2003 Blackwell Munksgaard

Oral Diseases (2003) 9, 24–28 All rights reserved 1354-523X/03

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Experimental Oral Pathology

Bone mineral density of the mandible in ovariectomized rats: analyses using dual energy X-ray absorptiometry and peripheral quantitative computed tomography S Kuroda1, H Mukohyama2, H Kondo1, K Aoki3, K Ohya3, T Ohyama4, S Kasugai1 1

Masticatory Function Control; 2Maxillofacial Prosthetics; 3Pharmacology; 4Removable Prosthodontics, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan

INTRODUCTION: Although previous studies have shown that maxillary molar extraction in ovariectomized (OVX) animals causes mandibular loss of bone, it is still questionable as to whether estrogen deficiency affects mandibles with functional occlusion. MATERIALS and METHODS: To answer this question, 13-week-old female Sprague–Dawley rats were bilaterally OVX or sham-operated. After 109 days, the bone mineral density (BMD) of the femurs and mandibles was measured using dual-energy X-ray absorptiometry (DEXA) and peripheral quantitative computed tomography (pQCT). RESULTS: In DEXA analysis, although the BMD of the total mandible of the OVX rats was similar to that of the sham-operated rats, the BMD of the condylar region in the OVX rats had decreased by 12.5%. In pQCT analysis, decrease in trabecular BMD of the mandibular bone was detectable but low in the molar region (maximal 13%), whereas no difference was seen in cortical BMD. In the femurs, the trabecular bone prominently decreased in OVX rats (30% decrease in pQCT analysis) as previously reported. CONCLUSION: This study revealed regional differences in the mandibular bone decrease in OVX rats. Although the mechanism of low susceptibility of the mandible to estrogen-deficient conditions remains unknown, it is likely that mechanical stress derived from functional occlusion is preventing bone loss in this pathological condition. Furthermore, this study demonstrated the advantage of pQCT in analyzing rat mandibular bone. Oral Diseases (2003) 9, 24–28

Correspondence: Shinji Kuroda, Masticatory Function Control, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan. Tel: +81-3-5803-4554, Fax: +81-3-58035934, E-mail: [email protected] Received 15 October 2001; revised 14 February 2002; accepted 7 October 2002

Keywords: bone mineral density of mandible; ovariectomized rat; dual energy X-ray absorptiometry; peripheral quantitative computed tomography

Introduction The quality and volume of the alveolar ridge bone is an important factor to be considered in prosthodontic treatment. We occasionally encounter difficult clinical cases because of insufficient bone. Bone is a tissue that is being constantly remodeled, and bone mass at any given time depends on the balance between the rate of osteoblastic bone formation and osteoclastic bone resorption. These cellular functions are controlled by various systemic and local factors. Estrogen deficiency in postmenopausal women elicits bone loss in the vertebrae and long bones resulting in bone fractures, and this condition is called postmenopausal osteoporosis (Wronski et al, 1985; Wronski, Cintron and Dann, 1988; Wronski et al, 1989b; Sharp et al, 2000). Compared with the vertebrae and long bones, considerably less information is available on bone loss of the mandibles under estrogen-deficient conditions. The bone mineral content of elderly females’ edentulous mandibles decreases with aging, but this is not evident in elderly men (Klemetti et al, 1993; Ulm et al, 1994; May et al, 1995). The ovariectomized (OVX) rat provides an experimental model of postmenopausal osteoporosis. The bone mineral content and mechanical properties of the mandibles of OVX rats are similar to those of sham-operated rats (Elovic, Hipp and Hayes, 1995b); however, maxillary molar extraction causes bone loss from the mandibles of OVX rats (Elovic, Hipp and Hayes, 1994; Klemetti and Vainio, 1994; Elovic, Hipp and Hayes, 1995a). These previous studies indicate that estrogen deficiency affects the mandible in both humans and experimental animals. However, it is still open to question as to whether estrogen deficiency

Mandibular BMD in OVX rats S Kuroda et al

affects mandibles with functional occlusion. To answer this question, we OVX rats, and then measured the bone mineral density (BMD) of mandibles. Dual energy X-ray absorptiometry (DEXA) and peripheral quantitative computed tomography (pQCT) are useful methods to measure patients’ BMD. DEXA is a 2-dimensional analysis whereas pQCT is a 3-dimensional analysis and dissection analysis is possible in pQCT. In the present study, we utilized DEXA and pQCT to measure BMD of rat mandibles because these two methods have not been simultaneously applied to measure mandibular BMD.

