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Differential responses of mandibular condyle and femur to oestrogen deficiency in young rats
PERGAMON
Archives of Oral Biology 43 (1998) 191±195
ARCHIVES OF ORAL BIOLOGY
Dierential responses of mandibular condyle and femur to oestrogen de®ciency in young rats T. Yamashiro, T. Takano-Yamamoto * Department of Orthodontics, Okayama University Dental School, 2-5-1 Shikata-cho, Okayama 700-8525, Japan Accepted 2 December 1997
Abstract This response of condyle and femur was evaluated by histomorphometry. Five female Sprague±Dawley rats, 6 weeks of age, were subjected to bilateral ovariectomy, and ®ve others were sham-operated. They were then killed 32 days later. Ovariectomy signi®cantly increased the total thickness of the condylar cartilage and distal femoral growth-plate cartilage in the young, growing rats. Ovariectomy caused a 2-fold increase in thickness of the proliferative layer and a 4-fold increase in thickness of the hypertrophic layer in the condylar cartilage, and a 1.3fold increase in thickness of the proliferative layer in the epiphyseal growth cartilage of the femur. Ovariectomy had no eect on the percentage of trabecular bone volume, the percentage of the bone surface covered by osteoblasts (Ob.S/BS) and osteoclasts (Oc.S/BS), and number of osteoclasts per bone surface (N.Oc/BS) in the mandibular condyle. On the contrary, ovariectomy caused a 68.5% decrease in bone volume, a 4-fold increase in Ob.S/BS, and 2-fold increases in Oc.S/BS and N.Oc/BS in the secondary spongiosa of the rat distal femur. Thus there was a prominent dierence in the response to oestrogen de®ciency between the mandibular condyle and femur in young growing rats. # 1998 Elsevier Science Ltd. All rights reserved. Keywords: Mandibular condyle; Femur; Ovariectomy; Bone histomorphometry; Rat
1. Introduction Cartilage increases its volume initially by cell proliferation and matrix formation, and then it is gradually replaced by bone (Silbermann et al., 1987). The mandibular condylar cartilage is actively involved in endochondral bone formation and contributes to the elongation of the mandibular ramus (Silbermann and Frommer, 1972), which is believed to play an important part in cranofacial growth (Koski and Lahdemaki, 1975; Sperber, 1976). Rabbit articular chondrocytes have speci®c receptors for 17 b-oestradiol (Dayani et al., 1988), suggesting that the direct regulation of cellular metabolism by sex hormones in chondrocytes is mediated through their own receptors. In the epiphyseal growth plate, ovariectomy increased the hyper-
* To whom all correspondence should be addressed.
trophic layer of cartilage (Ornoy et al., 1994), and oestrogen treatment of ovariectomized rats caused a decrease in thickness of the tibial growth plate (Turner et al., 1994). Ovariectomy had a similar eect on mandibular condylar cartilage (Okuda et al., 1996). The outcome of oestrogen treatment in vitro (Gray et al., 1987; Ernst et al., 1988; Fukayama and Tashjian, 1989) and in vivo (Takano-Yamamoto and Rodan, 1990) strongly suggests an anabolic eect of oestrogen on bone formation. High-anity nuclear binding sites for 17 b-oestradiol are described in human (Eriksen et al., 1988) and rat (Komm et al., 1988) osteoblast-like cells. In addition, Pensler et al. (1990) demonstrated oestrogen receptors in the nucleus of osteoclasts derived from membranous bone in children, whereby direct modulation of bone resorption by oestrogen may occur. In contrast, oestrogen de®ciency, which is important in the pathogenesis of postmenopausal osteoporosis, results in marked bone loss with
0003-9969/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved. PII: S 0 0 0 3 - 9 9 6 9 ( 9 8 ) 0 0 0 0 8 - 9
192
T. Yamashiro, T. Takano-Yamamoto / Archives of Oral Biology 43 (1998) 191±195
increased bone turnover in ovariectomized rats (Wronski et al., 1985; 1986; 1987; 1988); trabecular bone loss and an increase in bone turnover were observed in the femur. However, the skeletal alteration associated with ovariectomy varies with the region observed (Ma et al., 1994; Li et al., 1996). Mandibular condylar cartilage is a secondary or embryonic type of cartilage that diers in origin and histological organization from the epiphyseal growth plate of the long bones (Dibbets, 1990; Durkin, 1972). Therefore, it is of interest whether ovariectomy causes dierential eects on the mandibular condyle and femur. We have now sought to evaluate the eects of ovariectomy on the mandibular condyle and femur histomorphometrically in young rats.
