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4 감사의글 본논문이완성되기까지부족한저를항상격려해주시고사랑과관심으로논문지도를해주신김형준교수님께깊은감사를드립니다. 구강악안면외과학을사랑할수있게많은조언과따뜻한관심으로많은가르침을주신이충국교수님, 박형식교수님, 이상휘교수님, 강정완교수님, 정영수교수님, 민연숙교수님, 강상훈선생님, 김학진선생님께도진심으로감사드립니다. 논문심사를맡아주시고, 힘든수련생활을즐기며공부할수있게해주신차인호교수님, 남웅교수님께도감사드립니다. 많은격려와멋진수련생활을할수있게해주신윤정훈교수님께도감사드립니다. 바쁜와중에도실험및연구를도와주고, 함께고민하면서시행착오도많이겪었던믿음직한최세경선생님을비롯한동기들, 의국원선후배님들께감사드립니다. 또한실험에있어많은조언과많은도움을준정지희선생님에게도감사드립니다. 무엇보다도늘아낌없는사랑과헌신적인도움으로든든하고따뜻한버팀목이되어주는사랑하는부모님과할머니, 누나에게도진정으로사랑과고마움의마음을전하며, 항상가족처럼아껴주시는고재성부장님께도감사의마음을전합니다. 구강악안면외과의로부끄럽지않도록평생을노력하고, 많은가르침을주셨던교수님들과선배님들께자랑스러운제자가될수있는그때까지더욱좋은모습으로보답하겠습니다 년 12 월 박세현

5 목차 그림및표차례 i 국문요약 ii Ⅰ. 서론 1 Ⅱ. 연구대상및방법 3 1. 연구대상 3 2. 연구방법 3 Ⅲ. 결과 8 1. 육안적소견 8 2. 방사선학적소견 8 3. 조직형태학적소견 10 Ⅳ. 고찰 11 Ⅴ. 결론 20 참고문헌 32 영문요약 39

6 List of Table Table 1. Study design 4 List of Figure Figure 1. Operative procedure 5 Figure 2. Schematic drawing of operative procedure 6 Figure 3. Gross finding 22 Figure 4. Radiographic finding 23 Figure 5. Micro-CT finding(3d reconstruction image) 23 Figure 6. Micro-CT finding(longitudinal & Cross sectional view) 24 Figure 7. Comparison of the bone density : tibia vs. fibula 25 Figure 8. Histologic finding : Non-experimental area in 2weeks group 26 Figure 9. Histologic finding : Border area in 2weeks group 26 Figure 10. Histologic finding : Experimental area in 2weeks group 27 Figure 11. Histologic finding : Border area in 2weeks group 27 Figure 12. Histologic finding : Experimental area in 2weeks group 28 Figure 13. Histologic finding : Experimental area in 2weeks group 28 Figure 14. Histologic finding : Non-experimental area in 8weeks group 29 Figure 15. Histologic finding : Border area in 8weeks group 29 Figure 16. Histologic finding : Experimental area in 8weeks group 30 Figure 17. Histologic finding : Experimental area in 8weeks group 30 Figure 18. Histologic finding : Experimental area in 8weeks group 31 i

7 국문요약 웅성백서비골의임계크기분절성골결손에서 ErhBMP-2 와 absorbable collagen sponge 를 이용한골재생 < 지도교수김형준 > 연세대학교대학원치의학과 박세현 외상, 기형, 종양의절제등에기인한악안면부골결손수복에있어, 자가골이식의한계를극복하고자골유도인자인 ErhBMP-2를이용한골재생의연구가활발히진행중이다. 본연구는, E-coli system에서새롭게개발된 rhbmp-2를 absorbable collagen sponge(acs) carrier를이용해웅성백서비골의임계크기 (6mm) 분절성골결손부에이식한후골재생을평가하였다. 실험은 8 주령의 SD rat 14 마리를대상으로하였으며, 비골에약 6mm 의임계크기분절성골결손을형성한후 absorbable collagen sponge 만이식한군 ( 대조군 ) 과 rhbmp-2 를 absorbable collagen sponge 에 dipping 하여이식한군 ( 실험군 ) 으로나누었다. 모든 SD rat 은 2 주와 8 주경과후각각희생하였으며, 육안적, 방사선학적 (Standard dental X-ray, Micro-CT imaging), 조직형태학적검사로골재생을관찰하였다. 2주대조군에서는분절성골결손부에골재생이일어나지않은비유합상태가관찰되었고, 실험군에서는골재생으로완전유합이이뤄졌지만, 육안적소견상불규칙한피질골과과도한골형성이관찰되었다. 방사선학적으로, 대조군에서는골재생이일어나지않아비유합이관찰되었고, 실험군에서는육안적관찰과 ii

8 일치하는소견을보이며, 골재생으로완전유합되었지만, 재생된골의양이과도하고, 인접한정상비골에비해골질이낮은상태임이관찰되었다. 골밀도에있어, 실험군에서는피질골층의불연속성과기존의비골에비해낮은골밀도를보이며, 인접한정상경골의절반정도의골밀도를보였다. 조직학적으로재생된골의양이적고, 남아있던근위부정상비골과의경계가남아있음을확인할수있었다. 남아있던정상비골의판상골 (lamella bone) 과는달리콜라겐 (collagenous tissue) 과다수의혈관그리고미성숙골 (woven bone) 의형성이관찰되었다. 8주대조군에서도분절성골결손부골재생이일어나지않은비유합상태가관찰되었고, 실험군에서는골재생으로완전유합뿐만아니라, 재생된비골과남아있던근위부정상비골과의경계를확인할수없을정도의규칙적이고평활한골형성이관찰되는육안적소견을보였다. 방사선학적으로, 대조군에서는골재생이일어나지않아비유합이관찰되었고, 실험군에서는완전유합과함께기존의인접정상비골과유사한양상의골양및골질이관찰되었다. 골밀도에있어, 실험군에서는피질골층의연속성과기존의인접정상비골과유사한골밀도를확인할수있었다. 조직학적으로새롭게형성된골양이 2주실험군보다많이관찰되었고, 2주군에서보였던미성숙골이판상골로변화된것이관찰되었다. 또한기존에남아있던비골과의경계부를뚜렷이확인할수없었으며, 2주실험군에서골수강부위에형성되었던 woven bone이골수로대체되는것이관찰되었다. 8주실험군에서 2주실험군보다골개조과정을거친성숙된층판골형성이관찰되었지만, 2주, 8주실험군모든개체에서명확한골재생을확인하였다. E-coli system을이용하여새롭게개발된 ErhBMP-2는기존의 rhbmp-2보다적은농도와용량으로완벽한골재생을할수있다는점과, 다량생산이 iii

