Original Article. An Impact of Masticatory Muscle Function on IL-1Ç and SOX9 Expression in Condyle. Atith Manopinivate, Sawa Kaneko and Kunimichi Soma

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1 J Med Dent Sci 2006; 53: Original Article An Impact of Masticatory Muscle Function on IL-1Ç and SOX9 Expression in Condyle Atith Manopinivate, Sawa Kaneko and Kunimichi Soma Orthodontic Science, Department of Orofacial Development and Function, Division of Oral Health Sciences, Graduate School, Tokyo Medical and Dental University The importance of masticatory muscle function on the growth and differentiation of condyle was examined. The aim of this study was to investigate how reduced masticatory muscle function influences intrinsic regulatory factors which govern growth and differentiation of condylar cartilage. Masseter muscles of 3-week-old Wistar rats were resected bilaterally. Masseteric resected animals and corresponding control animals were sacrificed at 3, 6, 12 and 21 days post-resection. The condyles were then processed for histological and immunohistochemical analysis. The expression patterns of an inhibitory regulator (IL-1β) and a master regulator (Sox9) of chondrogenesis in condylar cartilage of growing rats were investigated. Quantitative analysis shows that masseteric resection significantly increased the number of IL-1β positive cells in proliferative layer. In contrast, the number of Sox9 positive cells was significantly decreased compared to the control animals. It can be concluded that the reduced articular function due to masseteric resection decreased condylar cartilage cell differentiation which led to the decrease in the thickness of condylar cartilage. Corresponding Author: Atith Manopinivate, DDS, Orthodontic Science, Department of Orofacial Development and Function, Division of Oral Health Sciences, Graduate School, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo , Japan Business Phone: Fax: luke.orts@tmd.ac.jp Received October 14; Accepted December 2, 2005 Key words: condylar cartilage, masseter muscle, IL-1Ç, Sox9 Introduction Many clinical studies have reported a close relation between masticatory muscle function and vertical dimension of facial morphology. A long facial height morphology with a high mandibular plane angle was found in individuals who have low muscular activity 1-4. These individuals also have small cross-sectional area of the masseter muscles 5-6. However, not many clinical studies have reported about the relation between masseter muscle function and the change of condylar growth. The significance of the masticatory muscle function on mandibular condylar cartilage growth was previously studied in various animal study models The growth rate of condylar cartilage was altered by resection of masseter muscle. In particular, a decrease in cartilage thickness was documented in conjunction with an inferior-posterior rotation of mandible expressed by the increase in mandibularpalatal angle Nevertheless, how intrinsic regulatory factors which govern growth, proliferation and differentiation of condylar cartilage cell change after masseteric resection has not yet been demonstrated. It is known that the growth of condylar cartilage is a well-programmed cascade of chondrocyte lineage, controlled by a host of orchestrated influences of various regulatory factors intrinsically synthesized by the cells in the condyle 13. The evidence that articular

2 68 A. MANOPINIVATE, S. KANEKO and K. SOMA J Med Dent Sci chondrocyte synthesizes IL-1Ç, an inhibitory regulator of chondrogenesis, has been reported in both gene and protein level 16. IL-1Ç inhibits chondrocyte proliferation 17 and differentiation by directly suppressing transcription of type II collagen gene, the main component of condylar cartilage matrix 18-20, and also inhibiting glycosaminoglycan production leading to decreased synthesis of aggrecan 19. Thus, these affect condylar cartilage formation and subsequently condylar growth. Besides, Sox9 is a high-mobility group (HMG) type transcription factor that controls the differentiation of chondrocyte in long bone 21 and condylar cartilage 13. This master regulator of cartilage growth is localized in proliferative layer of developing mandibular condyle. In knock-out gene experiments, Sox9-deficient cells demonstrated to be mesenchymal cells that do not express type II collagen, a chondrocyte-specific marker. Thus animals without Sox9 showed some defects in long bones and craniofacial areas 22. Moreover, a recent study