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Estrous-cycle-dependent Variation in Orthodontic Tooth Movement

¹ Division of Orthodontics, Department of Life-Long Oral Health Science, and
² Division of Pharmacology, Department of Oral Biology, Tohoku University Graduate School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan;

*corresponding author, igarashi{at}mail.cc.tohoku.ac.jp

	ABSTRACT

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Sex hormones, including estradiol, play important physiological roles in bone metabolism. The purpose of this study was to investigate whether there is estrous-cycle-dependent variation in orthodontic tooth movement, and, if so, to determine the mechanism. Ten-week-old female Wistar rats were used. They received repeated orthodontic force during specific phases in the estrous cycle. Tooth movement in animals that received force principally in estrus was about 33% greater than that in animals that received such force principally in pro-estrus (p < 0.05). Serum estradiol levels also varied according to the estrous cycle, with a peak during pro-estrus and a nadir during estrus, and were inversely related to tooth movement. Furthermore, there were negative correlations between estradiol and both serum TRAP activity and pyridinoline (r = -0.42, p < 0.05; r = -0.59, p < 0.001). These results suggest that cyclic changes in the estradiol level may be associated with the estrous-cycle-dependent variation in tooth movement through its effects on bone resorption.

KEY WORDS: estrous cycle • tooth movement • sex hormones • bone metabolic markers • rat

	INTRODUCTION

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It has become increasingly evident that sex hormones such as estrogen, progesterone, and androgen play important physiological roles in bone metabolism. These steroid hormones are essential for sexual dimorphism of the skeleton, skeletal maturation during growth, and maintenance of the bone balance in adults (Harris et al., 1996). It is well-known that estrogen deficiency is a major factor in the development of post-menopausal osteoporosis (Turner et al., 1994).

Previous animal and human studies have demonstrated that the levels of sex steroids, including estradiol and progesterone, fluctuate according to the estrous or menstrual cycle (Mishell et al., 1971; Butcher et al., 1974). In humans, it has been shown that there are some relationships between these hormones and serum markers of bone metabolism (Gorai et al., 1998; Zittermann et al., 2000).

Since mechanically induced bone modeling and remodeling are essential for orthodontic tooth movement, the responses to orthodontic force may vary depending on the phase of the menstrual cycle. In orthodontic clinics, there are more female patients than male patients. Furthermore, there is an increasing number of adult female patients seeking orthodontic treatment worldwide. Therefore, it is important to investigate whether the menstrual cycle affects orthodontic tooth movement, and, if so, to determine the mechanism. Such an approach may offer insight into the timing for effective treatment in female patients.

The purpose of this study was to determine whether there is any variation in orthodontic tooth movement depending on the phase of the estrous cycle. Overall, we investigated tooth movement in female rats that received repeated orthodontic force during a specific phase of the estrous cycle. Furthermore, we also investigated the behavior of the principal female sex hormones and markers of bone turnover in the serum during different phases of the estrous cycle, to understand the mechanism of possible estrous-cycle-dependent variation in tooth movement.

	MATERIALS & METHODS

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Experimental Design
We used 85 female 10-week-old rats (Wistar, Japan SLC Co., Shizuoka, Japan), weighing an average of 136 g each. The animals were adapted to a 12/12-hour light/dark cycle (with a light period from 07:00 to 19:00) for 2 wks. Laboratory chow (F2, Funabashi Farms Co., Funabashi, Japan) and de-ionized water were given ad libitum. Throughout the experimental period, vaginal smears, together with the measurement of vaginal impedance, were performed daily at fixed times (at 19:00 in the experiment on tooth movement and at 12:00 in the experiment on hormones and bone markers, respectively) so that the stage of the estrous cycle could be determined (Koto et al., 1987; Montes and Luque, 1988). We obtained the smears from each animal by rinsing the vagina with distilled water. After being dried, the smears were stained with hematoxylin for microscopic examination. Vaginal impedance was measured by means of a vaginal impedance checker (MK-10A, Muromachi Kikai Co., Tokyo, Japan). Based on the observation of vaginal smears, the estrous cycle could be divided into four distinct stages, i.e., pro-estrus, estrus, metestrus, and di-estrus (Fig. 1A). These stages were characterized by a moderate number of nucleated epithelial cells for pro-estrus, many cornified cells for estrus, leukocytes and cornified cells for metestrus, and nucleated epithelial cells with a larger number of leukocytes for di-estrus. As shown in Fig. 1B, vaginal impedance showed distinct cyclic variation, with the highest values in pro-estrus. Rats that showed vaginal impedance greater than 3.0 k were defined to be in pro-estrus. In rats, each stage lasts for approximately one day, resulting in a four-day estrous cycle (Butcher et al., 1974). In the present study, while about two-thirds of the animals exhibited a four-day estrous cycle, some rats followed a five-day cycle with a longer di-estrus phase that lasted for 2 days during acclimatization. In this study, animals were included unless they did not follow a stable four- or five-day estrous cycle. Two animals were disqualified due to unstable estrous cycles during acclimatization.

