The European Journal of Orthodontics Advance Access originally published online on January 13, 2006
The European Journal of Orthodontics 2006 28(2):190-194; doi:10.1093/ejo/cji093
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Influence of sex hormone disturbances on the internal structure of the mandible in newborn mice
Department of Orthodontics and Craniofacial Developmental Biology, Hiroshima University Graduate School of Biomedical Sciences, Japan
Address for correspondence Tadashi Fujita, Department of Orthodontics and Craniofacial, Developmental Biology, Hiroshima University Graduate School of Biomedical, Sciences, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan. E-mail: seven{at}hiroshima-u.ac.jp
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Summary |
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It has not yet been clarified how sex hormones affect craniofacial bone development immediately after birth. The purpose of this study was to examine the effects of sex hormone deficiency on craniofacial bone development immediately after birth, in terms of the internal structure of the mandible in newborn mice with orchiectomy (ORX) and ovariectomy (OVX). ORX, OVX and a sham-operation were performed on 40 five-day-old C57BL/6J mice. Eight weeks after surgery, each mandible was subjected to histomorphometric analysis of trabecular (Tr) and cortical (Ct) bone mineral density (BMD) by peripheral quantitative computed tomography (pQCT).
In the experimental groups, a significant reduction in BMD was found in comparison with the control groups. In histomorphometric analysis, the number of tartrate-resistant acid phosphatase (TRAP)-positive cells in the condyle and the thickness of the condylar cartilage layer was significantly greater in the experimental mice than in the controls. Trabecular bone volume of the condyle measured on azocarmine-aniline blue (AZAN) sections was significantly less in the experimental mice than in the controls. These results indicate that mandibular growth is inhibited by sex hormone disturbances and the relevant internal structures changed. The findings show that sex hormones are one of the key determinants of mandibular growth and development immediately after birth.
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Introduction |
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In orthodontic treatment, teeth move in the bone of the maxilla and mandible. Tooth movement is also influenced by the condition of the internal structure of the maxilla and mandible. Although sex hormones play an important role in maintenance of bone volume, a reduction causes osteoporosis. It has been demonstrated that ovariectomy (OVX) and orchiectomy (ORX) induce condylar bone loss, and that oestrogen and androgen are effective in the prevention of bone loss during adolescence (Fujita et al., 2001
). Oestrogen and androgen stimulate differentiation of the brain during embryogenesis (Swerdloff et al., 1992; Quadros et al., 2002); however, it has not yet been clarified how sex hormones affect craniofacial bone development immediately after birth.
Craniofacial growth shows great variation among individuals, and mandibular growth is related to various factors, such as growth hormones (Hwang and Cha, 2004), growth factors (Delatte et al., 2004), heredity (Oshikawa et al., 2004), and mechanical stress (Bresin et al., 1999). The effects of sex hormones on bone and muscle development are greater than those of genetic or environmental factors (Morishima et al., 1995). It has recently been reported, in an experimental study, that the suppression of sex hormone secretion during the pubertal growth phase inhibits craniofacial growth, particulary mandibular growth, and results in reduced craniofacial development (Fujita et al., 2004).
The purpose of this study was to examine the effects of sex hormone deficiency on craniofacial bone development immediately after birth, in terms of the internal structure of the mandible in newborn mice with ORX and OVX.
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Animals
Forty C57BL/6J 5-day-old mice (Jackson Laboratory, Bar Harbor, Maine, USA) were used in this experiment. The mice were divided equally into two experimental groups with ORX and OVX, and the corresponding sham-operation (control) groups. Under general anaesthesia with sodium pentobarbital, using a stereoscopic microscope (SZX9, Olympus Optical Co., Tokyo, Japan) 20 male and 20 female mice underwent ORX, OVX and the corresponding sham-operation five days after birth. All mice were sacrificed 8 weeks after surgery. The body weight was measured every four days (data given in Fujita et al., 2004). The animals were treated under the ethical regulations defined by the Ethics Committee, Hiroshima University Faculty of Dentistry.
Peripheral quantitative computed tomography (pQCT) measurements
After removing the surrounding soft tissue, the mandible was immersed in 70 per cent ethanol. Each mandible was then subjected to analysis of trabecular (Tr) and cortical (Ct) bone mineral density (BMD) by pQCT (XCT Research SA+, Norlandstratec, Pforzheim, Germany). Each mandible was scanned by three slices passing through a region 0.1 mm anterior and 0.1 mm posterior to the mesial root of the first molar. After scanning, the mandible was represented by 0.26 mm-thick cross sections, using a voxel size of 0.06 mm. The threshold used for cortical bone measurements was 690 mg/cm3 with a separation mode of 1.
Histomorphometric analysis
The mandibular condyles were fixed in 4 per cent formaldehyde, decalcified in EDTA (pH 7.4) for 2 weeks, dehydrated in an ascending ethanol series (70, 80, 90, 95, 99, 100 per cent), embedded in paraffin, and cut into 7 µm thick frontal sections. The sections were stained with tartrate-resistant acid phosphatase (TRAP), haematoxylin-eosin (H-E), and azocarmine-aniline blue (AZAN) for histological observation using an optical microscope (BH2-RFCA, Olympus Optical Co.).
The TRAP-stained sections were used to count the number of osteoclasts in the condylar head, and the H-E stained sections to measure the thickness of the condylar cartilage layers. The articular cartilage layers were divided into fibrous (articular), proliferative (chondrogenic), and maturative/hypertrophic (cartilaginous) zones. The sections stained with AZAN were used for histomorphometric analysis, which was performed in the subchondral area of the condyle, using the image analysis program NIH Image 1.59 (National Institutes of Health, Bethesda, Maryland, USA). On the sections passing through the centre of the mandibular condyle, the number of TRAP-positive cells was counted. For quantification of Tr bone volume, the area was measured on the frontal sections, and the means were calculated.
