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ii
iii
감사의글 모든순간저와함께하여주시며이끌어주신하나님께감사를드립니다. 본논문이완성되기까지부족한저를많은지도와격려로이끌어주신백형선교수님과이기준교수님께깊은감사를드립니다. 또한많은관심과조언으로심사를맡아주신황충주교수님, 국윤아교수님, 최성호교수님께감사를드리며많은가르침과사랑으로교정학의길을갈수있도록이끌어주신유영규교수님, 손병화교수님, 박영철교수님, 김경호교수님, 유형석교수님께진심으로감사를드립니다. 또한부족하지만교정의사생활을하는저에게항상모범이되시며자상하게이끌어주시는전윤식교수님, 최광철교수님, 성상진교수님께감사를드립니다. 논문이있기까지물심양면으로많은도움과격려를보내주신이철원교수님, 박재억교수님, 윤현중교수님, 김성훈교수님께감사를드리며실험과정에서많은도움을준성모병원수련의와교정과의국후배들에게이자리를빌어감사의마음을전합니다. 그리고누구보다도큰힘이되어주셨던부모님과사랑하는아내에게사랑과고마움의마음을전합니다. 2009 년 1 월 저자씀
차 례 그림차례 ii 국문요약 iii Ⅰ. 서론 1 Ⅱ. 연구내용및방법 4 1. 실험동물및장치장착 4 2. 조직표본의제작및염색 5 1) H-E(Hematoxylin-Eosin) 염색 5 2) PCNA(Proliferating Cell Nuclear Antigen) 면역조직화학염색 6 3) FGFR2(Fibroblast Growth Factor Receptor 2) 면역조직화학염색 6 4) Masson trichrome 염색 7 3. 치아이동거리측정 7 Ⅲ. 연구결과 9 1. 실험군및대조군에서치아이동거리의평가 9 2. 조직슬라이드관찰 10 1) Hematoxylin-Eosin 염색조직소견 10 2) PCNA(Proliferating Cell Nuclear Antigen) 면역조직화학염색소견 11 3) FGFR2(Fibroblast Growth Factor Receptor 2) 면역조직화학염색소견 12 4) Masson trichrome 염색소견 13 Ⅳ. 고찰 27 Ⅴ. 결론 32 참고문헌 33 영문요약 40 i
그림차례 Figure 1. Spring activated on rat incisor 4 Figure 2. Tissue section direction and region of interest 5 Figure 3. Suture width measurement 8 Figure 4. Tooth displacement in experimental groups 9 Figure 5. Young group Hematoxylin-Eosin stain 14 Figure 6. Adult group Hematoxylin-Eosin stain 16 Figure 7. Young group PCNA immunohistochemistry stain 18 Figure 8. Adult group PCNA immunohistochemistry stain 20 Figure 9. Young group FGFR2 immunohistochemistry stain 22 Figure 10. Adult group FGFR2 immunohistochemistry stain 24 Figure 11. Masson trichrome stain 26 ii
국문요약 백서전치에교정력적용시연령에따른 골막측치조골개조 교정적치아이동과관련된치근흡수, 골성열개 (bone dehiscence), bone fenestration 등의부작용은대개골막측치조골의골개조와관련되어발생하게된다. 그러나아직까지골막측치조골의골개조와관련된보상성골형성에대한지식은부족한실정이다. 이에 Young군 (12주) 과 Adult(8-12개월 ) 군백서절치에 double helical spring을사용하여 40g의측방력을가하였을때, 연령과시기에따른보상성골형성양상을조직형태학적으로비교하고자하였다. 연구결과 3주간의실험기간동안 Young군에서 Adult군에비해유의하게많은치아이동량을보였다. Young군의 7일군에서압박측 crestal 및 middle 치조골의골막측피질골표면은 scalloped surface를형성하며왕성한골개조양상을보였으나, Adult군에서는유사한변화가 crestal 부위에서만다소관찰되었다. PCNA 양성반응을보이는세포는 Young군에서 crestal 및 middle 치조골의골막측에서대조군에비해 3, 7, 14일군에서상당히증가한반면 21일군에서는감소되어주로 crestal 부위의치근인접면에서나타났으며, Adult군에서는 PCNA 양성세포가전기간에걸쳐주로 crestal 부위에국한되어나타났다. FGFR2 양성을보이는세포는 Young군에서대조군에비해더강하게 crestal 및 middle 치조골의골막측에서 3, 7, 14일군에서관찰된반면 Adult군의 3일군에서는같은부위에서증가되었으나, 14일군에서는주로 crestal 부위에서관찰되었다. Masson trichrome 염색결과 Young군과 Adult군모두에서대조군에비해장치활성화후왕성한 Type I collagen fiber 염색상이관찰되었다. 위의결과를통해교정적치아이동이골막측치조골표면에서의세포증식및골세포분화를촉진시키며, 특히 Adult군에비해 Young군에서치아이동에선행하는 iii
치조골의골개조가두드러지게나타나는것으로생각된다. 반면 Adult 군에서의 감소된골개조반응은교정치료시치조골협설측으로의과도한치아이동을제한하는 요인이될수있다고생각된다. 핵심되는말 : 보상성골형성, 골개조, 골막측치조골, 백서절치, 면역조직화학염색, 마손 - 트라이크롬염색 iv