2. Materials and methods Ten female Sprague±Dawley rats (Nihon-Animal Inc., Osaka), 6 weeks of age and weighing 150±170 g at the beginning of the study, were anaesthetized intraperitoneally with sodium pentobarbital (13 mg/kg body wt) and urethane (500 mg/kg body wt). The rats had been divided into two groups; ®ve were subjected to bilateral ovariectomy, and ®ve to a sham operation. Food and water were provided ad libitum to both groups. The rats were killed 32 days after the operations. The success of the ovariectomy was con®rmed by a failure to detect ovarian tissue by observation of the markedly atrophied uterine horns. For mandibular size, the following gross measurements were made on the right condyle with calipers: (1) anteroposterior length from the mesial surface of lower ®rst molar to the most posterior point on the condylar head (Fig. 1a); (2) anteroposterior length from the mesial surface of lower ®rst molar to the most posterior point on the angular process (Fig. 1b). Distal femurs and mandibular condyles from each group were removed and ®xed in 70% ethyl alcohol. Specimens were dehydrated in an ethanol series and embedded in methylmethacrylate. Serial sagittal sections of 5-mm thickness were cut and stained with Masson's trichrome. From the serial sections, the specimens containing the widest condylar process and distal femur were used for histomorphometry in mandibular condylar cartilage, distal epiphyseal growth cartilage, and the nearby trabecular bone. The histomorphometric variables were measured with a semiautomatic real-colour image analyser and an Olympus ¯uorescence microscope at 595 interfaced with a personal computer (PCs-9801 EX, NEC, Tokyo), using the software developed for bone histomorphometry (System Supply Co., Matsumoto). The image of the specimen was displayed on a video screen
Fig. 1. Schematic lateral view of the rat mandible: (a) distance between mesial surface of lower ®rst molar and mandibular condyle; (b) distance between mesial surface of lower ®rst molar and angular process.
through a high-resolution colour video camera (Flovel, Tokyo) mounted on the microscope. Each layer of mandibular condylar cartilage and distal femoral epiphyseal cartilage were measured perpendicular to the articular or mesial surface of the epiphyseal cartilage at the centre of cartilage. Mandibular condylar cartilage was histologically characterized as having four distinct layers (Fig. 2 A, B). The most super®cial layer is the articular, which is composed of ®brous tissue. Immediately inferior to the articular layer, the proliferative layer is composed of undierentiated mesenchymal cells. Inferior to the proliferative layer is the hypertrophic layer, which contains larger, spherical, maturing chondrocytes. The mineralized layer is the innermost layer of the hypertrophic layer. With Masson's trichrome the mineralized matrix is stained blue and the non-mineralized red. Distal femoral epiphyseal cartilage was histologically de®ned as having two distinct layers (Fig. 2 C, D). The proliferative layer is composed of mesenchymal cells superior to the hypertrophic layer. The hypertrophic plus mineralized layer is composed of non-mineralized and mineralized hypertrophic cells. The trabecular bone regions analysed histomorphometrically were located in the area extending between 0.5 and 1.0 mm from the calci®ed cartilage for a total area of 0.25 mm2 in the mandibular condyle, and in the secondary spongiosa extending between 1 and 2 mm from the epiphyseal growth plate for a total area of 1 mm2 in the femur. The following variables were quanti®ed at these standardized sampling sites: (1) osteoclast number per mm2 of bone surface; (2) active osteoclast surface, as the percentage of the bone surface covered with osteoclasts; (3) active osteoblast surface, as the percentage of the bone surface covered with osteoblasts; (4) trabecular bone volume (%). Osteoclasts were identi®ed as multinucleate cells containing round nuclei and a large cytoplasm that stained light red with Masson's trichrome, and were located immediately adjacent to the bone surface or in a resorption cavity. Osteoblasts were identi®ed by their size, cuboidal shape, red staining and position adjacent to the osteoid.
T. Yamashiro, T. Takano-Yamamoto / Archives of Oral Biology 43 (1998) 191±195
193
Fig. 2. Histological changes in mandibular condylar cartilage (A, B) and femoral growth cartilage (C, D): (a) articular layer, (p) proliferative layer, (h) hypertrophic layer, (m) mineralized layer, (h + m) hypertrophic layer + mineralized layer. (A) sham, (B) ovariectomized, (C) sham, (D) ovariectomized. Bar, 50 mm; 240.
ANOVA was performed to test for dierences between groups, and p-values of less than 0.05 were considered statistically signi®cant.
