성장기쥐의편측교근에주사한보툴리눔 A 형독소가하악골성장에미치는영향 곽소연 박기태 김지연 성균관대학교의과대학삼성서울병원치과진료부소아치과 국문초록 성장시기가다른쥐의편측교근에보툴리눔 A형독소를주사하여하악골성장에미치는영향을알아보고자하였다. 각기다른주령의쥐 (N=36, 1군 : 4주령, 2군 : 5주령, 3군 : 6주령 ) 를대상으로실험을시행하였으며각군은다시대조군, 편측주사군, 양측주사군으로나누어하악골의길이를계측하고다음과같은결과를얻었다. 1. 1군에서편측주사군의주사측과양측주사군모두하악체길이, 과두높이가대조군보다낮은값을보였다 2. 2군에서편측주사군의주사측과양측주사군모두하악전방부높이와과두높이에서대조군보다낮은값을보였다 3. 3군에서편측주사군의주사측과양측주사군모두하악체길이와과두높이, 과두돌기높이가대조군보다낮은값을보였다 4. 세군모두편측주사군의양측비교시통계적으로유의한차이는없었으나주사측은대조측보다낮은계측값을보이는경향이관찰되었다 (p>0.05). 주요어 : 보툴리눔 A 형독소, 하악골성장, 기능성기질 Ⅰ. 서론두개안면부의성장에관한이론은유전에의해서만성장이결정된다는 Brodie 1) 의유전설이발표된이후봉합부주도설 2), 비중격연골주도설 3), 기능성기질설 4), Van Limborgh 가설 5), servosystem theory 6) 등과같은여러가설들이발표된바있다. 기능성기질설은 Moss와 Rankow 4) 에의해서 1968년발표된이후두개안면의성장기전을밝히는데많은영향을주었다. 이가설에의하면골격과연관된비골격조직인기능성기질은성장에있어서일차적인역할을하며, 골격적요소는기능성기질에의해이차적, 보상적으로성장이일어난다. 기능성기질은크게골막성과낭성기질로나뉘는데근육은골막성기질의대표적인예이다. 저작근육의기능을변화시키기위해이전의많은연구들에서는기능하는치아를제거하는방법 7,8), 기능을변화시키는장치 를삽입하는방법 9), 섭취하는식이의경도를조절하는방법 10-12), 또는저작근을제거하거나 13-15) 저작근을지배하는신경을차단하는방법 16) 등다양한방법을사용하여악안면부의성장에미치는영향을평가하였다. 이번연구에서는이전연구들에서사용한방법들과다르게보툴리눔 A형독소를사용하여저작근의기능을변화시키고자하였다. 보툴리눔 A형독소는클로스트리디움보툴리눔에의해생산되는신경마비독소로콜린성신경말단에결합하여신경연접전부위에서아세틸콜린의분비를막아근신경전달을차단함으로써근육이수축하지못하도록한다 17). 보툴리눔 A형독소는 7가지보툴리눔독소의혈청형중상업적으로가장많이사용되는혈청형으로사시 (strabismus) 와안검경련 (blephrenospasm) 의치료목적 18) 으로처음소개된이후소아치과영역에는레트증후군 (Rett syndrome) 19), 정신지체 (mental retardation) 20), 무산소뇌병증 (anoxic encephalopathy) 21), 소뇌출혈 (cerebellar hemorrhage) 22) 등을 교신저자 : 김지연서울특별시강남구일원동 50 번지 / 삼성서울병원소아치과 / 02-3410-2409 / jychaee@skku.edu 원고접수일 : 2008 년 12 월 05 일 / 원고최종수정일 : 2009 년 05 월 25 일 / 원고채택일 : 2009 년 06 월 08 일 433
J Korean Acad Pediatr Dent 36(3) 2009 가진어린이의이갈이치료로소개된바있다 23). 이번연구에서는보툴리눔 A형독소를편측으로주사하였을경우, 이것이하악골성장에미치는영향을조사하고, 성장시기에따른하악골성장에대한영향을알아보고자하였다. Ⅱ. 연구재료및방법 1. 연구재료본연구는 AALAC International(Associaction for Assessment & Accreditation of laboratory Animal Care International) 인증기관인삼성생명과학연구소동물관리위원회의검토와승인을거쳤으며, 위원회의실험동물관리와사용에관한지침을준수하여시행하였다. 생후 4주, 5주, 6주령의 Sprague-Dawley계웅성백서각각 36마리를주령에따라 3군으로나누었다. 약제로는 Botox (Allegan, USA) 를사용하였다. -1 군 : 생후 4주령 (N=12) -2 군 : 생후 5주령 (N=12) -3 군 : 생후 6주령 (N=12) 각군을다시 3개의군으로나누었다. -대조군: 양측교근에생리식염수를 0.05cc씩주사 (N=4) -편측주사군: 한쪽은생리식염수주사 ( 대조측 ), 반대쪽은 Botox (0.05cc, 3U) 주사 ( 주사측 ) (N=4) -양측주사군: 양쪽에 Botox (0.05cc, 3U) 주사 (N=4) 각군에따라해당약물을천부교근의최대풍융부에투여하였다 (Fig. 1). 