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The effect of botulinum toxin injected into masseter muscle on condyle and masseter muscle of young beagle dogs Suk Joo Kim Department of Dentistry The Graduate School, Yonsei University

The effect of botulinum toxin injected into masseter muscle on condyle and masseter muscle of young beagle dogs A Dissertation Thesis Submitted to the Department of Dentistry and the Graduate School of Yonsei University in partial fulfillment of the requirements for the degree of Doctor of Philosophy of Dental Science Suk Joo Kim JUNE 2015

감사의글 이논문이완성되기까지세심한배려와아낌없는격려로저를지도해주시고, 부족한제가올바른학문의길로갈수있도록인도해주셨던황충주지도교수님께진심으로감사드립니다. 연구의시작에서부터함께고민해주시고, 논문이완성될때까지늘정성으로조언해주시고지도해주신김성택교수님께도진심으로감사드립니다. 또한논문을위해따뜻한조언을해주시고힘이되어주신유형석교수님, 차정열교수님, 조성원교수님께깊이감사드립니다. 교정학이라는학문의길에들어설수있게이끌어주시고, 지금도늘곁에서보살펴주시는박영철교수님께깊은감사의말씀을전합니다. 언제나따뜻하고도세심한조언으로함께해주시는백형선교수님, 김경호교수님, 이기준교수님, 정주령교수님께도진심으로감사드립니다. 치과대학학생때의생활뿐만아니라치과의사로살아가는데있어서늘관심과사랑으로이끌어주시는참스승이신김종관교수님께도감사드립니다. 그리고멀리서저를늘지켜봐주시고학문의길잡이가되어주시는교합학의스승이신김인권교수님과송영복교수님께도깊은감사를드립니다. 부족한저를응원해주시고언제나곁에서행복을위해함께해주시는연세여우치과동료원장이신김두형, 박준선, 이선복선배님들께도진심으로감사드립니다. 또한한결같은마음으로용기를주시는소성수선배님께도감사를드립니다. 연구의마무리에도움을준최윤정교수님, 실험실에서함께했던최성환, 김영훈, 이미림, 뭉해돌람, 이성일선생께도깊은감사를드립니다. 연구의어려움을격을때머나먼미국에서까지세심한조언을해준권혁제선생과격조있는영문교정에도움을준안하영선생께도감사드립니다. 그리고무엇보다도저에게세상의빛을보게해주시고정성과사랑으로바르게길러주셔서올바른한사람으로떳떳하게살아가게해주신, 세상에서가장존경하는아버지김덕영옹과사랑하는어머니김경숙여사께진심으로깊은감사를드리고영광을받칩니다. 저를늘아들처럼사랑해주시고감싸안아주시는장모님라분순여사와장인어른김성환옹께도진심으로감사드립니다. 든든한지원군치과의사동료이자사랑하는동생김정주에게도고마움을전합니다. 마지막으로, 연구를완성하기까지묵묵히늘곁에서힘이되어준저의아내와자랑스런세아들에게감사의마음을전합니다. 남편으로서늘부족한저를믿고, 언제나손을꼭잡고행복을위해함께해주는영원한인생의동반자인사랑하는아내김지현과늘온화한미소를품은듬직하고지혜로운똘똘이큰아들김성윤, 가족생각을누구보다많이하는정많고착하며영리한재주꾼둘째아들김성우, 늘가족들에게웃음을주는밝고애교많은똑똑한귀염둥이셋째아들김성민에게도진심으로고맙고영원히사랑한다는말을전합니다. 2015년 6월저자씀

Table of Contents List of Figures ii List of Tables ii Abstract iii I. Introduction 1 II. Materials and Methods 4 1. Materials 4 2. Methods 4 1) Animal preparation 4 2) Botulinum toxin injection 5 3) Collection of tissue samples 7 4) Histological procedure & analysis 8 5) Statistical analysis 8 III. Results 9 1. Condyle 9 2. Masseter muscle 13 IV. Discussion 16 V. Conclusion 23 VI. References 24 Abstract (Korean) 28 i

List of Figures Figure 1. Botulinum toxin (BoNT) injection 5 Figure 2. Time table of study protocol 6 Figure 3. Collection of sample specimen 7 Figure 4. The schematic image of condylar cartilage layers 9 Figure 5. Histological comparison image of condylar cartilage in groups 10 Figure 6. Comparison of thickness in four zones among three groups. 12 Figure 7. Histological analysis of the masseter muscles in groups 13 Figure 8. Comparison of muscle fiber cross sectioned area in groups 14 List of Tables Table 1. Mean thickness of four zones of the condylar cartilage for each groups 11 Table 2. Mean area of cross sectioned muscle fiber in groups 14 ii