View larger version (111K): [in this window] [in a new window]   Figure 1. Monitoring of the estrous cycle in rats and design of the experiment on tooth movement. (A) Vaginal smears. P (pro-estrus): The smear was characterized by a moderate number of nucleated epithelial cells. E (estrus): Many cornified cells appeared in the smear. M (metestrus): The smear contained leukocytes as well as cornified cells. D (di-estrus): The smear was characterized by nucleated epithelial cells and a large number of leukocytes. Bar = 200 µm. (B) Representative cyclic change in vaginal impedance and design of the experiment on tooth movement. Rats that showed vaginal impedance greater than 3.0 k were defined to be in pro-estrus. The animals were divided into 4 groups based on the stage of the estrous cycle when the force was mainly applied. Animals in the Estrus, Metestrus, Di-estrus, and Pro-estrus groups received force for 2 days during every estrous cycle from late pro-estrus, late estrus, late metestrus, and late di-estrus, respectively. Animals in each group received no force for the remaining 2 or 3 days in each estrous cycle. The animals were examined for 5 consecutive estrous cycles and received the force 5 times for 2 days in each estrous cycle.

Tooth Movement
Thirty-two animals were divided into 4 groups (8 animals in each group) based on the stage of the estrous cycle when the orthodontic force was applied. Animals in the Estrus, Metestrus, Di-estrus, and Pro-estrus groups received force for 2 days during each estrous cycle from late pro-estrus, late estrus, late metestrus, and late di-estrus, respectively. Animals in each group received no force for the remaining 2 or 3 days in each estrous cycle. Thus, the applied force was intermittent. The animals were examined for 5 consecutive estrous cycles and received the force 5 times for 2 days in each estrous cycle (Fig. 1B).

The method used to apply orthodontic force has been described previously in detail (Igarashi et al., 1994). Briefly, a uniform standardized expansive spring, made of 0.012-inch nickel-titanium wire (BF012C, Rocky Mountain Morita Corp., Tokyo, Japan), was placed in the animal's mouth between the right and left upper first molars. The spring initially generated an average expansive force of 125 mN on each side and was retained in the mouth by its own force. The appliances were set and removed at 19:00 under light ether anesthesia.

The expansive spring generated force on the right and left upper first molars to move buccally. When the appliance was removed, the movement of the molars was measured as described previously (Adachi et al., 1994).

The error of measurement was 8.0 x 10^-3 mm when a single investigator measured 15 randomly selected samples twice in a blind test. The error was calculated to be E = d²/2n (E, error; d, difference between 2 measurements; n, number of samples).

Hormones and Markers of Bone Turnover
Fifty-one animals were used for the assay for sex hormones, serum estradiol and progesterone, and their estrous cycles were monitored as described above. Biochemical markers of bone turnover, serum tartrate-resistant acid phosphatase (TRAP) activity, pyridinoline, osteocalcin, calcium, and phosphorus were also assayed in 31 of these 51 animals. Because of diurnal variations, experiments were performed at close to mid-day (12:00-14:00). Animals in a known estrous stage were killed by decapitation and bled into a glass test tube. The blood samples were allowed to clot at room temperature, and the serum was collected by centrifugation at 4°C. Aliquots were stored at -80°C until assayed.