백서전치에교정력적용시연령에따른 골막측치조골개조 ( 지도 : 백형선교수 ) 연세대학교대학원치의학과 모성서 Ⅰ. 서론 교정치료는치아의생물학적이동을통해심미성과기능적향상을이룸과동시에부작용을최소화할때성공적인치료라평가할수있다. 현재까지치아이동은주로압력-신장이론 1 에따라치근막과직접접해있는치주인대측치조골면에서의압박측의골흡수와, 신장측의골형성에의한다고알려져있다. 반면치근흡수, 골성열개 (bone dehiscence), bone fenestration 등의부작용이교정적치아이동에 동반될수있음이보고되었으며 2-7 이러한부작용들은대개상당한치아의이동과 동반되는골막측치조골 (periosteal side of alveolar bone) 의골개조 (bone modeling) 와관련되어있다. 즉순설측혹은협설측으로의치아이동은치조와내에서의골개조뿐아니라골막측치조골의상당한골개조를요구하며이러한조직반응은통상적으로보상성골형성 8-10 (compensatory bone formation) 이라알려져있다. 이러한보상성골형성에대해상당한협설측치아이동시에도적극적인골형성이관찰된다는긍정적인보고도있으나 8-11, Wehrbein 12,13 등은교정치료중 1
사망한환자의부검결과치아의이동에따른심각한골성열개를보고하는등 다소논란의여지가있다. 또한 Steiner 14 및 Engelking 15 등은원숭이를이용한 일련의실험에서상하악전치를전방으로이동시킨경우이동방향으로의치조골손실이발생되나다시후방으로재위치시킨경우순측골조직이상당량재생됨을보고하였으며, Karring 16 등도교정적으로만들어진치아의 giggling force에의하여만들어진골성열개가 giggling force의제거시회복될수있다고보고한바가있다. 그러나이러한연구는모두치아가원래의위치로되돌아갈때나타나는현상으로교정치료는원래의치아위치로의이동이아닌술자가계획한위치로치아이동을도모하는경우가대부분이므로이와는다른접근의연구가필요하다. Garib 17 등은성장기환자에서 RME(Rapid Maxillary Expansion) 를이용한상악확장시확장직후평균 7.1mm의골성열개를보고하였고, Sarikaya 18 등은전치를후방으로견인시구개측골두께의감소를보고하였고, Dorfman 19, Årtun 20, Yared 21 등은하악전치를전방으로경사시킬때골성열개와치은퇴축을유발시킨다고하는등골막측치조골의골개조능력이제한적이라고보고한반면, 일부연구 22-24 에서는이에동의하지않는등논란이있다. 특히 Ruf 25, Årtun 26 등은젊은 ( 아동또는청소년 ) 환자에서의하악전치의전방경사는골성열개와치은퇴축을야기하지않는다고보고하는등보상성골형성능력은연령에따른차이가있음을제시하였다. 연령에관련된치아이동에관한연구로 Jäger 27 등은연령이증가함에따라골형성능력과자극에대한반응성이낮아지며, Bridges 28 등은 Young군에서치조골의골밀도가낮아해부학적저항이낮아치아이동에유리하다고주장하고있다, 그러나일부연구 29-31 에서는교정력이적용될경우 Adult군과 Young군의조직학적인구성에큰차이가없다고보고하는등아직까지논란의대상이되고있는상황이다. 이러한논란을해소하기위해서는골막측치조골이어느시기에어떻게골개조혹은재형성이이루어지는지와성인과청소년기에서의골개조능력에차이가실제로나타나는지, 그렇다면그차이를유발하는요인은어떠한것인지알아볼 2
필요가있다. 이에본연구에서는백서의절치를교정적으로측방이동시연령과 시기에따른골막측치조골표면에서의골개조양상의차이를면역조직화학적 방법으로알아보고자하였다. 3
Ⅱ. 연구내용및방법 1. 실험동물및장치장착 연령에따라 Adult군 (8-12개월) 25마리와 Young군 (12주) 25마리, 총 50마리의 Male Sprague-Dawley rat을사용하였으며각연령군별로장치적용기간에따라대조 (control) 군, 3일, 7일, 14일및 21일실험군 ( 각각 5마리 ) 으로구분하였으며, 좌우절치에측방력을가하기위해 0.014 stainless-steel wire로 double helical spring을제작하였다. 실험군은럼푼 (rompun, Bayer, Korea) 0.05ml와졸레틸 (Zoletil 50, Virbac Lab Carros, France) 0.45ml를복강내주사하여마취후 high speed 1/4 round bur를사용하여좌우절치에 hole을형성하고제작된 spring을 hole에삽입하여장착하였다 (Fig 1.). Helical spring은 40g의힘으로좌우절치를측방으로이동시킬수있도록활성화하였으며실험기간동안재활성화하지않았다. 실험군에서 helical spring장치장착전과희생후 hole간의거리를버니어캘리퍼스를이용하여측정하였고실험기간종료후에도 spring의탄성이남아있음을확인하였다. Fig 1. Spring activated on rat incisor. 4
A B Fig 2. A; Tissue section direction, B; region of interest : * crestal area, ** middle area (magnification X40), scale bar 500 μm. 2. 조직표본의제작및염색 (1) H-E(Hematoxylin-Eosin) 염색 대조군과실험군은각시기별로과량의 ether로희생시킨후상악 premaxilla 부분을 dissection하였다. 분리된조직은 4% paraformaldehyde용액에 4일간고정시킨후 Calci-clear Rapid TM solution(pational diagnostics, Atlanta, USA) 을이용하여 24시간동안탈회하였다. 탈회시킨후치아의골막측치조골의관찰을위해 Fig 2-A와같은방향으로조직을절단하여파라핀포매후 5μm두께로 5
절단하여조직절편을만들었으며, 슬라이드상에준비된조직절편은자일렌으로탈파라핀한후순차적인저농도의알코올로처리하여함수처리한후통법에따라 H-E 염색을시행한후정중구개봉합부위의폭경을계측하고관심부위의조직학적소견을관찰하였다 (Fig 2-B,3,5,6). (2) PCNA(Proliferating Cell Nuclear Antigen) 면역조직화학염색 슬라이드상에준비된조직절편을자일렌을이용하여탈파라핀시킨후순차적인저농도의알코올로함수처리를하였고, 실온에서 0.5% Triton X-100용액으로 10분간 Antigen retrieval을시행하였다. Endogenous Peroxidase의비활성화를위하여 0.3% H 2 O 2 에 5분간 incubation을시행하였고, 5% Bovine serum Albumin으로 30분간 blocking을시행하였다. PCNA 염색을위하여 primary antibody로 FL-261(Santa Cruz Co., USA) 을 1:100으로희석하여실온에서 1시간 incubation후 4 에서 over-night incubation을시행하였으며 2nd antibodyconjugated polymer로 Envision kit(dako Co., Denmark) 를이용하였다. 