3. Results Ovariectomy resulted in a substantial reduction in uterine weight (data not shown). The body weight increased on average 8.5% in ovariectomized as compared to sham-operated rats. Gross mandibular dimensions are presented in Table 1. There was no signi®cant dierence between ovariectomized and sham-operated rats in the anteroposterior length from the mesial surface of the lower ®rst molar to the most posterior point on the condylar head (Fig. 1a) and that from the mesial surface of the lower ®rst molar to the most posterior point on the angular process (Fig. 1b). The results of the histomorphometric analysis of the condylar cartilage and epiphyseal growth plate are Table 1 Gross mandibular measurements in ovariectomized (OVX) and sham-operated rats Mandibular length (mm) First molar to condyle (a) First molar to angular process (b)
Sham
OVX
18.320.2
18.22 0.6
18.720.2
18.92 0.4
Results are the means2SD from ®ve rats per group. (a) and (b) as in Fig. 1.
shown in Table 2Table 3. Ovariectomy was associated with a signi®cant increase in the total thickness of the condylar and femoral growth-plate cartilages. Of the cartilage layers, the proliferative and hypertrophic were increased markedly in the ovariectomized rats. After ovariectomy there was a 2-fold increase in thickness of the proliferative layer and a 4-fold increase in thickness of the hypertrophic layer in the condylar cartilage (Table 2), and a 1.3-fold increase in thickness of the proliferative layer in the epiphyseal growth cartilage (Table 3). Bone volume, and the bone resorption and formation variables, for trabecular bone of the mandibular condyle were unaected by ovariectomy, as shown in Table 4. Ovariectomy was associated with a 68.5% decrease in bone volume, a 4-fold increase in active osteoblast surface, and 2-fold increases in active osteoclast surface and osteoclast number per mm of bone Table 2 Histomorphometry of condylar cartilage in ovariectomized (OVX) and sham-operated rats Thickness (m m) Total thickness Articular layer Proliferative layer Hypertrophic layer Mineralized layer
Sham 162 240 42 211 50 223 30 214 37 211
OVX 345261* 4826 97223* 15243* 6525
Results are the mean2SD from ®ve rats per group; *p < 0.05.
194
T. Yamashiro, T. Takano-Yamamoto / Archives of Oral Biology 43 (1998) 191±195 Table 3 Histomorphometry of distal femoral epiphyseal plate cartilage in ovariectomized (OVX) and shamoperated rats Thickness (m m)
Sham
OVX
Total thickness Proliferative layer Hypertrophic layer + mineralized layer
205 27 131 27 68 25
24026* 17327* 7427
we reported previously (Takano-Yamamoto and Rodan, 1990). Ovariectomy causes osteopenia, and increased bone resorption and formation, bone turnover in rats (Wronski et al., 1988). Furthermore, ovariectomy does not aect mandibular growth, although the length of the femur was increased after ovariectomy in rats of 4 and 10 weeks of age (Yamazaki and Yamaguchi, 1989). Thus, we here found a prominent dierence in the response to oestrogen de®ciency between the mandibular condyle and the femur in young, growing rats 32 days after ovariectomy. The reason for this dierential response to oestrogen de®ciency is unknown. Both the mandibular condyle and the femur are endochondral bones. However, the mandibular condylar cartilage is secondary or embryonic and the epiphyseal growth plate of long bone is primary cartilage (Dibbets, 1990; Durkin, 1972). The function of the epiphyseal growth plate is bone elongation. In contrast, although the mandibular condylar cartilage has growth potential, especially during the embryonal and early postnatal stages, its main function in rats older than 28 days is adaptive activity between the condylar and temporal bones (Copray et al., 1988). In addition, the condylar and articular cartilages increase their thickness in response to functional loading (Radin et al., 1978; Copray et al., 1988) and also react with an improvement of their structural mechanical properties (Bouvier and Zimny, 1987; Copray et al., 1988). The temporomandibular joint therefore has a more complicated anatomical structure and is exposed to more complex functional demands than other joints. Further studies are needed to under-
surface in the secondary spongiosa of the femur (Table 4).