reported that IL-1Ç inhibits the expression of the chondrocyte phenotypes by markedly down-regulating the expression of Sox9 23. The aim of this study was to examine how masticatory muscle function influences intrinsic regulatory factors, IL-1Ç and Sox9, which govern growth and differentiation of condylar cartilage. To investigate the impact of reduced masticatory muscle function by resecting masseter muscle on condylar growth and differentiation, cell expressing IL-1Ç and Sox9 and thickness of condylar cartilage were quantified and analyzed after masseteric resection. Materials and Methods Animals The animal protocols were approved by the Institutional Animal Care and Use Committee of the Tokyo Medical and Dental University. Forty four 3-weekold female Wistar rats (Sankyo Labo Service Co., Tokyo, Japan) were selected for this study. They were randomly divided into three groups: bilateral resection of the masseter muscle group (named as masseteric resection group) (n = 20), untreated control group (n = 20) and sham-operation control group (n = 4). Each group of the masseteric resection and the untreated control animals was later sub-divided into 4 subgroups (n = 5). The sham-operation control group was also later sub-divided into 4 subgroups (n = 1) according to the date of sacrifice and the observation periods (3, 6, 12 and 21 days post-resection). During the experimental period, all rats were weighed day-byday. Surgical procedures The surgical procedures were those used as previously reported by Monje F, et al. 11 Identical surgical procedures of masseter muscle resection were performed in all experimental animals. During surgical operation, they were deeply anesthetized with diethyl ether (Wako Pure Chemical Industries, Osaka, Japan) and intraperitoneal injection with a 4:1 mixture of ketamine hydrochloride and 20% xylazine hydrochloride (0.1 ml/ 100g body weight). The masseter muscle was exposed using a preauricular approach, the incision line was made with a scalpel along a line from a point anterior to the external ear to the lower border of the mandible at the angular process, the skin and fascia were carefully reflected, then the muscle was exposed and elevated by means of blunt dissection. The superficial and deep portions of the masseter muscle were removed bilaterally. The surgical sites were finally sutured with interrupted silk and applied with mercurochrome to prevent postoperative infection. The surgical procedures were performed with care under a stereomicroscope (Zeiss PO MI6, Oberkochen, Germany) to avoid any injuries to condyles, posterior blood vessels of condyles and facial nerves. Whereas, the sham-operation control animals were surgically operated following the above mentioned procedures but without removing masseter muscle, served as a surgical control group. The other group of animals was left unoperated as an untreated control group. Tissue acquisition After each interval of post-resection period, each experimental subgroup including the sham-operation control and untreated control subgroups were sacrificed by means of transcardiac perfusion with saline followed by ice-cold 4% paraformaldehyde with 0.21% picric acid in 0.1 M phosphate buffered saline (PBS), ph 7.4 under above mentioned anesthesia. For both histological and immunohistochemical analysis, the condyles were dissected out immediately and immersed en bloc overnight at 4 C in the same fixative solution. Subsequently, the tissue specimens

3 MASTICATORY MUSCLE INFLUENCES CONDYLAR DIFFERENTIATION. 69 were decalcified in 10% ethyl-enediamine tetraacetic acid-2na solution, ph 7.4, at 4 C for 5 weeks, dehydrated in graded ethanols and embedded in paraffin. Serial sections of 7-Òm thickness were made along the sagittal axis. The sections contained the mid-sagittal region of the condyle were stained with Toluidine blue for examination of cartilage thickness. Immunohistochemistry of IL-1Ç and Sox9 In each subject, three of the mid-sagittal sections of the condyles were immunohistochemically processed with antibodies either to IL-1Ç or Sox9. In brief, after the sections were dewaxed, rehydrated, and rinsed with 0.3% Triton X-100 in PBS and then treated with methanol containing 0.3% H2O2 to block endogenous peroxide activity, followed by preincubation with normal goat serum (G9023, 1:9 diluted with PBS, Sigma, MO, USA). After washing, the sections were incubated overnight at 4 C with primary antibodies either polyclonal rabbit anti-rat IL-1Ç (PR427B, 1:100, Endogen, MA, USA) or