9 가능하며, 경제적으로 BMP 생산비용을낮출수있다는장점이있다. E-coli system을이용하여새롭게개발된 ErhBMP-2와 absorbable collagen sponge의이식은분절성골결손부수복에유용하고, 자가골이식을대신할만한적절한방법중의하나로사료된다 핵심되는말 : 골형성유도단백질, 흡수성콜라겐스폰지, E-coli, 백서임계크기 분절성골결손 iv

10 웅성백서비골의임계크기분절성골결손에서 ErhBMP-2와 Absorbable collagen sponge를이용한골재생 < 지도교수김형준 > 연세대학교대학원치의학과 박세현 ` Ⅰ. 서론 골결손은외상, 선천성기형, 방사선골괴사, 종양의절제등의다양한원인에서기인하며, 이러한골결손부의재건을위한노력은지난수십년간계속되었다. 다양한수복방법이있지만자가골이식이악안면부결손부재건에는우선적으로선택이되고, 현재까지는 gold standard 로알려져있다 (Boyne et al., 1998; Hopp et al., 1989; Hausamen et al., 1992; Goldberg et al., 1987). 자가골이식은불편함, 경제적측면, morbidity, 수혜부크기에따른이식의한계등의문제점이있어최근에는미세혈관문합술을동반한자가골이식이선호된다. 미세혈관문합술을동반한자가골이식은술후즉시혈행공급으로큰수혜부에도이식의성공율을높일수있지만, 수술의난이도, 긴수술시간, 명백한 morbidity등의한계가있다 (Boyne, 1997; Esther et al., 2003; Anthony et al., 1995; Garrett et al., 2006; Suh et al., 2004; Kevin et al., 2007; Skoner et al., 2003). 이러한한계속에서악안면결손부의이상적인재건을위한다양한 1

11 시도가지속되고있지만, 아직까지확립된정설은없는실정이다 (Brian et al., 2003; Swennem et al., 2001, 2002; Paolo et al., 2006). 기존의골이식없이골유도인자인 Bone morphogenetic proteins(bmps) 를이용한재건술식이 head & neck 영역에큰파장을일으켰다. Bone morphogenetic protein (BMPs) 는 Transforming growth factor-β (TGF-β) superfamily에속하는성장인자로골과연골재생의강력한유도인자이다. BMPs는탈회된골편을 rat과 rabbit의피하조직에이식하여이식편주위로이소성골형성을관찰함으로써 Urist등에의해처음발견되었다 (Urist 1965; Urist et al., 1979). 그동안의많은연구를통해약 20여종의 BMPs가알려졌고, 생체실험에서 Recombinant human BMP-2(rhBMP-2) 의강력한골유도능력이입증되었다. 그러나, BMPs의임상적적용을위해서는보다효과적인 delivery system의개발이필요하며많은연구가진행되고있다 (Bostrum et al., 1996; Wang et al., 1990; Takaoka et al., 1991; Nakahara et al., 1988; Urist et al., 1984; Sigurdsson et al., 1996). 본연구는, E-coli system에서새롭게개발된 rhbmp-2를 absorbable collagen sponge(acs) carrier를이용해웅성백서비골의임계크기분절성골결손부에이식한후골재생을평가하고자한다. 2

12 Ⅱ. 연구대상및방법 1. 연구대상 본실험은 8주령의수컷웅성백서 (male Spraque-Dawley rat) ( g) 14마리의양쪽비골총 28 site를이용하였다. 암컷의경우호르몬의변화나임신등에의해결과에영향을미칠수있어수컷만을이용하였다. 실험동물은연세대학교의과대학임상의학연구센터의동물실험지침에따랐다. 2. 연구방법 1) 실험군 백서의분절성골결손부에 5x5x8mm, absorbable collagen sponge만이식한군을대조군으로, absorbable collgen sponge에 E-coli system에서채취한 0.1mg/ml 농도의 ErhBMP-2, 0.1ml를 micropipet을이용하여 dipping한 ErhBMP-2/ACS implant를삽입한군을실험군으로하였다. 백서우측비골을실험군으로, 좌측비골을대조군으로설정하였다. 각군은실험후각각 2주, 8주의치유기간후 CO 2 asphyxiation chamber를이용하여희생하여육안적, 방사선한적, 조직형태학적으로관찰하였다 (Table 1). 3

13 Table 1. Study design Group (n) Implants Control 2 wks (7) Absorbable collagen sponge only 8 wks (7) Absorbable collagen sponge only Experimental 2 wks (7) 8 wks (7) Absorbable collagen sponge loaded with ErhBMP-2 Absorbable collagen sponge loaded with ErhBMP-2 2) 실험방법 실험동물은 Zoletil (Virbac, Carros, France, 0.06mg/kg) 와 Rumpun (BayerKorea, Korea, 0.04mg/kg) 을복막주사하여전신마취하였다. 비골부위의털을깎은후 10% povidone-iodine 용액으로소독후고정하였으며, 1:100,000 epinephrine 함유된 lidocaine 주사후비골중앙부피부를절개하여비골을노출시켰다. 버니어캘리퍼스를이용하여무릎과발목관절의중앙부에서비골을절제하여약 6mm( 평균 6.75mm) 의분절성골결손을형성하였다. 각각의골결손부에앞에서서술한바와같이대조군에는 collagen sponge만을, 실험군에는 0.1mg/ml 농도의 ErhBMP-2, 0.1ml를 micropipet을이용하여 dipping한 ErhBMP-2/ACS implant를위치시키고, muscle cuff를유지하면서 4