모든실험동물은고형사료와함께분말사료를공급하여 4주간키웠으며, 매 1주경과때마다체중을측정하고각각의사료섭취량을측정하였다. 실험시작 4주후실험을종료하면서각실험동물의체중을측정하고과량의에테르로희생하였다. 희생된쥐의하악골을떼어낸후좌우를분리하여디지털카메라로일정거리에서촬 영하였다. 촬영된영상을컴퓨터에옮겨 1986년 Asano 24) 가발표한논문에서사용된계측점을이용하여컴퓨터상에서각계측치의길이를측정하였다 (Fig. 2). 2. 통계적분석 각군에서대조군, 편측주사군, 양측주사군간의하악골측정치, 체중, 섭취된사료총합의유의성을평가하기위하여 Kruskal-Wallis test(p<0.05) 를사용하였고, 유의성이있는결과를보인항목에대해서 multiple comparison test(p<0.05) 를시행하여각군간의비교를시행하였다. 각군에서편측주사군의주사측과대조측간하악골측정치차이의유의성은 Wilcoxon signed ranked test(p<0.05) 로분석하였다. Fig. 1. Superficial portion of the masseter muscle of the rat. (M: Superficial portion of the masseter muscle; P: Parotid gland; LI: Lower incisor; UI: Upper incisor) Fig. 2. Landmarks and measurements of the mandible used in this study. Me: most inferior point of mental protuberance Me : most inferior point of anterior alveolar bone Me-Me : tangent to the bottom of angular process through Me Go: most posterior tip of the angular process Cd: the central point of the condyle Cd : crossing point on Me-Me perpendicular to Me-Me from Cd Co: tip of coronoid process Co : crossing point ofn Me-Me perpendicular to Me-Me from Co 1. Me-Go: Mandibular body length 2. Me-Cd: Condylar Length 3. Me-Co: Coronoid process length 4. Me-Me : Anterior region height 5. Cd-Cd : Condylar height 6. Co-Co : Coronoid process height 434
Ⅲ. 연구결과 1. 1군, 2군, 3군내각각의편측주사군에서대조측과주사측간차이가있는가를 Wilcoxon signed rank test로검증한결과세군모두편측주사군에서양측간유의한차이는없었다 (p>0.05). 그러나수치상주사측은대조측보다낮은계측값을보이는경향이관찰되었다. 2. 4주령군에서의측정치평가 (Table 1) 4주령에실험을시작한 1군에서대조군, 편측주사군, 양측주사군간의하악골측정치를 Kruskal-Wallis test(p<0.05) 와 multiple comparison test(p<0.05) 를통하여비교한결과편측주사군의주사측과양측주사군모두하악체길이, 과두높이가대조군과비교시유의하게낮은값을보였다 3. 5주령군에서의측정치평가 (Table 2) 5주령에실험을시작한 2군에서대조군, 편측주사군, 양측주사군간의하악골측정치를 Kruskal-Wallis test(p<0.05) 와 multiple comparison test(p<0.05) 를통하여비교한결과양측주사군에서다른그룹에비해과두길이측정값이유의성있게낮은수치를보였고 (p<0.05), 편측주사군의주사측과양측주사군모두대조군에비해통계적으로낮은값을보였다 4. 6주령군에서의측정치평가 (Table 3) 6주령에실험을시작한 3군에서대조군, 편측주사군, 양측주사군간의하악골측정치를 Kruskal-Wallis test(p<0.05) 와 multiple comparison test(p<0.05) 를통하여비교한결과하악체길이와과두높이, 과두돌기높이가편측주사군의주사측과양측주사군모두에서대조군에비해낮은계측값을보였다 5. 