Serum estradiol and progesterone were measured by specific enzyme immunoassays (Assay Designs, Inc., Ann Arbor, MI, USA).

Serum pyridinoline was also measured by enzyme immunoassay (Metra Biosystems, Inc., Mountain View, CA, USA). Serum osteocalcin was measured by an enzyme-linked immunosorbent assay (Amersham Pharmacia Biotech K.K., Tokyo, Japan). Serum tartrate-resistant acid phosphatase (TRAP) activity and calcium concentration were determined with colorimetric test kits (Acid phospha B-test Wako and Calcium C-test Wako, respectively, Wako Pure Chemical Industries, Ltd., Osaka, Japan). Serum phosphorus concentration was also determined colorimetrically as previously described (Chen et al., 1956).

The minimum detection limits in the serum estradiol, progesterone, pyridinoline, and osteocalcin assays were 10 pg/mL, 3.1 pg/mL, 0.18 nmol/L, and 0.050 ng/mL, respectively.

The intra- and inter-assay coefficients of variation for estradiol, progesterone, pyridinoline, osteocalcin, and TRAP activity were 5.6 and 8.0%, 4.1 and 8.1%, 1.1 and 3.2%, 2.5 and 5.9%, and 2.2 and 4.2%, respectively.

Statistical Analysis
All of the data are expressed as means ± SEM. The data for tooth movement were subjected to two-way repeated-measures analysis of variance (two-way repeated-measures ANOVA). The Tukey-Kramer test was used to identify differences between groups. Pearson's correlation coefficient and linear regression analysis were used to assess interrelationships between the levels of sex hormones and biochemical markers of bone turnover. P < 0.05 was considered to represent a significant difference or correlation.

	RESULTS

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Estrous-cycle-dependent Variation in Tooth Movement
Eleven animals were excluded from the experiment on tooth movement due to failure of the appliance (4), disappearance of stable estrous cycles (5), or accidental death during anesthesia (2). The rest of the animals examined were all in good health and grew normally. There was no difference in body weight among the 4 groups at the end of the experiment. Although the total experimental period varied between rats with four-day and five-day estrous cycles, there was no significant difference in the amount of tooth movement in each of the 4 groups (p values in a two-way repeated-measures ANOVA were 0.11, 0.73, 0.82, and 0.19, for the Estrus, Metestrus, Di-estrus, and Pro-estrus groups, respectively). Therefore, the data for rats with four-day and five-day estrous cycles were combined.

There was a distinct variation in tooth movement among the 4 groups (p < 0.05 by two-way repeated-measures ANOVA). Fig. 2A shows mean tooth movement after the fifth cycle in all 4 groups. Tooth movement in the Estrus group was 32.6% greater than that in the Pro-estrus group, and this difference was statistically significant (p < 0.05 by the Tukey-Kramer test). Fig. 2B shows the time course of tooth movement in animals in the Estrus group and the Pro-estrus group. Tooth movement in both groups continued to increase significantly (p < 0.001 by two-way repeated-measures ANOVA). After the fourth cycle, tooth movement in the Estrus group was significantly greater than that in the Pro-estrus group (p < 0.05 by the Tukey-Kramer test).

View larger version (39K): [in this window] [in a new window]   Figure 2. Estrous-cycle-dependent variation in tooth movement. (A) The mean tooth movement after 5 consecutive estrous cycles in the 4 experimental groups. Each column represents the mean ± SEM. The number of animals for each column ranges from 4 to 7. *p < 0.05 by the Tukey-Kramer test. (B) Time course of tooth movement in rats in the Estrus and Pro-estrus groups. Each point represents the mean ± SEM of 4 to 7 animals. p < 0.001 for number of force applications and p < 0.05 for groups by two-way repeated-measures ANOVA. *p < 0.05 vs. the Pro-estrus group by the Tukey-Kramer test.

View larger version (41K): [in this window] [in a new window]   Figure 3. Fluctuations in serum estradiol and progesterone over the estrous cycle. (A) Estradiol. (B) Progesterone. Each column represents the mean ± SEM of 11 to 14 animals. *p < 0.05, **p < 0.01, by the Tukey-Kramer test.