2-3분간 DAB 발색후, Gill s Hematoxylin으로대조염색을시행하였으며알코올의농도를순차적으로높여탈수한후 mounting하여슬라이드를제작한후조직소견을관찰하였다 (Fig 7,8). (3) FGFR2(Fibroblast Growth Factor Receptor 2) 면역조직화학염색 슬라이드상에준비된조직절편을자일렌을이용하여탈파라핀시킨후순차적인 저농도의알코올로함수처리하였고, Proteinase K 로 36 에서 10 분간 Antigen retrieval 을시행하였다. Endogenous Peroxidase 의비활성화를위하여 0.3% 6
H 2 O 2 에 5분간 incubation을시행하였고 5% Bovine serum Albumin으로 30분간 blocking을시행하였다. FGFR2 면역조직화학염색을위하여 primary antibody로 Bek sc-122(santa Cruz Co., USA) 을 1:100으로희석하여실온에서 1시간 incubation후 4 에서 over-night incubation을시행하였으며 2nd antibodyconjugated polymer로 Envision kit(dako) 를사용하였다. 2-3분간 DAB develop를시행한후, Gill s Hematoxylin으로대조염색을시행한후알코올의농도를순차적으로높여탈수한후 mounting하여슬라이드를제작하였다 (Fig 9,10). (4) Masson trichrome 염색 슬라이드상에준비된조직절편을자일렌을이용하여탈파라핀시킨후순차적인저농도의알코올로함수처리한후 Weigert s iron hematoxylin으로 10분간핵염색후수세하였고, 1% HCl-alchohol용액으로탈색후수세하였다. Ponceau-acid fuchsin용액으로 10분간배경염색후 0.2% acetic acid용액으로수세하였으며 Phosphomolybdic acid - orange G용액으로 5분간매염과탈색후 0.2% acetic acid로수세하였다. light green 염색액으로교원섬유염색후 0.2% acetic acid를이용하여비결합 light green을탈색시킨후탈수및청명하여슬라이드를제작하여조직소견을관찰하였다 (Fig 11). 3. 치아이동거리측정 실험군 (3 일, 7 일, 14 일, 21 일 ) 에서 helical spring 장치장착전후의 hole 간의 거리를측정하였으며, 전치에장착된스프링에의하여치아의측방변위뿐 7
아니라정중구개봉합도이개되는양상을보여 (Fig 3.) 조직슬라이드상에서봉합의폭경을 Leica Application Suite(Leica Microsystem, Heerbrugg, Switzerland) 소프트웨어를이용하여계측하였으며 치아이동거리 = hole간의거리증가량 장치장착후봉합폭경 + 대조군의봉합폭경 을이용하여계측하였다. A B Fig 3. Suture width measurement. (magnification X40), scale bar 200 μm Mid palatal suture was widened by double helical spring and measured inter apex distance of palate with LAS software. A : before application, B : after application example of 14 days young group. 8
Ⅲ. 연구결과 1. 실험군및대조군에서치아이동거리의평가 실험군에서의치아이동거리는 Fig 4. 에표시하였다. 치아이동거리는 3일군에서는 Young군과 Adult군에서차이가없었으나, 시간이갈수록 Young군에서많은이동량을보여주었다. Young군에서는 7일, 14일군에서치아이동이지연되었고, 21일군에서다시치아이동량이증가하는양상을보였으나, Adult군에서는 21일군에서도치아이동의지연이계속되었다. Fig 4. Tooth displacement in experimental groups. In the young group more rapid tooth movement than adult group was observed. Tooth movement retardation occurred on 7 days and 14 days Young groups, but tooth movement was recovered on 21 days group. In the adult group tooth movement retardation was continued until 21 days group. 9
2. 조직슬라이드관찰 염색되어진슬라이드를 Fig 2-B 와같이관심지역을선정하고검경하였다. (1) Hematoxylin-Eosin 염색조직소견 a) 대조군 (control) Young군과 Adult군모두에서정상적인치주인대공간을보였으며 crest 정상부위에서조금넓어지는경향을보였다. Crestal 및 middle 부위모두에서골막측치조골은평탄한표면을보이며이에비해치주인대측골표면은다소의불규칙성을보였다 (Fig 5,6-A,F). b) 3일군 Young군과 Adult군모두에서 crestal 부위에서치주인대공간의감소와초자양화양상을보여세포수의감소를보였고, middle 부위의치주인대측치조골표면에는 lacuna가증가하였으며약간의 osteoclast가관찰되었다. Young군에서는골막측치조골 Crest 정상부위에서신생골의형성과표면의 irregularity가증가하였으며, Adult군에서는 crestal 부위의골막측치조골의골막주위의세포층이두꺼워졌다 (Fig 5,6-B,G). c) 7일군 Young군과 Adult군모두에서 crestal 및 middle 부위의치주인대측치조골에서다수의파골세포가관찰되며, crestal 부위에서는간접골흡수 (undermining bone resorption), middle 부위에서는직접골흡수 (direct bone resorption) 의소견을보였다. Young군에서 crestal 부위의골막측치조골에서는표면의 irregularity가 10