체중과사료섭취량분석 1군, 2군, 3군의각시점에서대조군, 편측주사군, 양측주사군간의체중의차이와섭취된사료의총량의차이를 Kruskal- Wallis test로검증한결과유의한차이가없었다 (p>0.05). Table 1. Mandibular measurements of 4-week-old rats group (Unit : mm) Control group : Bilateral Saline Unilateral group : BTX-A side Bilateral group : Bilateral BTX-A (Mean SD) (Mean SD) (Mean SD) Mn. Body length 20.57 0.13* 19.48 0.65 19.32 0.76 Condylar length 22.17 0.15 21.98 0.38 21.32 0.67 Coronoid process length 19.57 0.46 19.08 0.72 19.05 0.57 Ant. Region Ht. 4.71 0.10 4.48 0.13 4.47 0.19 Condylar Ht. 10.51 0.23* 9.98 0.47 9.12 0.37 Coronoid process Ht. 12.76 0.14 12.10 0.54 11.62 0.45 * : p<0.05, Kruskal-Wallis test, multiple comparison test Table 2. Mandibular measurements of 5-week-old rats group (Unit : mm) Control group : Bilateral Saline Unilateral group : BTX-A side Bilateral group : Bilateral BTX-A (Mean SD) (Mean SD) (Mean SD) Mn. Body length 20.98 0.65 20.57 0.38 19.91 0.93 Condylar length 22.96 0.29 22.93 0.36 21.77 *0.70 Coronoid process length 20.01 0.16 20.07 0.18 19.54 0.49 Ant. Region Ht. 4.88 0.08* 4.61 0.10 4.52 0.08 Condylar Ht. 11.17 0.46* 10.54 0.24 9.83 0.83 Coronoid process Ht. 13.26 0.37 12.67 0.23 12.33 0.88 * : p<0.05, Kruskal-Wallis test, multiple comparison test Table 3. Mandibular measurements of 6-week-old rats group (Unit : mm) Control group : Bilateral Saline Unilateral group : BTX-A side Bilateral group : Bilateral BTX-A (Mean SD) (Mean SD) (Mean SD) Mn. Body length 21.63 0.60* 20.39 0.19 20.62 0.25 Condylar length 23.46 0.40 23.20 0.27 23.06 0.19 Coronoid process length 20.80 0.56 19.98 0.79 20.12 0.42 Ant. Region Ht. 4.92 0.06 4.73 0.07 4.71 0.12 Condylar Ht. 11.17 0.24* 10.93 0.45 9.83 0.20 Coronoid process Ht. 13.98 0.14* 13.03 0.48 13.26 0.29 * : p<0.05, Kruskal-Wallis test, multiple comparison test 435
J Korean Acad Pediatr Dent 36(3) 2009 Ⅳ. 총괄및고찰두개안면골의성장과발육에관한이해는소아치과분야에서필수적이라할수있다. 특히하악골의성장부족이나과잉성장은부정교합을유발하고, 환자에게기능적및심미적으로큰영향을미치기때문에성장기동안하악골성장의조절에대한다양한연구가진행되었으며주로저작근육의기능을변화시키는방법으로연구들이시행되었다. 기능을변화시키는장치를삽입하는방법을통해하악골의성장을연구한박과권 9) 의연구에서백서하악골을기능적전방위시켰을때, 상악골길이에대한하악골길이의비가크게나타났으며, 하악과두의연골층이증가되었다고하였다. Baume 과 Derichsweiler 25) 는 44~55개월된원숭이에경사판을사용하여연구한결과과두연골은기능적치료에반응한다고하였다. 연식을섭취하게하여저작근의기능을저하시켜하악의성장을평가한연구들에서는하악지의길이에대한변화는관찰되었지만하악체의길이는유의한차이가없었고, 하악과두연골의증식층과전체두께가감소된결과를얻었다고보고하였다 10-12). 교근을절제하는방법을통한연구에서는교근의절제는두개안면성장에있어서보다수직적인성장패턴을유발한다하였지만 13-15), Monje 등 26) 은교근절제후하악의수평적, 수직적길이감소가모두일어난다는상이한결과를발표하였다. 