4. Discussion Ovariectomy is known to cause a signi®cant increase in the thickness of epiphyseal growth cartilage, particularly of the hypertrophic layer (Ornoy et al., 1994). We found similar changes, where 32 days after ovariectomy of young, growing rats the mandibular condyle and epiphyseal growth plate of the femur showed a signi®cant increase in cartilage thickness, particularly of the proliferative and hypertrophic layers. In the mandibular condylar cartilage we found a 2-fold increase in thickness of the proliferative layer and a 4fold increase in the hypertrophic layer; in epiphyseal growth cartilage we found a 1.3-fold increase in thickness of the proliferative layer. Oestrogen de®ciency thus had a much stronger eect on the mandibular condylar cartilage than on the distal femoral epiphyseal growth cartilage. Trabecular bone loss and increased bone turnover, which is shown typically in the femur, were not observed in the mandibular condyle in young, growing rats by 32 days after ovariectomy. On the contrary, ovariectomy aected these variables in the secondary spongiosa of the rat femur, causing a 68.5% decrease in bone volume, a 4-fold increase in the active osteoblast surface and 2-fold increases in the active osteoclast surface and the number of osteoclasts (as de®ned in Methods). These changes were similar to those that
Table 4 Bone histomorphometry of mandibular condyle and femur in ovariectomized (OVX) and sham-operated rats Mandibular condyle Variables Bone volume (%) Ob.S/BS (%) Oc.S/BS (%) N.Oc/BS (/ mm)
Femur
Sham
OVX
Sham
52.72 4.7 7.026.8 5.822.3 1.020.7
53.5 23.9 11.4 27.5 8.2 24.5 0.9 20.4
20.026.5 3.422.3 6.522.1 0.720.3
OVX 6.32 2.5* 12.62 2.1* 12.22 0.7* 1.62 0.2*
Results are the mean 2SD from ®ve rats per group; *p < 0.05. Ob.S/BS, active osteoblast surface; Oc.S/BS, active osteoclast surface; N.Oc/BS, number of osteoclasts (all in relation to bone surface, as de®ned in Methods).
T. Yamashiro, T. Takano-Yamamoto / Archives of Oral Biology 43 (1998) 191±195
stand the reason for the enhancement of the cartilage layer of the mandibular condyle without increased bone turnover associated with oestrogen de®ciency.
Acknowledgements This study was supported in part by Grants-in-Aid for Scienti®c Research from the Ministry of Education, Science and Culture of Japan (05771874 and 02454470).
References Bouvier, M., Zimny, M.L., 1987. Eects of mechanical loads on surface morphology of the condylar cartilage of the mandible in rats. Acta Anat. Basel. 129, 293±300. Copray, J.C., Dibbets, J.M., Kantomaa, T., 1988. The role of condylar cartilage in the development of the temporomandibular joint. Angle Orthod. 58, 369±380. Dayani, N., Corvol, M.T., Robel, P., Eychenne, B., Moncharmont, B., Tsagris, L., Rappaport, R., 1988. Estrogen receptors in cultured rabbit articular chondrocytes, in¯uence of age. J. Steroid Biochem. 31, 351±356. Dibbets, J.M., 1990. Mandibular rotation and enlargement. Am. J. Orthod. Dentofac. Orthop. 98, 29±32. Durkin, J.F., 1972. Secondary cartilage: a misnomer?. Am. J. Orthod. 62, 15±41. Eriksen, E.F., Colvard, D.S., Berg, N.J., Graham, M.L., Mann, K.G., Spelsberg, T.C., Riggs, B.L., 1988. Evidence of estrogen receptors in normal human osteoblast-like cells. Science 241, 84±86. Ernst, M., Schmid, C., Froesch, E.R., 1988. Enhanced osteoblast proliferation and collagen gene expression by estradiol. Proc. Natl. Acad. Sci. U.S.A. 85, 2307±2310. Fukayama, S., Tashjian, A.J., 1989. Direct modulation by estradiol of the response of human bone cells (SaOS-2) to human parathyroid hormone (PTH) and PTH-related protein. Endocrinology 124, 397±401. Gray, T.K., Flynn, T.C., Gray, K.M., Nabell, L.M., 1987. 17 beta-estradiol acts directly on the clonal osteoblastic cell line UMR106. Proc. Natl. Acad. Sci. U.S.A. 84, 6267±6271. Komm, B.S., Terpening, C.M., Benz, D.J., Graeme, K.A., Gallegos, A., Korc, M., Greene, G.L., O'Malley, B.W., Haussler, M.R., 1988. Estrogen binding, receptor mRNA, and biologic response in osteoblast-like osteosarcoma cells. Science 241, 81±84. Koski, K., Lahdemaki, P., 1975. Adaptation of the mandible in children with adenoids. Am. J. Orthod. 68, 660±665.