14 4-0 vicryl을이용하여 layer by layer suture를시행하였다 (Fig. 1). 실험후웅성백서는 21 플라스틱우리에서연세대학교의과대학임상의학연구센터지침에따라통상적인물과 pellet을공급받았다. 발사는일주일후시행하였고, 각 2주와 8주에 CO 2 asphyxiation chamber를이용하여희생하였다. Figure 1. Immobilization of the implant was facilitated by maintaining the muscle cuffs as shown by the arrows 5

15 Figure 2. Schematic drawing of ErhBMP-2/ACS implant inserted into the bony defect after fibular segmental ostectomy 3) 방사선학적평가 (Micro-CT imaging) 희생된 SD rat의 operative site인비골과경골을함께절제하여미세컴퓨터단층촬영 (Microcomputed tomography, Micro-CT) 을실시하였다. Micro- CT(SKYSCAN 1076, Belgium) 을이용해 2차원영상을얻었고, Nrecon reconstruction software를이용하여 3차원적재구성을시행하였고, dataviwer program을이용하여동부위경골과골밀도를상대적으로분석하였다. 6

16 (resolution=18um, Voltage (kv)=100, Current (ua)= 100, Filter=0.5 mm Aluminium, Exposure (ms)=1180, Rotation Step (deg)=0.500/360 Rotation) 4) 조직형태학적평가 절제한조직은 4 C 에서 4% paraformaldehyde/0.01m phosphatebuffered saline (PBS, ph 7.4) 에 24시간고정후, 실온에서 formic acidsodium formate로탈회하였다. 파라핀에포매후 7 μm 두께로절단하여 hematoxylin and eosin (H-E stain) 염색하여광학현미경으로관찰하였다. 7

17 Ⅲ. 결과 1. 육안적소견 1) 2 주군 대조군에서는분절성골결손부골재생이일어나지않아비유합이관찰되었고, 실험군에서는골재생으로완전유합이이뤄졌지만, 불규칙한피질골과과도한 (bulky) 골형성이관찰되었다 (Fig. 3; Aa-Ac). 2) 8 주군 대조군에서는분절성골결손부골재생이일어나지않아비유합이관찰되었고, 실험군에서는골재생으로완전유합뿐만아니라, 재생된비골을확인할수없을 정도의규칙적이고평활한골형성이관찰되었다 (Fig. 3;Ba-Bd). 2. 방사선학적소견 1) 방사선학적소견 (Standard dental X-ray) (1) 2 주군 대조군에서는골재생이일어나지않아비유합이관찰되었고, 실험군에서는 육안적관찰과일치하는소견을보이며, 골재생으로완전유합되었지만, 과도한 8

18 골양과기존의비골보다골질의저하가관찰되었다 (Fig. 4A). (2) 8 주군대조군에서는골재생이일어나지않아비유합이관찰되었고, 실험군에서는완전유합과함께기존의비골과유사할정도의골양및골질이관찰되었다 (Fig. 4B). 2) Micro-CT imaging (1) 3D reconstruction imaging 2 주군, 8 주군모두에서육안적, 방사선적소견과일치하였다 (Fig. 5). (2) Bone density 1 2 주실험군피질골층의불연속성과기존의비골에비해낮은골밀도를보이며 (Fig. 6), 상대적인골밀도비교한 graph에서인접한정상경골절반정도의골밀도를확인할수있으며, 재생된비골부위의피질골층과골수강부위의명확한경계가없음을알수있다 (Fig. 7). 2 8 주실험군피질골층의연속성과기존의비골과유사한골밀도를 Longitudinal & cross section을통해관찰되었고 (Fig. 6), 인접한정상경골과유사한정도의골밀도를보여주며, 재생된비골부위의피질골층과골수강부위가명확히구분됨을알수있다 (Fig. 7). 9

19 3. 조직형태학적소견 1) 2 주실험군 새롭게형성된골양이적으며, 기존에남아있던비골과의경계를확인할수있다 (x50, Fig 11). 상방의정상비골의 lamella bone과는달리 collagenous tissue와다수의혈관그리고미성숙 woven bone의형성이보인다 (x50, Fig 11). 재생중인골조직은활성화된 spindle type의 fibroblast가보이며, chondrocyte와연골형성부도관찰되며, 연골이 woven bone으로변하는부위도관찰된다 (x200, Fig 13). 활발한골형성을위한혈관형성이풍부한부위도관찰된다 (x100, Fig 12). 2) 8 주실험군 새롭게형성된골양이 2주실험군보다많이관찰되며, 2주군에서보였던 woven bone이 lamellar bone으로변화된것이관찰된다 (x200, Fig 18). 또한기존에남아있던비골과의경계부를뚜렷이확인할수없다 (Fig 14-16). 2주실험군에서골수강부위에형성되었던 woven bone이골수로대체되는것이관찰된다 (Fig 16-17). 10

20 Ⅳ. 고찰 rhbmps의골재생효과및안정성은여러동물모델을이용한임상전단계실험을통해입증되었고 (Marden et al., 1994; Bostrum et al., 1996; Mayer et al., 1996; Cook et al., 1994; Yasko et al., 1992; Toriumi et al., 1991; McKay et al., 2002), 최근에는임상적용또한보고되었다 (Boyne et al., 1997; Herford et al., 2008). 따라서 rhbmps에관한새로운테마는농도를최소화하면서골재생을최대로할수있는효과적인전달체의개발에초점이맞춰지고있다. 이상적인전달체는생체친화성, 쉬운조작성, 해당부위의연조직개입을막으며, 골형성이이뤄지기전까지공간유지능력등이요구된다. Biodegradable polymers, bone derivatives, Absorbable collagen sponge (ACS) (Boyne et al., 1997), Decalcified bone matrix (Toriumi et al., 1991), Hyaluronan (Hunt et al., 2001), Deproteinized bovine bone matrix (Jung et al., 2003), Hydroxyapatite (HA) (Koempel et al.1998), Calcium phosphates (Wikesjo et al., 2002), Polylactic acids (Weber et al., 2002), Polymethylethyl-methacrylate (Boyne, 2001) 등의전달체를이용한 rhbmps의사용은성공적으로골재생을할수있지만, 재현성의부족, 면역반응및바이러스전파가능성 (Asselmejer et al., 1993; Nemzek et al., 1994), 생체용해성의한계, 형태주조의어려움 (hydroxyapatite, tricalcium phosphate), cell infiltration이가능한다공성구조의부족, 강도의부족 (PMS/blood, collagen) 등의다양한문제점이보고되었다. 이런한계를극복하기위한노력이지속되고있음에도불구하고현재까지명확히규명된바는없다. 11