1986년 Sato 등 16) 은교근절제술대신교근신경에손상을가하여쥐의하악골성장을평가하여하악의높이와길이가모두감소된결과를얻은바있다. 그러나위에서언급한교근절제나신경손상방법은반흔조직을형성하고, 인접구조물손상을유발하는등성장에원하지않는영향을줄수있기때문에이번실험에서는인접구조물의손상없이근육의기능만변화시킬수있는보툴리눔 A형독소를사용하였다. 이번실험은백서를통한실험으로양측으로보툴리눔 A형독소를주사한이전실험에서하악의성장차이가관찰되었던결과를바탕으로보툴리눔 A형독소를편측으로주사하였을경우이것이하악골성장에미치는영향을조사하고, 성장시기에따른하악골성장에대한영향을알아보고자하였다. 보툴리눔 A형독소를투여한실험동물에서저작기능감소로인하여영양공급부족이나타날수있으므로모든실험동물에게고형사료와함께분말사료를혼합하여제공하였으며, 1군, 2군, 3군에서대조군, 편측주사군, 양측주사군간의체중의유의한차이는관찰되지않았다. 백서는생후 21일경에이유를시작하고그후로 incision, 저작등의하악운동을시작하며, 4~10주사이에하악골의성장이왕성하게일어난다. 이에본연구에서는 4주령, 5주령, 6주령의백서를사용하였으며, 주령을 3가지로나눈것은성장시기따라서유의성이있는차이를보이는수직적, 수평적계측항목의경향을알아보기위해서였다. 그러나본연구에서는각주 령에서유의한차이를보이는항목의규칙적인경향을관찰할수없었으며, 이는본연구의실험개체수가작았기때문이라고사료된다. 실험결과에서 4주령, 5주령, 6주령모두편측주사군의주사측과양측주사군의하악골계측치는통계적으로유의한차이를나타내지않았다. 또한각주령에서편측주사군의주사측과대조측을비교한결과한개체내에서통계적유의차를보이는항목은관찰되지않았으나주사측이대조측에비해계측치가더작게나타나는경향을보였다. Kwon 등 28) 은가토를이용하여본실험과유사하게연구를진행한결과특정부위의국소근육의기능저하로국소적성장변화를유도하여국소적인부위의형태변화를유도하였다고하였으며성장이완료된가토에서는이러한결과가나타나지않았다고보고하였다. 이번연구는백서를통한실험으로실험동물과사람은저작에관여하는체계가다르고해부학적구조물과각구조물의기능이상이하기때문에이번실험의결과를인간의안면부성장에그대로적용하여생각할수없을것이다. 그러나쥐와사람의안면구조는두개안면부복합체의여러요소들을통하여성장이일어난다는기본적인과정이유사하기때문에백서를이용하였다. 본실험에서편측주사군에서주사측이대조측에비해하악골계측치가작은듯이보였으나양측에서유의차를보이지않은것은개체수가작기때문에통계적유의성을증명할수없었을것으로추정할수있다. 그러나편측주사군의주사측과양측주사군사이에하악골계측치가유의차를보이지않은것으로미루어볼때하악의구조가한단위로연결되어있기때문에편측에만투여하여도반대측까지어느정도영향을주었을가능성을배제할수없을것이다. 이번실험의결과를토대로보툴리눔 A형독소의편측투여는국소적인구조변화를유도하여성장에영향을주는것으로사료된다. 현재소아과영역에서국소과긴장증 (Focal hypertonia) 을가지고있는어린이의치료에보툴리눔독소의사용이늘어나고있으며, 특히사시교정이나뇌성마비가있는아이들의자세나걸음걸이교정을위한보툴리눔독소의임상적인사용에대한연구또한많이이루어지고있다 18,29). 앞으로더많은개체수를대상으로한연구가필요하며동물실험에서신뢰할수있는결과가얻어진다면, 소아치과영역에서도어린이의근육의비대칭으로인한성장불균형의치료에보툴리눔 A형독소를이용한성장조절이가능할것이라사료된다. Ⅴ. 결론본연구는성장시기가다른쥐의편측교근에보툴리눔 A형 436
독소를주사하여근육을마비시켜이것이하악골성장에미치는영향을알아보고자하였다. 성장시기에따른보툴리눔 A형독소의효과차이를알아보고자각기다른주령의쥐 (1군: 4주령, 2군 : 5주령,3군: 6주령 ) 를대상으로실험을시행하였으며, 각군은다시대조군, 편측주사군, 양측주사군으로나누어하악계측치를측정하고다음과같은결과를얻었다. 1. 1군에서편측주사군의주사측과양측주사군모두하악체길이, 과두높이가대조군보다낮은값을보였다 2. 2군에서편측주사군의주사측과양측주사군모두하악전방부높이와과두높이에서대조군보다낮은값을보였다 3. 3군에서편측주사군의주사측과양측주사군모두하악체길이와과두높이, 과두돌기높이가대조군보다낮은값을보였다 4. 세군모두편측주사군의양측비교시통계적으로유의한차이는없었으나주사측은대조측보다낮은계측값을보이는경향이관찰되었다 (p>0.05). 참고문헌 1. Brodie A: The growth of alveolar bone and the eruption of the teeth. Oral Surg Med Oral Pathol, 1:334-341, 1948. 2. Sicher H: The growth of the mandible. Am J Orthod, 43:123-128, 1947. 3. Scott JH: Studies in the growth of the upper jaw. Dent Rec, 68:277-291, 1948. 