Statistical analysis
Analysis of variances (ANOVA) and pairwise comparisons (Fisher) were performed to examine the differences in values measured among the four groups with a confidence level greater than 95 per cent.
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Results |
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Analysis of BMD
In the pQCT scan, Tr-BMD and Ct-BMD of the mandible were significantly lower in the experimental mice than in the controls. Although Tr-BMD in the ORX mice was similar to that in the OVX mice, Ct-BMD was significantly lower in the ORX than in the OVX mice (Figures 1a and b).
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Number of TRAP-positive cells
The number of TRAP-positive cells in the condyle was significantly greater in the experimental mice than in the controls. No significant differences in the number of TRAP-positive cells were found between ORX and OVX mice (Figure 2a).
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Trabecular bone volume
Tr bone volume of the condyle, measured on AZAN sections, was significantly less in the experimental mice than in the controls. The Tr bone volume of the condyle had a negative correlation with the number of TRAP-positive cells. Tr bone volume was similar in the ORX and OVX mice (Figures 2b and 3).
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Thickness of the condylar cartilage layers
The total thickness of the articular cartilage layers was significantly greater in the experimental mice than in the controls. The ORX and OVX groups exhibited 1.7- and 2.1-fold larger values, respectively. Among the cartilage layers, the thickness of the proliferative and maturative/hypertrophic layers were significantly different between the experimental and control mice (Table 1).
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Discussion |
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In this experiment, the influence of sex hormone on the internal structure of the mandible immediately after birth was examined. An awareness of how the maxilla and mandible grow immediately after birth is of interest for orthodontists. It was assumed that sex hormones were closely related to craniofacial growth because oestrogen and androgen are mostly secreted from the ovary and orchis and the function of sex hormones is activated in adolescence when bone growth is at its highest. Thus, ORX and OVX mice were used in this investigation.
It has previously been reported, in a morphometric study using cephalometric analysis, that the disturbance of sex hormone secretion immediately after birth affects craniofacial growth (Fujita et al., 2004). In the present investigation, the histomorphometric changes were further examined, using pQCT, in terms of the internal structure of the mandible in ORX and OVX mice immediately after birth. This analysis showed that mandibular Tr-BMD and Ct-BMD were significantly lower in the experimental mice than in the controls. Although a decrease of BMD in patients with osteoporosis (Takagi et al., 1995) and in mice (Omi and Ezawa, 1995; Gaumet-Meunier et al., 2000) has been mainly found in Tr bone, in this study of ORX and OVX mice immediately after birth, Ct-BMD was significantly reduced analogous to the Tr bone. In these experiments, adult animals were used. There is limited information about changes in the internal structure of the mandible, measured by pQCT, immediately after birth.
The mandibular condyle is a centre for mandibular growth; however, growth of the mandible is not determined only by cartilaginous but also membranous growth (Berraquero et al., 1992). Ct-BMD reduction was found to be greater in the ORX than in the OVX mice in this study, and skeletal growth of ORX mice was inhibited more than that of OVX mice in a previous investigation (Fujita et al., 2004). Bone growth has been shown to be related to cortical bone density (Maki et al., 2000). This may be a reason why sex hormone secretion blockage immediately after birth causes poor development. These speculations, derived from the present findings, will hopefully be confirmed in future studies.
It is currently accepted that ORX and OVX enhances the turnover of long bones such as the femur or tibia. This phenomenon has also been demonstrated in the mandibular condyle (Fujita et al., 2001). However, the precise mechanism that causes this phenomenon remains to be elucidated. Furthermore, the influences on condylar modelling immediately after birth and subsequent growth still remain unclear.
With respect to bone remodelling affected by OVX, various studies have been carried out. Wronski et al. (1988) reported that the initial phase of rapid bone loss in the tibia of OVX rats was coincident with the maximal increase in bone turnover, and then both the bone loss and turnover decreased. Androgens stimulate normal skeletal development during puberty (Johansen et al., 1988), and the delay of puberty in humans has been associated with a lower peak in bone mass (Finkelstein et al., 1992). In the present study, the blockage of sex hormone secretion immediately after birth promoted bone resorption, and it was confirmed that Tr bone volume decreased. This finding is similar to the result of a previous experiment using eight-week-old mice (Fujita et al., 2001).
The influence of sex hormones on bone growth after adolescence is well documented. OVX increases hypertrophic cartilage, and the total amount of growth plate cartilage in OVX animals is decreased by oestradiol (Turner et al., 1994). In the present study, the experimental group exhibited significant differences in the thickness of the proliferative and maturative/hypertrophic layers from the controls, and Tr bone volume was less than that in the control groups. Therefore, sex hormone deficiency may disturb endochondral ossification. These results also indicate that sex hormones alter condylar remodelling, leading to degenerative changes in the temporomandibular joint.
These findings demonstrate that obstruction of the secretion of sex hormone causes changes in the internal structure of the condyle. In addition to previous morphometric results with lateral cephalograms (Fujita et al., 2004), the present study has demonstrated the influence of sex hormones on bone growth immediately after birth, and that sex hormones substantially influence craniofacial growth in newborn mice.
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Conclusions |
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TopSummaryIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
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Mandibular growth is inhibited by sex hormone disturbances and the relevant internal structures changed. These findings indicate that sex hormones are one of the key determinants of mandibular growth and development immediately after birth.
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Acknowledgement |
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This investigation was supported in part by a Grant-in-aid (No. 16791286) from the Ministry of Education, Science, Sports and Culture in Japan.
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References |
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