증가하였으며치조골은 3 일군보다더비후되었고, Adult 군에서는 crestal 부위의 골막측치조골에서신생골의형성과함께표면의 irregularity 가증가하였다 (Fig 5,6-C,H). d) 14일군 Young군과 Adult군의 creatal 부위의치주인대측치조골에서의간접골흡수양상이지속되었으며 middle 부위에서는골흡수가진행되어치주인대공간이증가하였고파골세포의숫자는 7일군보다감소하였다. Young군과 Adult군의 crestal 부위골막측치조골표면은 7일군에비해다소평탄한모습을보이며비후된양상을유지하고있으나, Young군에서는 middle 부위골막측치조골표면의 irregularity가증가하였다 (Fig 5,6-D,I). e) 21일군 Young군및 Adult군의 crestal 부위의치주인대측치조골에서파골세포가관찰되며초자양화된골조직의간접골흡수가지속되나, middle 부위의치주인대측치조골에서는파골세포가현저하게감소되어있으며표면의 irregularity 가감소되었고, Crestal 및 middle 부위의골막측치조골에서의 irregularity 도현저하게감소되었다 (Fig 5,6-E,J). (2) PCNA(Proliferating Cell Nuclear Antigen) 면역조직화학염색소견 Young군과 Adult군에서교정장치활성화전에는 crestal 부위의치조골골막측에서약간의 PCNA 양성세포가관찰되며치주인대공간에는다수의 PCNA 양성세포를관찰할수있었으나 (data not shown), 두군공히 middle 부위의골막측에서는 PCNA 양성세포가거의관찰되지않았다 (Fig 7,8-A,F). 11
3일군에서는 Young군에서 crestal 부위상방과골막측치조골주위에서 PCNA 양성세포의급격한증가가관찰되었으나, collapse된치주인대측에서는관찰되지않았고, Middle 부위에서는치조골의골막측과치주인대공간모두에서관찰되었다. Adult군에서 crestal 부위상방과치조골의골막측에서많은숫자가관찰되었으나, Young군보다는밀도가낮았으며 middle 부위의골막측에서는관찰되지않았다 (Fig 7,8-B,G). 7일군에서는 Young군에서 crestal 부위상방및 crastal 및 middle 부위의골막측에서 PCNA 양성세포가다수관찰되었다. 3일군에서보다치조골골막측의 crestal 부위에서감소하는양상을보이나, middle 부위쪽에서는증가하는양상을보인다. Adult군에서는 crestal 및 middle 부위의골막측에서 3일군보다 PCNA 양성세포가많이감소되었다 (Fig 6,7-C,H). 14일 Young군에서는골막측 crestal 부위의 PCNA 양성세포는많이감소하였으며, 골막측 middle 부위에서도감소하는양상을보여주었으나, Adult군에서는골막측 crestal 및 middle 부위모두에서 PCNA 양성세포가다시증가하였다 (Fig 7,8-D,I). 21일군소견으로 Young군에서 crestal 및 middle 부위의골막측모두에서 PCNA 양성세포는대조군수준으로감소되었고, Adult군에서 crestal 및 middle 부위의골막측에서일부 PCNA 양성세포가관찰되었으나, 14일군에비해현저하게감소하였다 (Fig 7,8-E,J). (3) FGFR2(Fibroblast Growth Factor Receptor 2) 면역조직화학염색소견 Young 군과 Adult 군에서교정력적용전치조골골막측과골내의골세포 (osteocyte) 에서 옅은 FGFR2 양성반응이관찰되었고 (Fig 9,10-A,F), 3 일군에서는 Young 군과 Adult 군모두에서 crestal 및 middle 부위의치조골의골막측에서강한 FGFR2 12
양성반응이관찰되었다 (Fig 9,10-B,G). 7일군에서는 Young군및 Adult군에서 crestal 및 middle 부위의골막측에서양성반응을보였으나, 3일군에비하여현저하게감소되었고 (Fig 9,10-C,H), 14일군에서는 Young군과 Adult군모두에서 crestal 및 middle 부위의골막측에서 7일군보다증가된반응양상을보이나, Adult군에서는주로 crestal 부위에집중되어반응이관찰되었다 (Fig 9,10-D,I). 21일군에서는 Young군과 Adult군모두의 crestal 및 middle 부위의치조골골막측에서대조군수준으로반응이약화되었다 (Fig 9,10-E,J). (4) Masson trichrome 염색소견 Masson trichrome 염색에서는 typeⅠcollagen fiber가 green색으로염색되는특징을지닌다. Young군과 Adult군에서교정력적용으로인하여골막측치조골에서의골양조직 (osteoid) 이증가되며신생골형성및치조골조직의재형성이증가되었다 32. Young군에서는활성화된골조직의반응이 21일군에서다시감소하는소견을보이나, Adult군에서는 Young군보다지연된골반응양상을보여준다 (Fig 11.). 13
* A B C F G H Fig 5. Young group Hematoxylin-Eosin stain. (magnification X100), scale bar 200 μm A E : respectively control, 3 days, 7 days, 14 days, 21 days group alveolar crestal area. F J : respectively control, 3 days, 7 days, 14 days, 21 days group alveolar middle area. On 3 days group (*)new bone formation on the periosteal side was observed and On 7 days group active bone modeling was observed with scalloped surface on the periosteal side of crestal area and on 14 days group scalloped surface spreaded on the periosteal side of middle alveolus at compressed side in the young group. 14
