Radiological Morphometric Analysis of the Mandibular Bone Structure after Ovariectomy in Mature Cynomolgus Monkeys

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1 Oral Science International, May 2005, p Copyright 2005, Japanese Stomatology Society. All Rights Reserved. Radiological Morphometric Analysis of the Mandibular Bone Structure after Ovariectomy in Mature Cynomolgus Monkeys Hideomi Asai, Yusuke Kozai, Yukiko Matsumoto, Ryota Kawamata, Satsuki Kumasaka, Takashi Sakurai and Isamu Kashima Division of Radiology, Department of Maxillofacial Diagnostic Science, Kanagawa Dental College Chief: Professor Isamu Kashima Department of Radiological Sciences, Faculty of Health Sciences, Komazawa University Chief: Professor Masao Takano Abstract: Purpose The effects of experimental osteoporosis on the trabecular bone structure of the mandible in cynomolgus monkeys were examined by radiological bone morphometric analysis. Methods Ovariectomy OVX was performed on twelve 10-year-old female cynomolgus monkeys, which were fed a controlled diet for 2 years. Twelve monkeys in a sham control group were fed under the same conditions. Using a microfocus tube and computed radiography, the removed mandibular bone samples were imaged by standardized magnification radiography, and two-dimensional digital imaging data were obtained. The structural parameters, such as skeletal area, perimeter, number, complexity, continuity and anisotropy, were measured using radiological bone morphometric analysis. The bone mineral density BMD was measured by dual energy X-ray absorptiometry. The width of the cortical bone was measured using magnified radiographic images. Results There were no significant differences between the OVX and the sham control groups in the skeletal structure indicated by the skeletal volume, number, width, perimeter, complexity, continuity, separation and spacing. However, there were significant differences between the two groups in the BMD of the mandibular body, cortical bone width, anisotropy and some parameters of the skeletal continuity. Among these parameters, the difference in the thinning of the cortical bone was most significant. Conclusions Using two-dimensional digital radiographic image data, this study suggests that the cortical bone width is more useful than the trabecular bone structure as the morphologic parameter for diagnosis of osteoporosis in the mandibular body. Key words : bone structure, digital radiography, mandibular bone, bone morphometry, cynomolgus monkey Received 3/25/05; accepted 6/2/05. Grant support: This research was partially supported by the Ministry of Education, Science, Sports and Culture, Grant-in-Aid for Scientific Research B 2, , Requests for reprints : Yusuke Kozai, Division of Radiology, Department of Maxillofacial Diagnostic Science, Kanagawa Dental College, 82 Inaoka-cho, Yokosuka-city, Kanagawa Japan, Phone: , FAX: , kozai@kdcnet.ac.jp Introduction Osteoporosis is defined as a systemic bone disease with a high risk of fracture caused by weakening of the bone due to reduction of the bone mineral density BMD and deterioration of the bone microstructure 1. Clinical fracture of the spinal bone, wrist bone and femoral neck commonly occur during falls within the osteoporotic population. Therefore, the BMD mainly in the lumbar