polyclonal rabbit anti-human Sox9 (sc , 1:200, Santa Cruz, CA, USA). After washing, secondary biotinylated goat anti-rabbit IgG (B-8895, 1:200, Sigma) was applied and then labeled with streptavidin complexed with biotinylated peroxidase. The sections were then developed in DAB (3, 3- diaminobenzidine, D-5637, Sigma) to observe final staining, and counterstained with hematoxylin and mounted with permanent aqueous mounting medium. Negative controls were obtained by replacing the primary antibodies with non-immune rabbit serum. Quantitative evaluation of condylar cartilage thickness and distribution of IL-1Ç and Sox9 positive immunostaining cells The central region of condylar cartilage in midsagittal sections of Toluidine blue stained sections, and IL-1Ç and Sox9 immunostained sections were selected due to the most prominent cellular responses found in this region, then photographed with a digital camera (Nikon DXm1200, Kanagawa, Japan) in order to analyze cartilage thickness and IL-1Ç and Sox9 immunostaining cell distribution, respectively. The entire condylar cartilage was regionalized into three areas: anterior half of the cartilage as the anterior region, posterior quarter of cartilage as the posterior region, and between these two areas as the central region 24. The condylar cartilage thickness of central region (determined by the mid-vertical line along the central region) was measured. The condylar cartilage Figure 1. An overview of the mandibular condyle of a 24-day-old rat shows histological staining of Toluidine blue. The condylar cartilage thickness (a double-headed arrow) was measured by a distance from the superior surface of the proliferative layer to the inferior surface of hypertrophic layer. G, glenoid fossa; D, articular disc; A, anterior region; C, central region; P, posterior region; Co, condyle. Bar = 500 Òm. thickness was defined as a distance from the superior surface of the proliferative layer to the inferior surface of hypertrophic layer 12,25 (Figure 1). The number of immunopositive cells representing the distribution of IL-1Ç and Sox9 in the representative area in proliferative layer of central region of condyle were recognized and quantified by the computer software based on the color, shade and contrast of the feature selected. IL-1Ç and Sox9 positive immunostaining cells were quantified separately under the fixed measurement frame of Òm in the proliferative layer. The data were estimated by Image-Pro Plus image analysis software (V 4.0, Media Cybernetics, MD, USA). Statistical analysis The results of the condylar cartilage thickness and the number of immunopositive cells of IL-1Ç and Sox9 were analysed by Mann-Whitney U-test with 95% of significant level using SPSS statistical software (V 11.0 for Windows, SPSS Inc., Ill, USA). Results Although the mean body weight of the masseteric resection group was slowly increasing compared to the untreated control and the sham-operation control

4 70 A. MANOPINIVATE, S. KANEKO and K. SOMA J Med Dent Sci Figure 3. The temporal pattern of the thickness of condylar cartilage during natural growth and after masseteric resection. Significant differences between control and experimental animals are marked with asterisk (*P 0.05). Figure 4. Immunohistochemistry shows IL-1Ç expression indicated by brown stains in proliferative layer of central region of condylar cartilage during natural growth (a, c; 3 and 6 days after the beginning of the experiment, respectively) and masseteric resection (b, d; 3 and 6 days after the beginning of the experiment, respectively). A box represents a measurement frame of Òm in the proliferative layer of central region. F, fibrous layer; P, proliferative layer; H, hypertrophic layer. Bar = 100 Òm. Figure 2. Histological staining of Toluidine blue shows the thickness of condylar cartilage in central region during natural growth (a, c, e, g; 3, 6, 12, 21 days after the beginning of the experiment, respectively) and masseteric resection (b, d, f, h; 3, 6, 12, 21 days after the beginning of the experiment, respectively). F, fibrous layer; P, proliferative layer; H, hypertrophic layer. Bar = 500 Òm. groups, there was no significant difference in the mean body weight among these groups throughout the experimental period. The sham-operation control and untreated control animals showed similar results of IL-1Ç and Sox9 expression and thickness of condylar cartilage with no significant difference. Therefore the results of shamoperation control animals are not shown in the result part.