D E I J Fig 5. continued 15
* A B C F G H Fig 6. Adult group Hematoxylin-Eosin stain. (magnification X100), scale bar 200 μm A E : respectively control, 3 days, 7 days, 14 days, 21 days group alveolar crestal area. F J : respectively control, 3 days, 7 days, 14 days, 21 days group alveolar middle area. On 7 days group (*)new bone formation and active bone modeling with scalloped surface of the periosteal side were observed only crestal alveolus at compressed side in the adult group. 16
D E I J Fig 6. continued 17
Δ Δ A B C F G H Fig 7. Young group PCNA immunohistochemistry stain. (magnification X400) Δ : PCNA positive cell (black nucleus), scale bar 50μm A E : respectively control, 3 days, 7 days, 14 days, 21 days group alveolar crestal area. F J : respectively control, 3 days, 7 days, 14 days, 21 days group alveolar middle area. In the young group, PCNA positive cells increased greatly on crestal and middle alveolus compared with those in the control group on 3, 7, 14 days groups, while they were decreased and remained on the crestal area adjacent to root on 21 days group. 18
D E I J Fig 7. continued 19
Δ Δ A B C F G H Fig 8. Adult group PCNA immunohistochemistry stain. (magnification X400) Δ : PCNA positive cell (black nucleus), scale bar 50μm A E : respectively control, 3 days, 7 days, 14 days, 21 days group alveolar crestal area. F J : respectively control, 3 days, 7 days, 14 days, 21 days group alveolar middle area. In the adult group, PCNA positive cells increased on 3 days and decreased on 7 days group, but on 14 days group they increased again. They were distributed principally on the crestal area in all duration. 20
D E I J Fig 8. continued 21
A B C Δ Δ F G H Fig 9. Young group FGFR2 immunohistochemistry stain. (magnification X400) Δ : FRFR2 positive cell (Hollow nucleus and brown cell membrane), scale bar 50μm A E : respectively control, 3 days, 7 days, 14 days, 21 days group alveolar crestal area. F J : respectively control, 3 days, 7 days, 14 days, 21 days group alveolar middle area. In the young group, the number of FGFR2 positive cells increased on creatal and middle area on 3 days group, decreased on 7 days group, increased again on 14 days group and decreased to the level of the control group on 21 days group. 22
D E I J Fig 9. continued 23
Δ Δ A B C F G H Fig 10. Adult group FGFR2 immunohistochemistry stain. (magnification X400) Δ : FRFR2 positive cell (Hollow nucleus and brown cell membrane), scale bar 50μm A E : respectively control, 3 days, 7 days, 14 days, 21 days group alveolar crestal area. F J : respectively control, 3 days, 7 days, 14 days, 21 days group alveolar middle area. In the adult group, FGFR2 positive cells were observed on crestal and middle alveolus on 3 days group, but on 14 days group they were principally observed on the crestal area. 24