Abstract The effect of botulinum toxin injected into masseter muscle on condyle and masseter muscle of young beagle dogs Suk Joo Kim, D.D.S., M.S. Department of Dentistry, Graduate School, Yonsei University (Directed by Prof. Chung Ju Hwang, D.D.S., M.S., Ph.D.) This study was based on the previous studies that muscular function can affect craniofacial and mandibular growth during growth period. There had been numerous attempts to regulate mandibular growth by controlling mandiblar function. Botulinum toxin (BoNT) had been studied as a treatment medicine for muscle hypertrophy. The purpose of this study is to analysis the histological effect of BoNT injected into the masseter muscle on the condylar cartilage and the masseter muscle in young beagle dog. Samples were divided into three groups, such as the saline injection, 10U BoNT injection and 20U BoNT injection. BoNT injection was carried-out twice every 4 weeks. After 10 weeks from the first injection, the histological differences in the condylar cartilage and masseter muscle were evaluated. In condylar cartilage the differences of the thickness in P zones were statistically significant between the two groups (group 10U & Control) and group 20U. H zones were significantly different among three groups. Especially in group 20U thinner P zone and H zone were shown. And Histological differences of masseter muscle between experimental groups (group 20U and group 10U) and control group (group Control) were observed. The shape of masseter iii

muscle fibers in group 10U and group 20U was different from that of group Control. And endomysium was seen clearly with the separated cell connections in group 10U and 20U. The area of cross sectioned muscle fiber among three groups showed a significant difference statistically (p<0.05) each other. Each muscle fiber per unit area of group 20U was the smallest. As a result of this, it was confirmed that BoNT injected into the masseter muscle could affect cell differentiation of condylar cartilage, which could ensure that consistent with the results of the previous studies made in lower animals, such as rat. If the further study that BoNT could affect the morphological changes in the mandible is carried out, this study could be a basis for the growth modification treatment of human mandible by using BoNT. Key words : botulinum toxin, condylar cartilage, masseter muscle, mandibular growth, beagle iv

The effect of botulinum toxin injected into masseter muscle on condyle and masseter muscle of young beagle dogs Suk Joo Kim, D.D.S., M.S. Department of Dentistry, Graduate School, Yonsei University (Directed by Prof. Chung Ju Hwang, D.D.S., M.S., Ph.D.) I. Introduction Botulinum toxin (BoNT) temporarily inhibits the acetylcholine release at the neuromuscular junction and decreases muscular contractions. BoNT based on this mechanism was used in the treatment of strabismus (Scott et al., 1989), and it has been shown to be effective in treating disorders characterized by local muscle hyperactivity. Such use has emerged in the field of dentistry. In dentistry region BoNT is used to treat the masticatory and facial muscle spasms, dystonias, orofacial dyskinesias, facial tics as well as pain disorders without a clear-cut motor hyperactivity basis in the field of the orofacial region including dentistry (Clark et al., 2007). It is also often used on patients with masseter muscle hypertrophy for esthetic improvement. The maximum bite force was significantly reduced after injection of BoNT for treating masseter muscle hypertrophy (Ahn and Kim., 2007). Other authors reported the effect of BoNT in reducing the masseter muscle, which was documented by ultrasonography and computed tomography (To et al., 2001). BoNT can reduce facial muscle thickness, and it can lead to changes in the facial contour (Kim et al., 2010). This therefore has been used for the esthetic effect in plastic surgery clinics. BoNT is also well known for relieving severe neurological disorders related bruxism. 1