4. Moss ML, Rankow RM: The role of the functional matrix in mandibular growth. Angle Orthod, 38:95-103, 1968. 5. Van Limborgh J: A new view on the control of the morphogenesis of the skull. Acta Morphol Neerl Scand, 8:143-160, 1970. 6. Charlier JP, Petrovic A, Herrmann-Stutzmann J: Effects of mandibular hyperpropulsion on the prechondroblastic zone of young rat condyle. Am J Orthod, 55:71-74, 1969. 7. Furstman L: The effect of loss of occlusion upon the mandibular joint. Am J Orthod, 51:245-261, 1965. 8. Hinton RJ, Carlson DS: Response of the mandibular joint to loss of incisal function in the rat. Acta Anat (Basel), 125:145-151, 1986. 9. 박효상, 권오원 : 백서하악골의기능적전방위가하악골성장에미치는영향. 대한치과교정학회지, 21:521-537, 1991. 10. Maki K, Nishioka T, Shioiri E, et al.: Effects of dietary consistency on the mandible of rats at the growth stage: computed X-ray densitometric and cephalometric analysis. Angle Orthod, 72:468-475, 2002. 11. 윤석희, 이상대, 김정욱등 : 성장중인쥐에서음식물의경도가하악과두의해면골에미치는영향 : 미세전산화단층촬영을이용한연구. 대한소아치과학회지, 31:228-235, 2004. 12. Bouvier M, Hylander WL: The effect of dietary consistency on gross and histologic morphology in the craniofacial region of young rats. Am J Anat, 170:117-126, 1984. 13. Gianelly AA, Ruben MP, Risinger R: Effect of experimentally altered occlusal vertical dimension on temporomandibular articulation. J Prosthet Dent, 24:629-635, 1970. 14. Horowitz SL, Shapiro HH: Modification of skull and jaw architecture following removal of the masseter muscle in the rat. Am J Phys Anthropol, 13:301-308, 1955. 15. Moore WJ: An experimental study of the functional components of growth in the rat mandible. Acta Anat (Basel), 85:378-385, 1973. 16. Sato Y, Ohmae H, Takano T, et al.: Influence of unilateral masseteric denervation on the growth of mandibular condylar cartilage: an autoradiographic study. J Osaka Univ Dent Sch, 26:177-186, 1986. 17. Silberstein S: Botulinum neurotoxins: origins and basic mechanisms of action. Pain Pract, 4:19-26, 2004. 18. Spencer RF, Tucker MG, Choi RY, et al.: Botulinum toxin management of childhood intermittent exotropia. Ophthalmology, 104:1762-1767, 1997. 19. FitzGerald PM, Jankovic J, Percy AK: Rett syndrome and associated movement disorders. Mov Disord, 5:195-202, 1990. 