21 Han등은, 손상부는숙주의정상적인치유과정에의해시작되기때문에전달체의디자인또한정상조직치유과정의구조와순서와유사하게고안되어야한다는이론적측면을바탕으로한가설을제창하였다. 정상치유과정의깊은이해가 BMPs의이상적인전달체개발의중요한단서가될수있으며, 골치유과정시작인 blood clot 형성에중요한역할을하는 heparin- fibrinfibronectin 복합체를이용한전달체계의개발이 BMPs 전달체계의새로운장을열것이라고보고하였다 (Han et al., 2008). 구강악안면영역에서 rhbmps 다양한전달체중 ACS에대한많은연구가보고되었고, 정형외과영역에서도임상전단계및임상적용한증례의보고가있었다 (McKay et al., 2002). 이같은노력으로 2002년에처음으로 1.5mg/cc 농도의 rhbmp-2/acs (INFUSE Bone Graft, Medtronic Spinal and Biologics, Memphis, TN) 가 interbody spinal fusion에서의사용이 FDA 승인을받았고, 2004년에는개방성경골골절에 intrarmedullary (IM) nail fixation에서의사용, 2007년 3월에는구강악안면영역에서최초로상악동거상술및치조골융기술 (Ridge augmentation) 에서의사용이승인을받게되었다. Boyne et al.(1997) 은 12 명의환자를대상으로상악동저거상술에있어서 0.43 mg/ml, rhbmp-2/acs 를이용한골유도의안정성및실행가능성을 평가하였다 (Total dose : 1.77 to 3.40 mg). 특기할합병증이나, 혈구수, 혈액화학적, 뇨분석의변화없이평균 8.51mm 의골형성을관찰함으로써 rhbmp-2/acs 의안정성및골이식을대체할만한이식재임을입증하였다 (Boyne et al., 1997). 12

22 Howell et al.(1997) 은 0.43 mg/ml rhbmp-2/acs를 ridge preservation & augmentation에적용해국소적, 전신적부작용이없었다는임상보고를하였고, rhbmp-2/acs는이술식에적용하기쉽고, 골형성및유지에유용하다는결론을내렸다 (Howell et al., 1997). Fiorellini et al.(2005) 은협측골벽결손으로 socket augmentation이필요한 80명의환자를대상으로발치후아무처치도시행치않은음성대조군, ACS만넣은양성대조군, rhbmp-2/acs 두농도 (0.75 mg/cc, 1.5 mg/cc) 를실험군으로 4개월후그효과를비교평가하였다. 실험군에서골높이의증가와골폭의증가를관찰할수있었고, 특히 1.5 mg/cc rhbmp-2/acs 그룹에서임플란트를위한적절한골높이및폭을얻었으며, 조직학적으로도새로형성된골이남아있던원래의골과차이가없었다고보고하였다 (Fiorellini et al., 2005). Boyne et al.(2005) 은상악동저거상술이필요한환자를대상으로 rhbmp- 2/ACS에관한임상적연구를보고하였다. 이연구에서, 골이식술을시행한군 (n=13) 과, 0.75 mg/cc rhbmp-2/acs (n=18), 1.5 mg/cc rhbmp-2/acs (n=17) 를이식한군을비교평가하였다 (Boyne et al., 2005). 골이식, 0.75 mg/cc rhbmp-2, 1.5 mg/cc rhbmp-2에서각각 mm, 9.47 mm, mm 골높이의변화가있었고, 4개월후새로이형성된골밀도는각각 350 mg/cc, 84 mg/cc, and 134 mg/cc 로통계학적차이가있었다. 또한재생된골에임플란트식립시 core biopsy를통한조직학적소견에서정상적인골성장및구조를확인하였고, functional loading후 6개월경과관찰하여 CT scan으로각군의골밀도를비교하였다. 1.5 mg/cc 농도의 rhbmp-2/acs로제작된 INFUSE Bone Graft(FDA approved) 를상악동저거상술에적용하여그효용성과안정성을평가한연구가 13

23 있었다 (INFUSE, 2007). 총 160명의상악동저거상술이필요한환자에서 82명은 rhbmp-2/acs 를, 78명은골이식을시행하였으며, 골이식군은자가골만을이용하거나자가골과동종골 (allogenic bone) 을혼합하여적용하였다. 술후 6개월에 INFUSE Bone Graft 를시행한군과골이식을시행한군에서 각각 7.83 mm 와 9.46 mm 의골높이의변화가있었다. 조직학적으로, 두군 모두에서생물학적, 구조적으로숙주의골과유사한새로운 trabecular bone 형성을관찰할수있었다. Functional loading 6개월후, INFUSE Bone Graft group 의임플란트 survival rate는 73%~ 79% 임을확인할수있었고, 12개월후의 implant success rates 는두그룹에서유사하였다. 기타임상적인부작용또한관찰되지않았다. 앞서언급한임상적연구들이 FDA 승인을받을수있었던원동력이되었고, ridge augmentation 에 1.5 mg/cc, rhbmp-2/acs 인 INFUSE Bone Graft 의 적용은임플란트의골유착및 functional loading 이가능한골형성에자가골 이식을대체할술식으로사료된다. 분절성골결손부에 rhbmps를적용한다양한연구가있었다. Marukawa et al.(2002) 은 6마리의원숭이하악에 30mm의분절성골결손부에 2mg/ml, 9 mg of rhbmp-2/poly-d, L-lactic-co-glycolic acid-coated gelatin sponge (PGS) 를 titanium plate를이용하여이식후골재생을평가하였다 (Marukawa et al., 2002). 술후 20주에, 재생된골은정상하악골의형태를유지하였고, 임플란트를식립하기에충분한폭과형태를관찰할수있었으며, 숙주골과구별하기어려울정도였다고보고하였다. 또한시간이지날수록방사선적골밀도증가, 정상하악골과유사한형태로 remodeling 및술후 30-52주사이에서 14