2 May, vertebrae, radius, and femoral head is used as a criterion for diagnosis and follow-up observation of treatment for osteoporosis. In the dental field, there have been clinical studies on the relationship between the mandible and osteoporosis. In particular, osteoporosis is considered to be a risk factor of periodontal diseases, temporomandibular disorders and denture instability due to alveolar ridge absorption 2 6. A number of basic studies using rats have reported the correlation between osteoporosis and reduction of the BMD in the mandibular body and head These studies suggest that general deficiency of estrogen affects the maxillofacial region as well as the long bones of the body. However, human mandibular bone has alveolar bone with teeth planted in its bone body that receive high occlusal force, which does not occur in long bones. Since the mandibular bone receives mechanical stress by masseter muscles, it has thick cortical bone and high BMD. These characteristics of mandibular bones are not observed in other long bones. There are many differences in the anatomical structure and function in the maxillofacial region between mice and humans. In particular, since lateral occlusal movement does not occur in rats, the effects of occlusion on rat mandibular bones are unlikely to be directly related to clinical evaluation with humans. In this study, using two-dimensional digital imaging data, we evaluated the effects of experimental osteoporosis on the mandibular body in cynomolgus monkeys, in which the occlusal movement is similar to that in humans. Materials and methods 1 Animals Both animal care and protocols for sampling were approved by INA Research Philippines Inc. Laguna, Philippines. Animal care and all experimental procedures conformed to the Principles of Laboratory Animal Care National Institute of Health NIH and to the appropriate national laws. Twenty-four female cynomolgus monkeys 10 years old were acquired from SI- CONBREC Manila, Philippines. Each animal was housed and fed in an area 60 cm 70 cm 77 cm that was subjected to a 12-hour light-dark cycle and maintained at about 25 with 45 80% humidity and ventilated 12 times per hour. The animals were given 100 g of artificial food containing 1% calcium certified primate Diet#5048, PMI Feed Inc., Richmond, USA and 30 g banana every day at 1 pm. Chlorinated well water was given ad libitum. The 24 female cynomolgus monkeys were fed for 60 days under the above conditions. General anesthesia was performed in the animals using 7-15 mg ketamine and 0.5 mg/kg xylazine. Bilateral ovariectomy OVX was performed in 12 animals, and sham surgery was performed in the remaining animals. They were divided into the sham control and the OVX groups. The groups were fed under the same conditions for 2 more years. The animals were sacrificed and the mandibular bones were removed. The mandibular bone was fixed in 10% formalin for 1 day, and then immersed in 70% alcohol. The BMD of the vertebrae and the bone of extremities were measured 2 years after surgery by dual energy X-ray absorptiometry DXA. The results showed that the BMD was lower in the lumbar vertebrae 14%, the proximal epiphysis of the femur 20%, the corpus femoris 13%, the radius 23% and the tibia 20% in the OVX group as compared to the sham control group Image Acquisition The mandibular bone was imaged by standardized direct 2.0-fold magnification radiography using an imaging plate IP, STVI type, Fuji Film Inc. and a microfocus tube with a focus size of 10 m 10 m P-70 II type, Pony Atomic Industry Inc.. Imaging was performed at a tube voltage of 30 kv, a tube current of 90 A, an irradiation time of 20 sec, a focus-object distance of 11 mm, and a focus-ip distance of 22 mm. Radiographic imaging data were read at a 100 m reading aperture size by the computed radiography system CR, CR AC-1 plus, Fuji Film Inc.. Structural parameters were measured by radiological bone morphometric analysis BONE, Idea Garden, Tokyo, Japan using the digital radiographic data. 3 Measurement of the BMD and the Cortical Bone Width The BMD in the mandibular bone was quantified by DXA. A bone body area, measuring 3 mm 23 mm, in the lower mandible without teeth or the cortical bone was established for measurement by area scanning. The measurement was performed three times, and the