20. Richmond G, Rugh JD, Dolfi R, et al.: Survey of bruxism in an institutionalized mentally retarded population. Am J Ment Defic, 88:418-421, 1984. 21. Pratap-Chand R, Gourie-Devi M: Bruxism: its significance in coma. Clin Neurol Neurosurg, 87:113-117, 1985. 22. Pollack IA, Cwik V: Bruxism following cerebellar hemorrhage. Neurology, 39:1262, 1989. 23. Tan EK, Jankovic J: Treating severe bruxism with botulinum toxin. J Am Dent Assoc, 131:211-216, 437
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Abstract THE MANDIBULAR GROWTH EFFECT OF THE BOTULINUM TOXIN TYPE A INJECTION INTO UNILATERAL MASSSTER MUSCLE OF GROWING RAT So-Youn Kwak, Ki-Tae Park, Ji-Yeon Kim Department of Pediatric Dentistry, Samsung Medical Center, Sungkyunkwan University School of Medicine The purpose of this study was to assess the effect on mandibular growth of botulinum toxin type A (BTXA) injection into the unilateral massester muscle of growing rats at three different growing stages. Thirty six male Sprague-Dawley rats were divided into three groups according to the age (group 1: 4 weekold, group 2: 5week-old, group 3: 6week-old). Then each group was randomly divided into 3 subgroups (control group, unilateral injection group, bilateral injection group). Experimental animals were sacrificed after 4 weeks. Then the jaw measurements were evaluated. The results were as follows: 1. In the group 1, mandibular body length, condylar height and coronoid process height of the unilateral group(btxa side) and the bilateral group were shorter than those of the control group 2. In the group 2, anterior region height, condylar height, coronoid process height of the unilateral group (BTXA side) and the bilateral group were shorter than those of the control group 3. In the group 3, mandibular body length, condylar height, coronoid process height of the unilateral group (BTXA side) and the bilateral group were shorter than those of the control group 4. There was no significant difference in mandibular measurements between the control side and the injection side in the unilateral group in all age groups (p>0.05). Key words : Botulinum type A toxin, Mandibular growth, Functional matrix 439