24 최소방사선적변화를관찰함으로써약 1년의 remodeling을위한기간을언급하였다. Toriumi et al.(1991) 은 250 mg rhbmp-2/inactivated, bone derived (dog) Matrix를이용해술후 6개월에하악골연속성을완성하였다고보고하였고 (Toriumi et al., 1991), Mayer 등 (1996) 은 200 mg rhbmp-2/poly(lactide-co-glycolide) 를이용하여술후 4개월에치조열수복을보고하였다 (Mayer et al., 1996). 2008년, Herford 와 Boyne 는큰하악골결손부에 rhbmp-2/acs의임상적용증례를보고하였다. 종양에의한골결손및 bisphosphonates, 방사선치료에의한골수염으로발생된골결손환자 14명의하악체와하악각결손부에 다른골이식재없이 rhbmp-2/acs (INFUSE ; Medtronics, Nashville, TN) 이식을시행하였다. 절제된골결손부의길이는 4-8cm( 평균 6.1 cm) 이었고, 용량은 4-8 mg로결손부크기에따라다르게적용하였으며, plate와 titanium mesh를공간유지를위해사용하였다. 술후 3-4개월에재생된골을촉진할수있었고, 5-6개월에는방사선학적으로확인이가능했다. 모든증례에서성공적인골재생을이뤘고, 임플란트및보철치료를시행하여교합기능을회복하였다고보고하였다 (Herford et al., 2008). Herford AS & Boyne PJ등은 ACS의구조적안정성의결여로인한연조직붕괴, 공간유지능력의부재, 과도한골재생을제한하기위해 titanium mesh나 plate를사용했다고보고하였다. 그러나본연구에서 conduit이나 plate 등의공간유지장치없이단지 muscle fascia를잘보존하여 layer by layer suture만시행하여도임상적, 조직형태학적으로연조직개입없는잘형성된골을확인할수있었다. 본연구 2주군에서는과도한골성장을관찰할수있었으나, 8주군에서는 remodeling으로인한 smoothening을볼수있듯이, rhbmps에의한골성장 15

25 조절로인한과정임을보여준다. 이를종합해보면, ACS 는연조직개입이나 공간유지능력이충분한적절한전달체로사료된다. 기존의포유류세포 (CHO cell) 를이용하여생산된 rhbmp는골형성에있어서효과적인것으로입증되었으나, 생산비용이높아일상적인임상시술에적용시키는데에는어려움이있다. 따라서, 새로운방식에의해 BMP를추출, 정제하기위한노력들이있어왔다 (Maruoka et al., 1995). 여러노력끝에 E- coli같은박테리아시스템을이용하여 BMP-2와 BMP-4가생산되어그활성이 in vivo와 in vitro에서확인되었고 (Kubler et al., 1998; Vallejo et al., 2002; Cho et al., 2008), Vallejo등 (2002) 은동물세포에서생산한 rhbmp-2와 E- coli에서생산한 rhbmp-2 homo-dimer의생물학적활성은차이가없다고보고하였으며, Cho KS 등 (2008) 은동물세포에서 E-coli에서생산한 rhbmp-2의활성이충분히나타났다고보고하였다 (Cho et al., 2008). E-coli system 에서의 BMP-2정제는 in vitro에서 inclusion body의 refolding 과정을통해서생산된다. 그러나이런 refolding 과정은복잡하고정제된양또한적으며, 비싼시약의필요로인한경제적측면에서도단점이있다. 여러연구끝에적은비용과간단한과정으로통해 E-coli system 에서 rhbmp-2 를정제하는새로운방법이보고되었다 (Cho et al., 2008). 따라서이런 E-coli 등의박테리아시스템에서생산된 rhbmp는다량생산이가능하며, 경제적으로 BMP의생산비용을낮추어 BMP의임상적용을보다쉽게할수있다는장점이있다. 본연구는웅성백서비골분절성골결손부에 E-coli system 에서새롭게 개발된 Recombinant human BMP-2(rhBMP-2) 를 absorbable collagen 16

26 sponge(acs) 전달매체로이식하여골재생효과를평가하였다. 웅성백서비골에 critical size defect인약 6mm의분절성골결손부를형성하여대조군에는 ACS만을, 실험군에는 ErhBMP-2를적용한 ACS를이식한후, 술후 2주와 8주에 CO 2 chamber를이용하여희생하여임상적, 방사선학적, 조직학적소견을비교관찰하였다. 본연구모델에서이용한 6mm의분절성결손은쥐의임계크기결손 (critical size defect) 으로, 이는자발적으로골치유가되지않는, 10% 이하의골재생을이루는가장작은결손을의미한다 (Choi et al., 2005; Schmitz et al., 1986; Hollinger et al., 1990). 임상적으로 2주실험군에서는골재생으로완전유합이이뤄졌지만, 불규칙한피질골과과도한 (bulky) 골형성이관찰되었고 8주실험군에서는골재생으로완전유합뿐만아니라, 재생된비골을확인할수없을정도의규칙적이고평활한골형성을관찰할수있었다. 방사선학적으로, 2주실험군에서는골재생으로완전유합되었지만, 과도한골양과기존의비골보다골질의저하및피질골층의불연속성을확인하였다. 8주실험군에서는완전유합과함께골양및골질이기존의비골과유사한양상및피질골층의연속성과기존의비골과유사한골밀도를확인하였다. 2주에재생된과도한골이 rhbmp-2의성장조절능력에의한 remodeling으로 8주에임상적, 방사선학적, 조직학적으로완벽한골재생을이루었다. 앞으로임상적용을위해서 remodeling이시작하여종료되는시기의확인이필요하며, 1주간격으로방사선학적분석과이를통한밀도를평가하는추가적인연구가필요할것으로사료된다. Yasko et al.(1992) 은백서 femora에 5mm 분절성골결손부에 guanidinehydrochloride extracted deminer alized rat bone matrix와각각다른농도 (1,4,11mg) 의 rhbmp-2를이식하여골재생을평가하였다. 그들은 11mg 17