View larger version (39K): [in this window] [in a new window]   Figure 4. Fluctuations in serum biochemical markers of bone turnover over the estrous cycle and their correlations with serum estradiol or progesterone. (A) TRAP activity. (C) Pyridinoline. (E) Osteocalcin. (G) Calcium. (H) Phosphorus. E, estrus; M, metestrus; D, di-estrus; P, pro-estrus. Each column represents the mean ± SEM of 6 to 8 samples. *p < 0.05, **p < 0.01, by the Tukey-Kramer test. (B) Correlation between TRAP activity and estradiol (r = -0.42, p < 0.05). (D) Correlation between pyridinoline and estradiol (r = -0.58, p < 0.001). (F) Correlation between osteocalcin and progesterone (r = 0.47, p < 0.01).

	DISCUSSION

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The present results clearly demonstrated that orthodontic tooth movement varies depending on the phase of the estrous cycle in rats. Tooth movement was the greatest in animals that received orthodontic force principally during estrus, whereas it was the least in those that received such force principally during pro-estrus. The present study also demonstrated that the serum estradiol level varied according to the phase of the estrous cycle, with a peak during pro-estrus and a nadir during estrus, and was inversely related to tooth movement. Estrogen is known to inhibit osteoclasts both directly and indirectly (Harris et al., 1996). Thus, consistent with previous human studies (Chiu et al., 1999; Zittermann et al., 2000), negative correlations were observed between estradiol and markers of bone resorption (TRAP and pyridinoline) in the present study. It has also been demonstrated that serum interleukin-1 and -6, which are known to play important roles in bone resorption, fluctuate during the menstrual cycle in humans (Cannon and Dinarello, 1985; Angstwurm et al., 1997). These findings suggest that there might be an estrogen-dependent cyclic variation in bone resorption during the estrous cycle in rats. Since the rate of orthodontic tooth movement strongly depends on the activity of osteoclasts that resorb bone (Rygh, 1986; Igarashi et al., 1994), cyclic changes in serum estrogen may account for the observed variation in tooth movement.

Progesterone, another important circulating sex hormone in females, also exhibited estrous-cycle-dependent fluctuation. However, this fluctuation did not seem to be related to tooth movement or to be significantly correlated with markers of bone resorption (Figs. 2A, 3B, 4A, 4C). However, there was a significant positive correlation between this hormone and serum osteocalcin, suggesting that bone-forming activity also varies depending on the progesterone level during the estrous cycle in rats. Although the presence of receptors for progesterone and its anabolic effects have been demonstrated in cells of osteoblastic lineage (Eriksen et al., 1988; Scheven et al., 1992), the role of this sex hormone in bone metabolism is less clear than that of estrogen.

In the present study, we examined estrous-cycle-dependent variations in the principal female sex steroids and biochemical markers of bone turnover, and possible relationships between them in rats, since there is little such information available for this species. The fluctuations of these hormones and markers were similar to those observed in humans (Gorai et al., 1998; Zittermann et al., 2000), although some of these variations were not statistically significant. The results suggest that these variations should be taken into account when female rats are used for future bone studies.

To our knowledge, there have been no reports on the effect of the estrous or menstrual cycle on orthodontic tooth movement, except for one clinical study performed in the middle of the last century (Storey, 1954). Based on observations of tooth movement in nine female patients, the author concluded that there was a cyclic variation in the rate of tooth movement in relation to the menstrual cycle, and that the rate increased during the second half of the cycle and fell before or at menstruation. However, the latter conclusion appears to be inconsistent with the results of recent human studies on the menstrual-cycle variation in bone turnover, which have revealed higher levels of bone-resorptive markers around menstruation. Therefore, further human studies are necessary to determine the effect of the menstrual cycle on orthodontic tooth movement in female patients.

	ACKNOWLEDGMENTS

 
We thank Prof. M. Kagayama for a careful reading of the manuscript and for providing valuable advice. This research was supported in part by a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Science, Sports, and Culture of Japan (No. 13672138).

Received October 8, 2001; Last revision March 6, 2002; Accepted March 18, 2002

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