Some BoNT study trying to figure out if it can be applied to the regulation of bone growth had been attempted, which was based on the functional matrix theory. According to the functional matrix theory (Moss and Rankow., 1968), craniofacial growth and development are not intrinsically regulated by bone or cartilage, but by the surrounding muscle. Thus, the induction of muscle hypofunction may influence facial growth. They have noted that muscle function is one of the most important epigenetic factors involved in guiding facial bone growth. Mandibular growth depends upon multiple factors and has therapeutic clinical importance for so-called functional orthopedic treatment which aims at targeted modification of growth processes in the viscerocranium (Schumacher and Dokládal., 1968; Schumacher., 1968). Mandibular growth is closely related to the occurrence and growth of temporomandibular joint. The secondary growth cartilage associated with temporomandibular joint development forms condylar cartilage. In some ways this is similar to that found in long bone epiphyseal cartilage during development stage. The condylar process of cartilage has proliferation layers of cells that can divide, which carries out role by progenitor cell for the cartilage growth. The cells become chondroblast and they form the extracellular matrix of cartilage by secreting type Ⅱ collagen and proteoglycan. After that they become chondorocytes. At the same time, the size of chondroblast is enlarged. After the cartilage formed, minerals are deposited on the cartilage, blood vessels come in the cartilage and the chondrocyte are destroyed. The differentiation of osteoblast takes place a series of processes such as formation of bone minerals deposited on the cartilage structure. The formation of bone through the differentiation of cartilage is subjected to these processes (Nanci., 2008). Many studies tried to figure out that the control of mandibular function can engage in such cartilage differentiation. Previous animal studies of masticatory hypofunction demonstrated 2

less growth of the mandibular ramus in both vertical and anterior-posterior dimensions. The angular and condylar processes were also dimensionally smaller (Kiliaridis et al., 1985, 1988; Kiliaridis and Shyu., 1988). Morphological changes during craniofacial growth have been investigated in animals using approaches such as altering food consistency and performing a myoectomy, myotomy, or denervation (von Wowern and Stoltze., 1978; Behrents and Johnston., 1984; Bouvier and Hylander., 1984; Navarro et al., 1995; Ulgen et al., 1997). Recent studies using BoNT (Kim et al., 2008; Tsai et al., 2010) have supported the theory. These studies included animal experiments designed to investigate changes in masticatory muscle function due to the effects of BoNT on skeletal development. They have shown that BoNT can have inhibitory effects on development rat mandible. In a study of rat mandibles injected with BoNT in bilateral masseter muscles, mandibular dimensions were reduced compared with those of saline-injected rats (Kim et al., 2008). Furthermore, unilateral injection of BoNT was associated with reduced mandibular growth on the BoNT injected side compared with the non-injected side. Localized BoNT injection may induce craniofacial growth changes (Park et al., 2015). The purpose of this study is to analysis the effect of BoNT injected into the masseter muscle on the condylar cartilage and the masseter muscle in young beagle dog. Whereas previous studies have made in the rat or rabbit, the experiment is significant that one step further made in the higher animals. 3

II. Materials and Methods 1. Materials Four male beagle dogs aged 15 weeks and weighing 10 11 kg were used for this study in accordance with protocols approved by the Animal Care and Use Committee, Yonsei Medical Center, Seoul, Korea. Botox R (Allegan. Inc. USA) was supplied as BoNT injection. 2. Methods Subjects were classified into three groups. They were divided into control group (group Control : 1 beagle) and two experimental groups (group 10U : 2 beagles, group 20U : 1 beagle). 1) Animal preparation Prepared animals were observed for 1 week during adaptation period. After 1 week of observation period, the first injection protocol was carried-out. The animals were then initially anaesthetized by a subcutaneous injection of atropine (Daewon Pharmaceutical Co. Ltd., Seoul, Korea) with a dose of 0.05 mg/kg body weight, followed by an intravenous injection of Zoletil (Virbac Korea Co. Ltd., Seoul, Korea) at 5 mg/kg body weight and Rompun (Bayer Korea Ltd., Seoul, Korea) at 0.2 mg/kg body weight. General anesthesia was then preserved by 2% enflurane. After checking that the animal was sedated, the injection procedure was performed. 4

2) Botulinum toxin injection Botulinum toxin mixture for injection was supplied as a freeze-dried powder. One vial (100U) of Botox R was mixed with 2 ml of saline. Injection points were marked at 5 mm, 7.5 mm height from mandible lower edge on both sides of beagle s masseter muscle (maximum prominent point). Each beagle belonging to experimental groups (group 10U & 20U) was injected with BoNT and saline mixture at 4 injection points each accordingly (Figure 1). Control group (group control) was injected with saline. group Control : 0.2ml of saline on each point group 10U : 0.1ml (5U) of mixture on each point (10U per side (20U in total)) group 20U : 0.2ml (10U) of mixture on each point (20U per side (40U in total) Figure 1. Botulinum toxin (BoNT) injection 5