Materials and methods Ovariectomy The animal experimental protocol in the present study was in agreement with the Tokyo Medical and Dental University standard. Twelve 13-week-old female Sprague–Dawley rats were obtained from a breeder. The six animals were bilaterally OVX and the others were shamoperated under Nembutal anesthesia. The rats of both groups were pair-fed and then sacrificed 109 days after the original surgical operation under chloroform anesthesia. The mandibles and femurs were dissected out and immersed in 70% ethanol. The uteri were also dissected out and weighed to check the effects of ovariectomy. Soft X-ray radiography and bone mineral density (BMD) measurement Soft X-ray images of the mandibles and femurs were taken with a soft X-ray radiographic apparatus (SPOM50; Sofron, Tokyo, Japan). Total femoral BMD was initially measured with a DEXA (DCS-600R; Aloka, Tokyo, Japan), and then calculated. Total mandible BMD was measured using DEXA, after which the BMD

of the incisal edge and condylar region of the mandible, as indicated in Figure 1, was measured. The trabecular and cortical BMD of the femurs were measured at cross sections 2 mm and 17 mm from the growth plate as shown in Figure 1, respectively, perpendicular to the long axis with pQCT (XCT 960 A; Norland & Stratec, Pforzheim, Germany). The positions are trabecular and cortically rich regions, respectively. Also, the mandibles were scanned from the medial plane to the distal plane of the molar region with pQCT as indicated in Figure 1, following which the trabecular BMD and cortical BMD of the mandibular sections, excluding the incisor and molar, were measured using computer software.

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Statistical analysis All values are represented as the mean ± standard division (s.d.). Statistical significance was determined using the unpaired Student’s t-test. P < 0.05 was regarded as statistically significant.

Results As a result of pair-feeding, there was no difference in body weight between OVX and sham-operated groups (data not shown). The femurs of OVX rats were more radiolucent than those of sham-operated rats, whereas radiolucency of the mandibles appeared similar in both groups (Figure 2). In OVX rats, both uterus weight and femoral total BMD in DEXA analysis decreased by 80.8 and 13.7%, respectively. In pQCT analysis, decrease of 30% at the 17-mm section in trabecular BMD of the femur was prominent in OVX rats whereas ovariectomy did not affect BMD of the cortical bone of the femur. In DEXA analysis, total BMD of the mandibles of OVX rats was similar to that of sham-operated rats

Figure 1 Areas and positions for BMD measurement. Total mandible BMD was measured by DEXA. Then, external incisal ridge and mandibular condyle in the areas surrounded with rectangle (4 · 6 mm) and square (2 · 2 mm) were to be scanned by DEXA, respectively (a). The sections including molars at interval of 0.5 mm from the medial plane of the first molar were scanned by pQCT (b). Trabecular and cortically rich regions of the femur were scanned at 2 mm and 17 mm from the growth plate, respectively, by pQCT (c) Oral Diseases

Mandibular BMD in OVX rats S Kuroda et al

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Figure 2 Soft X-ray Radiography. Femoral (a) and Mandibular (b) radiographies of Sham and OVX animals

Table 1 BMD of total mandible and condyle, and mineral density of incisor, scanned by DEXA

Total mandible Condyle Incisor

SHAM Mean ± s.d. (n ¼ 6)

OVX Mean ± s.d. (n ¼ 6)

98.35 ± 2.33 42.50 ± 3.18 94.35 ± 4.94

98.18 ± 5.34 37.20 ± 2.99* 94.58 ± 6.50

*Significant difference from SHAM (P < 0.05).

(Table 1); however, regional BMD measurement with DEXA demonstrated a BMD decrease of 14% in the condylar region in OVX rats (Table 1). In DEXA analysis, mineral density (mg ⁄ cm2, mean ± s.d.) of the incisors of OVX and sham-operated rats were similar (Table 1). Thus, ovariectomy did not affect mineral density of the rat incisors. In pQCT analysis of the molar region of the mandible excluding the molar, BMD decrease of maximal 13% at the eighth slice in trabecular bone was detectable in some slices (Figure 3a) whereas cortical BMD was not affected in any of the slices (Figure 3b).