3 56 Oral Science International Vol. 2, No. 1 mean was determined. The measurement of the cortical bone width at the inferior border of the mandible was performed on the digital radiographic images which were displayed on a cathode ray tube. The measurement region was established in the cortical bone in the inferior border of the mandible under the region of interest ROI of structural analysis. The measurement was performed in three regions, and the mean was determined. 4 Radiological Bone Morphometric Analysis Fig. 1 shows the block diagram of the radiological bone morphometric analysis used in this study. The ROI for analysis 20 mm 3 mm was established in the mandibular body region without teeth or the cortical bone in the inferior border of the mandible. Noise was removed from the digital radiographic imaging data of the trabeculae in the ROI by median filter treatment mask size, 3 3 pixels. Skeletal features of the trabeculae were extracted from the digital image data using a disk shaped single structuring element with a 5-pixel diameter, and images with the skeletal features were produced. The operation number n was 1-7, and the extraction of the skeletal features was performed in the trabeculae which had a width between 250 m and 1450 m. The equation used for thin analysis was a skeletal operation. After the skeletal gray scale images were binarized at a threshold level of 1.0, isolated skeletal components of less than 20 pixels were removed by a selective skeletal filtering process. The skeletal area Sk.Ar, the skeletal perimeter Sk.P, the skeletal thickness Sk.Th, the skeletal number Sk.N, the skeletal separation Sk.Sp, the skeletal spacing Sk. Spac, the trabecular bone pattern factor TBPf, the fractal dimension FD and the mean intercept length MIL were determined by morphometric indices calculated from the skeletal binary image data. Sk.Ar is a relative assessment of the quantity of skeletal structure in the entire tissue area T.Ar, and Sk.P is a relative assessment of the length of the skeletal perimeter in T.Ar. Sk.N shows the number of the skeletal elements; Sk.Th shows the mean width of the skeletal elements; Sk.Sp and Sk.Spac show the distance between the ends or centers of adjacent skeletal elements; TBPf shows the unevenness of the skeleton ; MIL shows the skeletal anisotropy. Continuity of the skeletal structures was evaluated by star volume analysis. In this system, the Fig. 1 Block diagram of radiological bone morphometric analyzing system. The system consists of median filter, morphological filter based on mathematical morphology, selective skeletal filter and bone morphometric analysis morphometric indices calculation, star volume analysis and node-strut analysis. trabecular star volume is replaced by the trabeculae skeletal star volume Vsk, and the marrow space star volume is replaced by the non-skeletal star volume Vsp. With increasing trabecular number and continuity, Vsk becomes larger as Vsp becomes smaller. Conversely, with decreasing trabecular number and continuity, Vsk becomes smaller as Vsp becomes larger. The intersecting line interval through the designated points in the image is 9 degrees through 360 degrees. Furthermore, skeletal features at a width of 1 pixel were extracted from the binary imaging data by a thinning process. Node-strut analysis was performed using the 1 pixel-wide skeletal imaging data. The node Nd number per tissue area T.Ar N.Nd/T.Ar and the terminus Tm number per tissue area N.Tm/T.Ar were determined as the parameters of the joint point number. The ratio of the total NdNd length to the

4 May, Fig. 2 Mean values of bone mineral density BMD in sham control and OVX groups. Fig. 3 Mean values of width of the cortical bone at the inferior border of the mandible in sham control and OVX groups. total strut length TSL NdNd/TSL, the ratio of the total TmTm length to TSL TmTm/TSL and the ratio of each strut length to T.Ar TSL/T.Ar, NdNd/T.Ar, TmTm/T.Ar were determined. The significant difference between the two groups of each parameter was analyzed with Student s t-test. Results Fig. 2 shows the BMD in the mandibular body region without teeth and the inferior border of cortical bone in the sham control and OVX groups. The BMD showed a significant decrease in the OVX group as compared to the sham control group. Fig. 3 shows the width of the cortical bone in the inferior border of the mandible. The mean width of the cortical bone was 3,000 m in the sham control group and 1,250 m in the OVX group, therefore, the mean width was more than twice the size in the sham control group as compared to the OVX group. Fig. 4 shows Sk.Ar, Sk.P, Sk.Th, and Sk.N. These parameters show almost the same value between the sham control and OVX groups, indicating no differences in the skeletal morphology. Fig. 5 shows Sk.Sp and Sk.Spac as the skeletal separation and distance between the ends or centers of adjacent skeletal elements. There were no significant differences in Sk.Sp and Sk.Spac between the sham control and OVX groups. Fig. 6 shows FD as the skeletal complexity. The result of FD showed almost the same values between the sham control and OVX group, and there was no significant difference. Fig. 7 shows Vsk and Vsp as the skeletal continuity. In the OVX group as compared to the sham control group, the result of Vsk was higher and the result of Vsp was lower. In other words, the OVX group showed higher skeletal continuity compared to the sham control group, however, there were no significant differences. Fig. 8 shows TBPf as the skeletal unevenness. There was no significant difference between the sham control and OVX groups. Fig. 9 shows MIL as the skeletal anisotropy. The result of MIL showed high skeletal anisotropy with a significant difference in the OVX group as compared to the sham control group. Fig. 10 shows the results of node-strut analysis as the evaluation of the skeletal continuity. The results of the Nd-related parameters and TSL/T.Ar were slightly higher in the OVX group than in the sham control group, and the results of NdNd/TSL and NdNd/T.Ar increased significantly in the OVX group as compared to the sham control group. The results of Tm-related parameters were slightly higher in the sham control group as compared to the OVX group, but there were no significant differences. Discussion Basic studies on the effects of osteoporosis on mandibular bones have been performed using ovariectomized rats. It has been reported that the BMD in the mandibular body and head regions was reduced by ovariectomy, suggesting that general deficiency of estrogen affects the maxillofacial region as well as the long bones However, since there are many differences