27 rhbmp-2를사용한군에서 4.5주에방사선학적으로연속성을확인할수있었고, 9주후에피질골층과 intramedullary canal의연속성을확인하였다고보고하였다 (Yasko et al., 1992). Marden et al.(1994) 은백서 craniotomy model에 guanidine-hydrochloride extracted deminer alized rat bone matrix와각각다른농도 (2.2 or 6.5mg) rhbmp-2를이식하여골재생을평가하였다. 약 2주후, 6.5mg rhbmp-2를이용한군에서완벽한재생을관찰할수있었다고보고하였다 (Marden et al., 1994). 위두연구에서, 같은실험동물과같은전달체를이용하였음에도불구하고다른농도의적용은주목할만하고, 이는 rhbmp-2가그수혜부에따라다른농도가적용되며, intramembranous skull CSD와 endochondral femur CSD간의발생학적차이가 rhbmp-2 재생능력에영향을미치고, 이는수혜부에따른 vascularity와 cellularity의차이또한영향을미친다고보고하였다 (Hollinger et al., 1998). 본연구에서는 Rat의 critical size calvarial defect model에적용한예비실험결과에준하여 0.1mg/ml의농도를선택하였다. 이는기존의연구뿐 아니라, FDA 승인받은 INFUSE (1.5mg/ml) 보다도훨씬적은농도로훌륭한 골재생을확인할수있었다. 또한 E-coli system에서새로이개발된 rhbmp- 2를이용해기존의연구에서보다적은용량 (0.1ml) 을사용했다는점도주목할만한점이다. 앞서언급한 Maden(1994) 의보고처럼수혜부의발생학적차이는분명 rhbmp-2 골재생에영향을줄수있고, 그수혜부에따른농도와용량의차이가존재할것으로생각된다. 본연구에서적은농도와적은용량으로훌륭한골재생을확인하였지만, 실험에서사용된 0.1mg/ml의농도와 0.1ml의용량이분절성골결손수복을위한적정농도와용량인가에대해서는추가적인연구가필요하다. 18

28 저자등은 E-coli system에서새롭게개발된 ErhBMP-2/ACS를비골의분절성골결손부에적용하여훌륭한골재생효과를확인하였다. E-coli system에서개발된 ErhBMP-2는기존의 rhbmp-2보다적은농도와용량으로완벽한골재생을할수있다는점과, 대량생산이가능하며, 경제적으로 BMP의생산비용을낮출수있다는장점이있다. 여러장점을갖춘 E-coli system에서개발된 ErhBMP-2를이용하여다른부위에서의골형성관찰등의추가적인연구가진행된다면머지않은시기에임상적용이가능할것으로사료된다. 19

29 Ⅴ. 결론 외상, 기형, 종양의절제등에기인한악안면부골결손수복에있어, 자가골이식의한계를극복하고자골유도인자인 rhbmp-2를이용한골재생의연구가활발히진행중이다. 본연구는, E-coli system에서새롭게개발된 ErhBMP-2를 absorbable collagen sponge(acs) carrier를이용해웅성백서비골의임계크기 (6mm) 분절성골결손부에이식한후골재생을평가하였다. 실험은 8주령의 SD rat 14마리를대상으로하였으며, 비골에약 6mm 의임계크기분절성골결손을형성한후 absorbable collagen sponge만이식한군 ( 대조군 ) 과 rhbmp-2를 absorbable collagen sponge에 dipping하여이식한군 ( 실험군 ) 으로나누었다. 모든 SD rat은 2주와 8주경과후각각희생하였으며, 육안적, 방사선학적 (Standard dental X-ray, Micro-CT imaging), 조직형태학적검사로골재생을관찰하였다. 2 주대조군에서는분절성골결손부에골재생이일어나지않은비유합상태가관찰되었고, 실험군에서는골재생으로완전유합이이뤄졌지만, 육안적소견상불규칙한피질골과과도한골형성이관찰되었다. 방사선학적으로, 대조군에서는골재생이일어나지않아비유합이관찰되었고, 실험군에서는육안적관찰과일치하는소견을보이며, 골재생으로완전유합되었지만, 재생된골의양이과도하고, 인접한정상비골에비해골질이낮은상태임이관찰되었다. 골밀도에있어, 실험군에서는피질골층의불연속성과기존의비골에비해낮은골밀도를보이며, 인접한정상경골의절반정도의골밀도를보였다. 조직학적으로재생된골의양이적고, 남아있던근위부정상비골과의경계가남아있음을확인할수 20

30 있었다. 남아있던정상비골의판상골 (lamella bone) 과는달리콜라겐 (collagenous tissue) 과다수의혈관그리고미성숙골 (woven bone) 의형성이관찰되었다. 8주대조군에서도분절성골결손부골재생이일어나지않은비유합상태가관찰되었고, 실험군에서는골재생으로완전유합뿐만아니라, 재생된비골과남아있던근위부정상비골과의경계를확인할수없을정도의규칙적이고평활한골형성이관찰되는육안적소견을보였다. 방사선학적으로, 대조군에서는골재생이일어나지않아비유합이관찰되었고, 실험군에서는완전유합과함께기존의인접정상비골과유사한양상의골양및골질이관찰되었다. 골밀도에있어, 실험군에서는피질골층의연속성과기존의인접정상비골과유사한골밀도를확인할수있었다. 조직학적으로새롭게형성된골양이 2주실험군보다많이관찰되었고, 2주군에서보였던미성숙골이판상골로변화된것이관찰되었다. 또한기존에남아있던비골과의경계부를뚜렷이확인할수없었으며, 2주실험군에서골수강부위에형성되었던 woven bone이골수로대체되는것이관찰되었다. 8주실험군에서 2주실험군보다골개조과정을거친성숙된층판골형성이관찰되었지만, 2주, 8주실험군모든개체에서명확한골재생을확인하였다. E-coli system을이용하여새롭게개발된 ErhBMP-2는기존의 rhbmp-2보다적은농도와용량으로완벽한골재생을할수있다는점과, 대량생산이가능하며, 경제적으로 BMP 생산비용을낮출수있다는장점이있다. E-coli system을이용하여새롭게개발된 ErhBMP-2와 absorbable collagen sponge의이식은분절성골결손부수복에유용하고, 자가골이식을대신할만한적절한방법중의하나로사료된다 21