Discussion Ovariectomy causes estrogen deficiency, and the OVX rat is a widely used animal model for postmenopausal osteoporosis. The remarkable decrease in the uteral weight of the OVX rats in the present study is a result of the bilateral removal of ovaries and the resultant estrogen-deficient condition. Ovariectomy stimulates food intake by the animals, and because mechanical stress applied to bone influences bone volume and structure, the accelerated food intake increases body weight and masticatory function, which would mask the direct effects of estrogen deficiency on bone. For this reason, we did a pair-feeding experiment, in which the amount of food intake of OVX and sham-operated groups was controlled. There was no difference in body weight between both groups. Oral Diseases

Figure 3 (a) Trabecular BMD of mandible by pQCT. Trabecular bone mineral density at the section shown in Figure 1b was measured by pQCT. X-axis shows slice number and the distance from the medial plane of the first molar. Values are presented as Mean ± s.d., n ¼ 6. *P < 0.05 was regarded as statistically significant. (b) Cortical BMD of mandible by pQCT. Cortical bone mineral density at the section shown in Figure 1b was measured by pQCT. X-axis shows slice number and the distance from the medial plane of the first molar. Values are presented as Mean ± s.d., n ¼ 6

Mandibular BMD in OVX rats S Kuroda et al

In OVX rats, both bone formation and resorption is accelerated; however, a lack of balance, in which more bone is resorbed than laid down, causes trabecular bone loss in the long bones and vertebrae (Otawara, Hosoya and Moriuchi, 1983; Wronski, Dann and Horner, 1989a; Wronski et al, 1989b; Toolan et al, 1992). In the present study, DEXA analysis showed that the total femoral BMD of OVX rats decreased. pQCT analysis showed that the BMD decrease in OVX rats was because of the decrease in trabecular bone; cortical bone was not affected. These results are similar to some previously reported by other investigators demonstrating that trabecular bone is more affected than cortical bone by OVX (Pastoureau, Chomel and Bonnet, 1995; Satoh, Soeda and Dokou, 1995; Breen et al, 1996). Elovic and collaborators have already reported that ovariectomy does not affect the BMD of the rat mandible (Elovic et al, 1995b). Likewise, DEXA analysis in the present study indicated that the total BMD of the mandible of OVX rats was similar to that of shamoperated rats. The incisors occupy a large area of the rat mandible, and total BMD of the rat mandible also includes the mineral content of the incisor and molars. However, we are able to rule out the effect of the teeth on mandibular BMD comparison between two groups, because ovariectomy did not change the mineral content of the incisor and it is unlikely that it changed the mineral content of the molars. Although no difference in the total mandible BMD between OVX and sham-operated rats was detectable, some regions of the mandible were clearly affected by exposure to estrogen-deficient conditions. First, BMD measurement of the condylar region demonstrated a BMD decrease in OVX rats of 14% compared with that of the sham-operated group. Percentage BMD decrease in this region was similar to the decrease in femoral BMD in OVX rats. Secondly, pQCT analysis revealed BMD of the trabecular bone of the molar region of the mandible to have decreased in OVX rats. Notably, the extent of this BMD decrease (maximal 13% decrease at the eighth slice in Figure 3a) was less than the decrease in trabecular bone of the femur in OVX rats (30% decrease at the 17-mm section in Figure 2). Thus, the susceptibility of the bone of the molar region of the mandible to estrogen-deficient conditions was low compared with the bone of the condylar region of the mandible and the femur. Mechanical stress applied to bone influences bone volume and structure by controlling bone remodeling. It is generally agreed that application of a sufficient degree of mechanical stress to bone is necessary to maintain bone volume and structure. In normal rats, extraction of teeth from the upper jaw causes osteoporotic changes in the trabecular bone of the mandible (von Wowern, Hjorting-Hsansen and Stoltze, 1979). Apparently, functional occlusion is crucial in maintaining the volume and structure of the mandible. In the present study, we found trabecular loss of bone in the molar region of the mandible in OVX rats. Although the precise mechanism of the lower susceptibility of the molar region of the mandible to estrogen-deficient conditions is not clear, it is likely that mechanical stress derived from functional

occlusion is preventing bone loss in this pathological condition. Recent studies demonstrated that maxillary molar extraction together with ovariectomy causes more bone loss in the mandible than does maxillary molar extraction alone (Elovic et al 1994; Klemetti and Vainio, 1994; Elovic et al, 1995a; Jahangiri, Kim and Nishimura, 1997), which supports this speculation. In the present study, we demonstrated that trabecular loss of bone in the molar region of the mandible under estrogen-deficient conditions was small but detectable when functional occlusion was present. Although the amount of this bone loss is small, a change in bone remodeling balance with increasing local osteolytic factors may be underlying this phenomenon. (Klemetti et al, 1993; Klemetti and Vainio, 1994; Elovic et al, 1994; Klemetti, Vainio and Kroger, 1994; Elovic et al, 1995a; Jahangiri et al, 1997) Furthermore, we demonstrated the advantage of pQCT in analyzing rat mandibular bone because the small and regional BMD change in the mandibles of OVX rats was detectable in pQCT, not in DEXA analysis.

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Acknowledgement This investigation was supported by Grant-in-Aid from the Ministry of Education (08457519).

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