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Li, M., Shen, Y., Qi, H., Wronski, T.J., 1996. Comparative study of skeletal response to estrogen depletion at red and yellow marrow sites in rats. Anat. Rec. 245, 472±480. Ma, Y.F., Ke, H.Z., Jee, W.S., 1994. Prostaglandin E2 adds bone to a cancellous bone site with a closed growth plate and low bone turnover in ovariectomized rats. Bone 15, 137±146. Okuda, T., Yasuoka, T., Nakashima, M., Oka, N., 1996. The eect of ovariectomy on the temporomandibular joints. J. Oral Maxillofac. Surg. 54, 1201±1210. Ornoy, A., Giron, S., Aner, R., Goldstein, M., Boyan, B.D., Schwartz, Z., 1994. Gender dependent eects of testosterone and 17 beta-estradiol on bone growth and modelling in young mice. Bone Miner. 24, 43±58. Pensler, J.M., Radosevich, J.A., Higbee, R., Langman, C.B., 1990. Osteoclasts isolated from membranous bone in children exhibit nuclear estrogen and progesterone receptors. J. Bone Min. Res. 5, 797±802. Radin, E.L., Ehrlich, M.G., Chernack, R., Abernethy, P., Paul, I.L., Rose, R.M., 1978. Eect of repetitive impulsive loading on the knee joints of rabbits. Clin. Orthop. 131, 288±293. Silbermann, M., Frommer, J., 1972. The nature of endochondral ossi®cation in the mandibular condyle of the mouse. Anat. Rec. 172, 659±667. Silbermann, M., Reddi, A.H., Hand, A.R., Leapman, R.D., Von der Mark, K., Franzen, A., 1987. Further characterization of the extracellular matrix in the mandibular condyle in neonatal mice. J. Anat. 151, 169±188. Sperber, G.H., 1976. Craniofacial Embryology. 2nd. John Wright and Sons, Ltd, Bristol. Takano-Yamamoto, T., Rodan, G.A., 1990. Direct eects of 17 beta-estradiol on trabecular bone in ovariectomized rats. Proc. Natl. Acad. Sci. U.S.A. 87, 2172±2176. Turner, R.T., Evans, G.L., Wakley, G.K., 1994. Reduced chondroclast dierentiation results in increased cancellous bone volume in estrogen-treated growing rats. Endocrinology 134, 461±466. Wronski, T.J., Lowry, P.L., Walsh, C.C., Ignaszewski, L.A., 1985. Skeletal alterations in ovariectomized rats. Calcif. Tissue Int. 37, 324±328. Wronski, T.J., Walsh, C.C., Ignaszewski, L.A., 1986. Histologic evidence for osteopenia and increased bone turnover in ovariectomized rats. Bone 7, 119±123. Wronski, T.J., Schenck, P.A., Cintron, M., Walsh, C.C., 1987. Eect of body weight on osteopenia in ovariectomized rats. Calcif. Tissue Int. 40, 155±159. Wronski, T.J., Cintron, M., Dann, L.M., 1988. Temporal relationship between bone loss and increased bone turnover in ovariectomized rats. Calcif. Tissue Int. 43, 179±183. Yamazaki, I., Yamaguchi, H., 1989. Characteristics of an ovariectomized osteopenic rat model. J. Bone Min. Res. 4, 13±22.
Archives of Oral Biology 43 (1998) 191±195
ARCHIVES OF ORAL BIOLOGY
Dierential responses of mandibular condyle and femur to oestrogen de®ciency in young rats T. Yamashiro, T. Takano-Yamamoto * Department of Orthodontics, Okayama University Dental School, 2-5-1 Shikata-cho, Okayama 700-8525, Japan Accepted 2 December 1997
Abstract This response of condyle and femur was evaluated by histomorphometry. Five female Sprague±Dawley rats, 6 weeks of age, were subjected to bilateral ovariectomy, and ®ve others were sham-operated. They were then killed 32 days later. Ovariectomy signi®cantly increased the total thickness of the condylar cartilage and distal femoral growth-plate cartilage in the young, growing rats. Ovariectomy caused a 2-fold increase in thickness of the proliferative layer and a 4-fold increase in thickness of the hypertrophic layer in the condylar cartilage, and a 1.3fold increase in thickness of the proliferative layer in the epiphyseal growth cartilage of the femur. Ovariectomy had no eect on the percentage of trabecular bone volume, the percentage of the bone surface covered by osteoblasts (Ob.S/BS) and osteoclasts (Oc.S/BS), and number of osteoclasts per bone surface (N.Oc/BS) in the mandibular condyle. On the contrary, ovariectomy caused a 68.5% decrease in bone volume, a 4-fold increase in Ob.S/BS, and 2-fold increases in Oc.S/BS and N.Oc/BS in the secondary spongiosa of the rat distal femur. Thus there was a prominent dierence in the response to oestrogen de®ciency between the mandibular condyle and femur in young growing rats. # 1998 Elsevier Science Ltd. All rights reserved. Keywords: Mandibular condyle; Femur; Ovariectomy; Bone histomorphometry; Rat
1. Introduction Cartilage increases its volume initially by cell proliferation and matrix formation, and then it is gradually replaced by bone (Silbermann et al., 1987). The mandibular condylar cartilage is actively involved in endochondral bone formation and contributes to the elongation of the mandibular ramus (Silbermann and Frommer, 1972), which is believed to play an important part in cranofacial growth (Koski and Lahdemaki, 1975; Sperber, 1976). Rabbit articular chondrocytes have speci®c receptors for 17 b-oestradiol (Dayani et al., 1988), suggesting that the direct regulation of cellular metabolism by sex hormones in chondrocytes is mediated through their own receptors. In the epiphyseal growth plate, ovariectomy increased the hyper-
* To whom all correspondence should be addressed.