D E I J Fig 10. continued 25
* * A B C D E F Fig 11. Masson trichrome stain. (magnification X100), Scale bar 200 μm Green color stain is type I collagen fiber. It means newly formed immature bone or active bone turn-over in Masson trichrome stain. A: control group, B: young 3 days group; green color increased, which meant bone activity increased and new bone formation(*) on the periosteal side of alveolar bone, C: Adult 7 days group; generally lower bone modeling activity than young group and new bone(*) formation was observed, D: Young 14 days group; most of crestal bone green, which showed alveolar bone was highly activated, E: Young 21 days group; most of crestal bone was red, which meant bone activity rapidly decreased, F: Adult 21 days group; most of crestal bone green, representing more delayed bone activity than in the young group. 26
Ⅳ. 고찰 골격성부정교합의절충치료를위해치조골범위를넘는과도한치아이동이계획될수있으며이경우좁은치조골내에서의치아이동량의증가는필연적으로치조골의피질골판의골개조를요구하나보상성골형성은연령에따라다를수있다는것이중론이다. 이에본실험에서는교정치료가가장활발히진행되는사춘기성장기무렵의청소년과, 30대성인군에해당되는 Young(12주 ) 군과 Adult(8-12개월 ) 군의 Sprague-Dawley rat을사용하였고 33, 전치와구치에서의협설측으로의치아이동과유사하게치아측방에얇은피질골판을가지고있는백서절치를이용하여측방으로확대하는디자인을적용하였으며 (Fig 1,2) 교정력은기존의 Engström 34 과 Akın 35 등의연구를참조하여 40g의교정력을적용하였다. 연령과관련되어교정력이적용된치아의이동속도에대한많은연구 28,36,37 에서 Adult군의치아이동속도가 Young군보다느리며그원인으로 Kyomen 36 등은연령과관련된세포활성도의감소, Abiko 37 등은연령과연관된치주인대공간의감소에따른치조골초자양화의증가, 연령과관련된골밀도의증가 28,38,39 등으로설명하고있다. 그러나 Jäger 40, Kabasawa 29 등은교정력이적용되면골아세포및파골세포의활성도및숫자에서 Young군과 Adult군에서차이가없다고주장하였으며, Bond 41, Melsen 42 등은 Adult군에서치아의초기이동이일어나기까지의반응시간에차이가있을뿐이며일단치아의이동이시작되면치아의이동속도에는차이가없다고주장하기도하였고, Ren 43 등은백서를이용한치아이동실험에서 4주이후부터는 Young군과 Adult군의치아이동속도에차이가없다고보고하기도하였고이와관련되어치아이동과관련된파골세포의숫자가 Young군에서장치장착 2주후가장많이관찰되나, Adult군에서는혈류를통한파골세포의증가로 4주이후에는차이를보이지 27
않는다는연구결과 44 를보고하기도하였다. 그러나이번실험결과는 3주까지의결과이지만치아이동속도는 Young군에서빠른것으로나타났으며 (Fig 4), H-E 염색소견상에서도 Young군에서보다왕성한골개조소견을보여주었다 (Fig 5,6). 이번연구에서교정력적용시골조직의반응양상을관찰하기위하여 PCNA 및 FGFR2 면역조직화학염색을시행하였다. PCNA 면역조직화학염색은분화되는세포종류에특이성이없이분열하는세포에서양성반응을나타내는특성을지녀 PCNA 양성세포가많다는것은자극에대한조직의반응이활발하다는것을의미하며, FGFR2 면역조직화학염색은골아세포 (osteoblast) 로분화하는 osteoprogenitor 세포에특이성을가지는면역조직화학염색법으로 45-47 PCNA 양성부위와 FGFR2 양성부위가상당부분일치하는것으로보아교정력에의하여치조골의골막측에서활발한골개조가진행되고있다는것을보여주며, 연령군에따른소견으로는초기양성반응을나타내는시기에있어서 Young군과 Adult군간의차이는없었으나, 발현되는정도에서는차이를보이며, 최대양성반응이나타나는시기는 Adult군이 Young군보다늦는등연령에따른조직활성도의차이를보여주었다. 이러한결과는연령의증가에따라인접조직에분포하는미분화세포의감소 48-50, 미분화세포의공급 (recruitment) 에따른반응의지연 36, 연령증가에따른골밀도의증가와 28,38,39 치주인대공간의감소 51, 치주인대의물성변화 52 등에따른골조직의변형 (deformation) 감소와이에따른골개조반응의감소등이원인으로생각된다. 현재교정력적용시치아의이동원리는주로압력-신장이론 1 에의해설명되어진다. 즉치관에교정력이가해지게되면치아는경사이동을시작하게되는데이때압박은주로치근부위와압박측치조정부위에집중되게되고, 그결과치조정부위의치주인대공간이 collapse되어부분적인산소분압의저하가발생하고, 일련의세포반응을거처파골세포가증가되어치주인대측치조골을흡수시켜치아가이동하게되면반대편인장측에신생골이형성된다는이론으로압박측의골흡수와인장측의골형성을특징으로하고있다. 이이론에따르면 28