31 Aa Ba Ab Bb Ac Bd Bc Figure 3. Gross findings of regenerative fibula. Aa : 2wks Experimental side, There is a complete union on the defect site with irregular cortex on the bulky regenerative area. Ab : 2wks Experimental side (Posterior view) Ac : 2wks Control side, Observed nonunion in defect site (lateral view) Ba : 8wks Experimental side, There is complete union on the defect site with regular cortex on the smooth regenerative area. Bb : 8wks Experimental side (Posterior view), There is remodeling of the irregular surface Bc : 8wks Experimental side (zoom) Bd : 8wks Control side, Observed nonunion in defect site (lateral view) ２２

32 A B Figure 4. Radiographic finding A : 2 weeks group, Images are similar to gross finding B : 8 weeks group, Images are similar to gross finding Aa Ab Ba Bb Figure 5. Micro-CT finding (3D reconstruction imaging) A : 2 weeks group, Images are similar to gross finding B : 8 weeks group, Images are similar to gross finding 23

33 2wks 8wks Figure 6. Micro-CT finding : Longitudinal & Cross sectional view There is a discontinuity of the cortical layer with low density of the bone marrow on the 2nd week group, and there is a continuity of the cortical layer with similar density of the bone marrow. 24

34 2wks Cortex Tibia Cortex Regenerative Bone marrow 8wks Cortex Tibia Cortex Regenerative Bone marrow Figure 7. Comparison of the bone density between neighboring and regenerative fibula at 2 weeks and 8 weeks post-op. Upper : Bone density on the 2 weeks experimental group was lower than that of half the density of the adjacent tibia Lower : Bone density on the 8 weeks experimental group was identical with adjacent tibia. 25

35 Figure 8. x50 magnification of 2weeks group : Non-experimental area(pre-existing bone) Figure 9. x50 magnification of 2weeks group : Border area New immature woven bone was seen. There was bony gap between the new bone and the adjacent fibula. 26

36 Figure 10. x50 magnification of 2weeks group : Experimental area New immature woven bone was seen. NB Figure 11. x50 magnification of 2weeks group : Border area There was definite margin between the new bone and the adjacent fibula. (NB : New bone, arrow : defect margin) 27

37 Figure 12. x100 magnification of 2weeks group : Experimental area New immature woven bone, collagenous tissue & rich blood vessels was seen. Figure 13. x200 magnification of 2weeks group : Experimental area Blue arrows : chondrocyte Red arrows : spindle type, fibroblast 28

38 Figure 14. x50 magnification of 8weeks group : Non-experimental area(pre-existing bone) Figure 15. x50 magnification of 8weeks group : Border area There was no noticable bony gap between the adjacent pre-existing fibula 29

39 Figure 16. x50 magnification of 8weeks group : Experimental area The woven bone that was observed at 2 weeks was replaced with bone marrow. Figure 17. x100 magnification of 8weeks group : Experimental area The woven bone that was observed at 2 weeks was replaced with bone marrow. 30

40 Figure 18. x200 magnification of 8weeks group : Experimental area Immature woven bone that observed at 2 weeks was replaced with lamellar bone 31

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44 Urist, M. R., Lietze, A., Dawson, E b-tricalcium phosphate delivery system for bone morphogenetic protein. Clin Orthop Relat Res 187 : Sigurdsson, T. J., Nygaard, L., Tatakis, D. N., Fu, E., Turek, T. J., Jin, L., Wozney, J. M., Wikesjö, U. M Periodontal repair in dogs: evaluation of rhbmp-2 carriers. Int J Periodontics Restorative Dent 16(6): Ahn, S. H., Kim, C. S., Suk, H. J., Lee, Y. J., Choi, S. H., Chai, J. K., Kim, C. K., Han, S. B. Cho, K. S Effect of recombinant human bone morphogenetic protein-4 with carriers in rat calvarial defects. J Periodontol 74(6) : Marden, L. J., Hollinger, J. O., Chaudhari, A., Turek, T., Schaub, R., Ron, E Recombinant bone morphogenetic protein-2 is superior to demineralized bone matrix in repairing craniotomies defects in rat. J Biomed Mater Res 28(10) : Mayer, M. H., Hollinger, J. O., Ron, E., Wozney, J Repair of alveolar clefts in dogs with recombinant bone morphogenetic protein and poly(ahydroxy acid). Plast Reconstr Surg 98 : Cook, S. D., Baffes, G. C., Wolfe, M. W., Sampath, T. K., Rueger, D. C Recombinant human bone morphogenetic protein-7 induces healing in a canine long-bone segmental defect model. Clin Orthop Relat Res 301 : Yasko, A. W., Lane, J. M., Fellinger, E. J., Rosen, V., Wozney, J. M., Wang, E. A The healing of segmental bone defects, induced by recombinant human bone morphogenetic protein (rhbmp-2). A radiographic, histological, and biomechanical study in rats. J Bone Joint Surg Am 74(5) :

45 Toriumi, D. M., Kotler, H. S., Luxenberg, D. P., Holtrop, M. E., Wang, E. A Mandibular reconstruction with a recombinant boneinducing factor. Functional, histologic, and biomechanical evaluation. Arch Otolaryngol Head Neck Surg 117(10) : McKay, B., Sandhu, H. S Use of recombinant human bone morphogenetic protein-2 in spinal fusion applications. Spine 27(16-1): S66-S85. Boyne, P. J., Marx, R. E., Nevins, M., Y, cols A feasibility study evaluation rhbmp-2/absorbable collagen sponge for maxillary sinus augmentation. Int J Periodont Restor Dent 17 : Herford, A. S., Boyne, P. J Reconstruction of mandibular continuity defects with bone morphogenetic protein-2 (rhbmp-2). J Oral Maxillofac Surg 66 : Asselmejer, M. A., Caspari, R. B., Bottenfield, S A review of allograft processing and sterilization techniques and their role in transmission of the human immunodeficiency virus. Am. J Sports Med 21(2) : Nemzek, J. A., Arnoczky, S. P., Swenson, C. L Retroviral transmission by the transplantation of connective-tissue allografts. An experimental study. J Bone Joint Surg Am 76(7) : Han, D., Liu, W., Ao, Q., Wang, G Optimal delivery systems for bone morphogenetic proteins in orthopedic applications should model initial tissue repair structures by using a heparin-incorporated fibrin-fibronectin matrix. Med Hypotheses 71(3):