After 4 weeks, the second injection was followed. The procedure was same with the first injection. During the experimental period all the animals were provided with dog meal mixed with water for soft diet. It was in order to exclude the variable to strengthen the function of the muscles during the experimental period. Total experiment was performed for 11 weeks (Figure 2). After 6 weeks from the second injection, the animals were sacrificed. Cardiac anesthesia followed by KCl (potassium chloride) intravenous injection was conducted. 1 week 5 weeks 11 weeks The phase of Observation The first phase of feeding The second phase of feeding First injection of BoNT Second injection of BoNT Sacrifice Figure 2. Time table of study protocol 6

3) Collection of tissue samples Both condyles of beagles were taken (Figure 3B). Specimens of masseter muscle were taken in the form of a 10 mm X 10 mm square column relative to most prominent point, based on the mandible lower edge (Figure 3C). The total of eight condyles and eight masseter muscle tissue specimens were collected. A B C Figure 3. Collection of sample specimen A. Condyle of beagle (the white arrow indicates condyle) B. Condyle specimen C. Masseter muscle specimen 7

4) Histological procedure & analysis The block specimens were rinsed in sterile saline. Each acquired samples were fixed by perfusion with a 10% formalin solution. The specimens of condylar cartilage were decalcified with 5% Nitric acid. After then, all the specimens were dehydrated in an ascending graded ethanol series (70%, 80%, 90%, 95%, and 100%) and cleared in xylene. These were then embedded in paraffin. Specimens of condylar cartilage were cut in serial sagittal sections and specimens of masseter muscle were cut in cross-sections. All sections were stained with hematoxylin and eosin (H-E) as a conventional method. The stained sections were evaluated with aid of a light microscope. The thickness of four zones such as F, R, P, H zones was measured at ten regions of condylar cartilage surface on each side (right and left side). Twenty measurements were obtained per a dog. All measurement sites were on point 2 mm away from the top of the condylar cartilage. And twenty five samples were extracted from the muscle fibers of each side randomly, and then the cross section area of the muscle fibers was measured. 'Image Pro' software (Media Cyberbetis Inc. USA) was utilized for the measurement of length and area. 5) Statistical analysis Comparative analysis of acquired data was performed with SPSS version 20 statistical software (SPSS Inc., Chicago, IL, USA). Data were evaluated by paired t-test, Wilcoxon signed rank test and one-way ANOVA for significance of differences. Statistical significance level for all was adopted in the p<0.05 level. 8

III. Results The differences in the tissue among the three groups were compared with each other. 1. Condyle The cell layers on condylar cartilage surface can be divided into the four following zones (Shen and Darendeliler., 2005). ; fibrous covering, reserve, proliferation and hypertrophic zones (Figure 4). Calcification zone and ossification zone are below the four zones (Figure 4). F zone (fibrous covering zone) R zone (reserve zone) P zone (proliferating zone) H zone (hypertrophic zone) Calcification zone Ossification zone Figure 4. The schematic image of condylar cartilage by layers F zone : The most superficial zone covering the articular surface is the articular fibrous layer, in which there are densely packed collagen fibers with fibroblasts. R zone : Beneath the F zone is the condylar cartilage. The superficial layer of the cartilage is a zone of reserve cartilage cells. The cartilage cells in this zone are small, and the amount of chondroid matrix is less, relative to the deeper layer. P zone : This deeper layer consists of mature cartilage with abundant intercellular cartilaginous matrix. The individual cartilage cells are relatively larger than in the overlying layer. H zone : The chondrocytes in this zone become highly mature. It should be noted that hypertrophic chondrocytes do not lose proliferative activity, at least during the embryonic period. Each tissue section was observed for these four zones mainly of condylar cartilage. The sagittal sections of condylar cartilage of subjects are shown in Figure 5. 9

A B H zone P zone F zone R zone C D E F Figure 5. Histological comparison image of condylar cartilage of group Control (A, B), group 10 U (C, D), and group 20 U (E, F). A, B Group Control shows normal condylar surface with multiple layers in beagle in growth period. : fibrous covering zone, reserve zone, proliferation zone, hypertrophic zone, calcification zone, and ossification zone C, D Group 10U shows no significant difference compared to group Control. E, F Group 20U shows markedly reduced proliferation and hypertrophic zone compared to group Control and group 10U. ( magnification. A, C, E : x40 ; B, D, F : x100 / scale bar. A, C, E : 500μm ; B, D, F : 200μm ) 10