trophic layer of cartilage (Ornoy et al., 1994), and oestrogen treatment of ovariectomized rats caused a decrease in thickness of the tibial growth plate (Turner et al., 1994). Ovariectomy had a similar eect on mandibular condylar cartilage (Okuda et al., 1996). The outcome of oestrogen treatment in vitro (Gray et al., 1987; Ernst et al., 1988; Fukayama and Tashjian, 1989) and in vivo (Takano-Yamamoto and Rodan, 1990) strongly suggests an anabolic eect of oestrogen on bone formation. High-anity nuclear binding sites for 17 b-oestradiol are described in human (Eriksen et al., 1988) and rat (Komm et al., 1988) osteoblast-like cells. In addition, Pensler et al. (1990) demonstrated oestrogen receptors in the nucleus of osteoclasts derived from membranous bone in children, whereby direct modulation of bone resorption by oestrogen may occur. In contrast, oestrogen de®ciency, which is important in the pathogenesis of postmenopausal osteoporosis, results in marked bone loss with
0003-9969/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved. PII: S 0 0 0 3 - 9 9 6 9 ( 9 8 ) 0 0 0 0 8 - 9
192
T. Yamashiro, T. Takano-Yamamoto / Archives of Oral Biology 43 (1998) 191±195
increased bone turnover in ovariectomized rats (Wronski et al., 1985; 1986; 1987; 1988); trabecular bone loss and an increase in bone turnover were observed in the femur. However, the skeletal alteration associated with ovariectomy varies with the region observed (Ma et al., 1994; Li et al., 1996). Mandibular condylar cartilage is a secondary or embryonic type of cartilage that diers in origin and histological organization from the epiphyseal growth plate of the long bones (Dibbets, 1990; Durkin, 1972). Therefore, it is of interest whether ovariectomy causes dierential eects on the mandibular condyle and femur. We have now sought to evaluate the eects of ovariectomy on the mandibular condyle and femur histomorphometrically in young rats.
2. Materials and methods Ten female Sprague±Dawley rats (Nihon-Animal Inc., Osaka), 6 weeks of age and weighing 150±170 g at the beginning of the study, were anaesthetized intraperitoneally with sodium pentobarbital (13 mg/kg body wt) and urethane (500 mg/kg body wt). The rats had been divided into two groups; ®ve were subjected to bilateral ovariectomy, and ®ve to a sham operation. Food and water were provided ad libitum to both groups. The rats were killed 32 days after the operations. The success of the ovariectomy was con®rmed by a failure to detect ovarian tissue by observation of the markedly atrophied uterine horns. For mandibular size, the following gross measurements were made on the right condyle with calipers: (1) anteroposterior length from the mesial surface of lower ®rst molar to the most posterior point on the condylar head (Fig. 1a); (2) anteroposterior length from the mesial surface of lower ®rst molar to the most posterior point on the angular process (Fig. 1b). Distal femurs and mandibular condyles from each group were removed and ®xed in 70% ethyl alcohol. Specimens were dehydrated in an ethanol series and embedded in methylmethacrylate. Serial sagittal sections of 5-mm thickness were cut and stained with Masson's trichrome. From the serial sections, the specimens containing the widest condylar process and distal femur were used for histomorphometry in mandibular condylar cartilage, distal epiphyseal growth cartilage, and the nearby trabecular bone. The histomorphometric variables were measured with a semiautomatic real-colour image analyser and an Olympus ¯uorescence microscope at 595 interfaced with a personal computer (PCs-9801 EX, NEC, Tokyo), using the software developed for bone histomorphometry (System Supply Co., Matsumoto). The image of the specimen was displayed on a video screen
Fig. 1. Schematic lateral view of the rat mandible: (a) distance between mesial surface of lower ®rst molar and mandibular condyle; (b) distance between mesial surface of lower ®rst molar and angular process.