5 58 Oral Science International Vol. 2, No. 1 Fig. 4 Comparison of skeletal area Sk.Ar, skeletal perimeter Sk.P, skeletal thickness Sk.Th and skeletal number Sk.N between sham control and OVX groups. There is no significant difference. Fig. 5 Comparison of skeletal separation Sk.Sp and skeletal spacing Sk.Spac. There is no significant difference. in bone metabolism between rodents and primates, limitations of ovariectomized rats as a standard osteoporosis model have been indicated In particular, closure of the epiphysial line growth line in long bones, which is observed in primates, does not occur in rodents, in which bone growth continues throughout their lifetime. In other words, remodeling, which occurs during bone metabolism in grown primates, is hardly ever observed in rodents. Furthermore, there are differences in the anatomical structure and function in the maxillofacial region between rats and humans. In particular, the occlusal and masticatory functions with anteroposterior and lateral movements of the human mandibles differ from those of rat mandibles. Studies on the relationship between occlusal pressure and the trabecular bone structure using humans and dogs indicated thin-

6 May, Fig. 6 Mean values of fractal dimension FD indicate skeletal complexity between sham control and OVX groups. There is no significant difference. ning and reduction of the trabeculae caused by loss of occlusal pressure However, similar studies using rats reported various results, such as significant reduction of the trabecula, less reduction of the trabecula, and increases in the trabecular density Monkeys, in which mechanical stress on bones is similar to that in humans because of their upright posture, are anatomically and functionally closer to humans than other experimental animals. Like human females, female monkeys have a sexual cycle The changes in bone metabolism in ovariectomized monkeys is the high turnover type as in humans, and the effects of ovarian hormones on bone metabolism in monkeys are considered similar to those in humans. Therefore, the bone strength and bone mass of the vertebra and the long bones in cynomolgus monkeys are reduced by ovariecto- Fig. 7 Comparison of skeletal star volume Vsk and non-skeletal star volume Vsp between sham control and OVX groups. There is no significant difference. Fig. 8 Mean values of trabecular bone pattern factor TBPf indicate skeletal roughness between sham control and OVX groups. There is no significant difference. Fig. 9 Mean values of mean intercept length MIL indicate skeletal anisotropy between sham control and OVX groups.

7 60 Oral Science International Vol. 2, No. 1 Fig. 10 Comparison of node-strut analysis N.Nd/T.Ar, NdNd/T.Ar, NdNd/TSL, N.Tm/T.Ar, TmTm/TSL, TmTm/T.Ar and TSL/T.Ar between sham control and OVX groups. There are no significant differences in N.Nd/T.Ar, N.Tm/T.Ar, TmTm/TSL, TmTm/T.Ar and TSL/T.Ar. my In the ovariectomized monkeys examined in this study, the BMD in the lumbar vertebrae, femur, radius, and cervical bone decreased significantly as compared to the monkeys in the sham control group. The BMD in the mandibular body region also decreased significantly in the OVX group as compared to that in the sham control group Fig. 2. Dental radiography and panoramic radiography are widely used for dental diagnosis. Therefore, clinical studies on the effects of osteoporosis on mandibular bones have been performed mainly by panoramic radiography. It has been reported that parameters showing morphological changes in the cortical bone, such as the mandibular cortical index, the ratio of the mandibular cortical width to the cancellous width and the mandibular inferior cortex, are useful for diagnosis of osteoporosis in mandibular bones This study demonstrated a significant difference in the mandibular cortical width between the OVX and sham control groups, which agreed with the clinical evaluation using panoramic radiography Fig. 3. It has been reported, however, that changes in osteoporotic bones occur more often in the cancellous bone than in the cortical bone 29. Using BMD alone, bone strength can be correlated in 64% of the cases, however, using both BMD and the trabecular bone structure, bone strength can be correlated in 94% of the cases 30. The NIH consensus development panel defines bone strength primarily reflects the integration of bone density and bone quality. Bone quality refers to architecture, turnover, damage accumulation eg, microfractures and mineralization 31 ; therefore, it is important to examine both the bone structure and the BMD in the evaluation of bone strength. In this study, we examined the changes in trabecular bone structure in mandibular bones with osteoporosis. The trabecular bone structure was analyzed by a morphological process which was based on mathematical morphology. This process is used for the selective extraction of trabecular components from the digital imaging data As a result, there were significant differences in MIL and in some Nd-related parameters for node-strut analysis between the OVX and the sham