Histological differences among three groups may describe as follows (Table 1, Figure 6). Table 1. Mean thickness of four zones of the condylar cartilage for each groups (unit : μm) Group Right Left Mean Fibrous covering zone Control 168.1±21.4 170.6±23.2 169.6±21.6 10U 175.2±10.6 177.7±12.2 176.4± 11.6 20U 173.6±19.9 176.2±19.8 173.6± 21.7 Reserve zone Control 81.6±16.0 83.6±12.8 82.8± 14.0 10U 83.2±8.4 82.4±6.4 82.9± 7.2 20U 88.0±9.6 89.2±9.2 88.8± 8.4 Proliferation zone Control 305.2±26.4 312.4±23.2 308.8±24.4 10U 241.2±21.2 246.0±23.2 243.6± 21.6 20U 88.8±21.6 87.6±22.0 87.2± 20.0 Hypertrophic zone Control 346.4±36.8 354.0±35.6 350.4± 36.0 10U 379.6±35.6 390.4±36.4 385.6± 36.1 20U 88.4±11.6 87.6±7.6 88.0± 9.6 11

450 400 350 300 250 Fibrous covering zone Reserve zone Proliferation zone Hypertrophic zone 200 150 100 50 0 Control 10U 20U Figure 6. Comparison of thickness in four zones among three groups In F zone and R zone of the condylar cartilage showed no significant difference among the three groups. However, in P zone and H zone the differences were able to observe between the groups. In P zones differences were not observed compared to the group 10U and Control. But differences of the thickness in P zones were statistically significant between the two groups (group 10U & Control) and group 20U (p<0.05). Table 2 and figure 6 show the differences. In group 20U thinner P and H zones were observed, and especially in H zone the difference was clear. And H zones were significantly different among three groups. There were no significant differences of the thickness in four zones between right and left in all groups. There were no differences in calcification zone and ossification zone among three groups. Bone trabecular patterns were irregular and severe bony defects were not detected in all groups. Areas occupied by ossifying bone per unit area were not statistically different among three groups. 12

2. Masseter muscle Masseter muscle samples of all groups were cut in cross-sections. Histological analysis of the masseter muscle in three groups is shown Figure 7. A B C D E F Figure 7. Histological analysis of the masseter muscles in group Control (A, B), group 10U (C, D), and group 20U (E, F). A, B Muscle fibers of group Control have polygonal form. C, D Group 10 U shows reduced size of muscle fiber compared to group Control. E, F Group 20U shows reduced size of muscle fiber compared to group Control and group 10U. ( magnification. A, C, E : x400 ; B, D, F : x1000 / scale bar. A, C, E : 50μm ; B, D, F : 20μm) 13

In the group Control, endomysium is unclear, and the adjacent muscle fibers are connected tightly. However, in the group 10U and 20U, endomysium was seen clearly with the separated cell connections. In group Control, muscle fibers had normal polygonal shape without necrosis or inflammation. The shape of muscle fibers of groups 10U and 20U was more round and diameter of the fiber decreased. Histological differences on area of cross sectioned muscle fiber among three groups may describe as follows (Table 2, Figure 8). Table 2. Mean area of cross sectioned muscle fiber in groups (unit : μm 2 ) Group Right Left Mean Control 1176.6±37.9 1180.9±44.4 1178.8±41.0 10U 839.8±49.5 842.3±48.5 841.1±48.5 20U 647.4±25.1 650.2±26.9 648.8±25.8 1400 1200 1000 800 600 400 200 0 Control 10U 20U Figure 8. Comparison of muscle fiber cross sectioned area in groups 14

Area occupied by each muscle fiber per unit area of group 10U and group 20U were smaller than that of group Control. Area measurement using Image Pro program shows area of each muscle fiber (Table 2 and Figure 8). There were no significant differences of the muscle fiber areas between right and left in all groups. The area of cross sectioned muscle fiber among three groups showed a significant difference statistically (p<0.05) each other. Each muscle fiber per unit area of group 20U was the smallest. 15