5 MASTICATORY MUSCLE INFLUENCES CONDYLAR DIFFERENTIATION. 71 Masseteric resection led to the significant increase in the expression of IL-1Ç in the proliferative layer of central region of condylar cartilage at day 3 and 6 after resection compared to the control animals (P 0.05; Figure 6). When the expression level was followed to the end of the experimental period, expression of IL-1Ç was still higher than control animals but with no statistical significance. On the other hand, quantitative analysis shows that masseteric resection led to the reduction in the expression of Sox9 in the same observation area at day 3 and 6 after resection but with no statistical significance. At day 12 and 21 post-resection, the expression levels of Sox9 decreased significantly compared to control animals (P 0.05; Figure 7). Figure 5. Immunohistochemistry shows Sox9 expression indicated by brown stains in proliferative layer of central region of condylar cartilage during natural growth (a, c; 12 and 21 days after the beginning of the experiment, respectively) and masseteric resection (b, d; 12 and 21 days after the beginning of the experiment, respectively). A box represents a measurement frame of Òm in the proliferative layer of central region. F, fibrous layer; P, proliferative layer; H, hypertrophic layer. Bar = 100 Òm. Histological findings The Toluidine blue stained sagittal sections of the condyle demonstrated a thinner condylar cartilage layer at the central region in the experimental animals compared to control animals at all time points in observation period (Figure 2). Quantitative analysis shows that the thickness of condylar cartilage in experimental animals significantly decreased compared to control animals at every time points in observation period (P 0.05; Figure 3). Immunohistochemical findings and quantitative analysis The inflammatory tissues and proximal tibia growth plate were used as a positive control for IL-1Ç and Sox9 staining, respectively. The expression of IL-1Ç and Sox9 was detected by immunohistochemistry in rat condylar cartilage. Throughout the experimental period, IL-1Ç immunoreactivity was consistently expressed by the cells in the proliferative layer (Figure 4), while the strong expression of Sox9 was also found to be localized in the cells in the proliferative layer (Figure 5). The characteristics of IL-1Ç and Sox9 immunoreactivity were in agreement with those which have been previously reported 13,16. Discussion In the present study, we showed the effect of resection of masseter muscle on the cascade of molecular events responsible for the differentiation of chondrocyte in condylar cartilage by investigating the temporal distribution of IL-1Ç and Sox9 expression. The expression level of IL-1Ç was significantly increased on experimental day 3 and 6 after masseteric resection compared to the control animals. The role of negative regulator of IL-1Ç in chondrogenesis has been reported It is of importance that the study on correlation between IL-1Ç and Sox9 showed that IL-1Ç inhibits Sox9 expression 23. Moreover, the significant reduction of Sox9 expression was found in the proliferative layer on experimental day 12 and 21 after masseteric resection compared to control animals. Therefore it could be explained that increased IL-1Ç expression in early experimental period (at day 3 and 6) was one of the factors being responsible for the decrease in expression of Sox9 on later experimental period (at day 12 and 21). This indicates the decreased differentiation of chondrocytes and reduced entry into chondrogenesis route, which possibly decreased the population of chondrocytes potentially available for chondrogenesis. Thus the above mentioned results of interaction of IL- 1Ç and Sox9 could cause reduction of cartilage thickness after masseteric resection in the later experimental period (at day 12 and 21). However, the decrease in cartilage thickness after masseteric resection compared to the control animals was found at all time points in observation period. Hence, there should be other factors controlling the