8 May, control groups Figs. 9 and 10. However, there were no significant differences between the two groups in structural parameters, such as the number and the width of the trabeculae, the complexity and the unevenness Figs These results suggest that the twodimensional digital imaging did not detect changes in the trabecular bone structure in the mandibular bones with osteoporosis because these changes were small. This study suggests that the analysis method does not have the capability to detect small changes in the trabecular bone structure of the mandible. On the other hand, since the mandibular body region is developed by intramembranous bone formation, it differs from long bone epiphysis and vertebra, which are developed by endochondral bone formation 41. The mandibular bone has a region which supports the teeth. The region is called the alveolar bone, and its bone absorption type differs from that of the long bones because it has continuous occlusal pressure upon it. In other words, to prevent loss of the tooth-supporting function due to severe bone absorption, unique alveolar bone proper surrounding the periodontal membrane, the mandibular canal and the cortical bone exists in order to maintain the mandibular bone. This suggests that there are large individual differences in the mandible receiving occlusal pressure via teeth. The alveolar bone, periodontal membrane, cementum, dental pulp and dentine are made from undifferentiated mesenchymal cells derived from the neural crest 42. The mandibular bone was once clinically considered to be similar to other bones. However, it has been reported that the response of mandibular bone cells receiving hormonal and mechanical stimulation clearly differs from that of long bone cells 43. This study suggested that the mandibular body region does not always show similar structural changes to those in long bones. Conclusions Experimental osteoporosis was produced by performing OVX in cynomolgus monkeys, and the effects of osteoporosis on the BMD in the mandibular bone, the width of the cortical bone, and the trabecular bone structure were examined using two-dimensional digital imaging data. As a result, there were significant differences in some parameters of the skeletal continuity and anisotropy between the sham control and the OVX groups, but there were no significant differences in the skeletal morphology, structural complexity, and skeletal unevenness between the two groups. However, the reduction of the BMD and the thinning of the cortical bone were significant in the OVX group as compared to the sham control group. It was suggested that morphological changes in the cortical bone are most useful as parameters for diagnosis of osteoporosis in the mandibular body region. Acknowledgements The authors express their sincere thanks to Dr. Takashi Sasano, Dr. Ken Inukai, Dr. Natsumi Chiba and Dr. Yuuki Murakami, Division of Oral Diagnosis & Radiology, Tohoku University Graduate School of Dentistry, for their useful advice and support regarding this work. This work was performed in the Kanagawa Dental College, Research Center of Advanced Technology for Craniomandibular Function and supported by a grant-in-aid Bioventure Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology. References 1 World Health Organization : Assessment of fracture risk and its application to screening for postmenopausal osteoporosis. WHO technical report series 843, Geneva: WHO: Wactawski-Wende J. : Periodontal diseases and osteoporosis: association and mechanisms. Ann Periodontal , Tezal M., Wactawski-Wende J., Grossi S. G., Ho A. W., Dunford R., and Genco R. J. : The relationship between bone mineral density and periodontitis in postmenopausal women. J Periodontol , Wowern N., and Gotfredsen K. : Implant-supported overdentures, a prevention of bone loss in edentulous mandibles? A5-year follow-up study. Clin Oral Implants Res , Biamchi A., and Sanfilippo F. : Osteoporosis : the effect on mandibular bone resorption and therapeutic possibilities by means of implant prostheses. Int J Periodontics Restorative Dent , Kribbs P.J., Smith D.E., and Chesnut C.H.3rd.: Oral findings in osteoporosis. Part II: Relationship between residual ridge and alveolar bone resorption and generalized skeletal osteopenia. J Prosthet Dent , Tanaka S., Shimizu M., Debari K., Furuya R., Kawawa T., and Sasaki T. : Acute effects of ovariectomy on wound healing of alveolar bone after maxillary molar extraction in aged rats. Ante Rec , Yamashiro T., and Takano-Yamamoto T.: Influences of

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