IV. Discussion The functional matrix theory (Moss and Rankow., 1968) states that craniofacial growth and development are by the surrounding muscle. Many studies have investigated the influence of muscle on craniofacial growth and development by changing function of masseter muscle to prove the functional matrix theory. Other studies attempted to control skeletal growth with the aim of restoring normal function and facial appearance by changing masticatory muscle function. They used invasive method to reduce function and force of masseter muscle. For example, there are myectomy, denervation procedure of the masseter muscle (Horowitz et al., 1955; Moore et al., 1973; Sato et al., 1986). But invasive method such as myectomy and surgical denervation may leave scar on tissue or damage other structures that can change growth patterns (Gardner et al., 1980). And other studies researched local mechanical regulations affecting the condyle including intermaxillary fixation, removal of incisors, alteration of dietary consistency, muscle resection, and application of orthodontic appliances (Bouvier and Hylander., 1984). Recent studies have used non-invasive methods to control the masticatory muscle. The most active research is the study by using the botulinum toxin (BoNT). BoNT is the paralytic neurotoxin from produced clostridim botulinum. It attaches at the cholinergic motor end plate, and cause paralysis of motor neuron (Borodic et al., 1994; Kim et al., 2003; Kwon et al., 2007). It is life-threatening toxin known to bind to receptors at the neuromuscular junctions of target nerves where they cleave soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, which are responsible for the process of vesicle-plasma membrane fusion (Aoki et al., 2002; Rosales et al., 2006). 16

Since a study verified that BoNT temporarily inhibits acetylcholine release at the neuromuscular junction and reduces muscular contractions, BoNT has been used as an effective method for reducing muscle activity and function. Because BoNT injection is a simple procedure, there is little room for experiment mistake. However, the accurate position during the injection of BoNT is important. There are many studies that examine the effect of BoNT injection for mandible growth control in rats. BoNT influences on inhibitory action of the developing mandible due to apoptosis at the proliferation stage of the reserve zone of the condylar cartilage in developing rat mandible (Kim et al., 2010). When the rats were injected with BoNT on masseter muscle during growth period, the rats after growing showed a decrease in mandibular height, but the mandibular length and intergonial distance were not affected (Huang JJ et al., 2010). Other study showed a facial morphology typical of a dolichofacial profile: short upper face accompanied by a long lower face with an extended mandibular length and ramus height and constricted bicoronoidal and bigonial widths which suggest that induction of localized masticatory muscle atrophy with BoNT alters craniofacial growth and development (Tsai CY. et al., 2010). In another study, BoNT injection into the temporalis and masseter muscles of growing rats induced reduction cortical bone thickness and BMD (Bone marrow density) of the skull and mandibular bone structure. The volumes of the temporalis and masseter muscles injected with BoNT were smaller (Tsai CY. et al., 2011). Apart from this, there were several studies on whether BoNT injection on masseter muscle induces mandibular asymmetry. Out of these studies, in most studies that were conducted on rat, morphological analysis indicates that facial asymmetry can be induced due to paralysis of masseter muscle. The muscles injected with BoNT were smaller than the sham or control muscles and anthropometric measurements 17

of the bony structures attached to the masseter muscle showed a significant treatment effect (Tsai CY et al., 2009). Unilaterally localized BoNT injection induced a change in craniofacial growth, and the skeletal effect was unilateral despite both sides of the mandible functioning as one unit (Park et al., 2015). In comparison to such results, in studies carried out on medium sized mammals such as rabbit, showed that although there could be subtle difference between structures (mandibular ramus length, zygomatic length, masseteric length, etc.) related to masseter muscle on two sides, it does not indicate that unilateral injection of BoNT caused significant facial asymmetry (Kwon et al., 2007). This suggests that, for more evolved mid-sized mammals with more complex mandible, activity of one side of normally functioning masseter muscle can influence the activity of masseter muscle on the other side. Furthermore, due variation for time and period for growth depending on type of animal, there can be difference in period for effect of BoNT to be maintained. Therefore, it is possible for results of experiments done on one side to be different to the other. Considering above previous results, in this study design, BoNT was injected to masseter muscles on both sides of beagle dog, a higher animal than rabbit, to eliminate any factors that could affect the results. We designed to evaluate the influence of BoNT on mandibular growth by comparing histological difference between BoNT injection (10U per one side, 20U per one side) groups and saline injection group. Previous studies with rats proposed the dose of BoNT injection. Those were about 0.02~0.05 U per gram for rat (Babuccu et al., 2009; Brodic et al., 1994; Kim et.al., 2003). We determined the dose for our experiment by reference to these studies. But because each animal might have different doses, we established two experimental groups (10U per one side, 20U per one side). Beagles weights were from 10 to 11Kg. 18