치아이동시치아와치주인대를사이에둔치주인대측치조골의반응을상당부분설명할수있으나, 치아이동에따른골막측치조골의보상성골형성반응을명확하게설명하지못하고있다. 치아이동시보상성골형성에대한실험으로 Shimpo 8 등은백서를이용한실험에서구치를설측으로경사시킬때설측피질골에서연령군에관계없이상당량의골생성을보고하였고, Wingard 11 등은원숭이실험에서상당량의협설측피질골의재형성을보고하기도하였다. 그러나많은임상연구에서치아의협설측이동시치조골의손실을보고하는실정이다 17-20. 이번실험에서 Young군에서는장치장착 3일군에서 crestal 부위의골막측치조골외측에서, Adult군에서는 7일군에서골막측치조골외측에서의보상성골형성을관찰할수있는데 (Fig 5,6,11) 이시기는 Shimpo 8, Verna 53 등의결과와같이보상성골형성반응은외력에대해빠르게반응하는것으로보인다. 이러한보상성골형성의기전에대하여명확하게알려지진않았지만 Melsen 54 등은원숭이를이용한실험에서치아가이동하는방향의압박측 ( 압력-신장이론에따르면흡수측 ) 전방에서골형성이일어난다고보고한바가있고이러한현상은 Frost의 mechanostat이론 55 으로잘설명될수있다. mechanostat 이론은외력에대한골조직의적응으로골조직이휘 (bending) 는방향 ( 피질골의 compression 방향 ) 에서골생성이일어나다는이론으로 Epker 56 는골조직이휘는방향에서골생성이일어난다는이론을치아이동에적용하여압력- 신장이론에서의인장측에서의골형성을설명하기도하였다. mechanostat이론은치아이동시의보상성골형성을잘설명할수있으며이번실험의조직학적결과와도유사한양상을보여주고있다. 이이론을토대로이번실험결과를해석해본다면 Young군에서치아가측방으로이동하게되면서 crestal 부위의치주인대측치조골면은초자양화되고그결과교정력은치조골로전달되어치조골의외측으로의휨현상 9,10 이발생하여 crestal 부위의골막측치조골측에서보상성골생성이일어났고, 7일군에서는 middle 부위의치주인대측에서직접골흡수 (direct bone resorption) 가진행됨에따라치주인대공간이증가하였고 29
이에따라 middle 부위의치조골이얇아지고골조직의 turn-over가증가함에따라골밀도가감소하여치조골의강도가감소되면서치조골의휨현상이 middle 부위까지확대되고, 그결과 middle area이치조골골막측까지골개조가진행되어골표면의 irregularity가증가양상을보여 14일군에서최고조에이르나, 21일군에서 crestal area의초자양회된조직의간접골흡수 (indirect bone resorption) 가진행됨에따라교정력이치조골로전달되지않으며 middle 부위의직접골흡수로치주인대공간이넓어져서더이상골흡수가진행되지않자골막측치조골의골개조가감소하면서평탄해지는모습을보여주고있다. Adult군에서도같은기전으로 7일군에서보상성골형성을보이나연령에따른조직활성도의감소에따른골조직의 turn-over의감소와연령에따른골밀도의증가 28,38,39 등으로 middle 부위까지활발한골개조가일어나지않은것으로보인다. 이번연구에서교정력에대한골막측치조골표면의반응으로 Fig 5,6처럼피질골표면에서의 irregularity 현상은 Reitan 9, Storey 10 등의연구에서도보고되고있으며, Storey는작용되는힘의크기에따라다른형태를보인다고설명하고있다. 이와 관련하여 Petrtýl 57 등은외력의적용시피질골표면에서외력방향으로함몰되는 현상은피질골 (cortical bone) 에서나타나는일반적인골재형성 (bone remodeling) 양상으로외력방향으로피질골표면을파골세포 (osteoclast) 가골을깊게흡수하여들어가며그뒤로많은수의골아세포 (osteoblast) 가따라가면서골조직을형성하는방법으로피질골의내부까지재형성되는과정으로설명하는데 Fig 5,6의골막측치조골표면에서보여지는 irregularity의형태와방향이이와유사한것으로생각된다. 이와같이외력에대한골막측치조골의신속한반응에대하여 Skerry 58, Klein-Nulend 59, Cowin 60 등은외력에의한골의변형에대하여골조직내부의골세포 (osteocyte) 들이 mechanosensor역할을함으로서주변의파골세포와골아세포의작용을조절하기때문으로설명하고있다. 이번연구결과치아이동에관하여치주인대측치조골의변화와함께혹은그보다도더신속한골막측치조골의변화가있으며, 보상성골형성을조직학적으로 30
관찰할수있었으며, 연령에따른차이도관찰할수있었다. 그러나연령에따른차이가없다 29,41 는주장에서도그전제조건은치아이동이시작되고난후로 Ren 43 등은 4주이상지난후부터차이가없다고주장하고있다. 따라서좀더장기간의연구가필요할것으로생각된다. 특히 Roholl 31 등은연령에따른골조직의세포성분의변화에서연령이증가하면골아세포의숫자가감소하지만파골세포의숫자는영향받지않는다고하였으며, 이번연구결과에서도 Adult군보다 Young군에서의 PCNA, FGFR2에서의반응의저하는연령의증가에따른골형성능력의저하를의미하며, 연령의증가에따르는치조골밀도의증가, 치주인대공간의감소, 치주인대물성의변화에따른 Adult군에서의치아이동속도의감소와함께치조골에서의평형관계 (homeostasis) 를유지하는중요한역할을하는것으로생각된다. 따라서연령증가에따른생물학적인반응과함께교정력의크기와시간등에대한추가적인연구가필요한것으로생각된다. 31
Ⅴ. 결론 교정력적용시시간과연령군에따른치아의이동량과골막측치조골의변화양상을관찰하기위하여백서절치에 40g의측방력을가한결과실험기간동안 Young군에서 Adult군에비해유의하게많은치아이동량을보였다. 보상성골형성은 Young군에서는 3일군, Adult군에서는 7일군에서관찰할수있었으며, Young군의압박측 crestal 및 middle 부위의골막측치조골표면은 scalloped surface를형성하며왕성한골개조양상을보였으나, Adult군에서는유사한변화가 crestal 부위에서다소관찰되었다. PCNA 양성반응을보이는세포는 Young군에서 crestal 및 middle 치조골의골막측에서대조군에비해 3, 7, 14일군에서상당히증가한반면 21일군에서는감소하여주로 crestal 부위의치근인접면에서일부관찰되었고, Adult군에서는 PCNA 양성세포가전기간에걸쳐주로 crestal 부위에국한되어나타났다. FGFR2 양성을보이는세포는 Young군에서대조군에비해더강하게 crestal 및 middle 치조골의골막측에서 3, 7, 14일군에서관찰된반면 Adult군의 3일군에서는같은부위에서증가되었으나, 14일군에서는주로 creatal 부위에서관찰되었다. Masson trichrome 염색결과 Young군과 Adult군모두에서대조군에비해장치활성화후왕성한 Type I collagen fiber 염색상이관찰되었다. 위의결과를통해교정적치아이동이골막측치조골표면에서의세포증식및골세포분화및골형성을촉진시키며이러한반응이특히 Young군에서 Adult군보다활발하게나타났고, Adult군에서의감소된골개조반응은교정치료시치조골협설측으로의과도한치아이동을제한하는요인이될수있을것으로생각된다. 32