through a high-resolution colour video camera (Flovel, Tokyo) mounted on the microscope. Each layer of mandibular condylar cartilage and distal femoral epiphyseal cartilage were measured perpendicular to the articular or mesial surface of the epiphyseal cartilage at the centre of cartilage. Mandibular condylar cartilage was histologically characterized as having four distinct layers (Fig. 2 A, B). The most super®cial layer is the articular, which is composed of ®brous tissue. Immediately inferior to the articular layer, the proliferative layer is composed of undierentiated mesenchymal cells. Inferior to the proliferative layer is the hypertrophic layer, which contains larger, spherical, maturing chondrocytes. The mineralized layer is the innermost layer of the hypertrophic layer. With Masson's trichrome the mineralized matrix is stained blue and the non-mineralized red. Distal femoral epiphyseal cartilage was histologically de®ned as having two distinct layers (Fig. 2 C, D). The proliferative layer is composed of mesenchymal cells superior to the hypertrophic layer. The hypertrophic plus mineralized layer is composed of non-mineralized and mineralized hypertrophic cells. The trabecular bone regions analysed histomorphometrically were located in the area extending between 0.5 and 1.0 mm from the calci®ed cartilage for a total area of 0.25 mm2 in the mandibular condyle, and in the secondary spongiosa extending between 1 and 2 mm from the epiphyseal growth plate for a total area of 1 mm2 in the femur. The following variables were quanti®ed at these standardized sampling sites: (1) osteoclast number per mm2 of bone surface; (2) active osteoclast surface, as the percentage of the bone surface covered with osteoclasts; (3) active osteoblast surface, as the percentage of the bone surface covered with osteoblasts; (4) trabecular bone volume (%). Osteoclasts were identi®ed as multinucleate cells containing round nuclei and a large cytoplasm that stained light red with Masson's trichrome, and were located immediately adjacent to the bone surface or in a resorption cavity. Osteoblasts were identi®ed by their size, cuboidal shape, red staining and position adjacent to the osteoid.
T. Yamashiro, T. Takano-Yamamoto / Archives of Oral Biology 43 (1998) 191±195
193
Fig. 2. Histological changes in mandibular condylar cartilage (A, B) and femoral growth cartilage (C, D): (a) articular layer, (p) proliferative layer, (h) hypertrophic layer, (m) mineralized layer, (h + m) hypertrophic layer + mineralized layer. (A) sham, (B) ovariectomized, (C) sham, (D) ovariectomized. Bar, 50 mm; 240.
ANOVA was performed to test for dierences between groups, and p-values of less than 0.05 were considered statistically signi®cant.
3. Results Ovariectomy resulted in a substantial reduction in uterine weight (data not shown). The body weight increased on average 8.5% in ovariectomized as compared to sham-operated rats. Gross mandibular dimensions are presented in Table 1. There was no signi®cant dierence between ovariectomized and sham-operated rats in the anteroposterior length from the mesial surface of the lower ®rst molar to the most posterior point on the condylar head (Fig. 1a) and that from the mesial surface of the lower ®rst molar to the most posterior point on the angular process (Fig. 1b). The results of the histomorphometric analysis of the condylar cartilage and epiphyseal growth plate are Table 1 Gross mandibular measurements in ovariectomized (OVX) and sham-operated rats Mandibular length (mm) First molar to condyle (a) First molar to angular process (b)
Sham
OVX
18.320.2
18.22 0.6
18.720.2
18.92 0.4
Results are the means2SD from ®ve rats per group. (a) and (b) as in Fig. 1.
shown in Table 2Table 3. Ovariectomy was associated with a signi®cant increase in the total thickness of the condylar and femoral growth-plate cartilages. Of the cartilage layers, the proliferative and hypertrophic were increased markedly in the ovariectomized rats. After ovariectomy there was a 2-fold increase in thickness of the proliferative layer and a 4-fold increase in thickness of the hypertrophic layer in the condylar cartilage (Table 2), and a 1.3-fold increase in thickness of the proliferative layer in the epiphyseal growth cartilage (Table 3). Bone volume, and the bone resorption and formation variables, for trabecular bone of the mandibular condyle were unaected by ovariectomy, as shown in Table 4. Ovariectomy was associated with a 68.5% decrease in bone volume, a 4-fold increase in active osteoblast surface, and 2-fold increases in active osteoclast surface and osteoclast number per mm of bone Table 2 Histomorphometry of condylar cartilage in ovariectomized (OVX) and sham-operated rats Thickness (m m) Total thickness Articular layer Proliferative layer Hypertrophic layer Mineralized layer
Sham 162 240 42 211 50 223 30 214 37 211
OVX 345261* 4826 97223* 15243* 6525
Results are the mean2SD from ®ve rats per group; *p < 0.05.