Histological examination of condylar cartilage and masseter muscle of beagle was carried out after 11 weeks of growth period and injection of BoNT twice. Masseter muscle specimens of all groups were cut in cross-sections. And the section of condyle specimen was sagittal section. Histological comparison of the condylar cartilage in the control, 10U injection, and 20U injection groups are shown in Figure 5. Points more than 2.0 mm away from the top of condylar cartilage were the distinction of the four zones which showed an irregular pattern. Around the top portion of the condyle, the differentiation of cells occurred actively and significant statistically differences were observed among groups. In P zones differences were not observed compared to the group 10U and Control. But differences of the thickness in P zones were statistically significant between the two groups (group 10U & Control) and group 20U (p<0.05). And H zones were significantly different among three groups. Especially in group 20U, where 20U was injected on each side, thinner proliferation zone and hypertrophic zone were shown. The thickness of the two layers of group 20U showed a significant difference statistically (p<0.05) compared to that of group 10U and Control. The average thickness of H zone in group 20U was less than one-third of that of group Control. But in F zone and R zone of the condylar cartilage showed no significant difference among the three groups. This result can be explained in two ways. This is either due to decrease in cell activity in proliferation zone or decrease in number of cells which induce proliferation. Study with rats showed that cell apoptosis in the proliferation zone of the BoNT group was increased compared with that of the control group. The cell death was detected in the proliferation zone adjacent to the reserve zone by Tunnel staining process. Cell death at the proliferation zone adjacent to the reserve zone is relative to the growth of the mandible and condyle in developing rat mandible (Kim et al., 2010). 19

The reserve zone has a growth potential (Kierszenbaum., 2002). It has also been known to play an important role in the proliferation of the cells. On our study we could not find significant difference of the reserve zone among groups. And there were no differences in calcification zone and ossification zone among three zones. Bony trabecular patterns were same and severe bony defects were not detected in all groups. Areas occupied by ossifying bone per unit area were not statistically different among three groups. Histological comparison of the masseter muscle in the control, 10U injection, and 20U injection groups are shown in Figure 7. The groups 10U and 20U showed the separated cell connections and cell degenerations with decrease in size of muscle fiber. The muscle fibers of the BoNT groups were more round and diameter of the fiber decreased. The area of cross sectioned muscle fiber among three groups showed a significant difference statistically (p<0.05) each other. Each muscle fiber per unit area of group 20U was the smallest and that of group Control was the largest. BoNT injection inhibits acetylcholine release at the neuromuscular junction and reduces muscular contractions. The formation of condensed muscle fibers in the BoNT injection group might be an expression of its degenerative atrophy (Akagawa et al., 1983). The histological assessment of muscle fiber implicate for reflection of muscle denervation after BoNT injection (Vilmann et al., 1990; Borodic and Pearce, 1994). The muscles injected with BoNT were smaller than control (Tsai CY et al., 2008). It has been studied that after injection of BoNT, the size of muscle fiber became smaller than the control group (Korfage et al., 2012). There was a study on the distribution of changes in fiber type in the muscle injected with BoNT. The investigation carried out on pigs showed that paralysed masseters displayed atrophic changes and the typical distributions of type IIa and IIb fiber 20

types in masticatory muscles were increased in the masseter muscles due to BoNT application (Gedrange., 2013). If the proliferation and differentiation of the cells occurs in condyle, these results lead to the formation of bone in accordance with endochondral bone formation. The gene expression patterns of chondrocytes during condylar growth have been categorized into two phases: maturation and mineralization (Inoue et al., 1995). The chondrocyte maturation is initiated with mesenchyme differentiation into pre-chondroblasts and terminated with highly matured hypertrophic chondrocytes. This process is well-manifested by cellular and phenotypic responses of chondrocytes, resulting in a unique zone-like packing of condylar cartilage (Shen., 2000). As a result, cell proliferation can be explained that led to bone formation and balance. Changes in the production of bone in growth may be a factor that influences the amount of growth can be confirmed in this previous study (Tsai CY et al., 2010; Park et al., 2015; Kim et al., 2010; Huang JJ et al., 2010). Thus the change in cell differentiation of the condylar cartilage due to the injection of BoNT can be a factor that can affect mandibular growth. As a result, the reduction in cell differentiation and proliferation caused by decrease of muscle function could make to reduce the bone formation of the condyle. According to these results, it is thought that it could reduce the growth of the mandible. In the past BoNT had been used for treatment purposes only confined to the muscles. But currently, various studies suggested possibility of influence of the bone growth by reducing the function of the muscles and the usage range of BoNT as a therapeutic agent is widened. Whilst most studies were limited to rat study, this experiment applies far higher animals such as dog to support the results of previous research. Based on the results of this experiment, further research should be carried out in the future to analyze morphologically how 21

histological changes on condylar surfaces have influence on mandibular growth in long-term. Moreover, further studies should be applied on monkeys, resembling human rather than dogs. Other treatments to control mandibular growth, especially which inhibit the overgrowth of mandible still have many issues in orthodontics. But we believe that BoNT could be applied to treatment for growth modification. Our experiment coupled with other studies so far can help to develop better treatment. 22