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Abstract Age-related periosteal bone modeling Induced by rat incisor movement Sung-Seo Mo Department of Dentistry Graduate School, Yonsei University (Directed by Professor Hyoung-Seon Baik D.D.S., M.S.D., Ph.D.) Orthodontic side effects induced orthodontic tooth movement such as root resorption, bone dehiscence and bone fenestration are usually associated with bone modeling in the periosteal side of alveolar bone. However, there have been few studies on the compensatory bone formation related to orthodontic tooth movement. This study was aimed to compare the pattern of bone modeling histologically in different age groups and duration of force application when orthodontic force(40g) was applied to the incisor of young (12 weeks) and adult (8-12 months) rat using double helical spring. During experimental period, the amount of tooth movement in the young group was significantly greater than that of the adult group. On 7 days group, active bone modeling represented with scalloped surface was observed on the periosteal side of the crestal and middle alveolus at compressed side in young group, while similar changes were observed only on 40
the crestal area in the adult group. In the young group, the number of PCNA positive cells increased greatly on the crestal area and middle alveolus compared with control group on 3, 7, 14 days groups, while they decreased on 21 days group. In the adult group, PCNA positive cells appeared localized on the crestal area throughout the time. In the young group, FGFR2 positive cells were observed more on crestal and middle alveolus than control group on 3, 7, 14 days groups. In the adult group these cells appeared on the crestal and middle alveolus on 3 days group, but mainly on the crestal area on 14 days group. In Masson trichrome stain, Type Ⅰcollagen fiber was observed a lot more after helical spring activation in both young and adult groups compared with control group. According to these results, it is speculated that orthodontic tooth movement may stimulate cell proliferation and differentiation on the periosteal side of compressed alveolar bone, and this response may lead to prominent bone modeling prior to tooth movement in the young group, compared to the relatively delayed and diminished response in the adult group. The decreased bone modeling response in the adult group may be considered as a factor which restrains the excessive bucco-lingual tooth movement. Key word : compensatory bone formation, bone modeling, periosteal side of alveolar bone, rat incisor, immunohistochemistry stain, masson trichrome stain 41