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T. Yamashiro, T. Takano-Yamamoto / Archives of Oral Biology 43 (1998) 191±195 Table 3 Histomorphometry of distal femoral epiphyseal plate cartilage in ovariectomized (OVX) and shamoperated rats Thickness (m m)
Sham
OVX
Total thickness Proliferative layer Hypertrophic layer + mineralized layer
205 27 131 27 68 25
24026* 17327* 7427
we reported previously (Takano-Yamamoto and Rodan, 1990). Ovariectomy causes osteopenia, and increased bone resorption and formation, bone turnover in rats (Wronski et al., 1988). Furthermore, ovariectomy does not aect mandibular growth, although the length of the femur was increased after ovariectomy in rats of 4 and 10 weeks of age (Yamazaki and Yamaguchi, 1989). Thus, we here found a prominent dierence in the response to oestrogen de®ciency between the mandibular condyle and the femur in young, growing rats 32 days after ovariectomy. The reason for this dierential response to oestrogen de®ciency is unknown. Both the mandibular condyle and the femur are endochondral bones. However, the mandibular condylar cartilage is secondary or embryonic and the epiphyseal growth plate of long bone is primary cartilage (Dibbets, 1990; Durkin, 1972). The function of the epiphyseal growth plate is bone elongation. In contrast, although the mandibular condylar cartilage has growth potential, especially during the embryonal and early postnatal stages, its main function in rats older than 28 days is adaptive activity between the condylar and temporal bones (Copray et al., 1988). In addition, the condylar and articular cartilages increase their thickness in response to functional loading (Radin et al., 1978; Copray et al., 1988) and also react with an improvement of their structural mechanical properties (Bouvier and Zimny, 1987; Copray et al., 1988). The temporomandibular joint therefore has a more complicated anatomical structure and is exposed to more complex functional demands than other joints. Further studies are needed to under-
surface in the secondary spongiosa of the femur (Table 4).
4. Discussion Ovariectomy is known to cause a signi®cant increase in the thickness of epiphyseal growth cartilage, particularly of the hypertrophic layer (Ornoy et al., 1994). We found similar changes, where 32 days after ovariectomy of young, growing rats the mandibular condyle and epiphyseal growth plate of the femur showed a signi®cant increase in cartilage thickness, particularly of the proliferative and hypertrophic layers. In the mandibular condylar cartilage we found a 2-fold increase in thickness of the proliferative layer and a 4fold increase in the hypertrophic layer; in epiphyseal growth cartilage we found a 1.3-fold increase in thickness of the proliferative layer. Oestrogen de®ciency thus had a much stronger eect on the mandibular condylar cartilage than on the distal femoral epiphyseal growth cartilage. Trabecular bone loss and increased bone turnover, which is shown typically in the femur, were not observed in the mandibular condyle in young, growing rats by 32 days after ovariectomy. On the contrary, ovariectomy aected these variables in the secondary spongiosa of the rat femur, causing a 68.5% decrease in bone volume, a 4-fold increase in the active osteoblast surface and 2-fold increases in the active osteoclast surface and the number of osteoclasts (as de®ned in Methods). These changes were similar to those that
Table 4 Bone histomorphometry of mandibular condyle and femur in ovariectomized (OVX) and sham-operated rats Mandibular condyle Variables Bone volume (%) Ob.S/BS (%) Oc.S/BS (%) N.Oc/BS (/ mm)
Femur
Sham
OVX
Sham
52.72 4.7 7.026.8 5.822.3 1.020.7
53.5 23.9 11.4 27.5 8.2 24.5 0.9 20.4
20.026.5 3.422.3 6.522.1 0.720.3
OVX 6.32 2.5* 12.62 2.1* 12.22 0.7* 1.62 0.2*
Results are the mean 2SD from ®ve rats per group; *p < 0.05. Ob.S/BS, active osteoblast surface; Oc.S/BS, active osteoclast surface; N.Oc/BS, number of osteoclasts (all in relation to bone surface, as de®ned in Methods).
T. Yamashiro, T. Takano-Yamamoto / Archives of Oral Biology 43 (1998) 191±195
stand the reason for the enhancement of the cartilage layer of the mandibular condyle without increased bone turnover associated with oestrogen de®ciency.
Acknowledgements This study was supported in part by Grants-in-Aid for Scienti®c Research from the Ministry of Education, Science and Culture of Japan (05771874 and 02454470).
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