V. Conclusion 1. In condylar cartilage the differences of the thickness in P zones were statistically significant between the two groups (group 10U & Control) and group 20U (p<0.05). And H zones were significantly different among three groups (p<0.05). Especially in group 20U thinner P zone and H zone were shown. 2. The shape of masseter muscle fibers in group 10U and group 20U was different from that of group Control. And endomysium was seen clearly with the separated cell connections in group 10U and 20U. 3. The area of cross sectioned muscle fiber among three groups showed a significant difference statistically (p<0.05) each other. Each muscle fiber per unit area of group 20U was the smallest. As a result of this, it was confirmed that BoNT injected into the masseter muscle could affect cell differentiation of condylar cartilage, which could ensure that consistent with the results of the previous studies made in lower animals, such as rat. If the further study that BoNT could affect the morphological changes in the mandible is carried out, this study could be a basis for the growth modification treatment of human mandible by using BoNT. 23

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국문요약 어린비글견의교근에주입된보툴리눔독소가 하악과두및교근에미치는효과 연세대학교대학원치의학과 ( 지도교수황충주 ) 김석주 하악의기능을통제함으로써하악성장을조절하려는많은시도들이있었다. 보툴리눔독소의주입은근육의기능을저하시킴으로써하악의기능을통제할수있는방법으로사용될수있음이이전의연구들을통하여밝혀졌다. 본연구는근육의기능이성장중에있는두개안면및하악의성장에영향을줄수있음을밝힌선학들의연구에기초하고있다. 본연구는성장기비글견의교근에보툴리눔독소를주입하여하악과두및교근의조직학적변화를살펴봄으로써보툴리눔의작용에따른교근의기능저하가하악과두부위의세포의분화와교근의근섬유에어떠한영향을주는지를알아보고자하였다. 총 4 마리의비글견을연구의대상으로하였으며, 실험군 (3 마리 ), 대조군 (1 마리 ) 으로나누어실험을진행하였다. 보툴리눔독소는 Allergan 사의 Botox R 를사용하였으며, 실험군중 2 마리는편측당 10U(group 10U), 1 마리는 20U(group 20U) 의보툴리눔독소를, 대조군은 saline 을 4 주간격으로양측교근에총 2 회주입하였다. 총 11 주간의사육기간후희생하여, 개체마다양측하악과두및교근의조직시편을채취하고 HE 염색을통한조직표본을분석하였다. 교근및하악과두의연골부위에서의조직학적비교분석결과, 다음과같은결과를얻었다. 28

1. 하악과두연골에서, group 20U의 P zone의두께는 group 10U와 group Control의 P zone 두께에비해얇았으며, 이러한두께의차이는통계학적으로유의한차이 (p<0.05) 를보였다. 또한 H zone의경우에는모든 group간에유의한차이 (p<0.05) 를보였다. 특히 group 20U에서더욱얇은 P zone과 H zone을관찰할수있었다. 2. group 10U 와 group 20U 의교근근섬유의횡단면은 group Control 의 근섬유의횡단면과는다른형태를가지고있었으며, 근섬유세포를 분리하는분명한근내막을관찰할수있었다. 3. 세군간에통계학적으로유의한근섬유의횡단면면적크기의차이 (p<0.05) 를보였으며, 근섬유의횡단면면적은 group 20U 에서가장 작았다. 본연구결과, 교근으로의보툴리눔독소주입이하악과두연골의세포분화에영향을준다는확인할수있었으며, 이는백서등의하등동물에서이루어진이전의연구들의결과에부합되는것이었다. 연구결과를토대로, 향후보툴리눔독소가하악구조의형태학적인변화에영향을끼칠수있음을더욱많은개체수에서증명한다면, 보툴리눔독소를이용한사람의하악골성장조절치료를위한중요한근거가될수있을것으로생각된다. 핵심되는말 : 보툴리눔독소, 하악과두연골, 교근, 하악성장, 비글견 29