant from the Japanese Ministry of Education, Culture, Sports, Science and Technology （Academic Frontier Project, 2004―2008,“High−Tech Research Center”Project, 2005―2009）. References 1）Cummings, S. R., Black, D. M. and Nevitt, M. C.： Bone density at various sites for prediction of hip fractures. Lancet 341：72―75, 1993. 2）Melton, L. J. Ⅲ., Actinson, E. J., O’Fallon, W. M., Wahner, H. W. and Riggs, B. L.：Long−term fracture prediction by bone mineral density assessed at different skeletal sites. J. Bone Miner. Res. 8：1227― 1233, 1993. 3）Akesson, K., Liunghall, S., Jonsson, B., Sernbo, I., Johnell, O., Gardsell, P. and Obrant, K. J.：Assessment of biochemical markers of bone metabolism in relation to the occurrence of fracture：A retrospective and prospective population−based study of women. J. Bone Miner. Res. 10：1823―1829, 1995. 4）Garnero, P., Hausherr, E., M.−C, Chapuy., Marcelli, C., Grandjean, H., Muller, C., Cormier, C., Breart, G., Meunier, P. J. and Delmas, P. D.：Markers of bone resorption predict hip fracture in elderly women： The EPIDOS prospective study. J. Bone Miner. Res. 11：1531―1538, 1996. 5）Specker, B. L.：Evidence for an interation between calcium intake and physical activity on changes in bone mineral density. J. Bone Miner. Res. 11：1539― 1544, 1996. 6）Ingle, B. M., Hay, S. M., Bottjer, H. M. and Eastell, R.：Changes in bone mass and bone turnover following distal forearm fracture. Osteoporos. Int. 10：399 ―407, 1999. 7）Margareta, H., Jan, S. and Nils, D.：Biochemical bone markers and bone density in hip fracture
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