6 72 A. MANOPINIVATE, S. KANEKO and K. SOMA J Med Dent Sci Figure 6. Quantitative analysis shows the temporal pattern of expression of IL-1Ç positive immunostaining in a fixed measurement frame in proliferative layer of central region of condylar cartilage from experimental day 3 to day 21. Significant differences between control and experimental animals are marked with asterisk (*P 0.05). Figure 7. Quantitative analysis shows the temporal pattern of expression of Sox9 positive immunostaining in a fixed measurement frame in proliferative layer of central region of condylar cartilage from experimental day 3 to day 21. Significant differences between control and experimental animals are marked with asterisk (*P 0.05). reduction of cartilage thickness in the early experimental period (at day 3 and 6). It has been pointed out that the regulation of condylar growth is composed of proliferation, differentiation and maturation of cartilage cells 26. Moreover, a decrease in cartilage thickness 27 and a marked reduction in proliferative activity 28 in mandibular condylar cartilage were found after reduced articular function by feeding the animals with soft diet. Therefore it could be assumed that the reduced proliferative activity of mesenchymal cells is one of the factors regulating the decrease of cartilage thickness in our study. Furthermore, the accelerated resorption of cartilage at the erosion front is another reasonable explanation for the findings in the early experimental period. Further investigation is required to confirm these assumptions. It seems that the time used by IL-1Ç to respond to the change of condylar loading induced by masseteric resection was shorter than that used by Sox9. We found a significant difference of IL-1Ç expression between experimental and control animals in the early period of experiment (at day 3 and 6), while the difference of Sox9 expression was found at later period (at day 12 and 21). These findings coincided with the previous in vitro study 29 which also found that the timing response of IL-1Ç after change in loading was shorter than that of Sox9. Nevertheless, the time course of IL- 1Ç and Sox9 expression between this in vitro study and our study cannot be compared to each other because of differences in experimental condition. It can imply that the increase of IL-1Ç expression was an early effect from masseteric resection on condylar cartilage differentiation and then the decrease of Sox9 expression was a following effect. Masseteric resection may result in abnormal loading on the tissues in and around the joint. Like other articular cartilage in the body, the condylar cartilage is a main load-bearing structure for induced biomechanical stress on the condyle. However, the most probable explanation for the abnormal loading on condyle caused by the masseteric surgery in this study could be a result of reduced articular function of condylar cartilage. In vitro, the application of mechanical strain promoted chondrogenesis associated with a down-regulation of IL-1Ç and up-regulation of Sox9 transcription factor 29. It was also documented that extrinsic mechanical stress is essential for chondroblast differentiation in the mandibular condyle 30. Furthermore mechanical forces not only affect the proliferative activities of the chondroprogenitor cells, but also have a great impact on their further differentiation and maturation 31. In contrast, lack of mechanical strain caused failure of chondrogenesis 32. The effect of reduced biomechanical stress on chondrocyte maturation 33, proteoglycan synthesis 34, collagen formation 35, and morphological change of condyle 27,36 has also been reported in the condyle. From the viewpoint of mechanical strain and chondrogenesis, it could be assumed that the decreased articular loading induced by masseteric resection caused the disturbance on the cascade of intrinsic molecular event that regulated the growth and differentiation process of the condyle. Our findings support the importance of influence of masticatory muscle function on the growth and differentiation of condyle especially in growing period of life.

7 MASTICATORY MUSCLE INFLUENCES CONDYLAR DIFFERENTIATION. 73 Therefore the optimum force that transmits to condylar cartilage from the function of masticatory muscle is essential for the growth and differentiation process of condylar cartilage. Conclusions Reduced articular loading on condylar cartilage caused by masseteric resection led to the molecular changes in up-regulation of IL-1Ç expression and down-regulation of Sox9 expression. This in turn led to less differentiation of chondrocytes in condyles, resulting in thinner condylar cartilage. Acknowledgments This study was financially supported by Grants-in-Aid for Scientific Research ( , and ) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. References 1. Bakke M, Michler L. Temporalis and masseter muscle activity in patients with anterior open bite and craniomandibular disorders. Scand J Dent Res. 1991;99: Ueda HM, Ishizuka Y, Miyamoto K, et al. 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