46 Howell, T. H., Fiorellini, J., Jones, A., Alder, M., Nummikoski, P., Lazaro, M., Lilly, L., Cochran, D A feasibility study evaluating rhbmp- 2/absorbable collagen sponge device for local alveolar ridge preservation or augmentation. Int J Periodontics Restorative Dent 17(2) : Boyne, P. J., Lilly, L. C., Marx, R. E., Moy, P. K., Nevins, M., Spagnoli, D. B., Triplett, R. G De novo bone induction by recombinant human bone morphogenetic protein-2 (rhbmp-2) in maxillary sinus floor augmentation. J Oral Maxillofac Surg 63(12) : Fiorellini, J. P., Howell, T. H., Cochran, D., Malmquist, J., Lilly, L. C., Spagnoli, D., Toljanic, J., Jones, A., Nevins, M Randomized study evaluating recombinant human bone morphogenetic protein-2 for extraction socket augmentation. J Periodontol 76(4) : Medtronic package insert no.m704819b INFUSE Bone Graft for Certain Oral Maxillofacial and Dental Regenerative Uses McKay, W. F., Peckham, S. M., Badura, J. M A comprehensive clinical review of recombinant human bone morphogenetic protein-2 (INFUSE Bone Graft). Int Orthop 31(6) : Marukawa, E., Asahina, I., Oda, M., Seto, I., Alam, M., Enomoto, S Functional reconstruction of the non-human primate mandible using recombinant human bone morphogenetic protein-2. Int J Oral Maxillofac Surg 31(3) : Maruoka, Y., Oida, S., Iimura, T., Takeda, K., Asahina, I., Enomoto, S., Sasaki, S Production of functional human bone morphogenetic protein -2 using a baculovirus/sf-9 insect cell system. Biochem Mol Biol Int 35(5) :

47 Kubler, N. R., Reuther, J. F., Faller, G., Kirchner, T., Ruppert, R., Sebald, W Inductive properties of recombinant human BMP-2 produced in a bacterial expression system. Int J Oral Maxiilofac Surg 27(4) : Vallejo L. F., Brokelmann, M., Marten, S., Trappe, S., Cabrera-Crespo, J., Hoffmann, A., Gross, G., Weich, H. A., Rinas, U Renaturation and purification of bone morphogenetic protein-2 produced as inclusion bodies in high-cell-density clutures of recombinant Escherichia coli. J Biotech 94(2) : Choi, K. H., Moon, K. O., Kim, S. H., Yun, J. H., Jang, K. L., Cho, K. S Purification and biological activity of recombinant human bone morphogenetic protein-2 produced by E. coli expression system. 대한치주과학회지 38 : Huh, J. Y., Choi, B. H., Kim, B. Y., Lee, S. H., Zhu, S. J., Jung, J. H Critical size defect in the canine mandible. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 100(3) : Schmitz, J. P., Hollinger, J. O The critical size defect as an experimental model for craniomandibulofacial nonunions. Clin Orthop Relat Res 205 : Hollinger, J. O., Kleinschmidt, J. C The critical size defect as an experimental model to test bone repair materials. J Craniofac Surg 1(1) : Hollinger, J. O., Schmitt, J. M., Buck, D. C.,, Shannon, R., Joh, S. P., Zegzula, H. D., Wozney, J Recombinant human bone morphogenetic protein-2 and collagen for bone regeneration. J Biomed Mater Res 43(4) :

48 Abstract Bone regeneration in a critical sized segmental defect in the SD rat fibula using a ErhBMP-2/ACS implant Se Hyun Park, D.D.S. Department of Dentistry, The Graduate School, Yonsei University (Directed by Professor Hyung Jun Kim, D.D.S., M.S.D., Ph.D.) Presently, there are many studies on bone formation by using rhbmp-2 in order to overcome the limitations of autogenous bone graft for reconstruction of maxillofacial bone defects. In this study, we examined the osteoinductive property of the newly pioneered ErhBMP-2 from E-coli system on the critical sized segmental bone defect of SD rat fibula by using absorbable collagenous sponge carrier. Segmented bone defect with critical sized of 6mm was formed on each fibula of 14 rats and they were divided into two groups. Experimental group was grafted with absorbable collagen sponge dipped with ErhBMP-2 and the control group was grafted with absorbable collagen sponge only. All SD rats were sacrificed after 2 weeks and 8 weeks. Bone formation was observed grossly, radiographically(by using standard dental X-ray and Micro-CT imaging) and histologically. 39

49 In the 2 weeks control group, there was no bone formation in the segmented bone defect which resulted in nonunion. In the experimental group, there was irregular and excessive cortical bone formation which resulted in union. Radiographically, there was no noticable bone formation in the control group, while there was irregular and excessive cortical bone formation which resulted in complete union. Bone density on the experimental group was lower than that of half the density of the adjacent tibia with discontinuity on cortical layer. Histologically, there was bony gap between the new bone and the adjacent fibula. Also, collagenous tissue, numerous blood vessels and woven bone formation was observed. In the 8 weeks control group, there was no bone formation in the segmented bone defect which resulted in nonunion. In the experimental group, there was regular bone formation which resulted in union. Radiographically, there was no noticable bone formation in the control group, while there was regular bone formation which was identical with the adjacent bone. Bone density on the experimental group was identical with adjacent tibia. Histologically, there was more newly formed bone in the 8 weeks group than 2 weeks group and lamella bone formation was observed in the 8 weeks experimental group. Also, there was no noticable bony gap between the adjacent fibula and the woven bone that was observed at 2 weeks was replaced with bone marrow. There was definite bone formation in the 2 weeks and 8 weeks experimental group, and there was mature lamella bone formation in the 8 40

50 weeks group compared to 2 weeks group. ErhBMP-2 from E-Coli system has advantage of better bone formation with less concentration compared to that of previous rhbmp-2. Also, it makes the cost of production decrease economically. EhrBMP-2 with absorbable collagen sponge could be considered as a new method that can substitute autogenous bone graft in the segmented bone defect. Keywords : rhbmp-2, Absorbable collagen sponge, E-coli, SD rat critical size segmental bone defect. 41